Download immunology syllabus 2013 - The University of Texas Medical School

IMMUNOLOGY
SYLLABUS
2013
University of Texas Medical School at Houston
“The Immune System is a Vital Organ System,
Necessary for Life.”
IMMUNOLOGY SYLLABUS 2013 - TABLE OF CONTENTS
Course Schedule
iii
Course Description
iv-vi
Essay Assignment + Exam/Essay Review Policy
vii-ix
Clinical Correlation: Reading List
x
Team Based Learning Exercise - Integrative Exercises
xi-xii
Lectures & Clinical Correlations
(Begins at Page Number 1)
OVERVIEW AND ELEMENTS OF THE IMMUNE SYSTEM
Medical Importance of the Immune System / How the Immune System Works
Cells and Organs of the Immune System
Innate Immunity/Inflammation
Syllabus Page
1
13
22
ANTIGENS, ANTIBODIES AND T CELL RECEPTORS - STRUCTURE AND ACTIVITIES
Immunogens & Antigens
Antibody Structure and Function I+II
38
49
COMPLEMENT
Complement
73
ANTIBODY, T CELL RECEPTORS, AND MHC – STRUCTURE AND ACTIVITIES
Genetic Basis of Ab Structure
Role of MHC in the Immune Response
The T Cell Receptor: Structure and Genetic Basis
Adaptive Immune Response: I+II
92
101
114
123
CELLULAR ACTIVITIES AND IMMUNE MEDIATION
Antigen-Antibody Interactions - ImmunoAssays
Antibody-Mediated Reactions
Cell-Mediated Reactions
145
162
173
IMMUNE SYSTEM AND INFECTIOUS DISEASE
Immunology of HIV Infection
Infection and Immunity
182
194
MEDICAL APPLICATIONS OF IMMUNOLOGY (Immunopathology)
Immune Regulation & Tolerance
Autoimmunity
Clinical Scenarios
Immunology of Cancer
Immunoprophylaxis (Vaccines) & Immunotherapy
Disorders of the Immune Response
Transplantation
Team Based Learning Exercise
Evolution of the Immune System
206
219
226
227
228
239
251
254
255
Timeline of Immunology
(located at end of syllabus)
(located at end of syllabus)
APPENDIX: Resource Information
i
Cover Description: Principles of Modern Immunobiology. B.H. Park and R.A. Good. 1974.
Lea & Febiger, Henry Kimpton Publishers, Philadelphia. p54.
The purpose of the Immunology course is to provide a basic knowledge of the immune response and
its involvement in health and disease. A series of lectures cover course components; additional
materials are presented through clinical correlations that focus on clinically applied immunological
concepts. An effort has been made to increase clinical relevance and problem-solving skills through
an essay assignment and through a team-learning exercise.
ii
SCHEDULE - IMMUNOLOGY 2013
Session
Date
Time
Instructor
OVERVIEW AND ELEMENTS OF THE IMMUNE SYSTEM
Jeffrey Actor
1 1/8/2013 10:00-10:50
2 1/8/2013 11:00-11:50
Jeffrey Actor
3 1/11/2013 11:00-11:50
Jeffrey Actor
ANTIGENS AND ANTIBODIES
4 1/15/2013 10:00-10:50
5 1/15/2013 11:00-11:50
6 1/18/2013 9:00-9:50
COMPLEMENT
7 1/22/2013 10:00-10:50
MEDIC web site
Topic
Medical Importance of the Immune System
Cells and Organs of the Immune Sytstem
Innate Immunity/Inflammation
Immunogens & Antigens
Antibody Structure and Function I
Antibody Structure and Function II
Sudhir Paul
Keri Smith
Keri Smith
Complement
Rick Wetsel
ANTIBODIES, T CELL RECEPTORS, AND MHC - STRUCTURE AND ACTIVITIES
Steven Norris
Genetic Basis of Ab Structure
8 1/22/2013 11:00-11:50
9 1/24/2013 9:00-9:50
Jeffrey Actor
Role of MHC in the Immune Response
10 1/24/2013 10:00-10:50
Jeffrey Actor
The T Cell Receptor: Structure and Genetic Basis
11 1/24/2013 11:00-11:50
Jeffrey Actor
Adaptive Immune Response 1
12 1/25/2013 10:00-10:50
Jeffrey Actor
Adaptive Immune Response 2
13 1/25/2013 11:00-11:50
Antigen-Antibody Interactions
Keri Smith
1/31/2013 1:00-3:00
1:00-3:00
Midterm Exam
CELLULAR ACTIVITIES AND IMMUNE MEDIATION
Steven Norris
14 2/5/2013 9:00-9:50
15 2/7/2013 10:00-10:50
Steven Norris
Antibody-Mediated Reactions
Cell-Mediated Reactions
IMMUNE SYSTEM AND INFECTIOUS DISEASE
Steven Norris
16 2/8/2013 10:00-10:50
17 2/8/2013 11:00-11:50
Jeffrey Actor
Immunology of HIV Infection
Infection and Immunity
MEDICAL APPLICATIONS OF IMMUNOLOGY (Immunopathology)
Shen-An Hwang
18 2/12/2013 11:00-11:50
19 2/14/2013 10:00-10:50
Sandeep Agarwal
Sandeep Agarwal and Jeffrey Actor
20 2/14/2013 11:00-11:50
21 2/18/2013 11:00-11:50
Priya Weerasinghe
22 2/19/2013 11:00-11:50
Semyon Risin
23 2/21/2013 11:00-11:50
William Shearer
24 2/25/2013 10:00-10:50
Wasim Dar
25 2/28/2013 8:00-9:50
Jeffrey Actor
26 2/28/2013 10:00-10:50
Adan Rios
3/7/2013 1:00-4:00
3/19/2013 Essay Assignment Due
1:00-4:00
Immune Regulation & Tolerance
Autoimmunity
Immunology: Clinical Scenarios
Cancer Immunology
Immunoprophylaxis (Vaccines) & Immunotherapy
Disorders of the Immune Response
Transplantation
Team Based Learning
Evolution of the Immune System
Final Exam
Essay must be submitted prior to 5:00pm
MEDICAL SCHOOL IMMUNOLOGY - 2013
COURSE DESCRIPTION
Course Director:
Jeffrey K. Actor, Ph.D.
Office Hours:
Fridays at 1:00-2:00p in MSB 2.214; or by appointment
LECTURERS
OFFICE
TELEPHONE
email
Jeffrey K. Actor, Ph.D.
Sandeep K. Agarwal, M.D., Ph.D.
Wasim A. Dar, M.D., Ph.D.
Shen-An Hwang, Ph.D.
Steven J. Norris, Ph.D.
Sudhir Paul, Ph.D.
Adan Rios, M.D.
Semyon A. Risin, M.D., Ph.D.
William T. Shearer, M.D., Ph.D.
Keri C. Smith, Ph.D.
Priya Weerasinghe, Ph.D.
Rick A. Wetsel, M.D., Ph.D.
MSB 2.214
BCM
MSB 6.256
MSB 2.2221E
MSB 2.278
MSB 2.230A
UPB-830
MSB 2.022
Texas Children's
MSB 2.248
MSB 2.408
SRB 430A
713-500-5344
713-798-3390
713-500-7400
713-500-5265
713-500-5338
713-500-5347
713-500-7766
713-500-5294
832-824-1274
713-500-2250
713-500-5275
713-500-2412
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
IMMUNOLOGY WEB PAGE: http://www.uth.tmc.edu/pathology/medic/immunology/immuntbl.htm
1) COURSE ORGANIZATION
The purpose of the Immunology course is to provide a basic knowledge of the immune response and its
involvement in health and disease. All lectures will be presented in MSB 2.006. An effort has been
made to increase clinical relevance and problem-solving skills through an essay assignment and facultypresented clinical correlations, and a team based learning exercise.
Any questions on the lecture material should be addressed to Dr. Actor or directly to that lecturer. If you
have general problems or comments regarding the course, your grades, or the faculty, please contact the
course director. If the problem is not resolved, you should make an appointment to see Dr. Robert L.
Hunter (Chairman of Pathology) at MSB 2.136 (500-5301) or, finally, Dr. Patricia Butler (Assoc. Dean
for Educational Programs) or Dr. Margaret McNeese (Assoc. Dean for Student Affairs).
2) COURSE MATERIALS
a)
Lectures. The student is responsible for all material covered in lectures and faculty presented
clinical correlations, as well as for any additional handouts or assignments (whether provided in this
syllabus or at a later time). Immunology is a rapidly advancing area, so the lectures may contain
new information not covered in the textbooks. Therefore you should make every effort to attend
lecture and take complete and accurate notes. Tapes of the lectures are available from the
Conference Operations Office (LRC) and can be used to verify your notes. Streaming video is
available on-line through the UT Med School student web pages.
iv
b) Reading. Two textbooks are required for the course. Chapter assignments are listed directly in
the syllabus chapter. Required Case Studies are listed separately in this syllabus.
R. Coico and Sunshine, G. Immunology: A Short Course. (6th Ed) John Wiley & Sons, Inc., 2009.
R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6th Ed)
Garland Publishing, New York, 2012.
The Coico et al. text was selected because it is well-organized, clearly and concisely written, and
contains chapter summaries, study questions, and case studies. The lecture schedule has been loosely
organized to match the Coico et al. book chapters. Knowledge of the assigned reading is required,
even if the material is not covered in the lectures. Modifications of the study questions may be used
in the exams. The Geha and Rosen text provides examples of the role of immunology in health and
disease, and is used extensively in the Clinical Correlations and as ‘Clinical Vignettes’ in the lectures.
Cases from Geha and Notarangelo presented (in part or in full) during lecture are considered
required reading. Please see the list of required associated cases for each lecture, located under
“Clinical Correlation Required Readings”.
c) MEDIC IMMUNOLOGY Web Page. You are encouraged to make use of the MEDIC Immunology
web site at: http://www.uth.tmc.edu/pathology/medic/immunology/immuntbl.htm . Materials are also on
Backboard. The website is actively updated during the course to include links for lecture materials and
information that will assist in understanding of course materials.
Alternative recommended texts available in the bookstore:





Actor, J.K. Elsevier’s Integrated Immunology and Microbiology (2nd Ed.), Mosby/Elsevier,
Philadelphia, 2012.
Parham, P. The Immune System. 3rd Edition. Garland Publishing, New York, 2009.
A. K. Abbas and Lichtman, A. H. Basic Immunology – Functions and Disorders of the Immune System,
3rd Edition (updated edition). Saunders-Elsevier. Philadelphia, PA. 2011.
Kindt, TJ, Goldsby, RA, Osborne, BA. Kuby Immunology (6th Ed.), W.H. Freeman and Company, New
York, 2007.
Murphy, K. Janeway’s Immunobiology (8th Ed.). Garland Publishing, New York, 2012.
Each of these texts may be found at the LRC or the HAM Library.
d) Essay Assignment. Students must turn in one essay assignment worth 10 points. Students must
attend one of the City-Wide Infectious Disease Rounds and provide a written review of one of the cases.
The assignment and due date are described in detail elsewhere in the syllabus, as well as on Blackboard.
e) Team Based Learning. There will be one team based learning exercise as a portion of the course.
The TBL is detailed later in the syllabus.
f) Clinical Scenarios. In addition to the regular lectures, we will have a Clinical Scenario session during
the semester. Past experience has shown that immunology (or any other medical topic) is easier to learn
and remember if it is presented as clinical cases involving 'real' patients. In each clinical correlation,
cases relevant to immunology will be discussed by faculty. The correlate scenarios are related to those
presented in the Geha and Notarangelo text, but may vary to accommodate additional learning materials.
g) AIDS Education Project. Students who participate in the AIDS Education Project (i.e. undergo the
training and participate according to guidelines) are eligible for two extra credit points. See your AIDS
Education Project representative for details.
h) Study Questions. Additional study questions are provided at the end of some lecture outlines. The
v
purpose of these questions is to test your knowledge and extend your learning beyond rote memorization
toward more 'cognitive' learning. The study questions will not be graded, but questions related to these
assignments will appear in the examinations. Answers are posted on the Immunology web site.
i) Streaming video/Video tapes. Lectures will be made available for viewing via streaming video over
the internet.
j) Office hours and other assistance. Students are encouraged to approach the lecturers if they need
assistance in understanding the course material. Dr. Actor is also available at his office (MSB 2.214) by
individual appointment or by phone or email.
3) GRADING
a) Examinations. There will be two major exams consisting of multiple choice, matching, and national
board format questions. The midterm exam will contain 60 questions (worth 40% of your grade), the
final exam will have 80 questions (worth 40% of your grade). Exam answers will be posted according to
accepted policies of the university. Sessions may be scheduled for question review after each exam.
b) Essay Assignment. The essay assignment is required and will be worth a possible 10 points.
Grading will be based on adherence to the format described in this syllabus, thoroughness, and
application of your budding medical knowledge and logic; you are not expected to 'know it all' at this
point. The due date is listed in the Essay instruction page in the syllabus, and posted on Blackboard.
Essays may be turned in early. Assignments turned in late will only receive a maximum of half credit (no
exceptions).
c) Team Based Learning. Questions answered for the TBL session will be worth a total of 10 points.
d) Extra credit. One opportunity for extra credit is available. The extra credit option is voluntary, and
is not a course requirement.
Participation in AIDS Education Project
2 points
e) Overall grade. The total possible points and grade assignments are given below. The total value of
points for the course is 100 points, plus 2 points possible through completion of the extra credit option.
Midterm
Final
Essay Assignment
Team Based Learning
40 points
40 points
10 points
10 points
(60 questions)
(80 questions)
Extra Credit
2 possible additional points
f) Final grade assignment. The final grade is based on percentage of points earned (max of 102 points)
as related to total possible points (max of 100).
Honors
High pass
Pass
Marginal Performance
Fail
90-100 %
85.5-89.99 %
69.5-85.49 %
65.5-69.49 %
65.49 % or below
vi
IMMUNOLOGY ESSAY ASSIGNMENT
The purpose of this assignment is to encourage you to explore an important form of information in
medicine: grand rounds presentations. You are not expected to master the analysis of the
information presented, but you should demonstrate that you have made an honest attempt to
understand and interpret it. The assignment is worth a possible 10 points (maximum).
The assignment is due any time on, or before, 5:00pm on March 19, 2013. Assignments turned in
late will receive a maximum of only half credit. Completed assignments are to be turned into the
Health Education Office (MSB 2.120). Assignments are not accepted via e-mail.
YOU MUST TYPE YOUR ESSAY. THE ASSIGNMENT SHOULD BE APPROXIMATELY
TWO-THREE PAGES FOR YOUR ANSWER. THIS IS NOT A SHARED ASSIGNMENT;
DUPLICATE ESSAYS (TURNED IN BY MORE THAN ONE STUDENT) WILL NOT BE
ACCEPTED.
Examples of the essay assignment can be found posted at:
http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/Ex1.Essay.Assignment.pdf
or on Blackboard.
______________________________________________________________________________
ASSIGNMENT: Grand Rounds Review. Attend one of the City-Wide Infectious Disease
Conferences held every Wednesday at Noon in the auditorium behind the elevators on the ground
floor of the DeBakey Building, Baylor College of Medicine (BCM Rm M112). (This building is the
white building next to the new Baylor Graduate School Building and across the street from the Jones
Library). Usually three cases are presented as unknowns, a differential diagnosis is made, and the
outcome and ramifications of the case are discussed. The presenters often provide handouts for the
case, but you may wish to take notes of the conference to help you glean out the information. Note
that the infectious disease and microbiology aspects are generally covered in detail, whereas
the immunology is often not discussed. Your job is to investigate the immunologic aspects of
the disease and incorporate them into your interpretation and case description. This is an
Immunology assignment; limit your discussion of Microbiology to pertinent information only.
It is not permitted to record or capture pictures/slides of presented materials. No picture
taking is allowed (due to HIPAA regulations, patient confidentiality, and proprietary
information).
Choose only ONE of the cases presented. The case must have an immunologic implication. (e.g. The
infection was cleared by immune mechanisms, or the patient was immunodeficient and thus
developed an unusual infection).
a) Briefly describe the case, concentrating on the clinical manifestations (patient's symptoms +
findings from examination and tests) that are most relevant. Use medical terms where you can, but
define them in a few words. Include the diagnosis, treatment, and outcome (if presented). USE
YOUR OWN WORDS. Include a copy of the handout for the case, if one was provided (see scoring
note below*).
b) Using your microbiology and immunology texts, describe the organism(s) which caused the
infection in this case. What is the normal course of disease, and how did they differ in this case?
What treatments are generally effective, and were they effective in this patient?
vii
c) The major portion of the essay should be devoted towards discussion of the immunologic
implications and principles of the case. Describe in as much detail as possible the normal
immune mechanisms to combat this infectious agent and how they affect the course of
infection (e.g. Macrophages phagocytose and process the antigen and present antigen
fragments in association with MHC Class II proteins to antigen-specific CD4+ helper T cells,
role of complement, cell phenotypes involved, etc.). Be specific and included details! How was
the immune response of this patient different than normal (if this is applicable)? Did the
patient have an underlying condition that contributed to the development of this infection? Did the
patient have cancer, AIDS, hereditary immunodeficiency or some other condition affecting the
immune response? How did the immune response (or lack thereof) affect the outcome of this case?
Did the immune response contribute to the pathogenesis of disease (i.e. is immunopathology
involved)? Describe immunization or other immunologic procedures (such as passive transfer of
antibodies) used in the prevention or treatment of this disease.
d) Cite references used in your analysis of the case. You will need to refer to published journal
articles to obtain specific background information or methods needed to comprehend the case.
Points will be subtracted if relevant citations are absent. You must include at least 2 primary
publications (meaning: journal articles) published within the past 3 years. Web pages are not
considered as primary references. You may also include syllabus chapters as references, but must
also include additional references that demonstrate you have expanded your discussion to materials
outside the course lecture presented materials. Syllabus chapters are NOT primary references. Up to
1 point is subtracted if the references are missing, incomplete, or inadequate to support your
discussion. Recommended: use PubMed to find related articles for the report.
e) *You must include a copy of the handout from the Grand Rounds session. 1 point is
subtracted if the handout is missing. Therefore, chose a case with a handout whenever possible.
f) The length of the essay should not exceed 3 pages (including references). 1 point is
subtracted for going over the set page limit.
In summary, make sure you:
 Describe clinical manifestations of the case.
 Discuss immunological aspects of case.
 Give full citations to cite your ideas, including use of current references from
journal articles.
 Attach a copy of the Grand Rounds handout for the case.
 Turn in your assignment on time.
Essays may be submitted anytime prior to the stated deadline. Late submitted essays will
automatically receive a 5 point deduction.
ESSAY GRADES: Essays will be returned to students as quickly as grading allows. Inquiries
regarding essay assignment grading must be submitted within one week after receipt of
returned assignments. Requests for review of essays past the one week period will be denied.
viii
Policy on Exam Grading (MS1 and MS2 courses) and Exam Review Sessions
Recommended by the Educational Policy Subcommittee: August 12, 2010
Approved by the Curriculum Committee: August 25, 2010
Revised by Educational Programs: August 31, 2010
Approved by the Curriculum Committee: September 15, 2010
Revised and Revisions Approved by the Curriculum Committee: May 18, 2011
The following policy delineates procedures related to exam grading and review/protest sessions
to be followed by all first- and second-year courses.
1. Course directors will score examinations through LXR and post results on MSGradebook as soon as possible.
2. Course directors will use item statistics generated by LXR to identify problematic
questions. Upon review, if a course director determines that a question was written
incorrectly (e.g. had more than one or no correct answer), then the director will give all
students credit for that particular question.
3. Large group post-exam review sessions may be held to provide feedback on difficult
examination topics, in a manner deemed appropriate by the course director. Copies of the
examinations will not be returned to the students during these sessions.
a. Course directors may meet with individual students to review examinations. The
format of these sessions, which may involve reviewing specific examination
questions, will be determined by the course director on a case by case basis.
ix
CLINICAL CORRELATIONS 2013
Required Readings
2013 Immunology Clinical Correlation Required Readings:
R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion.
(6th Ed) Garland Publishing, New York, 2012.
Reading for Lecture:
Required Readings
Assigned to lecture:
Cells and Organs
30. Congenital Asplenia
Assigned to lecture:
Innate Immunity
15. Chediak-Higashi Syndrome
25. Neutropenia
26. Chronic Granulomatous Disease
27. Leukocyte Adhesion Deficiency
Assigned to lecture:
Antibody Structure and
Multiple Myeloma (Blackboard file)
Function
46. Hemolytic Disease of the Newborn
Assigned to lecture:
Complement
32. Factor I Deficiency
33. Deficiency of C8
Assigned to lecture:
MHC
8. MHC Class II Deficiency
12. MHC Class I Deficiency
Assigned to lecture:
T cell receptor
7. Omenn Syndrome
T-Cell Lymphoma (Blackboard file)
Assigned to lecture:
Adaptive Immune Response
47. Toxic Shock Syndrome
Assigned to lecture:
Antibody-Mediated Reactions
41. Autoimmune Hemolytic Anemia
50. Allergic Asthma
52. Drug-Induced Serum Sickness
Assigned to lecture:
T cell
45. Acute Infectious Mononucleosis
Mediated Reactions
53. Contact Hypersensitivity to Poison Ivy
Assigned to lecture:
Infection and Immunity
28. Recurrent Herpes Simplex Encephalitis
48. Lepromatous Leprosy
Assigned to lecture:
Immunology of HIV Infections 10. Acquired Immune Deficiency Syndrome (AIDS)
Assigned to lecture:
Autoimmunity
36. Rheumatoid Arthritis
35. Systemic-Onset JID
40. Multiple Sclerosis
42. Myasthenia Gravis
Assigned to lecture:
Disorders of the Immune
1. X-linked Agammaglobulinemia
Response
4. CVID
9. DiGeorge Syndrome
16. Wiskott-Aldrich Syndrome
Assigned to lecture:
Transplantation
Kidney Graft Complications (Blackboard file)
11. Graft-Versus-Host Disease
Clin. Corr.
Class
Date
2/14
Time
11:00-11:50 AM
TBL
2/28
8:00-9:50 AM



Case Readings
36. Rheumatoid Arthritis
37. Systemic Lupus Erythematosus
Distributed Reading: Inflammatory Bowel Diseases (Crohn’s
Disease, Ulcerative Colitis, and Celiac)
39. Crohn’s Disease
44. Celiac Disease
Required readings complement lectures and presented materials. It is highly encouraged to view these clinical cases.
Case materials may not be covered in full during lectures, however, all required case study readings contain material that
may be tested on exams.
Assigned readings may be discussed in multiple lectures, in addition to the “assigned” lectures.
*Note: Most cases also appear in the previous edition: Geha and Rosen. Case Studies in Immunology. Garland Publishing, New
York, NY. 5th edition, 2007. Listed titles have different case numbers between book editions. Cases not included in the newer
edition are posted on Blackboard, or can be found in the LRC.
x
Spring Semester, 2013
Team Based Learning Exercise
The Immunology course will have one Team Based Learning exercise where
students will be required to address a clinically based scenario and provide answers
to related questions. Students will be assigned specific reading prior to the session,
which will assist in mastering of the material so as to allow participation in the group
activities. Materials will include new material in Immunology, as well as materials
already mastered in other courses. The format will be similar to the Clinical Applications
course.
The Team Based Learning Exercise is mandatory.
The Team Based Learning Exercise encompasses a graded set of exercises related to
multiple integrated aspects of a clinical scenario. The exercise is worth a maximum of 10
points towards your overall Immunology grade.
The session will utilize clinical scenario(s) to present problem(s). Students are divided
into teams; utilizing the groups already in place for the Clinical Applications course.
Approximately 5 problem questions arising from the clinical scenario are crafted to
foster discussion within the teams; each team is required to come to a consensus as to the
solution to the problem. Written justification may be required for the team solution, to
be prepared and handed in for grading at the end of the session.
Team Based Learning Exercise: February
Immunology
28th
8:00-9:50 a.m.
Persons missing the session must provide written notice explaining circumstances
for not attending. Written approval must be obtained from the Office of
Educational/Student Affairs prior to consideration for any makeup session or
alternate assignment.
xi
Spring Semester, 2013
Clinical Applications: Integrative Exercises
There will be a series of Clinical Applications (Integrative Exercises) throughout the first
year, up to three of which are scheduled for the Spring 2013 semester. These exercises
are designed to integrate content from the basic science courses and the ICM course and
to help students develop reasoning skills they will utilize in their clinical years. The
administration of these Exercises is held separate from the Immunology course, but
material from the exercises will be subject to assessment in all of the first year courses.
Attendance is required at these sessions and will be monitored.
The dates of the Clinical Applications Integrative Exercises during the Spring semester
are as follows; see Blackboard to confirm times and room assignments:
Students will be assigned to work in small groups of four to six students. These groups
will remain together for all seven of the Integrative Exercises throughout the year.
During the Integrative Exercises, each group will discuss a clinical problem that
integrates material from the current basic science courses and will develop a team answer
to a question regarding that clinical problem. The teams will then prepare a written
justification for their answer for one of these problems. These justifications will be
handed in for grading. Pre-reading and pretests may be posted to Blackboard as
necessary for each exercise. You will receive email notifications regarding any prereading or pretest assignments.
The graded responses from all of the sessions will contribute to the final grade in the
Integrative Exercise course. Each of the group members will receive the same score.
Students who have unexcused absences will receive a score of 0 for all responses for
that Integrative Exercise session.
Information presented within any Clinical Application Exercise
throughout the year is a potential source of testable material for exams
in any MSI class.
xii
MEDICAL IMPORTANCE OF THE IMMUNE SYSTEM
[HOW THE IMMUNE SYSTEM WORKS]
Jeffrey K. Actor, Ph.D.
MSB 2.214, 713-500-5344
(Special thanks to Gailen D. Marshall, Jr., MD, PhD, and Steven J. Norris, PhD)
Objectives
1. To appreciate the components of the human immune response that work together to
protect the host
2. To understand the concept of immune-based diseases as either a deficiency of
components or excess activity as hypersensitivity
3. To introduce the 7 main concepts of the course
KEYWORDS
immunodeficiency, hypersensitivity
Required Knowledge: Hypersensitivity Chart
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc,
New York, NY. 6th edition, 2009. Chapter 1.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/How2.htm
The chief function of immunity is to discriminate between self and non-self.
The immune cells and organs of the body comprise the primary defense system against
invasion by microorganisms and foreign pathogens. A functional immune system confers a
state of health through effective elimination of infectious agents (bacteria, viruses, fungi,
and parasites) and through control of malignancies by protective immune surveillance. In
essence, the process is based in functional discernment between self and non-self, a process
which begins in utero and continues through adult life.
Immune responses are designed to interact with the environment to protect the host against
pathogenic invaders. The goal of these chapters is to appreciate the components of the
human immune response that work together to protect the host. Furthermore, we will strive
to present a working clinical understanding of the concept of immune-based diseases
resulting from either immune system component deficiencies or excess activity.
Immunological memory as the basis for vaccine efficacy
Continued discrimination for health depends upon immunological memory where the
adaptive immune system can more efficiently respond to previously encountered antigen.
This results in resistance to repeated infection with pathogens and the resulting clinical
syndrome. This principle accounts for the clinical utility of vaccines which have done
more to improve mortality rates worldwide than any other medical discovery in recorded
history.
1
The clinical immunologist is a physician who has specialized in the diagnosis and treatment
of disorders of the immune system. Many other clinical specialties (such as oncology,
hematology, infectious diseases, transplant surgery, etc.) also deal with immunologicallybased diseases in their area of specialization. Much of the work of modern clinical
immunologists revolves around refining diagnostic techniques for greater clinical utility
and evaluating new therapeutic modalities such as recombinant cytokines and cytokine
modulators.
Protection against foreign pathogens
Normal physiologic functions of the immune system include the ability to discern self from
non-self, and recognition of foreign pathogens. This represents recognition of
environmental challenges in an attempt to preserve homeostasis while responding to
pathogenic agents. The goal is to respond with specificity, allowing sufficient intensity and
duration to protect the host without causing damage to self.
The Immune system protects against foreign pathogens, of which four major classes can be
defined. These include (1) Extracellular bacteria, parasites and fungi; (2) Intracellular
bacteria and parasites; (3) Viruses (intracellular); and (4) Parasitic worms (extracellular).
Immunodeficiency and dysfunction as the basis of disease
Immunological diseases can be grouped into two large categories – deficiency and
dysfunction. Immunodeficiency diseases occur as the result of the absence (congenital or
acquired) of one or more elements of the immune system. Immune dysfunction occurs
when a particular immune response occurs that is detrimental to the host. This response
may be against a foreign antigen or self antigen. It may also be an inappropriate regulation
of an effector response resulting in the absence of a protective response. Notwithstanding,
the host is adversely affected. A healthy immune system occurs as a result of balance
between innate and adaptive immunity, cellular and humoral immunity, inflammatory and
regulatory networks and small biochemical mediators (cytokines). Because specific
mechanism affects prognosis as well as therapeutic approaches, Gel and Coombs classified
these dysfunctional immune responses into hypersensitivity diseases.
Hypersensitivities will be discussed throughout the course, and in much greater detail in a
later chapter. There is considerable overlap in underlying mechanisms that contribute to the
hypersensitive responses. The major mechanisms are outlined on the following page.
2
Type I (also called immediate hypersensitivity) is due to aberrant production and activity
of IgE against normally nonpathogenic antigens (commonly called allergens). The IgE
binds to mast cells via high affinity IgE receptors. Subsequent antigen exposure results in
crosslinking of mast cell bound IgE with activation of mast cells that release preformed
mediators (eg. histamine, leukotrienes, etc.) and synthesize new mediators (i.e.
chemotaxins, cytokines). These mediators are responsible for the signs and symptoms of
allergic diseases. [A = Allergic]
Type II is due to antibody directed against cell membrane-associated antigen that results in
cytolysis. The mechanism may involve complement (cytotoxic antibody) or effector
lymphocytes that bind to target cell-associated antibody and effect cytolysis via a
complement independent pathway (Antibody dependent cellular cytotoxicity, ADCC).
Cytotoxic antibodies mediate many immunologically-based hemolytic anemias while
ADCC may be involved in the pathophysiology of certain virus-induced immunological
diseases. [C = Cytotoxic]
Type III results from soluble antigen-antibody immune complexes that activate
complement. The antigens may be self or foreign (i.e. microbial). Such complexes are
deposited on membrane surfaces of various organs (i.e. kidney, lung, synovium, etc). The
byproducts of complement activation (C3a, C5a) are chemotaxins for acute inflammatory
cells. These result in the inflammatory injury seen in diseases such as rheumatoid arthritis,
systemic lupus erythematosus, postinfectious arthritis, etc). I = Immune Comples]
Type IV (also called Delayed Type Hypersensitivity, DTH) involves macrophage-T cellantigen interactions that cause activation, cytokine secretion and potential granuloma
formation. Diseases such as tuberculosis, leprosy and sarcoidosis as well as contact
dermatitis are all clinical examples where the tissue injury is primarily due to the vigorous
immune response rather than the inciting pathogen itself. D = DTH]
EXPANDED FIGURES OF HYPERSENSITIVITIES INCLUDED IN APPENDIX.
3
Clinical suspicion for immunodeficiency may be made when patients present with chronic
infection or chronic inflammatory status, poor wound healing, constant fatigue and malaise,
or when unresponsive to vaccine administration. Certain infections with organisms may be
suggestive of deficiency in an immune related component. Alternatively, disruptions in
homeostasis may lead to immunodeficiency, such as those induced inadvertently by a
physician through medical treatment (iatrogenic).
The mechanisms for clinical immunodeficiency are varied, and will be examined (in part)
throughout the remainder of the course.
Therapeutic intervention for immune based diseases
Therapy for these diseases has historically been nonspecific, centering on repair of the
damaged tissues and inhibition of the aberrant immune responses with immunosuppressive
drugs. Recent work using such cutting edge techniques as recombinant DNA technology,
gene therapy, and stem-cell research have opened up an entire new avenue to address these
diseases by providing diagnostic and therapeutic modalities not previously available. For
immunodeficiency states, we have developed the g ability to replace elements through
marrow transplants, recombinant immune molecule administration and, soon, gene therapy.
Introduction to 7 Main Concepts towards Understanding Medical Immunology
1.
The chief function of the immune system is to distinguish between self and
non-self.
Health – effective elimination or control of health-threatening agents
Infectious agents – bacteria, viruses, fungi, parasites
Tumors – the immune system also plays an important role in the control of
malignant cells through a mechanism called immune surveillance
Hyporeactivity – inability to recognize and control health-threatening agents
(immunodeficiency)
Congenital immunodeficiency – immune defects due to genetic defects
Acquired immunodeficiency – caused by multiple agents, including
Human Immunodeficiency Virus and tumors
Malnutrition – severe malnutrition compromises the immune system
Young/Old Age – increased susceptibility to infection
Hyperreactivity – aberrant immune responses
Systemic autoimmunity – e.g. systemic lupus erythematosus
Organ-Specific autoimmunity – e.g. thyroiditis
4
Allergies and Asthma – aberrant immune response to environmental
allergens or chemicals
Immunopathology – general term for damage to normal tissue due to the
immune reaction to infectious agents or other antigens (e.g. rheumatic fever,
leprosy)
2.
Figure: Immune based diseases can be caused by lack of specific
functions (immune deficiency) or excessive activity (hypersensitivity).
The immune system consists of two overlapping compartments: the innate
immune system and the adaptive immune system.
Innate immune system
 Most primitive type of immune system; found in virtually all multicellular
animals (arguably also in plants!)
 Always present and active, constitutively expressed
 Nonspecific; not specifically directed against any particular infectious agent or
tumor
 Same every time; no ‘memory’ as found in the adaptive immune system
 First line of defense against infection
 Includes:
o Physical barriers (skin, mucus lining of gastrointestinal, respiratory and
genitourinary tracts)
o Phagocytic cells – neutrophils, macrophages
o Protective chemicals – acid pH of stomach, lipids on skin surface
o Enzymes – lysozyme in saliva, intestinal secretions; digests cell walls of
bacteria
o Alternate complement pathway – cascade of serum proteins that are
activated by bacterial cell wall components
5
Adaptive or acquired immune system
 Found only in vertebrates (fish, amphibians, birds and mammals)
 Must be induced to be active against infections or tumors
 Antigen-specific – adaptive immune responses recognize antigens, which can
be proteins, carbohydrates, lipids and nucleic acids.
 Memory – response against a given antigen is much stronger after the first
(primary) response. This heightened reactivity is called secondary responses,
and is due to increased numbers of memory B and T cells to that antigen
 Regulation – discriminates between self and non-self, prevents autoimmune
reactions in most individuals
o B lymphocytes – differentiate into plasma cells that produce antibodies
o T lymphocytes – subdivided into CD4+ and CD8+ populations
 Helper activity – help other lymphocytes respond to antigen
(mostly CD4+ T cells, subdivided into phenotypic responders)
 Delayed type hypersensitivity – activate macrophages to
phagocytose, kill pathogens (mostly CD4+ T cells)
 T cell-mediated cytotoxicity – cytotoxic T cells (mostly CD8+ T
cells) bind to and kill target cells (e.g. virus-infected cells and
tumor cells)
 Suppressor T cells/Treg cells – down-regulate the responses of
other lymphocytes
Table: Elements of Innate and Acquired Immune Responses
Innate
Adaptive
Rapid response (minutes to hours)
Slow Response (days to weeks)
PMNs and Phagocytes
B cells and T cells
Preformed effectors with limited variability
B cell and T cell receptors with highly selective
Pattern Recognition Molecules recognizing
specificities
structural motifs
Soluble activators
Antibodies (humoral)
Proinflammatory mediators
Cytokines (cellular)
Non-specific
Specific
No memory, no increase in response upon
Memory, maturation of secondary response
secondary exposure
6
3.
The antigenic specificity of the adaptive immune system is due to antigenspecific receptors.

Immunoglobulins (also called antibodies) – produced by B cell lineage
o IgM, IgD, IgG, IgA, and IgE subtypes
o Surface immunoglobulin (Ig) – antigen-specific receptor of B
lymphocytes
o Secreted immunoglobulin (Ig) – Ig molecules secreted by plasma cells

T cell receptor (TCR) – antigen-specific receptor of T lymphocytes
o  and  TCR subtypes
Coico and Sunshine, 2009. Fig. 1.3.

The basic reaction in immunology is the binding of antigen to an antigen-specific
receptor. The affinity of this interaction is similar to that of an enzyme binding to its
substrate.
Ag + Ab
AgAb

Typically, each antibody or T cell receptor molecule recognizes a single epitope, a
small region (e.g. 6-10 amino acids) of an antigen.
In a given B- or T-cell, the antigen-specific receptors of all are identical.
o Exception – IgM and IgD can be coexpressed on certain B cells
Each B cell and T cell has its own antigenic specificity, determined by the amino acid
sequence of its surface Ig or TCR. The region of the Ig or TCR that binds to the
antigen is called the paratope.
In each person, there are ~106 to 108 different Igs and TCRs, giving rise to an almost
endless supply of antigenic specificities. This is called diversity.



7
4.
The generation of antigen-binding diversity occurs prior to antigen exposure
through a DNA rearrangement process called VDJ joining.

The “business end” of an antibody or TCR is the variable region. This region contains
the antigen-binding site that binds to the epitope (meaning: the conformational shape
recognized).
Variable Region
Coico and Sunshine, 2009. Fig. 1.2.






The variable region is formed during B and T cell development. This process occurs
prior to exposure to a given antigen.
The DNA encoding the variable region is subdivided into V, D, and J gene segments.
There are multiple V, D, and J gene segments in the Ig and TCR genetic loci.
In most cells, these gene segments are spread out, so that all the V segments are
together, all the D segments are together, and all the J segments are together. This is
called the germline configuration, because it is the arrangement seen in sperm and ova.
The V, D, and J gene segments are brought together to form a contiguous exon
encoding the variable region. The V, D, and J segments are selected randomly in each
cell, giving rise to combinatorial diversity. This is similar to the “Pick 5” game in
Texas lotto, in which a large number of different number combinations exist.
The light chain gene locus (and some TCR genes) has only V and J regions.
There are several other mechanisms for generating diversity, as will be discussed in a
later lecture.
8
5.
To generate an active immune response against a certain antigen, a small
number of B and T cell clones that bind to the antigen with high affinity
undergo activation, proliferation, and differentiation into plasma cells (for B
cells) or activated T cells. This process is called ‘clonal selection’.





B and T cells are resting cells that lack functional activity until they undergo
activation, proliferation, and differentiation into plasma cells or activated T
cells. This process takes several days, which explains the lag between being
exposed to an infectious agent and eventually getting better when the immune
response ‘kicks in’.
Of the millions of different specificities of B and T cells produced, only a few will
have surface Ig or TCRs that bind the antigen with high affinity. However, we
produce B and T cells that will react with virtually any antigen, including those that
are man-made and are not found in nature (e.g. di-nitrophenol).
In nearly all cases, activation of a B or T cell requires two signals: binding of the
antigen-specific receptor to the antigen, and exposure to proteins called cytokines
expressed by helper T cells.
The blast cells resulting from activation undergo proliferation, resulting in a ~100fold expansion of the number of cells reactive to the antigen.
Some of these cells become effector cells (plasma cells and activated T cells that
express activities that help to eliminate the pathogen. Others become memory cells
that can give rise to secondary responses as described below.
…(106-108 clones)
Coico and Sunshine, 2009. Fig. 1.1.
9
6.





The adaptive immune system has memory, meaning that the response against
an antigen is much greater after the first exposure.
The first response to an antigen is called the primary response, and responses
thereafter are called secondary responses.
The different properties of secondary responses are due to memory cells generated
during the primary responses.
Secondary responses have
o Higher antibody levels
o Increased proportion of IgG and other immunoglobulin isotypes
o Shorter lag period
o Higher affinity for antigen
Vaccination is effective because it primes the immune system to provide secondary
responses when the individual is exposed to an infectious agent.
Each exposure to an antigen tends to increase the secondary response. This is why
booster immunizations are often used in vaccinations.
Coico and Sunshine, 2009. Fig. 4.12.
Figure: Primary and secondary antibody responses. The adaptive immune system
has memory, allowing for maturation of a rapid secondary immune response with
higher specificity and magnitude directed against foreign substances.
10
7.



The immune system is tightly regulated.
Self-reactive B and T cell clones are generated as a natural part of the random VDJ
recombination process.
The adaptive immune system has developed several mechanisms to eliminate or inhibit
self-reactive B and T cells.
o Elimination of self-reactive cells during their development through apoptosis.
o Permanent inactivation of self-reactive cells through a process called clonal
anergy.
o Inhibition of self-reactive cells by suppressor cells, inhibitory cytokines, and
other factors
Each immune response requires a combination of multiple factors, thereby limiting the
number of spurious responses.
SUMMARY – MEDICAL IMPORTANCE OF THE IMMUNE SYSTEM AND HOW
THE IMMUNE SYSTEM WORKS
Thus it can be said that the healthy immune system occurs as a result of balance – between
innate and adaptive immunity, cellular and humoral immunity, inflammatory and
regulatory networks and even cytokine modulators. Disease occurs when the balance is
altered either by deficiency or dysfunction.
Current and future research efforts center about defining exact hypersensitivity and/or
immunodeficiency mechanisms in specific diseases, developing diagnostic assays that have
individual patient relevance and finding more specific agents that can regulate or eliminate
aberrant immune responses while leaving the rest of the system intact. Research
opportunities abound in the broadening area of clinical immunology.
SUMMARY

The immune response is designed to interact with the environment to protect the
host against pathogenic invaders.

Immune-based diseases are either because of a lack of specific elements (immune
deficiency) or excess activity (hypersensitivity).

Hypersensitivity Chart: Know the differences between types of
hypersensitivity.
11
1. The chief function of the immune system is to distinguish between self and nonself.
2. The immune system consists of two overlapping compartments: the innate
immune system and the adaptive immune system.
3. The antigenic specificity of the adaptive immune system is due to antigen-specific
receptors.
4. The generation of antigen-binding diversity occurs prior to antigen exposure
through a DNA rearrangement process called VDJ joining.
5. To generate an active immune response against a certain antigen, a small number
of B and T cell clones that bind to the antigen with high affinity undergo
activation, proliferation, and differentiation into plasma cells (for B cells) or
activated T cells. This process is called ‘clonal selection’.
6. The adaptive immune system has memory, meaning that the response against an
antigen is much greater after the first exposure.
7. The immune system is tightly regulated.
12
CELLS AND ORGANS OF THE IMMUNE SYSTEM
Jeffrey K. Actor, Ph.D.
713-500-5344
Objectives: (1) Identify cell types involved in specific and non-specific immune responses. (2)
Present the developmental pathway of immune system cells. (3) Understand structure and function
of primary and secondary lymphoid organs.
Keywords: Reticuloendothelial System, Leukocytes, Myeloid Cells, Lymphocytes, Antigen
Presenting Cells (APC), GALT, MALT, BALT, Cluster of Differentiation (CD).
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York,
NY. 6th edition, 2009. Chapter 2; Geha and Notarangelo. Case Studies in Immunology. Garland
Publishing, New York, NY. 6th edition, 2012. Case 30: Congenital Asplenia.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/cellsimmsys.html
Immune system cells are derived from pluripotent hematopoietic stem cells in the bone marrow.
These cells can be functionally divided into groups that are involved in two major categories of
immune responses: innate (natural) and acquired. Innate immunity is present from birth and
consists of non-specific components. Acquired immunity by definition requires recognition
specificity to foreign (non-self) substances. The major properties of the acquired immune response
are specificity, memory, adaptiveness, and discrimination between self and non-self.
The acquired immune response is
subdivided into humoral and cellular
immunity, based on participation of
two major cell types. In Humoral
Immunity, B lymphocytes
synthesize and secrete antibodies.
Cellular Immunity (CMI) involves
effector T lymphocytes which
secrete immunoregulatory factors
following interaction with antigen
presenting cells (APCs).
Figure. The developmental
pathway of pluripotent bone
marrow stem cells.
Coico and Sunshine, 2009. Fig. 2.1.
13
Figure. The interrelationship between
innate and acquired immunity. An intricate
communication system allows components of
innate and acquired immunity to work in
concert to combat infectious disease.
Coico and Sunshine, 2009. Fig. 2.12.
Cluster of Differentiation (CD): Cell surface molecules are identifiable by monoclonal
antibodies. In humans, these molecules have been given number designations. The acronym CD
describes the cluster of antigens with which the antibody reacts; the number describes the order in
which it was discovered. As of 2010, the list of determinants officially identified 350 individual
and unique markers (link to Human CD Molecules). Surface expression of a particular CD
molecule may not be specific for just one cell or even a cell lineage. However, many are useful for
characterization of cells.
CD-specific monoclonal antibodies have been useful for 1) determining the functions of CD
proteins; 2) identifying the distribution of CD proteins in different cell populations in normal
individuals; 3) measuring changes in the proportion of cells carrying these markers in patients
with disease (e.g. decrease in CD4+ T cells is a hallmark of HIV infection); 4) developing
therapeutic measures for increasing or decreasing the numbers or activities of certain cell
populations.
Figure.
Nomenclature of
Inflammatory
Cells.
Reticuloendothelial System
Cells of the RES provide natural immunity against microorganisms by 1) a coupled process of
phagocytosis and intracellular killing, 2) recruiting other inflammatory cells through the
production of cytokines, and 3) presenting peptide antigens to lymphocytes for the production of
antigen-specific immunity. The RES consists of 1) circulating monocytes; 2) resident
macrophages in the liver, spleen, lymph nodes, thymus, submucosal tissues of the respiratory and
14
alimentary tracts, bone marrow, and connective tissues; and 3) macrophage-like cells including
dendritic cells in lymph nodes, Langerhans cells in skin, and glial cells in the central nervous
system.
Leukocytes
Leukocytes provide either innate or specific adaptive immunity. These cells are derived from
myeloid or lymphoid lineage. Myeloid cells include highly phagocytic, motile neutrophils,
monocytes, and macrophages that provide a first line of defense against most pathogens. The other
myeloid cells, including eosinophils, basophils, and their tissue counterparts, mast cells, are
involved in defense against parasites and in the genesis of allergic reactions. Cells from the
lymphoid lineage are responsible for humoral or cell mediated immunity.
Myeloid Cells
Neutrophils: Neutrophils are the most highly adherent, motile, phagocytic leukocytes and are the
first cells recruited to acute inflammatory sites. They ingest, kill, and digest pathogens, with their
functions dependent upon special proteins, such as adherence molecules, or via biochemical
pathways (respiratory burst).
Eosinophils: Eosinophils defend against parasites and participate in hypersensitivity reactions via
cytotoxicity. Their cytotoxicity is mediated by large cytoplasmic granules, which contain
eosinophilic basic and cationic proteins.
Basophils/Mast cells: Basophils, and their tissue counterpart mast cells, produce cytokines that
help defend against parasites, and also cause allergic inflammation. These cells display high
affinity surface membrane receptors for IgE antibodies, and have many cytoplasmic granules
containing heparin and histamine. The cells degranulate when cell-bound IgE antibodies are crosslinked by antigens, and produce low-molecular weight vasoactive mediators (e.g. histamine).
Monocytes/Macrophages: Monocytes and macrophages are involved in phagocytosis and
intracellular killing of microorganisms. Macrophages are differentiated monocytes, which are one
of the principal cells found to reside for long periods in the RES. These monocytes/macrophages
are highly adherent, motile and phagocytic; they marshal and regulate other cells of the immune
system, such as T lymphocytes; they serve as antigen processing-presenting cells.
Dendritic Cells: Dendritic cells provide a link between innate and adaptive immunity by
interacting with T cells in a manner to deliver strong signals for development of memory
responses. Dendritic cells recognize foreign agents and pathogens through a series of pattern
recognition receptors (non-specific), and are able to present antigen to both T helper and T
cytotoxic cells to allow those lymphocytes to mature towards functionality.
Lymphoid Cells
Lymphoid cells provide efficient, specific and long-lasting immunity against microbes/pathogens
and are responsible for acquired immunity. Lymphocytes differentiate into three separate lines: (1)
thymic-dependent cells or T lymphocytes that operate in cellular and humoral immunity; (2) B
lymphocytes that differentiate into plasma cells to secrete antibodies; and (3) natural killer (NK)
15
cells. T and B lymphocytes produce and express specific receptors for antigens while NK cells do
not.
B Lymphocytes: B lymphocytes differentiate into plasma cells to secrete antibodies. The genesis
of mature B cells from pre-B cells is antigen-independent. The activation of B cells into antibody
producing/secreting cells (plasma cells) is antigen-dependent. Mature B cells can have 1-1.5 x 105
receptors for antigen embedded within their plasma membrane. Once specific antigen binds to
surface Ig molecule, the B cells differentiate into plasma cells that produce and secrete antibodies
of the same antigen-binding specificity. If B cells also interact with T helper cells, they proliferate
and switch the isotype (class) of immunoglobulin that is produced, while retaining the same
antigen-binding specificity. T helper cells are thought to be required for switching from IgM to
IgG, IgA, or IgE isotypes. In addition to antibody formation, B cells also process and present
protein antigens.
T Lymphocytes: T lymphocytes are involved in the regulation of the immune response and in cell
mediated immunity, and help B cells to produce antibody. Mature T cells express antigen-specific
T cell receptors (TCR). Every mature T cell also expresses the CD3 molecule, which is associated
with the TCR. In addition mature T cells usually display one of two accessory molecules, CD4 or
CD8, which define whether a T cell will be a helper T lymphocyte, or a cytotoxic T lymphocyte
(CTL). The TCR/CD3 complex recognizes antigens associated with the major histocompatibility
complex (MHC) molecules on target cells (e.g. virus-infected cell).
Development of T lymphocytes
During differentiation in the thymus, immature T cells undergo rearrangement of their TCR  and
 genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for
further maturation only if their TCRs do not interact with self-peptides presented in the context of
self-major histocompatibility complex (MHC) molecules on antigen presenting cells.
T Helper Cells: T helper cells (Th) are the primary regulators of T cell- and B cell-mediated
responses. They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate
toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign antigen
complexed with MHC class II molecules on B cells, macrophages or other antigen-presenting
cells; and 4) aid effector T lymphocytes in cell-mediated immunity.
Currently, it is believed that there are two main functional subsets of Th cells, plus multiple other
helper subsets of importance. T helper 1 (Th1) cells aid in the regulation of cellular immunity,
and T helper 2 (Th2) cells aid B cells to produce certain classes of antibodies (e.g., IgA and IgE).
The functions of these subsets of Th cells depend upon the specific types of cytokines that are
generated, for example interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by Th1 cells; IL4, IL-6 and IL-10 by Th2 cells. Two other classes of T helper cells are thought to be involved in
oral tolerance and serve as regulators for immune function. Th17 cells, characterized by IL-17
secretion, are thought to be involved as effector cells for autoimmune disease progression, and
protect surfaces (skin, gut) from extracellular bacteria. Tfh cells (follicular helper T cells) also
provide help to B cells enabling them to develop into antibody-secreting plasma cells. They
function inside of follicular areas of lymph nodes. Finally, although no longer prevalent in the
literature, a subclass called Th3 cells were historically identified as secreting IL-4 and TGF- to
provide help for IgA production; they were thought to be suppressive for Th1 and Th2 cells.
16
T
Cytotoxic Cells: T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells infected
with intracellular pathogens. These cells 1) usually express CD8, and, 2) destroy infected cells in
an antigen-specific manner that is dependent upon the expression of MHC class I molecules on
antigen presenting cells.
T Suppressor/ T Regulatory Cells: T suppressor cells suppress the T and B cell responses and
express CD8 molecules. T regulatory cells (Tregs) also affect T cell response, with many cells
characterized as CD4+CD25+, TGF- secretors. Tregs regulate/suppress other T cell activities,
and help prevent development of autoimmunity.
Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells that
share properties of both T cells and natural killer (NK) cells. These cells recognize an antigenpresenting molecule (CD1d) that binds self- and foreign lipids and glycolipids. They constitute
only 0.2% of all peripheral blood T cells. The term “NK T cells” was first used in mice to define a
subset of T cells that expressed the natural killer (NK) cell-associated marker NK1.1 (CD161). It
is now generally accepted that the term “NKT cells” refers to CD1d-restricted T cells coexpressing a heavily biased, semi-invariant T cell receptor (TCR) and NK cell markers. Natural
killer T (NKT) cells should not be confused with natural killer (NK) cells.
Natural Killer Cells: NK cells are large granular “innate” lymphocytes that nonspecifically kill
certain types of tumor cells and virus-infected cells. NK cells share many surface molecules with
T lymphocytes. These circulating large granular lymphocytes are able to kill “self” in the absence
of antigen-specific receptors. NK cells are especially effective against viral infected cells, and
keep the expansion of virus in check until adaptive immunity kicks in. In this regard, they also
secrete interferon-gamma, which is an effective immunoregulator. NK cells can also kill via
antibody-dependent cellular cytotoxic mechanisms (ADCC) via their Fc receptors. NK cells
17
express a large number of receptors that deliver either activating or inhibitory signals, and the
relative balance of these signals controls NK cell
activity.
Antigen Presenting Cells (APCs) are found
primarily in the skin, lymph nodes, spleen and
thymus. They may also be present throughout
the diffuse lymphoid system. Their main role is
to present antigens to antigen-sensitive lymphoid
cells. APCs may be characterized by their ability
to phagocytose antigens, location in body, and
expression of Major Histocompatibility Complex
(MHC) related molecules.
Two main types of APCs are Dendritic Cells and
Macrophages. Of note, B cells are a special class
of APCs; because they have antigen-specific
antibody receptors they are enabled to internalize
and process targeted antigens.
Lymphoid Organs
The lymphatic organs are tissues in which lymphocytes mature, differentiate and proliferate.
Lymphoid organs are comprised of epithelial and stromal cells arranged either into discretely
capsulated organs or accumulations of diffuse lymphoid tissue. The primary (central) lymphoid
organs are the major sites of lymphopoiesis, where B and T lymphocytes differentiate from stem
cells into mature antigen recognizing cells. The secondary lymphoid organs, therefore, are those
tissues in which antigen-driven proliferation and differentiation take place.
Historically, the primary lymphoid organ was first discovered in birds, in which B cells undergo
maturation in the bursa of Fabricius, an organ situated near the cloaca. Humans do not have a
cloaca, nor do they possess a bursa of Fabricius. In embryonic life, B cells mature and
differentiate from hematopoietic stem cells in the fetal liver. After birth, B cells differentiate in the
bone marrow. Maturation of T cells occurs in a different manner. Progenitor cells from the bone
marrow migrate to the thymus where they differentiate into T lymphocytes. The T lymphocytes
continue to differentiate after leaving the thymus, and are driven to do so by encounter with
specific antigen in the secondary lymphoid organs.
18
Primary Lymphoid Organs
Thymus Gland: The lymphoid organ in which T lymphocytes are educated, mature and multiply.
It is a lymphoepithelial organ composed of stroma (thymic epithelium) and lymphocytes, almost
entirely of the T-cell lineage. This is where T lymphocytes learn to recognize self antigens as self,
and where these cells differentiate and express specific receptors for antigen. Only 5-10% of
maturing lymphocytes survive and leave the thymus.
Fetal Liver and Adult Bone Marrow: Islands of hematopoietic cells in the fetal liver and in the
adult bone marrow give rise directly to B lymphocytes.
Secondary Lymphoid Organs
The spleen and lymph nodes are the major secondary
lymphoid organs. Additional secondary lymphoid organs
include the tonsils, appendix, and Peyer’s patches.
Aggregates of cells in the lamina propria of the digestive
tract lining may also be included in this category, as well
as any tissue described as MALT (mucosa-associated
lymphoid tissue), GALT (gut-associated lymphoid
tissue) or BALT bronchus-associated lymphoid tissue).
Last but not least, the bone marrow can serve as an
important secondary lymphoid organ. In addition to
being a site of B cell generation, the bone marrow
contains many mature T cells and plasma cells.
Figure. Distribution of lymphoid tissues in the body.
Actor, Elsevier’s Integrated Immunology and Microbiology. 2012.
Lymph Node: Lymph nodes form part of the network which filters antigen from tissue fluid or
lymph during its passage from the periphery to the thoracic duct. Histologically, the lymph node is
composed of a B cell cortex containing primary and secondary follicles, a T cell paracortex, and a
central medulla which contains cords of lymphoid tissue.
Spleen: The spleen is a filter for blood, and is actively involved in the removal of dying and dead
erythrocytes. There are two main types of tissue; red pulp and white pulp. The white pulp
contains the lymphoid tissue, arranged around a central arteriole as a periarteriolar lymphoid
sheath (PALS). The PALS is composed of T and B cell areas, and contains germinal centers.
Dendritic reticular cells and phagocytic macrophages can be found in germinal centers where they
work to present antigen to lymphocytes.
19
Clinical Vignette - Congenital Asplenia (Case 30 in Geha and Notarangelo): Mr. and Mrs.
Vanderveer had five children. Their 10 month old daughter developed a cold, followed by upper
respiratory infection. The child became feverish, convulsive and died; the causative agent was
Haemophilus influenza which was isolated from the throat and cerebrospinal fluid. At autopsy she was
found to have no spleen.
How does the lack of a spleen affect B cell function, and what implications does this have towards
immune responses to infective agents? In adults? In children?
Review your histology chapters dealing with Hematopoiesis and the Immune System!
Table. Myeloid Leukocytes and Their Properties
Phenotype
Morphology
Circulating Differential Count*
PMN
Neutrophil
granulocyte
2-7.5x109/L
PMN
Eosinophil
granulocyte
0.04-0.44x109/L
PMN
Basophil
granulocyte
0-0.1x109/L
PMN
Mast Cell
granulocyte
Tissue Specific
Monocytes
monocytic
0.2-0.8x109/L
Macrophag
e
monocytic
Tissue Specific
Dendritic
Cell
monocytic
Tissue Specific
* Normal range for 95% of population, +/- 2 standard deviations
Table. Lymphoid Leukocytes and Their Properties
Total Lymphocytes
1.3-3.5x109/L
B Cell
monocytic
Adaptive
Plasma Cell
T Cell
Natural Killer T Cell
(NKT)
Natural Killer Cell (NK)
Effector Function
Phagocytosis and digestion of microbes
Immediate hypersensitivity (allergic)
reactions; defense against helminths
Immediate hypersensitivity (allergic)
reactions
Immediate hypersensitivity (allergic)
reactions
Circulating macrophage precursor
Phagocytosis and digestion of microbes;
antigen presentation to T cells
Antigen presentation to naïve T cells;
initiation of adaptive responses
monocytic
monocytic
Adaptive
Adaptive
Effector Function
Humoral immunity
Terminally differentiated, antibody
secreting B cell
Cell-mediated immunity
monocytic (rare)
monocytic
Adaptive
Innate
Cell-mediated immunity (lipids)
Innate response to microbial or infection
20
Summary: Cells of the Immune System
Immune system cells are derived from pluripotent hematopoietic stem cells. Immune responses by
these cells are divided into innate (natural) and acquired categories. Acquired immunity requires
recognition specificity to foreign antigens, and is subdivided, based on participation B
lymphocytes (humoral) and T lymphocytes (CMI). Surface molecules on human cells may be
defined according to designation of Cluster of Differentiation (CD) antigens, which are useful for
identifying different cell populations.
Cells of the RES provide natural immunity against microorganisms via phagocytosis and
intracellular killing, recruitment of other inflammatory cells, and presentation of antigens.
Leukocytes provide innate or specific adaptive immunity, and are derived from myeloid or
lymphoid lineage. Myeloid cells include highly phagocytic, motile neutrophils, monocytes, and
macrophages that provide a first line of defense against most pathogens. The other myeloid cells,
including eosinophils, basophils, and their tissue counterparts, mast cells, are involved in defense
against parasites and in the genesis of allergic reactions. Cells from the lymphoid lineage are
responsible for humoral or cell mediated immunity.
The major properties of the acquired immune response are specificity, memory, adaptiveness, and
discrimination between self and non-self. Lymphoid cells in these categories include T and B
lymphocytes and NK cells. T and B cells produce and express specific receptors for antigens while
NK cells do not. Receptor specificity is related to gene rearrangement of variable region
components during development, according to essential features for clonal selection.
B lymphocytes secrete antibodies; their activation is antigen-dependent following which they
differentiate into plasma cells. Upon interaction with T helper cells, they proliferate and switch the
isotype (class) of immunoglobulin produced, while retaining the same antigen-binding specificity.
B cells also process and present protein antigens; they have specific surface antigens (CD
molecules) necessary for response to foreign antigens.
T lymphocytes are involved in regulation of immune response and in cell mediated immunity.
During thymic differentiation, immature T cells undergo rearrangement of their TCR genes to
generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for further
maturation only if they recognize foreign antigens in the context of "self" molecules. Mature T
cells usually display one of two accessory molecules. CD4+ T helper cells are the primary
regulators of T cell- and B cell-mediated responses, and are further subdivided into subsets
dependent upon cytokines secreted. CD8+ T cytotoxic cells (CTLs) are cytotoxic against tumor
cells and host cells infected with intracellular pathogens. T suppressor cells suppress the T and B
cell responses and express CD8 molecules. T regulatory cells (Treg) are helper cells that suppress
other T cell activity and help prevent autoimmunity.
Natural Killer cells (NK) are large granular lymphocytes that nonspecifically kill certain types of
tumor cells and virus-infected cells. The NK cells are able to kill “self” in the absence of antigenspecific receptors. They kill via antibody-dependent cellular cytotoxic mechanisms (ADCC) via
their Fc receptors.
21
INNATE IMMUNITY and INFLAMMATION
Jeffrey K. Actor, Ph.D.
713-500-5344
Objectives: (1) Introduce innate immune defense mechanisms. (2) Define chemical mediators
involved in inflammation. (3) Review cell types involved in innate immune responses, and their
role in inflammation. (4) Define ADCC, chemokines, and Pattern-recognition receptors.
Keywords: Innate Immunity, Innate Defense Barriers, Neutrophils, Monocytes, Macrophages,
Natural Killer (NK) cells, Phagocytosis, APC, ADCC, Chemokines, Complement, PRRs,
Inflammasome.
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York,
NY. 6th edition, 2009. Chapters 2, 10 and 11; Geha and Notarangelo. Case Studies in
Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 15. Chediak-Higashi
Syndrome; Case 25. Neutropenia; Case 26. Chronic Granulomatous Disease; Case 27. Leukocyte
Adhesion Deficiency.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/Innate.html
Innate immune mechanisms provide the first line of defense from infectious disease. The innate
immune system is comprised of components which are present prior to the onset of infection and
constitute a set of mechanisms that are not specific for a particular organism. Rather, the innate
components recognize classes of molecules frequently encountered on invading pathogens, so as
to allow defensive measures while the specific immune response is either generated or
upregulated. Innate immune components are present from birth and consist of non-specific
components.
The innate defensive barriers can be divided into four major categories:
1. Anatomic - skin, mucous membranes
2. Physiologic - temperature, low pH, chemical mediators
3. Phagocytic and Endocytic - phagocytose to kill and digest microorganisms
4. Inflammatory - induction of vascular fluid leakage to area of tissue damage
Anatomic Barrier. The skin and mucous membranes provide an effective barrier against
microorganisms. The skin has the thin outer epidermis and the thicker underlying dermis to
impede entry, as well as sebaceous glands to produce sebum. Sebum is made of lactic acid and
fatty acids, which effectively reduce skin pH to between 3 and 5 to inhibit organism growth.
Mucous membranes are covered by cilia which trap organisms in mucous and propel them out of
the body.
Physiologic Barrier. The physiologic barrier includes factors such as temperature, low pH, and
chemical mediators. Many organisms can not survive or multiply in elevated body temperature.
Soluble proteins such as lysozymes, interferons and complement components play a major role in
innate immunity. Lysozmes can interact with bacterial cell walls; interferons alpha and beta are
natural inhibitors of viral growth; complement components use both specific and non-specific
immune components to convert inactive forms to active components that damage membranes of
pathogens. Low pH in the stomach discourages growth.
22
Phagocytic and Endocytic Barriers. Blood monocytes, tissue macrophages and neutrophils
phagocytose and kill microorganisms via multiple complex digestion mechanisms. Bacteria
become attached to cell membranes and are ingested into phagocytic vesicles. Phagosomes fuse
with lysosomes where lysosomal enzymes digest captured organisms.
Inflammatory Barriers. Invading organisms
cause localized tissue damage leading to
complex inflammatory responses. In 1600
BCE, Celsus described the four cardinal signs
of inflammation as rubor (redness), tumor
(swelling), calor (heat), and dolor (pain).
Later, Galen (2nd century) a fifth sign was
added; functio laesa (loss of function).
Inflammatory responses lead to (1)
Vasodilation causing erythema (redness) and
increased temperature; (2) increased capillary
permeability which allows exudates (fluid) to
accumulate leading to tissue swelling
(edema); and (3) influx of cells to site of
tissue damage. Once cells enter area of
damage, they release further chemotactic
factors to call in additional cells to damaged
area, leading to Chemotaxis, Activation,
Margination, Diapedesis (extravasation), and
finally recognition and attachment of these
cells to the damaged site.
-------------------------Chemical Mediators of Inflammation

23

Hageman factor: Plasma globulin (110 kD), blood clotting factor XII, which is activated
by contact with surfaces to form Factor XIIa, that in turn activates factor XI. Factor XIIa
also generates plasmin from plasminogen and kallikrein from prekallikrein. Both plasmin
and kallikrein activate the complement cascade. Hagemann factor is important both in
clotting and activation of the inflammatory process.

Thrombin: Protease (34 kD) generated in blood clotting that acts on fibrinogen to produce
fibrin. Consists of two chains, A and B, linked by a disulphide bond. Thrombin is
produced from prothrombin by the action either of the extrinsic system (tissue factor +
phospholipid) or, more importantly, the intrinsic system (contact of blood with a foreign
surface or connective tissue). Both extrinsic and intrinsic systems activate plasma factor X
to form factor Xa which then, in conjunction with phospholipid (tissue derived or platelet
factor 3) and factor V, catalyses the conversion.

Kallikrein: Plasma serine proteases normally present as inactive prekallikreins which are
activated by Hageman factor. Act on kininogens to produce kinins, to mediate vascular
reactions and pain.

Plasmin: Trypsin like serine protease that is responsible for digesting fibrin in blood clots.
Generated from plasminogen by the action of another protease, plasminogen activator. The
enzyme catalyses the hydrolysis of peptide bonds at the carbonyl end of lysine or arginine
residues. It also acts on activated Hageman factor and on complement.
24

Bradykinin: Vasoactive nonapeptide (RPPGFSPFR) formed by action of proteases on
kininogens. Very similar to kallidin (which has the same sequence but with an additional N
terminal lysine). Bradykinin is a very potent vasodilator and increases permeability of post
capillary venules, it acts on endothelial cells to activate phospholipase A2. It is also
spasmogenic for some smooth muscle and will cause pain.
Arachidonic Acid Metabolites: Inflammatory Role
Cell Types Involved in Innate Immunity
The cell types involved in innate immune responses include the polymorphonuclear cells
(neutrophils), monocytes and macrophages, eosinophils, and Natural Killer (NK) cells. Some of
these cells are capable of killing target cells via nonspecific (non-MHC dependent) through
release of lytic enzymes, perforin or TNF. Others are involved in phagocytic mechanisms that kill
via intracellular processes.
Neutrophils. Neutrophils are typically the first infiltrating cell type to site of inflammation.
Endothelial cells increase expression of E-selectin and P-selectin which are recognized by
neutrophil surface mucins (PSGL-1 or sialyl Lewisx). Chemoattractants (IL-8) trigger adhesion
and subsequent diapedesis. Multiple complement components (e.g. C5a) are chemotactic for
neutrophils, along with fibrinopeptides and leukotrienes. Activated neutrophils express high Fc
receptors and complement receptors to allow increased phagocytosis of invading organisms.
Activation of neutrophils leads to respiratory burst producing reactive oxygen and nitrogen
intermediates, as well as release of primary and secondary granules containing proteases,
25
phospholipases, elastases and collagenases, and lactoferrin. Pus, a yellowish white opaque creamy
matter produced by the process of suppuration consists of innumerable neutrophils (some dead
and dying) and tissue debris.
Figure. Cell membrane adhesion molecules and cytokine activation events associated with
neutrophil transendothelial migration. Left: Weak binding of selectin ligands on the neutrophil to Eselectin on the endothelial cells. Middle: IL-1 and TNF- upregulation of E-selectin, which facilitates
stronger binding. Right: The activation effects of IL-8 on
neutrophils cause a conformational change in the integrins
(e.g., LFA-1) to allow them to bind ICAM-1. Coico and
Sunshine, 2009. Fig 11.4.
Mononuclear Cells and Macrophages. Mediators such
as MIP-1 and MIP-1 attract monocytes to the site of
pathogenic infection. The monocytes express surface
ligands which recognize ligands (VCAM-1) on
endothelial cells, leading to diapedesis. Activated tissue
macrophages secrete IL-1, IL-6 and TNF- which further
increase expression of adhesion molecules on endothelial
cells to recruit neutrophils and more monocytes. These
molecules also increase release of acute-phase proteins
from the liver to assist in events leading to body
temperature increase.
Monocytes and macrophages ingest and destroy bacteria.
Multiple factors assist in preparing the particulate for
engulfment and targeting for destruction, including
various opsonins comprised of complement components.
Phagocytes bear several different receptors that
recognize microbial components and induce
phagocytosis. Five such receptors on macrophages are:
CD14, Toll-like receptors (such as TLR-4), the
macrophage mannose receptor, the scavenger receptor,
and the glucan receptor. All 5 receptors bind bacterial
carbohydrates. CD14 and CR3 are specific for bacterial
lipopolysaccharide (LPS). In addition, complement
receptors assist in this process.
Figure. Endocytosis and phagocytosis by macrophages.
Phagocytosed organisms are subjected to killing by
26
lysosomal enzymes in phagolysosomes. Killing of phagocytosed microbes is sone via ROS and
NO mediated mechanisms. These same substances can also be released to kill extracellular
microbes.
Figure. Important cytokines secreted by macrophages in response to bacteria and bacterial products
include IL-1, IL-6, CXCL8 (IL-8), IL-12, and TNF-a. TNF-a is an inducer of a local inflammatory response
that helps to contain infections. CXCL8 is also involved in the local inflammatory response, helping to
attract neutrophils to the site of infection. IL-1, IL-6, and TNF-a have a critical role in inducing the acutephase response in the liver and induce fever, which favors effective host defense in several ways. IL-12
may also activate natural killer (NK) cells.
The Inflammasome: Assembly and activation of the inflammasome is an essential process in
innate immune defense. The inflammasome is a cytosolic, multiprotein platform that allows
activation of pro-inflammatory caspases that cleave the precursor of interleukin-1β (pro-IL-1β)
into the active form. Secretion of active IL-1β helps to initiate a potent inflammatory response.
Antigen Presentation. Phagocytosed or pinocytosed antigens may then be presented to the
adaptive immune system cells. Dendritic cells, macrophages and monocytes are specifically good
at presenting antigens to T lymphocytes. In addition, B cells, are also extremely good APCs. Some
of the critical molecules which play a role in antigenic presentation by APCs to T cells are given
in the accompanying figure.
Figure from Immunology (6th ed). 2006. Goldsby, Kindt, Osborn and Kuby. WH Freeman Publisher.
27
NK Cells. NK cells are large granular lymphocytes that nonspecifically kill certain types of tumor
cells and virus-infected cells, and function as both cytolytic effectors and regulators of immune
responses. NK cells express a large number of receptors that deliver either activating or inhibitory
signals; the relative balance of these signals controls NK cell activity. NK cells are activated upon
detection of abnormalities in target cells such as the loss of antigen presentation molecules (MHC
class I expression) or up-regulation of stress-induced ligands. A variety of receptors trigger the
NK cytolytic activity directed toward certain tumor targets, virally infected cells, and even normal
immune system constituents such as immature dendritic cells. NK cells are also important
regulators of the adaptive immune system via their ability to secrete a number of cytokines in
response to immune activation.
Antibody-Dependent, Cell-Mediated Cytotoxicity (ADCC). ADCC is a phenomenon in which
target cells coated with antibody are destroyed by specialized killer cells. Among the cells that
mediate ADCC are NK cells, macrophages, monocytes, neutrophils and eosinophils. The killing
cells express receptors for the Fc portion of antibody coated targets. Recognition of antibody
coated target leads to release of lytic enzymes at the site of Fc mediated contact. In the case of NK
cells and eosinophils, target cell killing may involve perforin-mediated membrane damage.
Coico amd Sunshine, 2009. Fig.15.1
Clinical Relevance
Clinical Vignette – Case 15. Chediak-Higashi Syndrome: Chediak-Higashi syndrome is a rare inherited disorder
in which a severe immunological deficiency has been linked to deficits in NK cell function and to deficiency in
chemotactic and bactericidal function for neutrophils. Thus, these individuals are more susceptible to bacterial
infections. These individuals have characteristic giant lysosomes within neutrophils. Bone marrow transplantation
is the only effective therapy.
28
Chemokines. Chemokines are specialized cytokines that are chemotactic for leukocytes. They
are small polypeptides that are synthesized by a wide variety of cell types. They act through
receptors that are members of the G-protein coupled signal transducing family. All chemokines
are related in amino acid sequence and their receptors are integral membrane proteins that are
characterized by containing seven membrane-spanning helices.
Chemokines fall mainly into two distinct groups. The CC chemokines have two adjacent cysteine
residues (hence the name "CC"). The CXC chemokines have an amino acid between two cysteine
residues. Each chemokine reacts with one or more receptors, and can affect multiple cell types.
Chemokines and their functions will be covered again in greater depth in the Adaptive
Immunity chapter.
Properties of selected chemokines.
Chemokine
CCL2 (MCP-1)
CCL3 (MIP-1)
CCL5 (Rantes)
CCL11 (Eotaxin)
CXCL8 (IL-8)
Major Cell Source
Monocytes and Macrophages,
Fibroblasts
Monocytes, T cells, Fibroblasts,
Mast cells
T cells, Endothelium
Monocytes and Macrophages,
Endothelium and Epithelium
Monocytes and Macrophages,
Fibroblasts, Endothelial cells
Cell Type Attracted
Chemoattractant for monocytes
Chemoattractant for neutrophilic granulocytes
Chemoattractant for Eosinophils and Basophils, Monocytes and
Dendritic cells, and T cells
Chemoattractant for Eosinophils
Chemoattractant for Neutrophils
Additional Chemokines are listed in the Appendix.
29
Complement Components. The activation of complement is an important component of innate
immunity. This will be discussed in detail in further lecture. A brief introduction to complement
follows:
Activation of the complement system results in the production of several different
polypeptide cleavage fragments that are involved in five primary biological functions of
inflammation and immunity.
1. Direct Cytolysis of foreign organisms (e.g. bacteria): Antibodies recognizing pathogenic
determinants form the basis of a physical structure to which complement components interact.
Specifically, complement component C1 interacts with the Fc portion of IgM and IgG (except
IgG4) binding to the surface of bacteria. The binding of C1 initiates a cascade of events whereby a
membrane attack complex (MAC) is built upon the cellular surface. Synthesis of the MAC
structure culminates in assembly of a pore channel in the lipid bilayer, causing osmotic lysis of the
cell. MAC formation requires prior activation by either the classical or alternative pathways, and
utilizes the proteins C5b, C6, C7, C8, and C9.
2. Opsonization of foreign organisms. Complement components (e.g. C3b or inactivated C3b;
iC3b) bind to pathogens. Interaction with receptors (CR1, CR2, CR3, and CR4) on the surface of
macrophages, monocytes, and neutrophils leads to enhanced phagocytosis and targeted destruction
of organisms.
3. Activation and directed migration of leukocytes. Proteolytic degradation of C3 and C5 leads
to production of leukocytes chemotactic anaphylatoxin. For example, C3a is chemotactic for
eosinophils. C5a is a much more potent chemokine, attracting neutrophils, monocytes and
macrophages, and eosinophils. Interaction of C3a, C4a or C5a with mast cells and basophils leads
to release of histamine, serotonin, and other vasoactive amines, resulting in increased vascular
permeability, causing inflammation and smooth muscle contraction.
30
4. Solubilization and clearance of immune complexes. One of the major roles complement plays
is the solubilization and clearance of immune complexes from the circulation. First, C3b and C4b
can covalently bind to the Fc region of insoluble immune complexes, disrupting the lattice, and
making them soluble. C3b and C4b bound to the immune complex are recognized by the CR1
receptor on erythrocytes facilitating their transport to the liver and spleen. In the liver and spleen
the immune complexes are removed and phagocytosed by macrophage-like cells. The RBCs are
returned to the circulation.
5. Enhancement of humoral immune response. Coating of antigens with C3d (a breakdown
product of C3) facilitates their delivery to germinal centers rich in B and follicular dendritic cells.
Clinical Relevance
Clinical Vignette – Factor I Deficiency (Case 32, Geha and Notarangelo): The alternative pathway
of complement activation is important in innate immunity. Deficiency in Factor I (as well as deficiency
in Factor H) affects cleavage of C3b, and therefore leads to reduced C3bi. The nonproduction of iC3b
results in defective opsonization, which is critical for removing and destroying invading bacteria.
31
Receptors of the Innate Immune System. Receptors of the innate immune system recognize
broad structural motifs that are highly conserved within microbial species [ called PathogenAssociated Molecular Patterns (PAMPs)]. Such receptors are referred to as Pattern-Recognition
Receptors (PRRs). In a similar manner, Damage/Danger-Associated Molecular Pattern molecules
(DAMPs) initiate immune activity as part of the noninfectious inflammatory response.
Engagement of any of these receptors leads to triggering of signal pathways that promote
inflammation.
Receptor (location)
Target (source)
Effect of Recognition
Receptors of the Innate Immune System. [Table adapted from Immunology (2002) by Goldsby,
Kindt, Osborne and Kuby - W.H.Freeman, et al., NY.]
32
FIGURE 2.6. (A)
Pattern recognition
receptors called
TLRs binding to
molecules with
specific pattern
motifs expressed
by various
pathogens. (Coico,
2009)
33
Link Between Innate and Adaptive (Acquired) Immunity
 Recognition of pathogen via Pattern Recognition Receptors (PRR) or Toll-Like Receptors
(TLR) leads to activation and maturation of APCs.
 APCs process antigen and present to naïve T cells. Specifically, dendritic cells form a major
bridge between cells of the innate and adaptive immune responses.
 Presentation is accompanied by secretion of cytokines to assist development of T cell
response (e.g. Th1 maturation via presence of IL-12).
 Absence of internal activation signals can leads to Th2 development (MyD88 regulated).
Figure 6-4. Link between innate and adaptive (acquired) immunity. Pathogen recognition through
pattern recognition receptors is an important bridge between innate and adaptive immune function.
Recognition leads to activation and maturation of the presenting cell. Here, dendritic cells are
depicted as primary presenting cells, which assist in dictating subsequent responses. Processed
antigen is presented to naive T cells, accompanied by secretion of cytokines to assist development
and maturation of T-cell phenotypic response (e.g., T helper cell-1 maturation via presence of
interleukin-12). Inset box shows important Toll-like receptors and specific ligands involved in
pathogen recognition. At least 15 different Toll-like receptors have been identified. A more
complete list of Toll-like receptors and ligands is provided in the Appendix.
34
Summary: Innate Immunity
Immune system cells are derived from pluripotent hematopoietic stem cells. Immune responses of
the innate immune system provide natural immunity against microorganisms via phagocytosis and
intracellular killing, recruitment of other inflammatory cells, and presentation of antigens.
Leukocytes that provide innate immunity are derived from myeloid lineage. These cells include
highly phagocytic, motile neutrophils, monocytes and tissue macrophages, eosinophils, and
Natural Killer (NK) cells. These cells provide a first line of defense against most pathogens.
Innate defense barriers include (1) anatomic barriers, (2) physiologic barriers, (3) Phagocytic
barriers, and (4) inflammatory barriers. Damage to tissue caused by invading pathogens can lead
to rubor, tumor, calor, dolor, and functio laesa. Tissue damage leads to an influx of inflammatory
cells through chemotaxis, activation, margination and diapedesis. The inflammatory process is
initiated and controlled via multiple chemical mediators.
Neutrophils are usually the first cell type to arrive at the site of tissue damage. Activation leads to
respiratory bursts and release of granules to control bacterial growth. Mononuclear cells and
macrophages engulf organisms via multiple mechanisms, leading to control and destruction within
intracellular phagosomes. NK cells are large granular lymphocytes that kill targets via ADCC or
through lysis using perforin-induced mechanisms.
Chemokines and complement components are critical for activation of innate immune functions.
Defects may lead to severe clinical complications.
Pattern Recognition Receptors present on innate immune system cells assist in the recognition of
bacteria and virions. Recognition by PRRs of PAMPs leads to activation of multiple facets of
cellular response. In a similar manner, damage/danger associated DAMPS can function to elicit
innate inflammatory functions in non-infectious situations.
35
A general summary chart of innate components, effectors and function:
Component
Effectors
General Function
Anatomic and
Skin and Mucous Membranes
- Physical barriers to limit entry, spread
Physiologic
Temperature, Acidic pH, Lactic acid
and replication of pathogens
Barriers
Chemical Mediators
Inflammatory
Mediators
Hageman factor
Thrombin
Kallikrein
Bradykinin
Leukotrienes and Prostaglandins
Complement
Cytokines and Interferons
Lysozymes
Acute Phase Proteins and Lactoferrin
- Clotting, activation of inflammation
- Protease acting to produce fibrin
- Mediating vascular reactions and pain
- Vasoactive nonapeptide; spasmogenic
for some smooth muscle and will cause
pain
- Vasodilation and increased vascular
permeability
- Direct lysis of pathogen or infected cells
- Activation/Mediation of other immune
components
- Bacterial cell wall destruction
- Mediation of response
Inflammatory
Mediators
Complement
Cytokines and Interferons
Lysozymes
Acute Phase Proteins and Lactoferrin
Leukotrienes and Prostaglandins
- Direct lysis of pathogen or infected cells
- Activation of other immune components
- Bacterial cell wall destruction
- Mediation of response
- Vasodilation and increased vascular
permeability
Cellular
Components
Polymorphonuclear Cells
 Neutrophils, Eosinophils
 Basophils, Mast Cells
Phagocytic-Endocytic Cells
 Monocytes and Macrophages
 Dendritic Cells
Other Cells
 NK cells
- Phagocytosis and intracellular
destruction of microorganisms
36
- Presentation of foreign antigen to
lymphocytes
- ADCC
IMMUNOGENS AND ANTIGENS
Sudhir Paul, PhD
OBJECTIVES
To learn the molecular attributes and properties of compounds which render them
immunogenic and antigenic.
KEYWORDS
Immunogen, antigen, hapten, epitope, adjuvant.
READING
Chapter 3 of the Coico, et al textbook. Geha and Notalangelo, 2012. Case Studies in
Immunology, 6th Ed., 46. Hemolytic Disease of the Newborn. Multiple Myeloma (On file
on Blackboard/LRC). Web Resource:
http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/immantigen.html
ANTIGEN OR IMMUNOGEN?
IMMUNOGEN - Agent capable of binding immune receptors AND inducing an
immune response by B cells and T cells
ANTIGENS - Agent that binds with varying degrees of specificity to immune
receptors (antibodies on B cells; T cell receptor on T cells)
All immunogens are antigens, but not all antigens are immunogens.
IMPORTANCE OF IMMUNOGENICITY
 Germfree colostrum-deprived piglets are immunologically "virgin" and extremely
susceptible to microbial infection due to lack of passive maternal immunity. They
are, however, highly immunologically competent as determined by their excellent
immune response to various immunogens. An immunogen is the inducer of
specific antibody formation. The initial step in the primary immune response is
priming of multipotent uncommitted cells ("virgin" X cells) to committed
monopotent cells (Y cells). Y cells proliferate and differentiate into antibodyforming cells (Z cells). Adapted from Y.-B. Kim 1975
 Vaccines are the cornerstone of eradicating microbial disease – many available
vaccines. See slide.
 New vaccines are needed for emerging diseases. See slide.
EPITOPES RECOGNIZED BY T OR B CELLS
Epitopes are the three dimensional arrangements of atoms (sites) on the surface of
an antigen that bind to the paratope of an antibody OR the linear peptides that bind the
MHC molecules/T cell receptor. Epitopes recognized by B cells generally differ from
those recognized by T cells.
38
B cells can mount specific antibody responses without or with help from T cells (Tindependent or T dependent cells).
PHYSICOCHEMICAL FORCES INVOLVED IN ANTIGEN-IMMUNE
RECEPTOR BINDING
Antibody binding to antigen does not involve covalent chemical bonds. Instead, several
weaker types of molecular interactions are utilized. Thus, the reactions are reversible.
There are four kinds of forces that stabilize antigen-antibody interactions:
1. Electrostatic interactions. Usually due to the attraction between the charged
amino acid residues in proteins such as lysine, arginine, glutamic acid and aspartic
acid, for instance. The number of such interactions will enhance the affinity of the
interaction dramatically.
2. Hydrogen bonding. Electrostatic binding with covalent character. Example, -H
atom shared by electronegative N and O atoms.
39
3. Van der Waal’s forces. Attractive and repulsive forces between induced
oscillating dipoles in the electron clouds of two adjacent atoms. The force is
proportional to the 7th power of the distance separating two molecules. This is a
weak force, but is additive, and there are many van der Waal’s contacts in antibodyantigen complexes.
4. Hydrophobic bonding. Usually involves non-polar amino acids, e.g., leucine,
isoleucine.
MAJOR CLASSES OF ANTIGENS/IMMUNOGENS
The following major chemical classes of compounds may be
antigenic/immunogenic:
1. Proteins or glycoproteins. Most proteins or glycoproteins are excellent
antigens. The greater the complexity and molecular weight, the better it is as
an antigen.
2. Carbohydrates or polysaccharides. Bacterial capsules (i.e. pneumococci)
are powerful antigens. The ABO blood group epitopes are carbohydrates.
3. Lipids. Are not routinely antigenic, but if used as a hapten, immune
responses can be elicited, i.e. sphingolipids.
4. Nucleic Acids. Are poorly immunogenic themselves, but as haptens are good
antigens. Antibodies to DNA are important in patients with systemic lupus
erythematosus.
SEQUENTIAL AND CONFORMATIONAL EPITOPES
Two general classes of epitopes can be distinguished. They are best described as
they exist on protein antigens, but other classes of antigens (i.e. carbohydrates and
nucleic acids) can also express antigenic/immunogenic epitopes under some
circumstances.
1. Conformational (Non-Sequential) Epitopes
Conformational epitopes require the native 3-dimensional configuration of the
molecule to be intact for their expression. Denaturation of the molecule destroys
these kinds of epitopes and antibodies specific for conformational epitopes will
not bind denatured antigens. Conversely, denaturing the molecule prior to
injection of the animal (cooking an egg) will alter the conformation of the
molecule and the antibodies elicited that are specific for antigens on the denatured
form will not react with the undenatured form of the molecule.
2. Sequential Epitopes
Sequential epitopes are short stretches of amino acids (4-7 in length) which can
be recognized MHC molecules in short peptides or by antibodies in short peptide
40
regions within larger antigens. Thus, the only requirement is that the right
sequence of amino acids is expressed.
EXAMPLE OF CLINICAL RELEVANCE
Two cases in Geha & Notarangelo, 6th edition, emphasize the influence of genetic factors in immunogenicity of infectious agents
and immunogenicity of administered vaccines.
Case 12—MHC Class I Deficiency—This case describes the consequences of a failure of antigen processing for
protection from infectious agents. Tatiana Islayev, age 17, had been chronically ill since age 4. She had a history of repeated
sinus, lung, and middle ear infections due to a variety of respiratory viruses. Her 7-year old brother Alexander had a similar
history. The parents and 3 other children were healthy. Tatiana and Alexander had received oral polio vaccinations as well as
DPT and BCG vaccinations and tolerated them well. WBC analysis showed a profound deficiency of CD8 T cells. Further
studies showed that both Tatiana and Alexander had a nonsense mutation in their TAP-2 genes, a gene coding for a protein that
transports peptide fragments into the lumen of the endoplasmic reticulum where it binds to MHC class I molecules and this
complex is then transported to the surface of the cell to be recognized by a CD8 T cell.
Case 8—MHC Class II Deficiency—This case illustrates a genetically acquired susceptibility to pyogenic and
opportunistic infections. Helen Burns was the second child born to her parents. She had received routine polio and DPT
vaccinations at 2 months of age. At 6 months of age she developed pneumonia in both lungs, accompanied by a severe cough
and fever. Pneumocystis carinii was isolated from a tracheal aspirate and she was treated with pentamidine and seemed to
recover fully. Since P. carinii was found (an opportunistic pathogen), severe combined immunodeficiency (SCID) was
suspected. Her T cells were found incapable of responding to tetanus toxoid and her serum Ig concentrations were very low.
Her CD4 T cells were very low but her CD8 cell count was normal (ruling out a diagnosis of SCID). While working up her sib
and parents for possible bone marrow donation, it was found that Helen’s B cells did not express MHC Class II molecules. Her
mother was selected as the best donor of bone marrow. The graft was successful and normal immune function was restored.
41
REQUIREMENTS FOR IMMUNOGENICITY
Four characteristics that contribute to the immunogenicity of a substance:
1. Size, dose, route
Usually, compounds of less than 1,000 daltons are non-immunogenic.
Compounds between 1,000 and 6,000 daltons may or may not be immunogenic.
Those greater than 6,000 daltons are generally immunogenic. Intermediate dose is
most immunogenic. Immunogenicity is also a function of route of administration.
2. Chemical Composition
Physicochemical complexity is usually necessary for a compound to be
immunogenic. Homopolymers of amino acids usually are not immunogenic (i.e.
B. anthracis poly-gamma-D-glutamic acid, 50,000 daltons).
3. Foreignness
Foreignness was once considered to be an absolute requirement for
immunogenicity. It is now clear that certain self-components can be
immunogenic to the individual. Foreignness is an excellent general guideline as
to whether something might be immunogenic, but it is not a definitive
requirement for immunogenicity. Particulate and denatured antigens are often
more immunogenic.
4. Adjuvants/Degradability
Adjuvants enhance immune responses by inducing cytokine release or antigen
processing. T-dependent immunogens must be enzymatically degraded in order
to be immunogenic. Peptides of D-amino acids are non-immunogenic whereas
their L-isomers usually are immunogenic. Genes mapping to the Major
Histocompatibility Complex (MHC) can profoundly affect the degree of
immunogenicity of any substance.
HAPTENS
Haptens are low molecular weight compounds that are non-immunogenic by themselves
but become immunogenic after conjugation to high molecular weight carrier substances
that are immunogenic. The figure below illustrates coupling non-immunogenic p-aminoarsonic acid to a carrier to make it immunogenic.
Clinical Relevance—The hapten concept has been adapted to modern vaccine technology with great
success. There are several vaccines licensed that are based on covalently coupling isolated epitopes to
carrier molecules, usually tetanus toxoid. Hapten-type vaccines for pneumococcus and for
Haemophilus are currently available. Several others are in development. Such an approach provides a
much safer type of vaccine compared to using whole immunogen molecules or killed viral preparations.
42
MULTIVANT ANTIGENS
Macromolecules usually have multiple unique or repeat epitopes. The former type of
immunogens induce heterogenous immune responses (mixtures of antibodies or T cells
directed to individual antigens). The latter type of immunogens are often T-independent.
Both types of antigens can form large complexes with multiple antibodies, a phenomenon
that can cause pathological immune complex deposition, particularly in the kidney.
IMMUNOLOGIC SPECIFICITY AND CROSS-REACTIVITY
The forces mediating antigen-antibody recognition allow for a high degree of specificity.
That is, antibodies specific for one epitope or hapten can easily distinguish that epitope or
hapten from other similar structures. However, this specificity is not absolute because
antibodies specific for one epitope can bind with structurally similar, but non-identical
epitopes although with a lower affinity. Specificity and cross-reactivity can be
distinguished by inspecting the following table reporting antibody reactivity with various
structurally defined carbohydrate epitopes:
43
CROSS-REACTIVITY
Cross reactivity refers to the situation where the cell receptor or antibody can react with
two molecules because a) they share one or more identical epitopes or b) the epitope in
question is similar enough in sequence or in shape to bind to the receptor with a weaker,
yet functional, affinity.
Examples:
1. Toxoids—Antibodies elicited with toxoids react with native toxins
(Clinical Application—vaccination with tetanus toxoid and with diphtheria toxoid).
2. ABO Blood Group Antigens—Antibodies elicited by certain
environmental carbohydrate antigens react with the human A or B blood group antigens.
3. There are 4 strains of the flavivirus that causes Dengue. The virus
infects cells of the monocyte-macrophage lineage. Infection with one strain elicits
antibodies reactive with a common epitope on all 4 strains. Upon infection with a
different strain, the antibody to the common epitope reacts by cross-reaction and
facilitates phagocytosis by macrophages, thus helping the virus gain entry and the second
infection is typically much more severe than the first due to this cross-reactivity.
4. Yet another example of cross-reactivity is the ability of antibodies to
bacterial antigens to attack host tissues, causing autoimmune disease.
44
ADJUVANTS
Definition: An adjuvant is a substance, which when mixed with an immunogen,
enhances the immune response against the immunogen. The adjuvant itself is not
usually immunogenic.
Examples of Immunologic Adjuvants
1. Freund’s Complete Adjuvant. This is a mixture of a petroleum
based oil, an emulsifying agent and killed Mycobacteria. A water-inoil emulsion is formed with microdroplets of antigen solution
surrounded by the oil. This works by slowly releasing antigen over a
long period of time while inducing a delayed hypersensitivity reaction.
It is used experimentally, but not in humans.
2. Lipopolysaccharide (LPS). Is experimental
3. Muramyldipeptide. Is experimental
4. Synthetic Polynucleotide (Poly AU). Is experimental
5. Aluminum Hydroxide (alum precipitate). Is used in humans.
Functions to enhance the ingestion and eventual processing of antigen.
6. Cytokines. Are experimental
Currently, adjuvant research is a high priority research area for enhancing the
immunogenicity of the new genetically engineered vaccines. Aluminum hydroxide is
currently the only FDA licensed adjuvant in the US. There are several new adjuvants in
phase III clinical trials but the FDA has not yet licensed any of these. Some other
adjuvants are licensed in other countries, but are not available in the US. Adjuvant MF59
is licensed in Europe, but not in the US. MF59 consists of stable droplets (<250 nm) of
the metabolizable oil squalene and two surfactants, polyoxyethylene sorbitan monooleate
and sorbitan trioleate, in an oil-in-water emulsion.
45
SUMMARY
1. Immunogens elicit immune responses; antigens bind to antibodies, lymphocyte
receptors or MHC molecules.
2. The properties of foreignness, size, chemical complexity and degradability all
contribute to the degree of immunogenicity.
3. Haptens are low molecular weight substances that only become immunogenic upon
covalent coupling to an immunogenic molecule. Haptens are models for epitopes.
4. Major classes of antigens include carbohydrates, lipids, nucleic acids and proteins
or glycoproteins.
5. Linear epitopes require only the primary linear structure to be intact but
conformational epitopes require that the 3D integrity of the molecule is intact.
6. Antibody binding to antigen does not utilize covalent interactions—multiple weak
interactions stabilize the binding such as electrostatic interactions, hydrogen
bonding, hydrophobic interactions and Van der Waal’s forces.
7. Immunologic specificity is dependent on stereochemical positioning or spatial
arrangement of chemical groupings and on the chemical nature of the group
(mass and charge).
8. Immunologic cross-reactions can be due to antigens sharing identical epitopes or
having epitopes with similar but non-identical chemical groupings.
9. Adjuvants enhance the magnitude of the resulting antibody response when mixed
with an immunogen before injection. Aluminum hydroxide (alum) is the only
licensed adjuvant for human use in the USA. Several others are currently in phase III
clinical trials and some other adjuvants (ie MF59) are licensed in foreign countries.
46
Self Study questions for Antigens and Immunogens
1. Name two common immunogens used in human medicine.
2. Name one hapten now used for human vaccination purposes.
3. Name one disease where DNA and/or RNA is a known antigen.
4. Name some molecular pairings that illustrate hydrophobic interactions, electrostatic
interactions, and hydrogen bonding as might be found in a typical epitope-paratope
reaction.
5. What is immunologic cross-reactivity?
6. Given a peptide consisting of 7 amino acids that had been shown (in preliminary
studies) to be a linear epitope from botulinum toxin, describe the series of steps that
would be needed to determine if the peptide could be used successfully as a hapten for
eliciting antibodies reactive with intact toxin.
7. Describe how you would set up an experiment to show grade school science students
what you mean by “Immunogenicity”. Do it by using antigen X and antigen Y injected
into mice. X & Y have different molecular weights.
Answers can be found at: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/immantigen.html
47
IMMUNOLOGY
Antibody Structure and Function
Dr. Keri C. Smith
OBJECTIVES
Develop an understanding of how the structural and molecular features of antibody molecules
mediate both the protective and the pathologic functions of the different classes of antibodies.
KEYWORDS
Immunoglobulin (Ig), isotype, allotype, idiotype, opsonin.
READING
Chapter 4 in the Coico et al textbook, 2009.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/antibodies.html
INTRODUCTION
The production of circulating antibodies is one of the major functions of the immune system.
Antibodies belong to the general class of glycoproteins called globulins, due to their property of
being insoluble in half-saturated ammonium sulfate solutions. Subsets of antibodies have been
discovered and they are now known collectively as immunoglobulins, abbreviated Ig. Studies of
the molecular structure of the various Ig have clarified many of the properties such as specificity,
cellular reactivity, complement fixation, placental transfer and anaphylactic activity after mast cell
activation.
ISOLATION AND CHARACTERIZATION
Ig are found in large quantities in blood serum. The bulk of Ig migrates in the gamma region when
subjected to electrophoresis at pH 8.2. Tiselius and Kabat proved in 1939 that the gamma region
contained most of the antibodies in an immunized animal:
49
The shape of the gamma peak, being rather broad, suggested very early that the antibody
population was a heterogeneous collection of molecules with slightly different charges. This
heterogeneity was a major early obstacle in attempts to determine the structure of antibody
molecules and relate structure to function. This problem was partially solved by the discovery
of myeloma proteins that are homogeneous Ig produced by cells in a type of cancer called
multiple myeloma, a cancer of plasma cells.
STRUCTURE OF LIGHT AND HEAVY CHAINS
Enzymatic fragmentation and chemical reduction studies showed the basic 4-chain structure
of the predominant Ig, originally called gamma globulin. Pepsin and papain fragments of
Ig were used, along with studies of the reductive products to establish the 4-chain structure
with each molecule consisting of 2 identical heavy chains and 2 identical light chains. Amino
acid sequences then were used to further distinguish constant portions of the chains from
variable portions. The domain concept, hinge region and carbohydrate locations were
determined
.
50
Light Chains
Studies of Ig from many species showed that nearly all species studied had two types of
Light chains, called κ and λ. The difference between the two types of light chains is in the
amino acid sequence of the constant region domain. The overall ratio of the two Light chain
types varies between species (mice have 95% of their Ig with κ chains whereas human Ig has
60% κ and 40% λ).
Heavy Chains
There are 5 different classes or ISOTYPES of heavy chains. Each class of Heavy chain has a
characteristic amino acid sequence that distinguishes it from the other four classes but all five
classes have significant percentages of amino acid sequence similarities. The five Heavy
chain Ig classes are IgG, IgA, IgM, IgE and IgD. The different Heavy chains corresponding
to their class are given Greek letter designations: γ, α, μ, ε and δ. In many species, there are
two or more subclasses of some heavy chains that differ from one another by only a few
amino acids. Humans have 4 subclasses of the IgG isotype called IgG , IgG , IgG , and
1
IgG . IgA has two subclasses IgA and IgA .
4
1
2
3
2
Domains
Amino acid sequence studies of Ig shows that there is regularity to the structure in which
there are disulfide-bridged loops of approximately 60 amino acids for each 100-110 amino
acids. This is the case for both Heavy and Light chains. These are called domains.
There are two domains on both κ and λ Light chains and either 4 or 5 domains on heavy
chains. The amino acid sequences in the first domain on both Light and Heavy chains are
highly variable from molecule to molecule, and are referred to as the V or V
L
H
domains, respectively. The other Light chain domain is constant in its amino acid sequence
for the κ or λ type of chain and is referred to as the C domain.
L
The constant domains of Heavy chains are numbered from the amino terminal end toward the
carboxy terminal end as C 1, C 2, C 3 and (for IgM and IgE) C 4.
H
H
H
H
The following shows some specific biological functions carried out by Ig domains.
DOMAIN FUNCTIONS OF HUMAN IgG
------------------------------------------------------------------------------------------------------------Function
Domain(s)
V +V
Antigen Binding
H
L
C 1+C
Spacer between antigen-binding and effector functions
C 2
Binding C1q
H
L
H
Control of catabolic rate
Interaction with Fc receptor on macrophage/monocyte
C 3
H
C 1+C 3
H
H
Bind to Protein A
Interact with Fc Receptors on placental syncitiotrophoblast, neutrophils, and
cytotoxic K-cells
51
Hinge Region
IgG, IgA and IgD genes each have an exon coding for a short span of amino acids that
occupy the space between the C 1 and C 2 domains. This segment is rich in Cys and Pro
H
H
and permits significant flexibility between the two Fab arms of the antibody and the area is
called the hinge region, accordingly. Since this stretch is open to solvent, it is highly
susceptible to protease cleavage.
Variable Region or Variable Domain
Kabat and Wu developed their variability plot to try to predict which amino acids in the
Variable regions actually contacted antigen. They found that the greatest variability in
sequence between molecules occurred at 3 distinctive regions in Light and Heavy chains.
These were then called hypervariable regions. They were separated by sequences called
framework regions. It has been formally proven that the amino acids comprising the
hypervariable regions are the contact residues for antigen. Since they form the region of
structural complementarity for Ag epitopes, they are termed complementarity-determining
regions (CDRs).
52
In this plot the term VARIABILITY is defined as the ratio of the number of different amino
acids at a given position to the frequency of the most common amino acid at that position.
For example, in the original research at position 7, 63 different light chains had been
sequenced and Serine occurred 41 times so its frequency was 41/63 or 0.65. In all, 4 amino
acids were found at position 7. Thus the Variability value was 4/0.65 or 6.15.
IMMUNOGLOBULIN VARIANTS
Isotypes
The five major classes of Ig (IgG, IgA, IgM, IgE, IgD) are isotypic variants of
immunoglobulins or isotypes. Structural variations in the heavy chains distinguish the
isotypes from one another. The structural elements that define one isotype are the same for
each species. There are subclasses of IgG and IgA.
TABLE 4.2. Important Differences Between Human IgG Subclasses
IgG1
IgG2
IgG3
IgG4
Occurrence (% of total IgG)
70
20
7
3
Half-life
23
23
7
23
Complement binding
+
+
+++
—
Placental passage
++
±
++
++
Binding of monocytes
+++
+
+++
±
ALLOTYPES
Definition: Ig allotypes are defined as structural variants of the constant regions of L or H
chains of Ig that are coded by germ line genes.
These differences are detected serologically. Specific antibodies are available for analyzing
these differences from one individual to another. The differences are usually due to a single
amino acid difference in the light or heavy chain. Sometimes is it more than one amino acid
difference. The best examples of allotype differences in human Ig are the three allelic
variants of human kappa light chains.
Allotype
Amino acid differences
Valine @ 153, Leucine @ 191
Alanine @ 153, Leucine @ 191
Alanine @ 153, Valine @ 191
Km(1)
Km(1,2)
Km(3)
Mendelian co-dominant autosomal allelic genes code Allotypes. There are 4 subclasses of
human IgG and two subclasses of IgA (described earlier).
Importance: Allotypes are used forensically in cases of disputed parentage or in analysis of
blood. The allotypic variation has no influence on antibody specificity.
53
Clinical Vignette
Results of Pediatric vaccine trials published in 1985 and 1986 show some
correlation between severity of Hemophilus influenzae type b infections and
the IgG2 allotype of the patient (Ambrosino et al, J. Clin. Invest. 75, 19351942, 1985; Granoff et al, J. Infect. Dis. 154, 257-264, 1986). Briefly, they
showed that children with the G2m(23) allotype have higher levels of
immunity than the G2m(23) negative children and Km(1) allotype is more
protective in black children than other L chain allotypes.
IDIOTYPES
Idiotypes are epitopes found in V regions of antibody molecules. They are defined
serologically.
The idiotypic epitopes (Idiotopes) are thought to be located near or even within the
paratope site on the antibody. It is clear that several different kinds of anti-idiotypic
antibodies exist based on the type of idiotope that they recognize. The two most common are
shown in he following diagram:
54
Summary of the properties of human immunoglobulin isotypes.
55
Schematic structures for the 5 major isotypes of immunoglobulins are shown below:
STRUCTURAL FEATURES OF IgG
Important Structural Features of the human IgG isotype include:
a) Two domains in the L chain and 4 domains in the H chain.
b) The Inter-H chain disulfides hold the two large halves together in the area called
the HINGE region (see above) due to its flexibility. L→H disulfides covalently
connect the L chains to the H chains.
c) The single carbohydrate moiety is located in the C 2 domain. This carbohydrate
H
may have important structural and/or functional properties. The general function
of carbohydrate on the Ig’s as well as other glycosylated polypeptides seems to be
that it plays a role in cellular transport and in secretion.
56
BIOLOGIC PROPERTIES OF IgG
AGGLUTINATION AND PRECIPITATE FORMATION
IgG antibodies can cause agglutination of particulate antigens like bacteria. With soluble
antigens such as toxins, IgG antibodies can form insoluble precipitates. The mechanism will
be discussed in the lecture on Antigen-Antibody reactions. Both types of reactions make the
complexes easier for macrophage scavenger cells to ingest and destroy the antigen.
PLACENTAL PASSAGE
In humans, IgG is selectively passed through the placenta (but NOT IgG ) to provide passive
2
rd
th
immunity to the fetus. This begins in the 3 or 4 month of pregnancy in humans. Resistance of
the neonate to most common infections is almost exclusively via this passive IgG. Active transfer
is mediated by the Fc region of IgG molecules.
While this passive immunity is crucial for neonatal protection, some transferred antibodies can be
destructive. Erythroblastosis fetalis, hemolytic disease of the newborn, is mediated by the
passage of anti-Rh (D) antibodies of the IgG class across the placenta in an Rh (D)-negative
mother carrying an Rh (D) positive fetus. She can be sensitized to the Rh antigen by a previous
pregnancy. These antibodies can destroy fetal red blood cells and cause disease that can vary
from mild to fatal, depending on the maternal titer of anti-Rh (D).
Case 46—Hemolytic Disease of the Newborn—Mrs. Waymarsh was 31
and pregnant for the third time. Her blood type was A, Rh-negative. Her
husband was also type A, but Rh-positive. Her first child was born healthy
nd
and she was given RhoGAM following delivery. During her 2 pregnancy,
she developed a 1:16 indirect Coombs titer of 1:16 and a healthy girl was
induced at 36 weeks and she was again given RhoGAM. Five years later in
the third pregnancy, her Coombs titer was 1:16 at 14 weeks and bilirubin
was found in increasing amounts in the amniotic fluid . A low fetal
hematocrit (6.2%, normal is 45%) was detected in fetal blood. 85 ml of type
O, Rh-negative packed RBC were transfused into the umbilical vein. At 30.5
weeks another transfusion of 75 ml was given, and at 33.5 weeks 80ml was
transfused. At 34.5 weeks, labor was induced and a normal female infant
was born.
OPSONIZATION
IgG is a powerful opsonizing antibody (from the Greek opsonin, to prepare for eating). The
antibody reacts with microbial epitopes and its Fc region is then efficiently bound by specific
Fc receptors on macrophages and/or polymorphonuclear cells. The net effect is engulfment of
the bacteria into the phagocyte. The figures on the following page illustrate both the
mechanism of opsonization.
ANTIBODY DEPENDENT CELL MEDIATED CYTOTOXICITY (ADCC)
Certain large granular lymphocytes [also called Natural Killer (NK) cells] have Fc receptors
on their surface and when antibodies bind cellular antigens (for instance on tumor cells) NK
cells can then bind to the antibody via the Fc portion. The NK cell can kill the tumor cells
due to the secretion of substances by the NK cell that are cytotoxic to the tumor cells. Only
57
IgG antibodies are implicated in this type of activity and the importance of ADCC in host
defense or tissue damage is still controversial.
COMPLEMENT ACTIVATION
IgG antibodies are efficient activators of the Complement system. Complement will be
covered in a separate lecture. Briefly, many antigen-antibody interactions trigger a series of
enzymes collectively known as Complement. Some of the by-products of these reactions can
act as opsonins and other components are chemotactic (attract phagocytic cells). IgG
antibody activation of Complement can have profound biological effects, some positive and
some negative. Details will be presented in the lecture on Complement.
BACTERIAL IMMOBILIZATION
Motile bacteria can have their movement arrested by IgG and IgM antibodies by crosslinking their flagella or clumping them via their flagella. The antibody can function in this
regard like handcuffs in stopping the waving of flagella. The result is that the bacteria are less
invasive and less efficient in spreading through tissue.
VIRAL NEUTRALIZATION
Most viruses utilize some form of cellular receptor for initial binding that results in the virus
gaining entry into the cell or moving its DNA or RNA into the cell. IgG and IgM antibodies
specific for those structures on the virus that bind to the cell receptors will inhibit or
eliminate initial binding to the cell, thereby protecting the cell from viral entry. The binding
of IgG also facilitates phagocytosis of the organism.
TOXIN NEUTRALIZATION
Bacterial toxins usually are toxic to cells because the toxin binds to specific cellular receptors
to gain entry to the cell and then toxic effects occur intracellularly. The strategy that the
immune system employs to protect the animal from toxins is to make a variety of antibodies
specific for many different epitopes on the toxin to immobilize it in the form of an antigenantibody aggregate and stop the toxin from reaching the cell receptor. The Ab-Ag aggregates
can be easily phagocytosed and the toxins degraded and rendered non-toxic by acid proteases
in the phagosomes. The problem is in finding a way to use the toxins to trigger an immune
response without killing the host with toxin. This has been done with diphtheria and tetanus
58
toxins very successfully by treating the toxins with formaldehyde. Formaldehyde treatment
eliminates the toxic properties of the molecules without significantly affecting the antigenic
makeup of them (review the section on Cross-reactions in the lecture and chapter on
Immunogens and Antigens). These TOXOIDS then can be used to make vaccines that will
elicit a strong immune response with no toxic effects.
STRUCTURAL FEATURES OF IgM
IgM antibodies are called macroglobulin antibodies because of their high molecular weight.
Important structural features of IgM include:
a) the molecules are polymers of 5 four-peptide subunits each bearing an extra CH
domain as compared to IgG or IgA molecules.
b) polymerization of the subunits into a pentamer depends upon the presence of J
chain whose function may be to stabilize the Fc sulfhydryl groups during Ig
synthesis so that they remain available for cross-linking the five subunits.
c) the free molecule assumes a “star” or “wagon wheel” shape in free solution, but
when bound to antigens on membranes it adopts a “crab-like” shape.
d) the net paratope valency is 10, but with larger antigens, the effective valency falls
to 5 and this is attributed to steric restriction due to the lack of flexibility in the
molecule.
e) the hinge region is not nearly as flexible as the hinge in IgG.
f) the IgM antibodies tend to be of relatively low affinity as measured against haptens
but, because of their multivalency, they bind with high avidity to antigens with
multiple epitopes.
59
g) there are 5 distinct CHO moieties, compared to one in IgG.
h) there is an intersubunit cysteine bridge in the CH3 domain bridging the 4-chain
subunits in the molecule.
i) the Complement binding site is in the CH3 domain.
BIOLOGIC PROPERTIES OF IgM
Most of the IgM is found in intravascular spaces and the concentration in serum is low in
normal circumstances. IgM does not pass the placenta so elevated levels of IgM in the fetus
are indicative of congenital or perinatal infection. It is the first isotype to appear in serum
after vaccination with most antigens (T-dependent antigens) and elevated levels of IgM in
adults is also indicative of recent antigen exposure
AGGLUTINATION
IgM antibodies are highly efficient in aggregating or agglutinating particulate antigens such
as bacteria or red blood cells.
ISOHEMAGGLUTININS
The IgM population of antibodies includes the natural isohemagglutinins, the naturally
occurring antibodies reactive with the red blood cell antigens of the ABO series. These
antibodies are thought to be elicited by bacteria that have carbohydrate epitopes similar to, or
identical to, the antigens of the A or B blood group. The following table shows the kinds of
isohemagglutinin antibodies normally found in patients with the various blood groups:
Blood Type
A
B
AB
O
Isohemagglutinin Normally Present
Anti-B
Anti-A
None
Anti-A and Anti-B
COMPLEMENT ACTIVATION
Its pentameric form coupled with the correct amino acid sequence for binding complement
make IgM the most efficient isotype on a mole/mole basis for complement activation. It has
been shown experimentally that the binding of one IgM antibody on the surface of a red
blood cell is sufficient to lyse the cell but that in excess of 100 IgG antibodies are needed to
lyse a red blood cell.
STRUCTURAL FEATURES OF IgA.
IgA appears selectively in the sero-mucous secretions such as saliva, tears, nasal fluids,
sweat, colostrum and secretions of the lung, genitourinary and gastrointestinal tracts where it
has the job of defending the exposed external surfaces of the body against attack by microorganisms from the environment. It is present in these fluids as a dimer stabilized against
proteolysis by combination with another protein, secretory component, which is the cleaved
portion of the polymeric Ig receptor (PIgR) that allows for transport across the epithelial cell
layer (See figure below). sIgA is a single peptide of molecular weight 60,000
Important structural features of the IgA molecule include:
60
a) A larger number of disulfides compared to IgG
b) Two intrachain disulfides on the H chain in the CH2 and CH3 domains providing
for intermolecular bonding and J chain binding, respectively.
c) Three sites of glycosylation
Subclasses of IgA. Two subclasses of IgA exist. They are designated IgA1 and IgA2. The
momomeric form of IgA1 is the major subclass found in serum, whereas dimeric IgA1 and
IgA2 are found equally in mucosal secretions. The IgA1 and IgA2 subclasses differ in the
length of their hinge regions, and the IgG2 sublcass contains a disulfide bond at the C
terminus of the light chain
BIOLOGIC PROPERTIES OF IgA
Serum IgA I is monomeric and has no known biological function and a short (5.5 day) halflife. Secretory IgA (sIgA) is always in the dimeric form Most IgA is transported to
epithelial surfaces (see figure below) where it functions as a first-line barrier of protection
for these sensitive surfaces.
61
IgA ROLE IN MUCOSAL INFECTIONS
One of the primary roles of IgA on mucosal surfaces is to protect these surfaces from
infection. LOTS of sIgA is made in the intestine – estimated 3 g/day, also found in nasal
passages, saliva, and breast milk.
IgA ROLE IN BACTERICIDAL ACTIVITY
IgA antibodies will not fix Complement. The IgA molecule has bactericidal activity for
gram-negative organisms, but only in the presence of lysozyme that is normally present in the
same secretions where IgA is found. The secretory component that remains attached to
dimeric IgA may provide some protection against bacteria.
IgA ROLE IN VIRICIDAL ACTIVITY
IgA is an effective viricidal agent, preventing attachment of viral structures to specific
cellular receptors and, due to its multivalency in the dimeric form, is an efficient agglutinator
of viruses.
PASSIVE IMMUNOTHERAPY
sIgA can be transferred from mothers’ breast milk to the intestinal tract of the infant.
Provides protection against pathogens (i.e. rotavirus, cholera)
STRUCTURAL FEATURES OF IgD.
IgD is only found in trace quantities in serum. Its primary biological role is in triggering of
lymphocytes. It is co-expressed on the surface of certain subsets of lymphocytes along with
IgM.
Important structural features of the IgD molecule include:
a) IgD is not synthesized and secreted by mature plasma cells.
b) The constant region of the δ chain is comprised of 383 amino acids making it
longer than γ and α chains, but shorter than μ and ε which have 4 constant region
domains.
c) The hinge region of IgD is the largest of any Ig hinge with 64 amino acids in two
structurally distinct segments of about 30 residues each. This allows for
maximum flexibility in contacting antigen.
BIOLOGIC PROPERTIES OF IgD
IgD is nearly non-detectable in serum. IgD is found on the surface of mature B cells that
have not been antigen stimulated and is thought to be involved in maturation of these cells.
This will be discussed in more detail in the lecture on “Biology of B and T Cells”.
STRUCTURAL FEATURES OF IgE.
IgE molecules are normally only present in serum in trace amounts. The Fc part of the IgE
molecule has a recognition site for IgE receptors on the surface of mast cells. When IgE
62
antibodies of a given specificity encounter their antigen while bound to the surface of mast
cells, degranulation of the mast cells ensues with large amounts of histamine and other
vasoactive amines are released that have dramatic physiological consequences. IgE can be
thought of as the “allergy” or reaginic antibody. IgE is responsible for the symptoms of hay
fever and of extrinsic asthma when patients with atopic allergy come into contact with the
allergen, e.g. grass pollen, ragweed pollen, penicillin etc.
Important structural features of IgE include:
a) the large number of carbohydrate moieties.
b) the 5 distinct domains in the H chain (reminiscent of IgM). The cytotropic regions
appear to be the C 2 and C 3 domains.
H
H
BIOLOGIC PROPERTIES OF IgE
IgE IN HYPERSENSITIVITY REACTIONS
Type I hypersensitivity is mediated by IgE antibodies and occurs when an IgE response is
directed against innocuous antigens, such as pollen. The resulting release of pharmacological
mediators such as histamine by the IgE-sensitized mast cells produces an acute inflammatory
reaction with symptoms such as asthma or rhinitis. The most characteristic manifestation of
these types of reactions is hay fever; swollen eyes, runny nose, etc. However, if sensitization
is systemic and antigen is introduced IV (bee or wasp sting, injection of antibiotics, ingestion
of allergen orally) the symptoms can proceed rapidly through hives to cardiac arrest.
Mast cells and their circulating counterpart the basophil display a high affinity receptor for
the Cε2:Cε3 junction area in the Fc region of the IgE molecule. Local or even distant
production of IgE molecules specific for any antigen can then coat these cells with specific
antibody molecules of the IgE class. Arrival of antigen then causes:
a) cross-linking of the receptors
b) the breakdown of phosphatidyl inositol to inositol triphosphate
c) generation of diacylglycerol
d) an increase in intracytoplasmic free calcium.
63
Since inhibitors of methyltransferases and serine esterases inhibit all these events and
mediator release, it is assumed that activation of these enzymes by receptor bridging is the
initial event. Phospholipase c activation generates both IP3 (which mobilizes intracellular
Ca2+), and diacylglycerol which activates protein kinase c. The biochemical cascade
produces membrane-active “fusogens” such as lysophosphatidic acid that may facilitate
granular membrane fusion and degranulation, and the series of arachidonic acid metabolites
formed by the cyclooxygenase and lipxygenase pathways.
64
CLINICAL VIGNETTES
The first clinical vignette is on reserve in the LRC. It is a story entitled “How it feels to
die” written by a former editor of Life Magazine. Briefly, he self-prescribed some
penicillin tablets when he woke up one morning. The story describes his experience with
severe anaphylactic shock. It describes his treatment by his family physician who (lucky
for him) lived right next door. She had the right things with her to treat him and he lived.
Case 49—Acute systemic Anaphylaxis—A life-threatening immediate hypersensitivity
reaction to peanuts. John was a healthy 22 month-old who developed swollen lips while
eating a cookie containing peanut butter. A month later he ate another of the same kind of
cookie. He started to vomit, became hoarse, had difficulty breathing, started to wheeze
and developed a swollen face. He was rushed to the ER but became lethargic and lost
consciousness en route. Upon arrival at the ER his blood pressure was 40/0 (normal
80/60), pulse was 185 (normal 80-90) and respiration was 76 (normal 20). Epinephrine,
saline, anti-histamine, and corticosteroid were administered. Within one hour he was
responsive and after an overnight hospital stay including further epinephrine and antihistamine treatments, he was discharged. The parents were instructed to avoid giving him
any foods containing peanuts or peanut extract. Further tests were scheduled in the
Allergy Clinic.
IgE IN PARASITIC INFECTIONS
Elevated levels of IgE have been detected during the course of infection with certain
parasites. For example, infections with ascaris (a roundworm) routinely raises the detectable
levels of IgE antibodies more than other isotypes and immunization with ascaris antigens
selectively induces the formation of IgE antibodies. It is possible that IgE originally arose in
evolution as a specific means of protection from parasites.
The degranulation process releases histamine, heparin, eosinophil and neutrophil chemotactic
factors, and platelet activating factor, while leukotrienes B4, C4 and D4, prostaglandins and
thromboxanes are all newly synthesized. When there is a massive release of these mediators,
their bronchoconstrictive and vasodilatory effects predominate and become life
threatening. The figures below summarize the triggering mechanisms and effects of IgEantigen crosslinking on mast cells.
Typical immune responses in humans, as defined by detection of antibodies in serum specific
for the injected immunogen, have 4 clearly defined phases. Each phase correlates with known
mechanisms involved in immune responses.
Primary Response
1. Latent or Lag phase is long
2. Exponential Production phase-moderate rate
3. Steady state reached at modest Ab concentrations
4. Declining phase
65
Secondary (Anamnestic) Response
1. Lag phase is shorter (sometimes MUCH shorter!)
2. Exponential Production phase is steeper
3. Higher concentrations of antibody are detectable (sometimes MUCH higher!)
4. Production of antibody continues for a longer time (Slower declining phase)
In anamnestic responses, class switching from IgM to IgG production is much quicker and
antibodies of the IgA and IgE class are more likely to be detected than in primary responses.
Affinity maturation of the IgG produced is usually detected, that is, the IgG produced after
secondary immunization has a higher affinity for antigen than IgG synthesized during the
primary response.
THE IMMUNOGLOBULIN SUPERFAMILY
Structural features found in Immunoglobulin heavy and light chains are also seen in other
proteins, frequently in membrane bound glycoproteins. Due to the structural similarities,
these proteins are classified as members of the Immunoglobulin Superfamily. One of the
more important members of the Ig superfamily is the T cell receptor. It will be discussed in
detail in a subsequent lecture.
66
Case—Multiple Myeloma (posted on Blackboard), a malignancy of terminally
differentiated B lymphocytes. Isabelle Archer was a 55 yo housewife who began to
experience excessive fatigue. Routine blood tests showed an elevated sedimentation
rate. The elevated sed rate prompted measurement of her serum Ig levels. IgG was 3790
mg/dl (normal is 600-1500), IgA was 14 mg/dl (normal is 150-250) and Igm was 53
mg/dl (normal is 75-150). Electrophoresis screening of her serum showed a monoclonal
protein in the gamma region which was shown to be IgG with kappa light chains. Over
the next 3 years her IgG level rose to 6312 mg/dl and she experienced destruction of the
second thoracic vertebral body with extrusion of a plasmacytoma. She was treated with
hemotherapy and radiation and when her IgG level rose to 8200 mg/dl she was treated
with cyclophosphamide, etoposide and decadron, which lowered her IgG level to 6000
mg/dl. She now remains fully active requiring occasional blood transfusions for anemia
and complains at times of bone pain. Her serum IgG is stable at 6200 mg/dl.
The electrophoresis result, shown here, shows normal serum in lane 1, 3 and 5, Mrs.
Archer’s serum in lanes 2, 4 and 6. Lanes 1 & 2 are stained just for protein, lanes 3 and
4 were stained with anti-lambda antiserum and lanes 5 and 6 were stained with antikappa antiserum.
ANTIBODY ENGINEERING
Presents a way to make “custom made” antibodies for therapeutic use. “Recapturing in
recombinant form the naturally developed diversity of antibodies”
Single chain Fv (ScFv): V domains of expressed antibodies linked with a flexible peptide.
VL and VH domains are cloned into a plasmid vector, then the recombinant protein is
secreted by bacteria. Can also be expressed as Fab fragments containing full length light
chains linked by disulfide bonds to the VH-CH1 fragment of the heavy chain. Half life is
extremely short (hours).
“Humanized” antibodies: V domains of mouse antibodies against a specific target are cloned
into vectors encoding the constant regions of human antibodies. Resulting antibody may be
specific for antigen, and the potential for antigenicity is decreased.
Immunotoxins: conjugate antibodies with toxins to be able to specifically bind to target cells
and deliver toxin to a specific area (minimizes side effects).
67
CDR affinity
engineering
VL-VH
combinatorial diversity
VL
VH
C
D
R
C
D
R
CL
C
D
R
CH
1
CH2
CH3
CH3
Fc receptor
binding
CH2
Complement
activation
68
C domain
vectors
C
D
R
C
D
R
C
D
R
Fv
SUMMARY (Structural Features)
1. Antibodies are collectively defined as Immunoglobulins (Ig). Were originally found in
the globulin fraction of serum and migrate in electrical fields in the gamma region.
2. The basic Ig unit consists of 2 Light chains and 2 Heavy chains, all covalently bound via
disulfide bridges. Domains of the molecule responsible for unique biological functions
are also disulfide bonded.
3. There are two types of Light chains in humans, κ and λ, distinguished by different amino
acid sequences in the constant domain.
4. There are five classes or isotypes of heavy chains, γ, α, μ, δ, and ε corresponding to the
heavy chains of IgG, IgA, IgM, IgD, and IgE, respectively.
5. The hinge region in Ig allows for flexibility of the molecule.
6. The variable region or domain contains the short stretches of amino acid sequences that
make contact with antigen. These areas are called hypervariable regions or
complementarity-determining regions of the molecule.
7. Distinguishing features of IgG molecules include 4 domains on each of the two γ heavy
chains and two domains on the light chains, inter-H chain disulfide bridges between the
H chains, a single carbohydrate moiety in the C 2 domain. The IgG subclasses differ
H
structurally, in the H chains. It is normally the most abundant Ig.
8. Distinguishing features of IgM molecules include 5 domains on each of the two μ chains,
the soluble form of IgM is a pentamer of 4-chain subunits with a J chain attached
with a total paratope valency of 10 giving it high avidity. There are 5 separate
carbohydrate moieties on each H chain, the Complement binding site is in the CH
3
domain.
9. Distinguishing features of IgA molecules include 4 domains on each of the two α chains, a
larger number of disulfides compared with IgG, three sites of glycosylation, two
subclasses of IgA exist (IgA1 and IgA2) and two allotypic forms of IgA2.
10. Distinguishing features of IgD include 4 domains on each of the two δ chains with an
extended (64 amino acid) hinge region. Like IgM and IgA, IgD is more highly
glycosylated than IgG. IgD is found in only trace amounts in serum, most is bound to
surfaces of mature lymphocytes.
11. Distinguishing features of IgE include 5 domains on each of the two ε chains. Like IgM,
IgA, and IgD, IgE is more highly glycosylated than IgG. The C 2 and C 3 domains are
H
H
cytotropic for mast cells. Only trace amounts are found in serum (except in some
allergy patients) as most is attached to mast cells.
69
12. Allotypes are structural variants of L or H chains. Are germ line encoded Mendelian
autosomal codominant genes. Usually consist of one or a very few amino acid
differences.
13. Idiotypes are epitopes found in the V regions of specific antibodies. The idiotype
defining each antibody specificity is different.
70
SUMMARY (Biological Properties)
1. Class and subclass differences among the Ig’s for mediating biological functions such as
placental transfer, complement fixation, half life, location of function (secretions,
intravascular) depend on structural features of the molecules.
2. Antibodies can be either protective or destructive.
3. IgG antibodies efficiently agglutinate or precipitate antigens, cross the placenta,
opsonize bacteria for phagocytosis, mediate ADCC, neutralize bacterial toxins,
immobilize motile bacteria and neutralize viruses.
4. IgA is transported to epithelial surfaces in a dimerized form and protects such surfaces
from infection. IgA is uniquely bactericidal only in the presence of lysozyme, is
virucidal by preventing attachment of viruses to cell receptors.
5. IgM is concentrated intravascularly and does not pass the placenta. It is a highly effective
agglutinator of particulate antigens due to its pentameric structure, is the principle
isohemagglutinin (barrier to blood transfusion), and is highly efficient for fixing
complement.
6. IgD is found on the surface of mature antigen sensitive B cells, only in trace amounts in
serum and has no significant known protective properties.
7. IgE causes allergies. Binds to the surface of mast cells and then cross-linking by antigen
triggers the mast cell to secrete several pharmacologically active substances that cause
the symptoms of allergies. Functionally protective in parasitic infections.
71
Antibodies
Table 3-1. Classes of Antibody Isotypes and Functional Properties*
Immunoglobulin Class
IgG
IgE
Isotype
IgM
IgD
Structure
Pentamer
Monomer
Monomer
Monomer
IgA
Monomer,
dimer
Heavy chain
designation
μ
δ
γ
ε
α
Molecular weight (kDa) 970
184
146-165
188
160 × 2
Serum
concentration(mg/mL)
1.5
0.03
0.5-10.0
<0.0001
0.5-3.0
Serum half-life (days)
5-10
3
7-23
2.5
6
J chain
Yes
No
No
No
Yes
Complement activation Strong
No
Yes, except
IgG4
No
No
Bacterial toxin
neutralization
Yes
No
Yes
No
Yes
Antiviral activity
No
No
Yes
No
Yes
Binding to mast cells
and basophils
No
No
No
Yes
No
Additional properties
Effective
agglutinator of
particulate
antigens, bacterial
opsonization
Found on surface
of mature B cells,
signaling via
cytoplasmic tail
Antibodydependent
cell
cytotoxicity
Mediation of
allergic
response,
effective
against
parasitic worms
Monomer in
secretory fluid,
active as dimer
on epithelial
surfaces
72
COMPLEMENT
Rick A. Wetsel, Ph.D.
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc,
New York, NY. 6th edition, 2009. Chapter 13; Geha and Notarangelo. Case Studies in
Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 32. Factor I
Deficiency; Case 33. Deficiency of C8.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/complement.html
WHAT IS COMPLEMENT?
Complement is a system of more than 30 serum and cell surface proteins that is
involved in numerous functions in inflammation and immunity. In conjunction with
specific antibodies, it acts as the primary humoral defense system against bacterial and
viral infections. Complement activity is heat labile and can be destroyed by heating
serum to 56O C 30 minutes (which inactivates the C3 and C4 proteins as well as other
complement components).
Most of the complement proteins in the serum are
produced by liver hepatocytes. C3 is the most abundant serum complement protein
with a normal range of 1.0 to 1.5 mg/ml in healthy individuals. Some of the complement
components (e.g. C3, factor B) are acute phase proteins and can increase in
concentration two to three fold. Many of the complement proteins have shared
sequences, indicating that they evolved by gene duplication and recombination. The
complement genes are scattered throughout the human genome; the genes for the
proteins C4, C2, and factor B are located within the Major Histocompatibility Complex
on chromosome 6.
COMPLEMENT FUNCTIONS
Activation of the complement system results in the production of several different
polypeptide cleavage fragments that are involved in five primary biological functions of
inflammation and immunity.
1. Cytolysis of foreign organisms (e.g. bacteria)
2. Opsonization and phagocytosis of foreign organisms
3. Activation of inflammation and directed migration of leukocytes
4. Solubilization and clearance of immune complexes
5. Enhancement of humoral immune response
73
THE COMPLEMENT CASCADES
Complement activation involves the sequential activation of complement proteins, either
by protein-protein interactions or by proteolytic cleavage. At each step, the number of
protein molecules activated increases, amplifying the reaction. This sequential reaction
is call the complement cascade.
Many complement proteins are present as zymogens which are activated either by
conformational changes or by proteolytic cleavage by other complement proteins.
Activation of these zymogens results in specific serine protease activities that are
capable of cleaving other complement proteins, producing the complement cascade.
ACTIVATION
Complement activation is initiated by the presence of antigen-antibody complexes
(Classical Pathway), foreign cell surfaces (Alternative Pathway), or by mannose on
pathogenic organisms (Lectin Pathway).
FIGURE 1. OVERVIEW OF COMPLEMENT CASCADES
74
Space for notes:
75
CLASSICAL PATHWAY ACTIVATION
-Primarily by IgG and IgM immune complexes
-IgM > IgG3 > IgG1> IgG2
-IgG4, IgA, IgD, and IgE do not activate
Activation of the classical pathway requires the local reaction of antibodies with two or
more antigenic sites. These Ag-Ab complexes may consist of a single IgM molecule
bound to two or more antigenic sites, or two or more human IgG molecules (IgG1, IgG2,
or IgG3) bound to epitopes. Such a complex could (for example) occur on a bacterial
cell surface or in an aggregate of antibodies with soluble antigens. Ag-Ab reaction
causes conformational changes in CH2 of IgG and CH3 of IgM, permitting the binding of
C1q. Binding of two or more arms of C1q causes conformational changes that lead to
cleavage and activation of the bound zymogens C1r and C1s.
FIGURE 2.
Bridging of two membrane-bound IgG molecules by the C1
component. Binding distorts the C1 molecule and triggers activation.
Activated C1s can cleave C4 and C2 into large (C4b and C2b) and small (C4a and C2a)
fragments. C4 is cleaved first, and approximately 1% binds to a nearby surface via a
covalent linkage. C2 can complex with surface bound C4b and can be cleaved by C1s.
The resulting C4bC2b complex is the classical pathway C3 convertase, and has the
ability to specifically cleave C3 into large (C3b) and small (C3a anapylatoxin) fragments.
C3 is the most abundant complement protein and plays a pivotal role in complement
activation. Many molecules can be cleaved into C3b and C3a. Cleavage results in
exposure of the labile thiolester bond in C3b, permitting some to bind covalently to
proteins and carbohydrates on cell surfaces. The C3a anaphylatoxin is released into
the blood and mediates many important inflammatory activities that will be discussed
76
later. Some of the C3b binds to C4bC2b to form C4b2b3b, which is the C5 convertase.
This complex by the C2b protease subunit will cleave C5 into big (C5b) and small (C5a
anaphylatoxin) subunits. C5a is released into the blood and as C3a mediates many
important inflammatory activities. C5a on a molar basis is 100 times more potent than
C3a.
FIGURE 3. The classical pathway of complement activation generates a C3
convertase that deposits large numbers of C3b molecules on the surface of the
pathogen.
FIGURE 4. Cleavage of C3 and C4 exposes a thiolester bond that causes the
resulting large fragments, C3b and C4b, to bind covalently to nearby molecules
on bacterial or viral surfaces.
The classical pathway of complement can also be activated by the serum mannose
binding lectin complex (MBL-MASP). This complex is structurally similar to the C1
complex. However, instead of binding to immune complexes it binds to directly to
polysaccharides on gram-negative bacteria. The mannose binding lectin is C1q-like in
structure and the MBL associated proteases (MASP) are similar to C1r and C1s. MBLMASP on binding bacterial surfaces can cleave C4 and C2 thereby activating the
remainder of the classical pathway.
77
ALTERNATIVE PATHWAY ACTIVATION
The main difference between the classical and alternative pathways is that the initiation
of the classical requires an activating substance. The alternative pathway, by
contrast, runs continuously and spontaneously at low levels in the blood plasma. The
alternative pathway activation occurs when C3b binds to a surface that lacks inhibitors
that block complement activity, such as most bacterial cell surfaces. Certain plastic
surfaces, like those initially used in heart-lung machines and dialysis machines, also
activate the alternative pathway with obvious deleterious effects. Because antibody is
not necessary, the alternative pathway represents an innate immune response and can
react as soon as bacteria enter the body. The alternative pathway is also important in
amplifying reactions initiated by the classical pathway.
The low level activation of C3 that allows the alternative pathway to be activated is
called the tick-over model. The thiolester bond in C3 is spontaneously hydrolyze at
low rates yielding a C3b-like [C3(H2O)] molecule that now has a binding site for factor B
exposed. The bound factor B is attacked by factor D, which cleaves it into Ba and Bb
fragments. The Ba fragment is released, while the Bb fragment remains noncovalently
associated with C3(H2O), forming an initial C3 convertase. The Bb subunit of this
convertase has serine protease activity specific that can now specifically cleave
additional C3 molecules into C3a and C3b fragments. If a activator surface is nearby,
such as a bacterial surface, then the newly formed C3b molecule can covalently attach
and bind factor B. The bound factor B is cleaved by factor D and the surface-bound C3
convertase (C3bBb) then attacks another native C3 molecule and so on. The activating
surface (bacteria) thus accelerates a reaction that in its absence occurs at a slow rate.
FIGURE 5. THE TICK-OVER MODEL
78
LYTIC PATHWAY-FORMATION OF THE MEMBRANE ATTACK COMPLEX (MAC)
After the C3 convertase cleaves C3 to generate C3b, the next step in either the
classical, lectin, or alternative pathways is the binding of C3b to the C3 convertase
complex, changing it to a C5 convertase, which catalyzes the proteolytic cleavage of
the C5 protein. Cleavage of C5 to C5a and C5b represents the first step of the lytic
pathway. The small C5a fragment is released into the blood and is the most potent
complement anaphylatoxin. The large C5b molecule binds proteins C6 and C7. The
complex C5b67 has hydrophobic regions that permit it to insert into the lipid bilayer
nearby cell membranes. Subsequent binding of C8 permits some leakage of cell
contents, causing slow lysis. This process is accelerated by binding of multiple C9
molecules, which assemble to form a protein channel through the membrane. C9 is
analogous to perforins produced by cytolytic T cell and NK cells. C5b6789 is called the
Membrane Attack Complex (MAC). MAC formation is an important mechanism for
eliminating bacteria resistant to intracellular killing by phagocytes, such as Neisseria
species.
FIGURE 6. FORMATION AND REGULATION OF THE MEMBRANE ATTACK
COMPLEX (MAC)
REGULATION OF COMPLEMENT ACTIVATION
Complement activation is a tightly regulated series of reactions, that without control
would result in the inappropriate activation on normal host cells. This would result in
excess inflammatory mediators and by direct lysis of host cellular membranes by MAC.
79
This does not normally happen because there exist several complement inhibitors in
serum as well as on the surface of host cells.
C1-inhibitor (C1INH)
serum protein binds to activated C1r,C1s, removing it
from C1q
C4-binding protein (C4BP)
serum protein that binds C4b displacing C2b;
co-factor for C4b cleavage by factor I
Complement Receptor 1 (CR1;CD 35) Binds C4b displacing C2b, or
C3b displacing Bb; cofactor for I
Factor H (H) serum protein binds C3b displacing Bb; cofactor for I
Decay Accelerating Factor (DAF;CD55)
Membrane protein displaces Bb
from C3b and C2b from C4b
Membrane Cofactor Protein (MCP;CD46)
membrane protein that
promotes C3b and C4b
inactivation by I
CD59 (Protectin)
Prevents formation of MAC on homologous cells.
Widely expressed on membranes
S Protein (Vitronectin)
serum protein binds C5b-7 prevents insertion
into membrane
Clusterin (SP-40-40)
serum protein binds C5b-7 prevents insertion
into membrane
Complement Receptors
There are several characterized complement receptors that are involved in binding
complement activation and degradation products. They are expressed on various cell
80
types and are involved in mediating many of the biological functions attributed to
complement.
TABLE II DISTRIBUTION AND FUNCTION OF RECEPTORS FOR COMPLEMENT
PROTEINS ON SURFACE OF CELLS
C5a Receptor (C5aR;CD88) is a seven transmembrane G-protein coupled receptor
expressed primarily on neutrophils and macrophages. Also found on hepatocytes and
various tissue epithelial cells. Causes smooth muscle contraction, histamine release
from mast cells and vasodilation. Will modulate the hepatic acute phase response. It
also is a potent chemoattractant for neutrophils, monocytes, macrophages, and
eosinophils.
C3a Receptor (C3aR) also seven transmembrane receptor. Tissue distribution currently
being worked out. Causes smooth muscle contraction, histamine release from mast
cells, and vasodilation. Chemoattractant for eosinophils but not neutrophils.
81
BIOLOGICAL FUNCTIONS OF COMPLEMENT
1. CYTOLYSIS OF FOREIGN ORGANISM BY C5B-9 MAC COMPLEX
2. OPSONIZATION AND PHAGOCYTOSIS
C3b, C3bi is coated on microorganisms (opsonization)
Receptors for C3b (CR1) and C3bi (CR3) on macrophages and neutrophils can then
bind the complement coated bacteria facilitating the phagocytosis reaction
3. ACTIVATION OF INFLAMMATION AND CHEMOTAXIS OF LEUKOCYTES BY
COMPLEMENT ANAPHYLATOXINS (C3A, C4A, C5A)
82
All three peptides mediate: 1. smooth muscle contraction 2. histamine release from
mast cells and 3. increase vascular permeability.
C5a on binding C5aR mediates chemoattraction of neutrophils, monocytes,
macrophages, and eosinophils. C3a is a chemoattractant for eosinophils but not
neutrophils.
4. SOLUBILIZATION AND CLEARANCE OF IMMUNE COMPLEXES
One of the major roles complement plays is the solubilization and clearance of immune
complexes from the circulation. First, C3b and C4b can covalently bind to the Fc region
of insoluble immune complexes, disrupting the lattice, and making them soluble. C3b
and C4b bound to the immune complex is recognized by the CR1 receptor on
erythrocytes facilitating their transport to the liver and spleen. In the liver and spleen the
immune complexes are removed and phagocytized by macrophage-like cells. The
RBCs are returned to the circulation. Individuals deficient in the early complement
components cannot make C3b and C4b. They are therefore predisposed to immune
complex diseases such as systemic lupus erythematosus.
83
5. ENHANCEMENT OF THE IMMUNE RESPONSE
CR2 (CD21) is expressed on B-cells and follicular
dendritic cells. This receptor binds the C3d fragment
of C3. C3b coated on antigens will be broken down
eventually to C3d and C3c fragments by factor I. The
C3c fragment is released into the blood with no know
function. The C3d fragment remains covalently bound to
the antigen. Coating of antigens with C3d facilitates
their delivery to germinal centers rich in B and follicular
dendritic cells.
CR2 also is part of the B-cell coreceptor complex. Binding of C3d coated antigens to
CR2 leads to signaling through CD19. Animals deficient
in C3 have an impaired immune response to T
dependent antigens.
COMPLEMENT DEFICIENCIES AND ASSOCIATED ABNORMALITIES
Human deficiencies in many of the complement proteins have been described. These
deficiencies are usually attributable to inherited mutated genes. Genetic deficiencies in
classical and alternative pathway components, including C1q, C1r, C4, C2, properdin,
and factor D have all been described. C2 deficiency is the most common of the
complement deficiencies.
Deficiencies in the early classical pathway proteins
predispose individuals to the development of systemic lupus erythematosus (SLE). The
reason for this is not completely clear, but it is at least partly do to the inability of these
individuals to clear immune complexes readily. Because of its central importance in
killing bacteria, homozygous C3 deficiency can be lethal, especially in young children if
it is not diagnosed.
Deficiencies in the terminal components predispose these
individuals to recurrent bacterial infections with Neisserial species.
84
Table III. Complement Deficiencies and Associated Diseases
85
Dear Immunology students,
There is confusion between designation of the correct term used for the
complement C3 convertase, with discrepancies between the lecturer and
what is printed in the Coico, 2009 text. We will use the nomenclature
provided by the lecturer and use what is listed in the syllabus.
Here is our understanding. The nomenclature for complement has
undergone revision so that the large, target-bound fragment is consistently
given the 'b' designation, while the small, soluble fragment is called 'a'.
Thus over the last 10 years texts have begun to reverse 2a and 2b, which
were initially named incorrectly by this convention. According to the
current (but not universally accepted) nomenclature, the C3 convertase is
C4b2b and C4a and C2a are the released fragments; Coico, et al. apparently
have not yet adapted this change.
You will see other opinions in nomenclature, primarily from older texts that
have not adopted the new naming structure.
Therefore, due to a change in nomenclature in order to maintain the
a=smaller and b=larger scheme, the correct C3 Convertase is C4b2b.
86
SUMMARY
1.
The complement system is a group of over 30 serum proteins and cell
surface molecules that act as an important part of the overall immune
system.
2.
The activities of complement include: 1) cytolysis of foreign organisms,
2) opsonization and phagocytosis of foreign organisms, 3) activation of
inflammation and directed migration of leukocytes, 4) solubilization and
clearance of immune complexes, and 5) enhancement of humoral
immune response.
3.
The complement cascade is a series of reactions involving complement
proteins. It can be divided into two phases: activation and lysis (MAC
formation).
Activation involves protein-protein interactions and
proteolytic cleavage, whereas the lytic pathway involves protein-protein
interactions.
4.
There are three activation pathways: the classical, lectin, and alternative.
Classical pathway activation requires antigen-antibody complexes
(containing IgM or certain IgG subclasses). The Lectin pathway is a
newly described pathway that activates the classical pathway
independent of antibody. The Mannose Binding Lectin complex is
substituted in place of C1 and recognizes polysaccharides on bacterial
surfaces.
The alternative pathway is an innate system in which
complement components react directly with foreign substances.
Classical pathway activation involves the proteins C1,C4,C2, and C3,
and the alternative pathway utilizes the proteins C3, B, D, and P.
5.
Complement activation results in the release of anaphylatoxins
(C3a,C4a, and C5a). They are important mediators of inflammation,
causing recruitment and activation of neutrophils, macrophages,
and
other cell types. Activation also produces cleavage products (C3b, C3bi,
and C4b) which serve as opsonins, enhancing phagocytosis. The C3d
cleavage fragment is involved in enhancing the immune response.
6.
MAC formation produces a channel in the cytoplasmic membrane of
bacteria or other cells, leading to cell lysis. It requires prior activation by
either the classical or alternative pathways, and utilizes the proteins C5b,
C6, C7, C8, and C9.
7.
The complement system is regulated by several inhibitors, including C1
inhibitor, Factor H, C4 binding protein, CD 59, and Decay Accelerating
Factor.
87
8.
Deficiencies in complement components result in increased susceptibility
to bacterial infections and can lead to autoimmune diseases, including
systemic lupus erythematosus.
STUDY PROBLEMS
1.
Watch the Complement Video in the Learning Resource Center (VT 803).
2.
From memory, draw the classical, lectin, and alternative pathways of
complement.
3.
Understand how each is activated.
4.
Name 5 biological functions mediated by complement activation.
5.
Name the complement activation products that mediate each function.
6.
What are the three complement anaphylatoxin peptides?
7.
How does complement clear immune complexes from the circulation?
8.
What complement activation products bind covalently to cell surfaces?
9.
Know how the complement activation pathways are affected if a certain
component is missing.
10.
If a patient is missing a particular complement protein, what disease(s) are they
predisposed?
11.
On the figure shown on the next page, assign the activation fragments
responsible for that function.
88
Study Problem 11.
89
Complement Component Table I
90
Actor. 2012. Figure 6.6. Activation of complement through the classic pathway (antigen-antibody
complexes), the alternative pathway (recognition of foreign cell surfaces), or the lectin pathway (or
mannose-binding pathway) promotes activation of C3 and C5, leading to construction of the membrane
attack complex..
91
GENERATION OF ANTIBODY DIVERSITY
Steven J. Norris, Ph.D.
Recommended Reading: Actor, 2012, Chapter 3.
WebResource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/geneab.html
I. General principles
1. Ag-binding diversity
results from differences in
the Variable domains due to –
 Multiple V gene segments
 V(D)J joining
 Random assortment of H, L
chains
 Junctional/insertional diversity
 Somatic mutation
2. Functional diversity
is due to differences in
the Constant domains
 IgM  C’ fixation
 IgG  C’ fixation, opsonization
through Fc receptors
 IgA  secreted Ig
 IgE  allergic reactions
Changes in these Ig isotypes occur through isotype switching
3. Generation of Ag-binding diversity occurs during B or T cell development before
exposure to antigens; isotype switching occurs in B cells after antigen exposure.
4. Both the generation of Ag-binding diversity and isotype switching involve DNA
rearrangement.
5. Each B cell and all of its progeny produce only one type of heavy and light chain V
region, and thus have a single antigen specificity. This specificity can be ‘fine tuned’ by
point mutations (so-called somatic hypermutation) that occurs after antigen exposure.
Development of Ag-binding
Diversity (VDJ rearrangement)
(IgM/IgD producing B cells) 
Exposure to Ag + T cell
cytokines results in
isotype switching (B cells
change expression from IgM
to other isotypes)
92
II. The heavy chain Ig locus and VDJ rearrangement
1. The heavy chain Ig locus is on chromosome 14 in humans. It encodes the IgM and IgD
heavy chains ( and , respectively) that are expressed by B cells initially, as well as
the other heavy chain isotypes (1, 2, 3, 4, 1, 2, and ) that are expressed after
antigen exposure. In all cells except B cells, it is found in the so-called germline
configuration, i.e. the same arrangement that is found in ova and spermatozoa; this
germline form of the locus is hence passed from one generation to the next.
2. In the germline form, the Ig heavy chain locus contains multiple V, D, J , and C gene
segments:
Abbreviation:
V
D
J
C
Meaning:
“Variable”
“Diversity”
“Joining”
“Constant”
Number:
~50
~20
~6
9*
Size:
Function:
~95 aa
~3-6 aa
Form part of Variable Domain
~13 aa
~110 aa
Form the Constant Regions
per Domain
*One for each heavy chain isotype; each constant region contains 3 or 4 C domains
(see figure on proceeding page)
3. Stem cells in the bone marrow or fetal liver are stimulated to differentiate into B cells.
These B cell precursors (pro-B cells) are then stimulated to undergo VDJ
rearrangement. The pro-B cells begin to express Rag-1 and Rag-2, which direct the
recombination of the Ig genes in B cell precursors and the TCR genes in T cell
precursors. In B cells, the first step is D-J joining, in which the DNA between
randomly selected D and J regions is looped out and the intervening sequence is
deleted (see diagram). (This process involves the recognition of 7-bp and 9-bp
sequences next to the D and J regions; the same type of recognition occurs in all Ig
and TCR VDJ rearrangements.)
4. Following D-J joining, a similar looping out and deletion mechanism occurs between
the V and D regions, resulting in V-D joining. The resulting contiguous V,D, and J
gene segments have no intervening introns and form the Variable Region Exon. If the
93
14 undergoes
rearrangement. If this recombination is unsuccessful, the cell undergoes
apoptosis and dies.
Coico et al., 2009 Fig. 6.3. V(D)J rearrangement. V-J joining in the V locus is shown
5. After successful rearrangement, the 
chain is transported to the surface of
the cell along with the surrogate light
chain proteins, VpreB and 5. The
presence of this complex on pre-B
cells triggers the initiation of light
chain rearrangement.
Coico., 2009, Fig. 7.2. (A) IgM-like receptor on
Pre-B cells; (B) Surface IgM on mature B cells.
6. In mature B cells, the  (IgM) and  (IgD) heavy chains can both be expressed on the
same cell by the alternative splicing of RNA as shown in Fig. 6.4 (preceding page).
Clinical vignette – B cell maturation, from Geha and Notarangelo, “Case Studies in Immunology”.
Case 1
III.
X-linked Agammaglobulinemia – medical student Bill Grignard has normal T cell
function, but has almost no B cells or antibodies due to a defect in signaling at the pre-B
cell stage.
Light chain rearrangement.
1. In humans and most other mammals, there are two light chain loci called kappa () and
lambda (). These are located on two different chromosomes. The germline
arrangement is similar to that of the heavy chain locus, except there are no D gene
segments. Also, for the kappa locus there is only one constant region, whereas the
lambda locus has multiple constant regions, each with its own J gene segment.
2. Once successful heavy chain rearrangement occurs, the pre-B cell proceeds with
kappa gene rearrangement. In this case, randomly selected V and J segments in one
chromosome join together to form the Variable Region Exon; no D segments are
involved.
3. If a functional kappa chain is produced, V(D)J rearrangement stops and the cell
becomes a immature B cell that expresses only IgM on its surface. The surface IgM
will be anchored by a hydrophobic ‘tail’, and will look like the molecule shown in Fig.
94
7.2 (see above). Later, the cell can coexpress both IgM and IgD and thus become a
mature B cell.
4. If the first rearrangement does not produce a kappa chain, then the second ‘sister’
chromosome will undergo rearrangement. If that is unsuccessful, then the two lambda
loci will rearrange one after the other. If those are nonproductive, the cell will undergo
apoptosis and die, as was the case for the heavy chain locus.
5. The end result of this process is an immature B cell expressing only IgM with either
kappa or lambda light chains. As the cell leaves the bone marrow, it begins to express
both IgM and IgD and thus becomes a mature B cell. This difference is important
because immature B cells are more readily tolerized (made nonresponsive to
antigen) than are mature B cells.
6. The steps of B cell development are summarized in the following figure (Coico, 2009,
Fig. 7.1). B cell tumors may be ‘frozen’ at different stages of this maturation process;
the less mature tumor types tend to be more aggressive and to have a poorer
prognosis.
95
IV.
Mechanisms of Ag-binding diversity
It is estimated that each individual is capable of producing B and T cells with 1015 to 1018
different antigen binding sites, each with a different (but perhaps overlapping) antigen
specificity! As a result, there are very few compounds that will not induce an immune
response, even if they are synthetic and have never been found in nature previously.
Equally amazing is that most of the diversity is generated during V(D)J rearrangement,
prior to antigen exposure. Thus each individual randomly produces a huge “repertoire” of
B and T cells, only a small proportion of which (<1%) will respond to any one antigen or
infectious agent.
How is this Ag-binding diversity created?
As described in the Antibody Structure and Function lecture, the portions of the Variable
Region that participate in antigen binding are called Complementarity Determining
Regions or CDRs for short. All differences in antigen binding are thus due to differences
in these sequences. Two of the CDRs (CDR1 and CDR2) are ‘hard-wired’ into the V gene
segment, and thus depend upon the V segment selected during rearrangement. CDR3
consists of the junction of the V, D, and J gene segments and hence has a high degree of
variability. The CDRs of both the heavy and light chain participate in the formation of the
antigen binding pocket or paratope.
V
D J
Coico et al., 2009, Fig. 4.4
Coico et al., 2009, Fig. 4.5
There are 5 major mechanisms for generating antibody diversity in humans:
1. Availability of multiple V gene segments – as indicated above, there are ~50 V gene
segments in the heavy chain locus, and about 40 V gene segments each in the kappa
and lambda light chain loci. Thus there are about 50 heavy chain and 80 light chain
sequences encoding CDR1 and CDR2.
2. Combinatorial diversity (different VDJ and VJ combinations) - the V, D, and J
regions in heavy chains (and the V and J regions in light chains) are selected randomly
during V(D)J rearrangement (“joining”). For example, one cell could express a heavy
chain with VH3, DH1, and JH5, while another could express VH43, DH3, JH1; Thus 50 x 20 x
6 or 6000 different heavy chain VDJ loci can occur. A lesser degree of variation can
occur in the light chain recombinations, because there are no D regions; there are ~200
and ~160 different VJ combinations in kappa and lambda, respectively. This
combinatorial diversity affects the sequence of CDR3.
96
3. Assortment of heavy and light chains. Because the heavy and light chain loci
recombine independently, each B cell will contain a different combination of H and L
chains. This raises the total number of possible V, D, and J combinations to ~2 x 106.
4. Junctional and insertional diversity. The recombination between, say, D and J
segments is not precise, i.e. may occur a few base pairs in one direction or the other.
This ‘sloppiness’ causes differences in the amino acid sequence and leads to
junctional diversity.
Insertional diversity results from the activity of terminal deoxynucleotide
transferase (TDT), an enzyme that is expressed during heavy chain rearrangement.
TDT adds nucleotides randomly at the V-D and D-J junctions.
Both junctional and insertional diversity affect CDR3.
All of the above mechanisms occur during B cell development, before antigen
exposure.
5. Somatic hypermutation – the V regions of the antibody heavy and light chain genes
undergo a >10,000 higher rate of mutation than ‘regular’ DNA. This somatic
hypermutation occurs only after antigen stimulation. Some of these mutations
increase the affinity of antibody for antigen, and those B cells expressing antibody with
higher affinity will be selectively stimulated, increasing the proportion of high affinity
antibody in secondary responses. This process is called affinity maturation.
Altogether, these mechanisms produce almost an endless variety of antibody
specificities.
V.
Isotype switching
Isotype switching (also called class switching) results in the changing of the isotype of
antibody expressed by a given B cell, e.g. from IgM to IgG3. Here are some features of
isotype switching.
1. The constant region gene expressed is always the one immediately downstream of the
V region (exception: both IgM and IgD can be expressed by ‘niave’ B cells that have
not been stimulated by antigen).
2. The V region does not change during isotype switching; therefore the same antigenic
specificity is retained.
3. Isotype switching results when antigen-stimulated B cells receive a cytokine signal from
T helper cells. For example, IL-4 stimulates B cells to switch to IgE or IgG1.
4. Switching involves the deletion of intervening DNA between specific recombination
sites called switch regions (see figure below). Because the intervening DNA is lost,
the B cell cannot ‘switch back’ to an isotype that has already been deleted.
5. The V region and C regions are transcribed together, and RNA splicing and translation
results in expression of the ‘new’ isotype.
97
VI.
Membrane vs. secreted Ig expression
1. B cells express only membrane-bound immunoglobulins.
2. Plasma cells express only secreted immunoglobulins.
3. The difference between membrane vs. secreted Ig is the presence or absence of a
hydrophobic ‘tail’ at the carboxy terminus of each heavy chain.
4. Regulation of membrane vs. secreted Ig expression is due to alternative splicing of
the RNA transcript.
1. Membrane-bound form – RNA splicing results in retention of the M exons, which
encode the hydrophobic amino acid region that anchors the Ig in the membrane.
2. Secreted form – the RNA transcript is cleaved before the M exons, resulting in a
readily secreted, hydrophilic form of the heavy chain.
5. Differentiation of a B cell into a plasma cell results in this change.
VII.
Regulation of Ig expression.
1. After VDJ joining, the promoter 5’ to the V region is brought within a few thousand base
pairs of the enhancer element between the J region and the constant region. The
enhancer greatly increases the rate of transcription, increasing Ig production.
2. Ig production is further increased in the differentiation of B cells into plasma cells.
VIII. The immunoglobulin gene superfamily
How did this fantastic mechanism evolve in the first place?
1. Antibodies are present in some form in all vertebrates, but are not found in invertebrate
animals. However, vertebrates and invertebrates express a large number of closely
related cell surface proteins, which are collectively called the immunoglobulin gene
superfamily. It is thought that antibodies and T cell receptors evolved from cell
surface receptors used for other functions, such as cell-cell interactions.
98
2. Domain structure. All members of the immunoglobulin (Ig) gene superfamily contain
structures called Ig domains. An example of the two domains found in Ig light chains is
shown below. Domains have the following features:
 Primary amino acid sequence similarity (often only 20-30 percent)
 About 100-110 amino acids in length
 Beta-pleated sheet structure
 Commonly have an intrachain disulfide bond
3. Members of the Ig gene superfamily are
important in both immunologic and nonimmunologic systems, and include the
following:
 Immunoglobulins
 T cell receptors
 Major histocompatibility class I and II
proteins
 Many receptors specific for
leukocytes (e.g. CD3, CD4, and
CD8)
 Many additional cell-surface
receptors not exclusively involved in
the immune system (e.g. intercellular
adhesion molecule 1 [ICAM1],
neuro-cellular adhesion molecule
[NCAM], and chorioembryonic
antigen [CEA])
Coico et al., Fig. 4.14
4. The ‘Big Bang’ theory – John Marchalonis at University of Arizona and others have
proposed that the genes encoding recombinase proteins RAG1 and RAG2 were
obtained by horizontal transfer of DNA from fungi or bacteria, resulting in the sudden
acquisition of the antibody system in early vertebrates.
99
SUMMARY – GENERATION OF ANTIBODY DIVERSITY
1. Generation of antigen-binding diversity results from V(D)J recombination during B
cell development and somatic mutation after antigenic stimulation. Functional
diversity of antibodies results from isotype switching, allowing expression of the 9
different Ig isotypes. Each B cell and all of its progeny express only one heavy
chain and one light chain V region sequence, and thus all have the same antigenic
specificity.
2. During B cell development, rearrangement of the heavy chain locus occurs first. DJ recombination is followed by V-D recombination, resulting in formation of the V
domain exon comprised of V, D, and J gene segments. This process requires
pairing of 7-bp and 9-bp sequences, after which the intervening DNA is ‘looped out’
and deleted permanently from the chromosome.
3. Light chain rearrangement occurs through a similar process, in which randomly
selected V and J gene segments in the kappa or lambda light chain loci are joined.
4. V(D)J recombination proceeds through a hierarchy of heavy chain locus  kappa
locus  lambda locus. Functional heavy and light chains must be produced, or the
developing B cell undergoes apoptosis and dies.
5. There are five sources of antibody diversity: 1) presence of multiple V gene
segments; 2) Combinatorial diversity, resulting from random recombination of V, D,
and J segment combinations; 3) junctional and insertional diversity, resulting in
changes in the V-D and D-J junctions; 4) co-expression of different H and L chain
pairs; and 5) somatic hypermutation.
6. Isotype switching occurs after antigenic stimulation and requires cytokines produced
by T cells. DNA is deleted between switching regions, so that a different constant
region gene is juxtaposed close to the V domain exon. Expression of different Ig
isotypes results.
7. B cells express only the membrane-bound form of Ig, whereas plasma cells express
only the secreted form. This results from differential termination of heavy chain
transcription.
8. Ig gene expression is upregulated by enhancer elements and other factors;
expression by plasma cells is much higher than in B cells.
9. Immunoglobulins and T cell receptors are members of the immunoglobulin gene
superfamily, which apparently first evolved a large set of cell surface receptors.
100
THE ROLE OF THE MHC IN THE IMMUNE RESPONSE
Jeffrey K. Actor, Ph.D.
713-500-5344
Objectives: (1) Understand genetic organization of the major
histocompatibility complex. (2) Present an overview of differential processing
of antigens in the MHC class I and class II pathways. (3) Discuss MHC
restriction as related to presentation of antigens. (4) Present an overview of
disease association with MHC type. (5) Introduce CD1 non-peptide lipid
presentation.
Keywords: HLA, H-2, Class I, Class II, 2-microglobulin, APC, Polymorphism, CD1
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York,
NY. 6th edition, 2009. Chapter 8; Geha and Notarangelo. Case Studies in Immunology. Garland
Publishing, New York, NY.6th edition, 2012. Case 8: MHC Class II Deficiency.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/MHC.html
The Major Histocompatibility Complex (MHC) is a locus on a chromosome comprised of multiple
genes encoding histocompatibility antigens that are cell surface glycoproteins. MHC genes encode
both class I and class II MHC antigens. These antigens play critical roles in interactions among
immune system cells; class I participates in antigen presentation by macrophages to CD8+
lymphocytes (CTL), class II molecules participates in antigen presentation by macrophages to
CD4+ lymphocytes (T helper). MHC genes are very polymorphic. The locus also encodes a third
category of MHC genes, those of the class III type. The class III MHC molecules include
complement proteins, tumor necrosis factor, and lymphotoxin. In man, the MHC locus is
designated as HLA (Human Leukocyte Antigen).
MHC molecules gain their name because they were first identified as the targets for rejection of
grafts between individuals. When organs are transplanted across MHC locus differences between
donor and recipient, graft rejection is prompt. In 1980 the Nobel Prize was awarded to Baruj
Benacerraf, Jean Dausset and George D. Snell, for their work involving the major
histocompatibility complex and rejection of skin grafts using inbred strains of lab mice. In mice,
the MHC locus is designated as H-2. It has since been determined that the function of the MHC is
the presentation of antigen fragments (epitopes) to T cells.
Organization and Structure of the MHC Genes and Gene Products
MHC molecules are organized into 3 classes. Class I molecules are found on all nucleated cells.
The class II molecules are found on B-cells and macrophages. Class III genes encode for various
soluble proteins that include certain complement components.
Human MHC: The HLA locus in humans is found on the short arm of chromosome 6. The class I
region consists of HLA-A, HLA-B, and HLA-C loci and the class II region consists of the D
region which is subdivided into HLA-DP, HLA-DQ, and HLA-DR subregions. Class I molecules
are important in presentation of intracellular antigen to CD8+ t cells as well as for effector
functions of target cells. Class II molecules are important in the induction of an immune response,
since antigen-presenting cells must complex an antigen with the class II molecules to present it in
101
the presence of cytokines to CD4+ lymphocytes. Class III molecules encoded by genes located
between those that encode class I and class II molecules include complement components.
Coico and Sunshine, 2009. Fig. 8.1
Polymorphism of Class I and Class II MHC genes
Each chromosome 6 encodes three class I molecules B, C and A and three class II proteins DR,
DP and DQ. All six of these MHC molecules show a high level of allotypic polymorphism, i.e.
certain regions of the molecules differ from one person to another. The chance of two unrelated
people having the same allotypes at all twelve sets of genes that encode MHC molecules is very
small. There is a 25% chance of having identical genes that encode MHC molecules with each
sibling, but only six of the twelve sets of genes that encode MHC molecules are inherited from
each parent. MHC class I and class II molecules that are not possessed by an individual are seen as
foreign antigens upon transplantation and are dealt with by the recipient's immune system
accordingly. The highly polymorphic class I and class II MHC products are central to the ability of
T cells to recognize foreign antigen and the ability to discriminate "self" from "non-self".
The Class I MHC molecules are each somewhat different from one another with respect to aminoacid sequence, and all three are co-dominantly expressed in the membrane of every nucleated cell
in an animal - but, depending on the organ involved, at different levels of expression (as high as 5
x 105 molecules per cell on lymphocytes). The term "co-dominantly expressed" means that
each gene encoding these proteins on each parental chromosome of the diploid cells is
expressed. MHC class I molecules expressed on progeny (F1) cells match maternal or paternal
class I molecules since only the  subunit genes exhibit species-specific polymorphisms. MHC
class II molecules expressed on F1 cells include homologous and heterologous  dimer mixtures
since both  and  subunit genes exhibit species-specific polymorphism. Homologous dimers
match class II molecules expressed on either parental cell type while heterologous dimers are
unique to the F1 genotype and are functionally non-equivalent to parental class II molecules.
Structure of the MHC Class I Molecule
Each class I locus codes for a transmembrane polypeptide of molecular weight approximately 45
kDa, containing three extracellular domains (1, 2, 3). The molecule is expressed at the cell
surface in a noncovalent association with an invariant polypeptide called 2-microglobulin (2M)
102
of 12 kDa. 2M is a member of the Ig superfamily, the complex of class I and 2M appears as a
four-domained molecule with the 2M and 3 domain of class I juxtaposed near the cell surface
membrane.
Figure. View of MHC class I showing how
a T cell receptor interacts with the class I
molecule/2-M with peptide bound in the
peptide binding groove.
103
Figure. Schematic representation of an
intact class I antigen in the plasma
membrane. MHC class I showing the
association of a class I molecule with
2M.
Structure of the MHC Class II Molecule
Class II molecules have 2 transmembrane polypeptide chains ( and , 30-34 and 26-29 kDa
respectively); the peptide-binding site is shared by the two domains furthest from the cell
membrane. The overall structure of the peptide-binding site is very similar for both class I
and class II MHC molecules; the base is made of -pleated sheet, as in an immunoglobulin
domain – the sides of the groove that holds the peptide are -helices. Peptides bind within
the allele specific pockets defined by the 2 transmembrane polypeptide chains, where they
are presented to the TCR for recognition. The extracellular domain shows variability in
amino acid sequences, yielding grooves with different shapes. These grooves cradle the
processed antigen for interaction with the T cell receptor. The CD4 molecule assists in the
recognition process, and binds to the invariant portion of the MHC class II molecules.
Like class I genes, class II genes also exhibit polymorphism with multiple allelic forms
expressed. In humans, allelic forms are designated different from the mouse. For examples,
human class II genes are given numbers such as HLA-D4 or HLA-D7.
Figure. View of MHC class II showing
how a T cell receptor interacts with the
class II molecule with peptide bound in
the peptide binding groove.
Figure. Schematic representation of an
intact class II antigen in the plasma
membrane. MHC class II showing the two
chain class II molecule.
104
MHC Class III Molecules: Class III HLA genes encode complement components that
show no structural similarity to either class I or class II molecules. These genes, along
with genes encoding tumor necrosis factor (TNF), separate HLA class II and class I genes
on the chromosome.
MHC and Antigen Presentation
There are two major classes of presented antigen (Ag) called endogenous and exogenous
Ag. MHC class I presents endogenous Ag epitopes to CD8+ T cells and MHC class II
present exogenous Ag epitopes to CD4+ T cells. All nucleated cells are capable of
presenting MHC class I, but only specialized cells present Ag epitopes on MHC class II.
These are macrophages, dendritic cells and B cells. When exogenous Ag enters the body
it is phagocytosed, digested and the resulting fragments are presented on MHC class II.
When the CD4+ T cell receptor binds Ag-MHC class II it is activated to proliferate and
secrete cytokines which in turn activate the other immune competent cells to generate
humoral and/or cellular immunity. When CD8+ T Cell (CTL) receptor binds Ag-MHC
class I it is activated to produce and secrete toxin that kills the cell to which it is bound.
The cells that ingest, digest and present exogenous Ag epitopes on MHC class II are
called antigen presenting cells (APCs), and the process of ingestion, digestion and
presentation is called antigen processing and presentation. All nucleated cells can display
MHC class I, but only APCs display MHC class II.


CD8+ T Cell recognize Ag-MHC class I and CD4+ T Cell recognize Ag-MHC
class II.
Antigen is recognized in conjunction with proteins of the major
histocompatibility complex (MHC). Different antigen degradation and
processing pathways produce MHC-peptide complexes where "endogenous"
peptides associate with class I molecules and "exogenous" peptides associate
with class II molecules.
105
Endogenous (cytoplasmic) antigen processing and MHC class I presentation: MHC
class I molecules bind peptide fragments derived from proteolytically degraded proteins
endogenously synthesized by a cell. Small peptides are transported into the endoplasmic
reticulum where they associate with nascent MHC class I molecules before being routed
through the Golgi apparatus and displayed on the surface for recognition by cytotoxic T
lymphocytes. MHC class I molecules bind small antigenic peptides that are 8-10 amino
acid residues in length.
Coico and Sunshine, 2009. Fig. 8.7.
106
Exogenous (endosomal) antigen processing and MHC class II presentation: MHC
class II molecules bind peptide fragments derived from proteolytically degraded proteins
exogenously internalized by "antigen presenting cells," including macrophages, dendritic
cells, and B cells. The resulting peptide fragments are compartmentalized in the
endosome where they will associate with MHC class II molecules before being routed to
the cell surface for recognition by helper T lymphocytes. MHC class II molecules bind
larger antigenic peptides usually 13-18 amino acid residues in length (but may be longer).
Like class I molecules, class II MHC molecules are synthesized in the RER. The class II
 and  chains reside there as a complex with an additional polypeptide called the
invariant chain (Ii). The invariant chain blocks the groove of the class II molecule and
prevents endogenous antigens from binding there. The MHC/invariant chain complex is
transported to an acidic endosomal or lysosomal compartment that contains a degraded
antigen peptide. The invariant chain comes off the complex, exposes the groove of the
class II molecule, and allows the antigen peptide to slip into the groove. The class
II/antigen peptide complex is then transported to the surface of the APC where it is
available for interaction with CD4 TH cells.
FIGURE 8.5. Processing of exogenous antigen in MHC class II pathway, (Ii = invariant
chain; CLIP = fragment of Ii bound to MHC class II groove.)
Coico and Sunshine, 2009. Figure 8.5.
107
Coico and Sunshine, 2009. Table 8.1.
108
The role of the MHC in Thymic Education
The education process by which T cells in the thymus learn to recognize antigenic
peptides in the context of self-MHC molecules is a two-step process involving both
positive and negative selection.

Step 1: Initially, immature thymocytes within the thymic cortex express low
levels of TCR, but high levels of both CD4 and CD8 (double-positive cells).
They interact with thymic epithelial cells that express high levels of both class I
and class II MHC molecules. Thymocytes with moderate affinities for these selfMHC molecules are allowed to develop further, while thymocytes with affinities
too high or too low for self-MHC are induced to die by apoptosis. The
thymocytes that survive are said to have been "positively selected" through their
interaction with self-MHC.

Step 2: The positively selected thymocytes then begin to express high levels of
TCR, some of which recognize self components other than self-MHC. These
cells must be deleted to prevent autoimmune destruction of healthy host tissues.
Negative selection is the elimination of T cells reactive with self components
other than the MHC. Negative selection occurs in the deeper cortex, at the
corticomedullary junction, and in the medulla of the thymus. The thymocytes
interact with antigen processed and presented by interdigitating cells and
macrophages. Only thymocytes that fail to recognize self antigens are allowed to
survive and proceed along the maturation process, with the remainder undergoing
apoptosis. Eventually, T cells that survive the negative selection process lose
either CD4 or CD8, becoming "single positive" cells. Fewer than 5% of
thymocytes survive selection and leave the thymus to take up residence in the
secondary lymphoid organs.
109
Role of MHC in activation of T lymphocytes
The binding between the TCR and the MHC/antigen peptide complex is highly specific
and acts as the first signal to induce T cell activation. T cells do not respond to either
self-MHC alone or to free peptide. Activated T cells differ from resting T cells in that
they proliferate and secrete lymphokines and/or lytic substances. The affinity of the TCR
for the MHC/antigen complex is often too low to fully activate the T cell; there are
numerous accessory molecules that increase avidity between the T cell and APC by
performing an adhesive function.
Cytotoxic T lymphocytes: CTLs are able to kill target cells directly by inducing
apoptosis. Nucleases and other enzymes activated in the apoptotic process may help
destroy the viral genome, thus preventing the assembly of virions and potential infection
of other cells. CTL induce apoptosis only in the target cell; neighboring tissue cells are
not affected. Two mechanisms for induction of apoptosis have been identified:


Preformed perforins are released at the target cell surface which generate
transmembrane pores in the target cell, through which a second protein,
granzyme, can gain entry to the cytosol and induce the apoptotic series of events.
Apoptitic signaling via membrane-bound molecules can occur via Fas on the
target cell surface and Fas ligand on the CTL surface. The processes of antigen
recognition, CTL activation and delivery of apoptotic signals to the target cell can
be accomplished within 10 minutes. The apoptotic process in the targeted cell
may take 4 hours or more and continues long after the CTL has moved on to
interact with other tissue cells.
T helper lymphocytes: The initial interaction between T lymphocytes and the APC is
mediated by adhesion molecules. Interactions occur between LFA-1, CD-2 and ICAM-3
on the T-cell, and with ICAM-1, ICAM-2, LFA-1 and LFA-3 on the APC. These
molecules synergize in binding of lymphocytes to the APCs. This transient binding
allows the T-cell to sample the large numbers of MHC molecules on the surface of the
APCs for their specific peptide. If a T-cell recognizes its peptide ligand bound to MHC,
signaling via the T-cell receptor complex is induces more conformational changes,
eventually leading to the production of T-cell cytokines. T cells require co-stimulation
through binding of the CD-28 ligand with the CD-80/CD-86 ligand of the APC.
T-dependent B cell activation: B cells can also specifically take up antigen via binding
through their surface Ig. This is internalized, broken down to peptides and the peptides
are presented on the B cell surface held in the peptide binding grooves of MHC class II
molecules. If this B cell interacts with a primed T cell that recognizes the peptide/class II
complex on the B cell then the T cell may transiently express accessory ligands. This
results in cells going into cell cycle and the secretion of cytokines. For the B cell, this
action, in concert with correctly released T cell cytokines, will drive isotype switching as
well as maturation of the B lymphocyte into a plasma cell.
110
Association of Disease with MHC Haplotype
Particular MHC alleles are associated with better protection against certain infections.
Certain alleles are associated with a greater chance of developing autoimmunity. Some
diseases are distinctly more common in individuals with a particular MHC allele or MHC
haplotype. Diseases with a strong association with certain MHC alleles include insulindependent diabetes and Graves' disease. Expression of HLA-DR4 is associated with
rheumatoid arthritis. Nearly 90% of people with ankylosing spondylitis carry the HLAB27 allele. Expression of HLA-DR2 is associated with multiple sclerosis.
The Association of HLA serotype with susceptibility to autoimmune disease will be
covered in the AutoImmunity Lecture. An updated list of genetic polymorphisms
associated with increased susceptibility to disease will be presented during that lecture.
It is hypothesized that in some cases MHC molecules serve as receptors for the
attachment and entry of pathogens into the cell; this makes individuals with a certain
HLA type more susceptible to infection by a particular intracellular pathogen using that
HLA molecule as a receptor. Alternatively, an infectious agent might possess antigenic
determinants that resemble MHC molecules (molecular mimicry). Such resemblance
might allow the pathogen to escape immune detection because it is seen as "self," or it
may induce an autoimmune reaction. Because MHC molecules differ in their ability to
accommodate different peptides, some individuals who express certain MHC genes may
lack the ability to present microbial epitopes capable of inducing protective T cell
responses. Finally, there may simply be no T cells capable of recognizing a particular
MHC/antigen combination, leading to a "hole in the T cell repertoire."
Coico and Sunshine,
2009. Figure 8.6.

111
Clinical Vignette - The case of Helen Burns (Case 8 in Geha and Notarangelo): Helen Burns
was 6 months old when she developed pneumonia, caused by the opportunistic pathogen
Pneumocystis carinii. Helen was tested for severe combined immunodeficiency; it was found that
Helen's T cells could be stimulated with mitogen (phytohemagglutinin), but could not respond to
specific antigenic stimuli. It was further established that Helen had low overall immunoglobulin
levels and decreased CD4 cells. Her CD8 cell counts were within normal range. Helen's white blood
cells were examined for expression of MHC Class I and Class II molecules. She was diagnosed with
MHC Class II deficiency. A bone marrow transplant was performed using Helen's mother as a
donor. The graft was successful and immune function was restored.
How does MHC Class II deficiency selectively
affect CD4 T cell function, and what implications
does this have towards immune responses to
infective agents?
Geha and Notarangelo, 2012. Figure 8.3.
112
Summary: Role of HLA in the Immune System
MHC molecules are organized into 3 classes. Class I molecules are found on all nucleated
cells. The class II molecules are found on B-cells and macrophages. Class III genes encode for
various soluble proteins that include certain complement components. Human class I region
genes consists of HLA-A, HLA-B, and HLA-C loci and the class II region genes consists of
the D region which is subdivided into HLA-DP, HLA-DQ, and HLA-DR subregions. All MHC
molecules show high allotypic polymorphism.
Class I molecules are important in presentation of Ag epitopes to CD8+ T cells as well as for
effector functions of target cells. Each class I locus codes for a transmembrane polypeptide
containing three extracellular domains (1, 2, 3), which is expressed at the cell surface in
a noncovalent association with 2-microglobulin (2M). All nucleated cells express Class I
molecules. Class II molecules present antigen in the presence of cytokines to CD4+
lymphocytes. Class II molecules have 2 transmembrane polypeptide chains. Peptides bind
within the allele specific pockets defined by the 2 transmembrane polypeptide chains, where
they are presented to the TCR for recognition.
Different antigen degradation and processing pathways produce MHC-peptide complexes
where "endogenous" peptides associate with class I molecules and "exogenous" peptides
associate with class II molecules. MHC class I molecules bind small antigenic peptides that are
8-10 amino acid residues in length; MHC class II molecules present slightly larger peptides.
T cells in the thymus learn to recognize antigenic peptides in the context of self-MHC
molecules by a two-step process involving both positive and negative selection.
The binding between the TCR and the MHC/antigen peptide complex acts as a signal to
induce T cell activation. T cells do not respond to either self-MHC alone or to free peptide.
Accessory molecules increase avidity between the T cell and APC by performing an adhesive
function.
CTLs recognize Ag in the context of MHC class I, and kill target cells directly by inducing
apoptosis. They release preformed perforins at the target cell surface to generate
transmembrane pores in the target cell, through which a second protein, granzyme, gains
entry to the cytosol to initiate an apoptotic series of events. CTLs can also deliver apoptitic
signals via surface bound molecules.
T helper lymphocytes recognize Ag and MHC class II on the APC in a manner mediated by
adhesion molecules. The recognition is specific and requires co-stimulation through ligand
interactions on the APC. Activation of the T helper cell leads to specific cytokine release. B
cells are good antigen presenters to T cells.
Certain MHC alleles are associated with a greater chance of protective immune responses to
pathogens, as well as towards developing autoimmunity. Some diseases are distinctly more
common in individuals with a particular MHC allele or MHC haplotype. Possible reasons for
this are discussed.
113
T CELL RECEPTOR:
Structure and Genetic Basis
Jeffrey K. Actor, Ph.D.
713-500-5344
Objectives: (1) Present an overview of the T receptor structure and organization of the gene loci
encoding for the T cell receptor chains; (2) explain mechanisms underlying generation of T cell
receptor diversity; (3) examine the stages in thymic selection of T lymphocytes; and (4) compare
and contrast the T cell receptor with the B cell receptor.
Keywords: T cell receptor (TCR).
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York,
NY. 6th edition, 2009. Chapter 9; Geha and Notarangelo. Case Studies in Immunology. Garland
Publishing, New York, NY. 6th edition, 2012. Case 7: Omenn Syndrome.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/TCR.html
The acquired immune response is subdivided, based on participation of two major cell types. B
lymphocytes originate in the bone marrow, and synthesize/secrete antibodies. This is termed
humoral immunity. T lymphocytes mature in the thymus, and secrete immunoregulatory factors
following interaction with antigen presenting cells; this is termed cellular immunity (CMI).
Lymphocyte Biology
Lymphoid cells provide efficient, specific and long-lasting immunity against microbes/pathogens
and are responsible for acquired immunity. This lecture will primarily examine the biology of two
classes of lymphocytes: (1) thymic-dependent cells or T lymphocytes that operate in cellular and
humoral immunity; and (2) B lymphocytes that differentiate into plasma cells to secrete
antibodies. T and B lymphocytes produce and express specific receptors for antigens.
The major properties of the acquired immune response are specificity, memory, adaptiveness,
and discrimination between self and non-self. All of these properties are related to the random
selection of variable region components during the development of both B cells and T cells.
The lymphatic organs are tissues in which lymphocytes mature, differentiate and proliferate. The
primary (central) lymphoid organs are those in which B and T lymphocytes mature into antigen
recognizing cells. In embryonic life, B cells mature and differentiate from hematopoietic stem
cells in the fetal liver. After birth, B cells differentiate in the bone marrow. Maturation of T cells
occurs in a different manner. Progenitor cells from the bone marrow migrate to the thymus where
they differentiate into T lymphocytes. The T lymphocytes continue to differentiate after leaving
the thymus, and are driven to do so by encounter with specific antigen in the secondary lymphoid
organs.
The secondary lymphoid organs are those tissues in which antigen-driven proliferation and
differentiation take place. The spleen and lymph nodes are the major secondary lymphoid organs.
114
Additional secondary lymphoid organs include the tonsils, appendix, and Peyer’s patches.
Aggregates of cells in the lamina propria of the digestive tract lining may also be included in this
category, as well as any tissue described as MALT (mucosa-associated lymphoid tissue), GALT
(gut-associated lymphoid tissue) or BALT bronchus-associated lymphoid tissue).
T Lymphocytes: T lymphocytes are involved in regulation of immune response and cell mediated
immunity. They provide necessary factors to help B cells produce antibody. Mature T cells
express antigen-specific T cell receptors (TCR). Every mature T cell expresses the CD3 molecule,
which is associated with the TCR. The TCR/CD3 complex recognizes antigens associated with the
major histocompatibility complex (MHC) molecules on target cells (e.g. virus-infected cell). The
TCR is also expressed on the cell surface in association with co-receptor or accessory molecules
(CD4 or CD8).
The structure of the T-cell receptor (TCR) complex showing the
predominant form of the antigen-binding chains,  and , and the
associated signal transduction complex, CD3 (, , and  chains)
plus  (zeta) or eta) or  (theta. (-) and (+) represent
electrostatic interactions.
T Cell Receptor: The TCR is a transmembrane heterodimer composed of two disulfide-linked
polypeptide chains. T lymphocytes of all antigenic specificities exist prior to contact with antigen.
Each lymphocyte carries a TCR of only a single specificity. T Lymphocytes can be stimulated by
antigen to give rise to progeny with identical antigenic specificity. Lymphocytes reactive with
“self” are deleted or inactivated to ensure that no immune response is mounted against self
components.
The vast majority of T lymphocytes express alpha [] and beta [] chains on their surface. Cells
that express gamma [] and delta [] chains comprise only 5% of the normal circulating T cell
population in healthy adults. Each chain (, or ) represents a distinct protein with
approximate molecular weight of 45 kDa. An individual T cell can express either an  or a 
heterodimer as its receptor, but never both.
The TCR recognizes antigen in the form of peptides which are bound in the groove on MHC
molecules (reviewed in detail in lecture: Role of MHC in Immune Response). The interactions
between heterodimers create three hypervariable regions called complementarity determining
regions (CDRs 1, 2, and 3).
115
The interaction of TCR, MHC, and peptide.
The complementarity determining regions
(CDRs) of the TCR V regions and peptide
bound in the peptide-binding groove of an
MHC class I molecule are depicted. [Based
on the crystal structure described by K. C.
Garcia et al. (1998): Science 279: 1166.]
The T cell receptor genes are closely related members of the immunoglobulin gene superfamily.
Each chain consists of a constant (C) and a variable (V) region, and is formed by a gene-sorting
mechanism similar to that found in antibody formation. The repertoire is generated by
combinatorial joining of variable (V), joining (J), and diversity (D) genes, and by N region
diversification (nucleotides inserted by the enzyme deoxynucleotidyl-transferase). Unlike
immunoglobulin genes, genes encoding TCR do not undergo somatic mutation. Thus there is no
change in the affinity of the TCR during activation, differentiation, and expansion.
116
TCR-CD3-complex: The TCR heterodimer is tightly associated with six independently encoded
CD3 subunits (, , , ,  and ) required for efficient transport to the cell surface. CD3 subunits
possess long intracellular tails and are responsible for transducing signals upon TCR engagement.
Genes Coding for T-Cell Receptors: Genes which code for the T cell receptor and the
mechanisms used to generate TCR diversity are similar to those of immunoglobulins.
 The TCR V, D, and J genes are mixed together in a more complicated manner than found for
immunoglobulin genes.
  and  uses only V and J gene segments.
  and  use V, D, and J gene segments.
 There are many more V and V genes (50-100) than V and V genes (5-10) present in germ
line.
 The  and  chain genes are mixed together in one locus. The genes encoding the  chain are
entirely located between the cluster of V and J gene segments.
The top and bottom
rows show germline
arrangement of the
variable (V), diversity
(D), joining (J), and
constant (C) gene
segments at the T-cell
receptor  and  loci.
During T- cell development, a V-region
sequence for each chain
is assembled by DNA
recombination. For the
chain (top), a V gene
segment rearranges to a
J gene segment to
create a functional gene
encoding the V domain.
For the  chain
(bottom), rearrangement of a D, a
J, and a V gene
segment creates the
functional V-domain
exon.
Geha and Notarangelo, 2012. Figure 7.1.
Order of TCR Gene Rearrangement:
 The earliest cell entering the thymus has its TCR genes in the germ line configuration
(unrearranged).
 Both  and  chain genes then begin to rearrange, more or less simultaneously.
 If the  chain genes rearrange successfully, then  chain genes also start to rearrange. If both 
and  genes rearrange functionally, no further gene rearrangement takes place and the cell
remains a  T cell.
 If  and/or  rearrangements are not functional, then  gene rearrangement continues followed
by  gene rearrangement. In this manner, a  product appears, and the cell becomes an  T
cell.
117
The Process of Recombination: Recombination of V, D, and J gene segments is coordinated by
recombinase-activating genes RAG-1 and RAG-2. The enzymes recognize specific DNA signal
sequences consisting of a heptamer, followed a spacer of 12 or 23 bases, and then a nonamer. If
either RAG gene is impaired or missing, homologous recombination events are abolished. This
gives rise to severe combined immunodeficiency (SCID). Mutations which result in partial
enzymatic activity can also occur, and can give rise to immunodeficiency diseases. An example of
such disorder is Omenn Syndrome, discussed in detail in the Case Studies in Immunology (Geha
and Notarangelo, chapter 7) text.
Generation of T-Cell Receptor Diversity:
The overall level of diversity is greater for T cell receptors than that for immunoglobulins. This is
primarily due to additional junctional diversity in possible TCR gene rearrangements. Most of the
variability in the TCR occurs within junctional regions encoded by D, J and N nucleotides. This is
118
the region that corresponds to the CDR3 loops that form the center of the antigen binding sites.
So, while the center of the binding site is highly variable, the remaining portion of the heterodimer
is subject to relatively little variation.
Number of V gene pairs
Junctional diversity
Total Diversity
Immunoglobulins
~2 - 3.4 x 106
~3 x 107
~1014
T cell : Receptors
5.8 x 106
~2 x 1011
~1018
Development of T lymphocytes
During differentiation in the thymus, immature T cells undergo
rearrangement of their TCR  and  genes to generate a diverse
set of clonotypic TCRs. Immature thymocytes are selected for
further maturation only if their TCRs do not interact with selfpeptides presented in the context of self-major histocompatibility
complex (MHC) molecules on antigen presenting cells. Different
signals lead to the alternate developmental outcomes of maturation
or apoptosis (positive versus negative selection). Positively
selected thymocytes undergo alternate commitment to either the T
killer or T helper lineages, which correlate precisely with a cell's
TCR specificity towards MHC class I or II molecules,
respectively. Lineage commitment is marked phenotypically by
the loss of expression of one of the co-receptor molecules, CD8 or
CD4. Immature thymocytes express both co-receptors (double
positive), while T killer or T helper cells express only CD8 or
CD4, respectively (single positive CD8+ or CD4+).
Figure. Changes in surface molecules of thymocytes at different
stages of maturation.
The majority of peripheral blood T lymphocytes express the  and  form of the TCR. In healthy
adults, less than 5% express a heterodimer comprised of the  and  chains. Virtually all the cells
that express the TCR- are CD4+CD8- (T helper) or CD4-CD8+ (T cytotoxic or T suppressor).
Almost all cells expressing TCR- are CD4-CD8- (double negative). While the TCR-
expressing lymphocytes are known to function as helper and cytotoxic cells, the function of the
TCR- cells is not well understood.
Figure. Main stages in the development of a T lymphocyte.
119
++, CD4+CD8+ cell interacts
with Thymic epithelial cell
Interaction = Pos Selection
No interaction =
MHC + self
CD4+CD8+ cell DEATH
MHC + non-self
++, CD4+CD8+ cell
interacts with interdigitating cell
High affinity interaction =
DELETION
Low affinity interaction =
SURVIVAL
Commitment CD4+ or CD8+
Figure. Main stages in Thymic Selection.
T Helper Cells: T helper cells (Th) are the primary regulators of T cell- and B cell-mediated
responses. They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate
toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign antigen
complexed with MHC class II molecules on B cells, macrophages or other antigen-presenting
cells; and 4) aid effector T lymphocytes in cell-mediated immunity.
Currently, it is believed that there are two main functional subsets of Th cells, plus other helper
subsets of importance. T helper 1 (Th1) cells aid in the regulation of cellular immunity, and T
helper 2 (Th2) cells aid B cells to produce certain classes of antibodies (e.g., IgA and IgE). The
functions of these subsets of Th cells depend upon the specific types of cytokines that are
generated, for example interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by Th1 cells; IL4, IL-6 and IL-10 by Th2 cells. Two other classes of T helper cells are thought to be involved in
oral tolerance and serve as regulators for immune function. Th3 cells secrete IL-4 and TGF- and
provide help for IgA production, and have suppressive properties for Th1 and Th2 cells. Th17
cells, characterized by IL-17 secretion, are thought to be involved as effector cells for autoimmune
disease progression.
T Cytotoxic Cells: T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells
infected with intracellular pathogens. These cells 1) usually express CD8, and, 2) destroy infected
cells in an antigen-specific manner that is dependent upon the expression of MHC class I
molecules on antigen presenting cells.
T Suppressor/ T Regulatory Cells: T suppressor cells suppress the T and B cell responses and
express CD8 molecules. T regulatory cells also affect T cell response, with many cells
characterized as CD4+CD25+, TGF- secretors.
120
 T Cells: Not all T cells express  TCRs. An alternative is to express  chains of the TCR.
Generally,  cells lack CD4, although some  cells do express CD8. The functions of  cells are
not well understood.  T cells can function in the absence of MHC molecules. They home to the
lamina propria of the gut, and are thought to assist in protection against microorganisms entering
through epithelium at mucosal surfaces. Their range of response to antigens is limited. 
expressing cells have been found to be active towards mycobacterial antigens and heat shock
proteins. They have the ability to secrete cytokines like their  counterparts.
Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells that
share properties of both T cells and natural killer (NK) cells. hese cells recognize an antigenpresenting molecule (CD1d) that binds self- and foreign lipids and glycolipids. They constitute
only 0.2% of all peripheral blood T cells. The term “NK T cells” was first used in mice to define a
subset of T cells that expressed the natural killer (NK) cell-associated marker NK1.1 (CD161). It
is now generally accepted that the term “NKT cells” refers to CD1d-restricted T cells
coexpressing a heavily biased, semi-invariant T cell receptor (TCR) and NK cell markers. Natural
killer T (NKT) cells should not be confused with natural killer (NK) cells.
-------------------------Comparison of the B cell and T cell receptors:
Both BCRs and TCRs share these properties:
 they are integral membrane proteins
 they are present in thousands of identical copies exposed at the cell surface
 they are made before the cell ever encounters an antigen
 they are encoded by genes assembled by the recombination of segments of DNA
 allelic exclusion ensures only one receptor with a single antigenic specificity
 they demonstrate N region addition during gene rearrangement
 they have a unique binding site
 this site binds to a portion of the antigen called an antigenic determinant or epitope
 the binding, like that between an enzyme and its substrate depends on complementarity of
the surface of the receptor and the surface of the epitope
 the binding occurs by non-covalent forces (again, like an enzyme binding to its substrate)
 successful binding of the antigen receptor to the epitope, if accompanied by additional
"signals", results in:
1. stimulation of the cell to leave G0 and enter the cell cycle
2. repeated mitosis leads to the development of a clone of cells bearing the same
antigen receptor; that is, a clone of cells of the identical specificity.







BCRs and TCRs differ in:
their structure
the genes that encode them
the type of epitope to which they bind
TCRs do not somatically mutate
TCRs do not undergo isotype switching
TCR gene recombination exhibits far greater junctional diversity than Ig genes
TCRs are never secreted from the T cell
121
Clinical Vignette - Omenn Syndrome (Case 7 in Geha and Notarangelo): Patients with Omenn
syndrome demonstrate severe immunodeficiency characterized by the presence of activated, anergic,
oligoclonal T cells, hypereosinophilia, and high IgE levels. There is a body of evidence to indicate that
the immunodeficiency manifested in patients with Omenn syndrome arises from mutations that decrease
the efficiency of V(D)J recombination. These individuals bear missense mutations in either the RAG-1 or
RAG-2 genes that result in partial activity of the two proteins. In many cases, amino acid substitutions
map within the RAG-1 homeodomain and decrease DNA binding activity, while others lower the
efficiency of RAG-1/RAG-2 interaction.
Summary: T Lymphocytes
T lymphocytes are involved in regulation of immune response and in cell mediated immunity.
Every mature T cell expresses CD3, which is associated with the TCR. During thymic
differentiation, immature T cells undergo rearrangement of their TCR  and  genes to generate a
diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation only if
they recognize foreign antigens in the context of MHC molecules. Mature T cells usually display
one of two accessory molecules. CD4+ T helper cells are the primary regulators of T cell- and B
cell-mediated responses, and are further subdivided into functional subsets dependent upon
cytokines secreted. CD8+ T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells
infected with intracellular pathogens. T suppressor cells suppress the T and B cell responses and
express CD8 molecules.
Summary: T Cell Receptor: Structure and Genetic Basis
Mature T cells express antigen-specific TCR in a complex with CD3 molecules. The TCR is a
disulfide-linked heterodimer composed of either  or  chains. T cells express either  or 
chain heterodimers, but never both.
T cell receptor genes are closely related members of the immunoglobulin gene superfamily and
derive part of their structural diversity form recombination of different V, D, and J gene segments.
The mechanisms for T cell receptor gene switching are similar to those of immunoglobulin genes,
but T cell receptor genes do not have somatic mutations.  chains of the TCR have only V and J
segments, and join to  chains.  chains of the TCR have genes for V, D, and J segments. The
process of recombination is coordinated by recombinase-activating genes RAG-1 and RAG-2.
If  rearrangements are unsuccessful on both chromosomes,  chains join to  chains to give 
phenotypic T cells.  chains have only V and J segments; chains have V, D, and J segments.
122
ADAPTIVE IMMUNE RESPONSE I and II
Jeffrey K. Actor, Ph.D.
MSB 2.214, 713-500-5344
Required Reading: Coico and Sunshine, 2009. Chapters 7, 9, 10, 11. Geha and Notarangelo.
Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 47.
Toxic Shock Syndrome.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/Biologybt.html
OBJECTIVES
1. Distinguish between innate and adaptive immune responses on the basis of
antigen specificity, HLA restriction and memory.
2. Understand role of immune mediators including cytokines, chemokines,
costimulatory and adhesion molecules in the development of adaptive immune
responses.
3. To describe the various effector and regulatory functions of T and B cells.
4. To demonstrate the molecular events associated with T cell and B cell activation.
5. Compare and contrast effector cells in cytotoxic mediated immunity.
6. To develop a practical understanding of mechanisms and clinical relevance of T dependent and - independent antibody responses.
KEY WORDS
cytokine, T cell receptor, B cell receptor, helper T cell, cytotoxic T
lymphocyte, NK cell, NKT cell, T-dependent antibody, Tindependent antibody response.
I. OVERVIEW OF THE IMMUNE RESPONSE
The acquired immune response is subdivided, based on participation of two major cell
types. B lymphocytes originate in the bone marrow, and synthesize/secrete antibodies.
This is termed humoral immunity. T lymphocytes mature in the thymus, and secrete
immunoregulatory factors following interaction with antigen presenting cells; this is
termed cellular immunity (CMI).
A.
Purpose – maintain homeostasis
B.



C.
Discriminates
Self from nonself (foreign, effete)
Pathogenicity – ability to cause disease
Intracellular vs. extracellular pathogen
Begins in utero
123
D.
Remembers previous encounters
E.
Goal is proper specificity, intensity, and duration
The major properties of the acquired immune response are specificity, memory,
adaptiveness, and discrimination between self and non-self. All of these properties
are related to the random selection of variable region components during the
development of B cells and T cells. The essential features for clonal selection of these
cells include:




B and T lymphocytes of all antigenic specificities exist prior to contact with antigen.
Each lymphocyte carries specific surface molecules (immunoglobulin or T cell
receptor) of only a single specificity.
Lymphocytes can be stimulated by antigen under appropriate conditions to give rise
to progeny with identical antigenic specificity.
Lymphocytes potentially reactive with “self” are deleted or inactivated to ensure that
no immune response is mounted against self components.
II. MOLECULAR COMPONENTS
A.
Cytokines
When confronted with above challenge, the host immune response will determine
appropriate degree of antigen-specific cell mediated vs. humoral response. In order to
accomplish this, various regulatory networks controlled by cytokines are activated.
1.
Physical and Biological Properties

small mol weight peptides and glycopeptides

produced by a variety of cell types
accessory cells
leukocytes
somatic cells – endothelium, fibroblasts, etc.

short plasma half lives (makes determining levels clinically difficult)

modulate immune/inflammatory responses by stimulating/inhibiting
various cell populations (inflammatory, epithelial, fibroblast)

one cell type can make multiple cytokines and a single cytokine can be
made by a variety of cell types
124
2.

redundancy – multiple cytokines can have the same biological activity

pleomorphism/pleotropic – the same cytokine can have multiple
activities depending upon the target cell, concentration and/or
presence of other cytokines

action can be endocrine, paracrine and/or autocrine
Classification – Biological Activity
 Interferons (,, – interfere with viral replication but also have
immunomodulatory properties

Colony Stimulation Factors – support growth of WBC elements of bone
marrow; mediate various inflammatory reactions

Tumor Necrosis Factors – produce hemorrhagic necrosis of tumors in
mice; major mediator of inflammation and is elevated in sepsis
syndrome

Chemokines – groups of molecules that mediate chemotaxis of various
inflammatory cells

Interleukins – various immunoregulatory functions between (inter)
various leukocyte (leukin) populations
AN EXPANDED LIST OF CYTOKINES, INTERFERONS
CHEMOKINES ARE INCLUDED IN THE APPENDIX.
125
AND
III. T LYMPHOCYTES
T lymphocytes are involved in the regulation of the immune response and in cell
mediated immunity, and help B cells to produce antibody. Mature T cells express
antigen-specific T cell receptors (TCR). Every mature T cell expresses the CD3
molecule, which is associated with the TCR. In addition mature T cells usually display
one of two accessory molecules, CD4 or CD8. The TCR/CD3 complex recognizes
antigens associated with the major histocompatibility complex (MHC) molecules on
target cells (e.g. virus-infected cell).
Important T cell markers
Surface Markers of T cells. Additional markers include: CD45RO, Leukocyte common
antigen for memory T cells (activated). CD45RA, Leukocyte common antigen for naive
T cells (resting). Coico and Sunshine, 2009. Figure 9.4.
TCR-CD3-complex: The TCR heterodimer is tightly associated with the CD3 coreceptor made up of independently encoded subunits (, , , and two  chains). The
CD3 complex is required for efficient transport of the TCR to the cell surface. CD3
subunits possess long intracellular tails and are responsible for transducing signals
upon TCR engagement with MHC presented antigen.
A. T Helper cells
T helper cells (Th) are the primary regulators of T cell- and B cell-mediated responses.
They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate
126
toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign
antigen complexed with MHC class II molecules on B cells, macrophages or other
antigen-presenting cells; and 4) aid effector T lymphocytes in cell-mediated immunity.
1.
Paired Interactions between the APC and CD4+ T cell
(Immunological synapse)



Antigen receptor
 MHC II : TCR - antigen binding
 MHC II : CD4 molecule - the co-receptor
Costimulatory pairs - second signals
 CD40:CD40L (CD154)
 CD28/CTLA-4:B7 (CD80, CD86)
Adhesion molecules
 CD58(LFA-3):CD2
 CD54(ICAM-1):CD11a/CD18 (LFA-1)
127
2.
The TH1/TH2 paradigm
The TH1/TH2 paradigm was first proposed by Mossman and Coffman to explain the
differential effects of T cell help – i.e. T cells helping B cells and T cells helping other T
cells. These cells were distinguished functionally rather than morphologically by the
differences in cytokine patterns that they produced.

Differential production of specific cytokine patterns by subpopulations of
CD4+ cells

TH1 help other T cells develop immunity
(mostly T cell-mediated)

TH2 help B cells (and other WBC) develop immunity against extracellular
pathogens (mostly through IgE, mast cells and eosinophils
against intracellular pathogens
Dendritic Cell
Currently, it is believed that there are multiple functional subsets of Th cells. In addition
to the ones mentioned above: Th17 cells, characterized by IL-17 secretion, are thought
to be involved as effector cells for autoimmune disease progression, and protect
surfaces (skin, gut) from extracellular bacteria. Tfh cells (follicular helper T cells) provide
help to B cells in germinal areas enabling them to develop into antibody-secreting
plasma cells; they function inside of follicular areas of lymph nodes.
The functions of all the subsets of Th cells depend upon the specific types of cytokines
that are generated, for example interferon-gamma (IFN-gamma) by Th1 cells and IL-4
by Th2 cells, and IL-17 by Th17 cells.
128
Regulatory T cells (Treg) represent subpopulations of T helper cells that trigger
suppressive activities following engagement of their T cell receptor with presenting
antigen occupying the MHC on an antigen presenting cell. They typically secrete
molecules such as TGF-β, which function to suppress other T helper cell type activity.
They usually express CD4 and CD25 on their cell surface, and express the transcription
factor Foxp3.
T-cell Regulation: Treg receive a
signal via CTLA-4 which induces
their suppressive activity. Treg may
also receive a signal triggering their
suppressive activity following
interaction with an MHC class II
molecule. Treg may then suppress
the activation of CD4+ T cells by
secreting TGF-  (and IL-10).
FIGURE 12.4. From Coico, 2009.
129
130
3.
Intracellular Events – receptor mediated transcription of cytokine
genes by a sequence of molecular events
FIGURE. Intracellular
events in CD4+ T-cell
activation. The result of
activation events is
enhanced transcription and
increased stabilization of IL2 mRNA. Coico, 2009.











MHCII/peptide binds TCR
TCR activates CD3
CD3 transduces activation signal across membrane
Tyrosine kinases (Fyn, Lck) activated by CD45
Fyn, Lck cluster with ITAMs and phosphorolate them
ZAP-70 (another tyrosine kinase) binds to ITAMs
Activated ZAP-70 binds to phospholipase c- (PLC-)
PLC- splits PIP2 into DAG and IP3
 DAG activates PKC >>>>> NF- (transcription factor)
 IP3 increases iCa++ >>> activated calcineurin >>> NF-AT
Transcription factors enter nucleus and bind to chromosomes
Upregulate T cell activation genes (cytokine, cytokine receptors)
Upregulate adhesion molecules on surface to promote further
activation events.
131
132
4.
Other events of T cell activation



Decreased expression of selectins molecules to allow homing to lymph
nodes
Requirement for multiple signals from APC to activate cell
Function of costimulatory pairs – promote the T cell activation process
 CD40: CD154(CD40L)
 CD80/CD86 (B7.1,B7.2):CD28
 CD80 binding upregulates TH1
 CD86 binding upregulates TH2
 CD28 binding upregulates IL-2 production
 Lack of CD28 binding induces tolerance
 CD80/86:CTLA-4
 downregulates IL-2 production
 negative activation signal – tolerance
 induces memory cell formation
133
B. Role of Cytotoxic Cell-Mediated Immunity in Host Defense
Host defenses against extracellular infectious agents (e.g., bacteria, protozoa, worms, fungi)
typically utilize (1) Antibody, (2) Complement, and/or (3) activated Phagocytes. However,
these mechanisms are not adequate for defense against intracellular infectious agents (an
infectious agent that invades a host cell). Therefore a different defense system is required.
The mechanisms used are those referred to as cytotoxic cell mediated immunity.
Induction of helper function for cytotoxic cell mediated immunity. In many cases, first
CTL encounter with antigen must have help from Helper T cells. The helper cells must
recognize antigen presented by MHC Class II molecules on an APC (antigen presenting
cell) (dendritic cell or macrophage). The activated Th1 cell secretes IL-2 and IFN-,
which activates CTLs.
Activation of Th1 cells also triggers the activation of NK cells and macrophages which
then target specific cells.
Generation of CD8+ T
cells effector cells and
target cell killing. (A)
dendritic cells activate
CD8+ T cells directly.
(B) One pathway for
CD4+ T cells to activate
CD8+ T cells. (C) Target
cell killing by a CD8+
effector T cell. Coico
and Sunshine, 2009. Fig
10.10.
134
Effector cells in Cytotoxic Cell Mediated Immunity. Both innate and adaptive cells play a
role in cytotoxic cell mediated immunity. The major cell players and their properties are
listed and summarized in the table below.

CTLs – Antigen specific and MHC Class I restricted.
i. CTLs express CD8.
ii. CTLs kill their targets by using Perforin, Granzymes, Cytokines,
Fas and Fas ligand.

NK cells - nonspecific (they do not use a T cell receptor).
i. Morphologically large granular lymphocytes (LGLs);
ii. Non-T and non-B lymphocytes lacking surface CD3, CD4, CD8 and
CD19. They do not express immunoglobulins or TCRs.
iii. NK cells express CD16 and CD56.
iv. NK cells kill by releasing perforin, granzymes and cytokines (IFN-
and TNF).

Lymphokine activated killer cells (LAK cells) are
i. Morphologically LGLs.
ii. Non-T non-B lymphocytes.
iii. Reaction –nonspecific.

NK-ADCC
i. antibody-dependent cellular cytoxicity (ADCC).
ii. Have Fc receptors (CD16) that recognize Fc portion of IgG.
Table 1: Effector Cells in Cytotoxic Cell Mediated Immunity
Effector Cell
CD markers
Effector
MHC
Molecules
recognition
CTL
TCR,CD3,CD8,CD2 Perforin,
required
cytokines (TNFClass I
β, IFN-)
NK cell
CD16,CD56, CD2
Perforin,
no
cytokines (TNFβ, IFN-)
NK cell
CD16,CD56, CD2
Perforin,
no
ADCC
cytokines (TNFβ, IFN-)
LAK cell
CD16,CD56, CD2
Perforin,
no
cytokines (TNFβ, IFN-)
Macrophage CD14
TNF-α,
no
enzymes, NO, O
radicals
135
Antigen
recognition
specific TCR
nonspecific
specific IgG
nonspecific
nonspecific
Cytotoxic cells (CTLs) directly kill tumor cells and host cells infected with intracellular
pathogens. These cells 1) usually express CD8, and, 2) destroy infected cells in an
antigen-specific manner that is dependent upon the expression of MHC class I
molecules on antigen presenting cells.
1.



2.


3.


General Considerations
Adaptive host defense against intracellular pathogens
CD8+ CTL is MHC I restricted
Is affected by TH1 cells which are also antigen-specific but MHC II
restricted
Development of CTL
TCR interacts with MHC I – antigen complex
o In association with CD8
o Also involves costimulatory molecules
IL-2R upregulated
o IL-2 from Th cells cause clonal proliferation
o IFN causes activation of CTL
Killing of Target cells by CTL
IFNupregulates perforin formation
o Perforins form transmembrane channels that kill target
o Similar to complement-mediated lysis
IFNupregulates granzyme formation
o Serine proteases
136
o Pass to target through the perforin-induced channels
o Activate target cell apoptosis




Fas/FasL (CD95/95L) interaction
o FasL expression on T cell upregulated in activated CTL
o Initiates apoptosis in target through formation of capsases
CTL releases “doomed” target to kill more target cells if available
As response is regulated, CTLs themselves undergo apoptosis
Remnant is antigen-specific memory CTL
Table 2: Cytotoxic Products of Activated CTLs
Cytotoxic
Product
Perforins
TNF-
Fas ligand
Nucleases
Serine
proteases
Effect on Target cell
- Polymerize in the membrane of the target cell to form
poly-perforin channels that allow cytosol to leak out and
toxic molecules to enter the cell.
- Degrades proteins in cell membrane
- Initiates apoptosis
- Degrades DNA and RNA in the cell
- Degrade proteins in the cell membrane
137
C. Recognition of “Different” Antigens by T cell receptors
1. Presentation of Lipids and Glycolipids
Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells
that share properties of both T cells and natural killer (NK) cells. These cells recognize
an antigen-presenting molecule (CD1d) that binds self- and foreign lipids and
glycolipids. They constitute only 0.2% of all peripheral blood T cells. The term “NK T
cells” was first used in mice to define a subset of T cells that expressed the natural killer
(NK) cell-associated marker NK1.1 (CD161). It is now generally accepted that the term
“NKT cells” refers to CD1d-restricted T cells co-expressing a heavily biased, semiinvariant T cell receptor (TCR) and NK cell markers. Natural killer T (NKT) cells should
not be confused with natural killer (NK) cells.
Upon activation, NK T cells are able to produce large quantities of interferon-gamma, IL4, and granulocyte-macrophage colony-stimulating factor, as well as multiple other
cytokines and chemokines (such as IL-2 and TNF-alpha). NKT cells seem to be
essential for several aspects of immunity because their dysfunction or deficiency has
been shown to lead to the development of autoimmune diseases (such as diabetes or
atherosclerosis) and cancers. NKT cells have recently been implicated in the disease
progression of human asthma. The clinical potential of NKT cells lies in the rapid
release of cytokines (such as IL-2, IFN-, TNF- α,
and IL-4) that promote or suppress different immune
responses.





CD1- antigen presenting molecules
present lipid and glycolipids derived from
microbial antigens to T cells.
These
molecules
are
non-MHC
restricted and nonpolymorphic.
They are distinct from MHC class I and
II. Similar structure to MHC class I,
having three extracellular domains and
expressed in association with 2
microglobulin on APC.
Binds hydrophobic region of lipid with
polar bound by TCR.
Binds to a variety of T cells including
NK1.1 (CD4+) cells. (NKT cells).
o Induces NK1.1 to secrete large
amounts of IL-4
o May be important in generating TH2 activities
 Overall, the CD1 molecules bind antigen in a deep, narrow hydrophobic pocket, with
ligands interacting via hydrophobic interactions rather than hydrogen bonding.The
role of CD1 in pathogenesis has not yet been fully determined.
138
2. Superantigens
 Activate T cells expressing a specific Vsegment as part of TCR
 Presented by Class II molecules on MHC but not in peptide groove
 Several organisms have components that function as superantigens
o Staphylococcus
o Rabies virus
 Activate large numbers of T cells (possible mechanism for toxic shock
syndrome)
SUPERANTIGENS
•Superantigens bind
directly to T-cell
receptors and MHC,
without processing.
•Usually involves
direct interaction to
V region of TCR.
V VD
V VD
J
J
J
J
C
C
C
C
3. Mitogens
 Polyclonal activators of T cells by activating widespread mitosis
 Derived from plant lectins
 Phytohemagglutinin (PHA)
 concanavalin A (conA)
 pokeweed mitogen (PWM)
 Other mitogens
 Endotoxin (lipopolysaccharide) – mouse B cells,
monocytes/macrophages
 AntiCD3 – polyclonal T cell activator
139
human
VI.
B CELL ACTIVATION AND FUNCTION
B Lymphocytes: The genesis of µ and delta chainpositive, mature B cells from pre-B cells is antigenindependent. B cell development is characterized
by recombinations of immunoglobulin H and L chain
genes and expression of specific surface
monomeric IgM molecules. At this stage of
development, B cells are highly susceptible to the
induction of tolerance. Cells bearing only
monomeric IgM are referred to as immature. These
cells may undergo deletion (death by apoptosis),
anergy (long term inactivation, or receptor editing
(reactivation via V-D-J gene recombination).
Once these cells acquire IgD molecules on their
surface, they become mature B cells that are able
to differentiate after exposure to antigen into
antibody-producing plasma cells. Mature B cells
can have 1-1.5 x 105 receptors for antigen
embedded within their plasma membrane.
The activation of B cells into antibody producing/secreting cells (plasma cells) is
antigen-dependent. Once specific antigen binds to surface Ig molecule, the B cells
differentiate into plasma cells that produce and secrete antibodies of the same antigenbinding specificity. If B cells also interact with T helper cells, they proliferate and switch
the isotype (class) of immunoglobulin that is produced, while retaining the same
antigen-binding specificity. This occurs as a result of recombination of the same Ig VDJ
genes (the variable region of the Ig) with a different constant (C) region gene such as
IgG. T helper 2 cells are thought to be required for switching from IgM to IgG, IgA, or
IgE isotypes. The generation of memory B cells is associated with class switching; this
process occurs in the spleen or lymph node.
In addition to antibody formation, B cells also process and present protein antigens.
After the antigen is internalized it is digested into fragments, some of which are
complexed with MHC class II molecules and then presented on the cell surface to CD4+
T cells.
B cells secrete antibody upon antigenic stimulation, a multi-step process involving
interactions with T cells. B cells express many surface molecules which assist in the
process of antibody production through delivery of various activation signals. Some of
these costimulatory molecules are depicted in the figure below. Fc receptors are
important in "feedback" mechanisms to deliver negative signals to the cell.
140
Surface Markers of human and murine peripheral B cells. Remember that B cells carry the HLA-D
(and I-A/I-E), class II restricted major histocompatability marker, as well as have specific receptors for
complement receptors. Coico and Sunshine, 2009. Figure 7.7.
A. T cell - B cell cooperation


T dependent antigens
 Require CD4+ help for B cells to make antibody
 Must be to same antigen but different epitopes (linked recognition)
 B cell epitope - hapten
 T cell epitope - carrier
T – B interactions
 For primary response, requires APC (dendritic cell the best)
 For secondary response, no APC necessary
 Requires cytokines for B cell growth (IL-4), proliferation (IL-6)
 Isotype switch from IgM to
o IgG (IFN
o IgA (IL-5)
o IgE (IL-4,13)
 External Ag on B cell bound by surface IgM
 Internalized, processed and presented via MCH II to TCR in
association with CD4 molecule
 Costimulation (CD40:CD154; CD28:CD80/86)
 Adhesion (CD58:CD2; ICAM-1:LFA-1; CD72:CD5)
 Result is cytokine production by T cell that binds via receptor to B cell
141
Coico and Sunshine. 2009. Figure 10.9.
B.
T independent Responses




Do not need T cell help to make antibody
Antigen is typically polymerized molecules (such as polysaccharides)
Only generate IgM responses
Do not generate memory
142
C.





B Cell Activation Pathways
Surface IgM is crosslinked
CD19,21,81 are coreceptors for BCR
Tyrosine kinases activated (Lyn, Fyn, Blk, Lck)
Phosphorylates the ITAMs of Ig/Ig molecules associated with surface Ig
Syk then activated



Activated Syk activates PLC-which splits PIP2 into DAG and IP3
 DAG>>PKC>>>multiple kinases
 IP3 >>calcineurin
Both pathways activate transcription factors – NF-, NF-AT
Result in nucleus is upregulation of cytokine receptor and Ig genes
143
SUMMARY

The major properties of the acquired immune response are specificity, memory,
adaptiveness, and discrimination between self and non-self.

Lymphoid cells in these categories include T and B lymphocytes. T and B cells
produce and express specific receptors for antigens. Receptor specificity is
related to gene rearrangement of variable region components during
development, according to essential features for clonal selection.

Cytokines are small molecular weight glycopeptides with a variety of cellular
origins and functions, both effector and regulatory.

Helper T cells (TH) provide assistance to B cells to make antibody and other T
cells to become cytotoxic by production of specific cytokines, expression of co
stimulatory and adhesion molecules molecular mechanisms involving
transcription factors

Cytotoxic T lymphocytes (CTL) are for host defense against intracellular
pathogens and induce death of the target cells by various mechanisms

T cell antigen receptors can be activated by a variety of molecules such as
proteins, lipids/glycolipids, superantigens and mitogens

B cells make antibodies, the quantity and isotype of which is dependent upon
whether the T cell is involved (T –dependent) or not (T-independent) and relates
to both the nature of the antigen and the underlying immunological capabilities of
the host
144
IMMUNOLOGY
ANTIGEN-ANTIBODY INTERACTIONS, IMMUNE ASSAYS, EXPERIMENTAL
SYSTEMS
Dr. Keri C. Smith
OBJECTIVES
The objective of these lectures is to learn how the exquisite specificity of
antibodies can be used in the clinical laboratory for diagnostic assays that measure either
antibodies or antigens and review experimental systems that will be discussed later in the
course.
KEYWORDS
Affinity, agglutination, prozone, zeta potential, precipitation, immunoelectrophoresis,
radial immunodiffusion, nephelometry, radioimmunoassay, ELISA.
READING
Chapter 5 of the Coico et al textbook, 2009.
Case 46 in Case Studies in Immunology, 6th Ed.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/antibodies.html
INTRODUCTION
It is clear that antibodies play a major role in protection from a variety of
diseases, toxins, viruses, parasites, etc. In addition, once antibodies have been made,
they can be used for a variety of diagnostic assays in the laboratory to detect the presence
of absence of a particular antigen or bacterium or virus in a sample. The use of
antibodies specific for red blood cell antigens has made routine transfusions possible and
opened up the whole area of transplantation.
The reaction of antigen with its homologous antibody is a two-stage phenomenon.
The initial or primary binding reaction can occur invisibly. The secondary manifestation
of that interaction is dependent on several factors such as:
a) Isotype of the antibody
b) Valence of antigen
c) Form (particulate or soluble) of the antigen
The type of assay used depends vitally on these factors. For example, determination of a
patient’s red blood cell type is done using intact red cells and so the assay called
agglutination is used. The kinds of assays used to detect soluble antigens such as growth
hormone cannot be used for red cell typing because of the particulate nature of the red
cell.
Review of Figure 5.1 on page 60 in the textbook will demonstrate many of the
features of antigens and of antibodies and fragments of antibodies that can dictate design
of specific assays. It is clear that valency of both antigen and antibody can be important.
Please review this figure thoroughly.
145
PRIMARY INTERACTIONS BETWEEN ANTIBODY AND ANTIGEN
Antigens and antibodies interact as the result of multiple weak, non-covalent
reactions. You should now review these interactions from the “Immunogens and
Antigens” lecture. Due to the relative weakness of these forces, Ab-Ag reactions can be
readily dissociated by:
a) low or high pH
b) by high salt concentrations
c) by chaotropic ions.
ASSOCIATION CONSTANT
The strength of the primary interaction between one paratope and its epitope can
be precisely measured by using the law of mass action since the reaction is noncovalent.
The binding of an antigen univalent epitope such as a free hapten (H) to a paratope can
be represented by the equation:
Ab + H  AbH
The association constant is then defined by the expression:
K= [AbH]/[Ab][H]
The K value represents the intrinsic association constant or the Affinity for monoclonal
antibodies and will represent an average association constant for polyclonal antibodies
AFFINITY AND AVIDITY
Definition: The intrinsic association constant, the reaction between a single
paratope and its epitope, is termed the affinity. Affinity measurements cannot account
for the overall efficiency of binding because having more paratopes/molecule will
enhance the overall efficiency of binding since each paratope on each molecule is
identical in its affinity. Thus, antigen multivalency enhances antibody-binding
efficiency. This enhancement due to antigen multivalency is called avidity. Affinity and
avidity are illustrated in the following figure:
146
SECONDARY INTERACTIONS BETWEEN ANTIBODY AND ANTIGEN.
AGGLUTINATION REACTIONS
Definition: The term agglutination infers aggregation of insoluble particles.
Aggregation of red blood cells or bacterial cells is routinely used for estimation of the
concentration of antibodies in a serum taken from a patient or experimental animal.
Definition: The term titer is used to describe the highest dilution of that serum
that will agglutinate a standard amount of the cells (i.e. 50 ul of a 1% suspension).
Clinical Wisdom
A set of sera can be compared for their titers, but the titer determined by another
laboratory or another technician on the same set of sera could vary significantly from
those found earlier. This happens routinely in blood banking, but the results reported by
the same technician within a given laboratory over time are usually internally
consistent. It is imperative to understand that titers of sera or of monoclonal antibodies
are not quantitative measurements.
PROZONE-Agglutination reactions can sometimes exhibit the phenomenon of
prozone. This occurs because very high concentrations of antibodies can totally saturate
147
all epitopes on each cell added so that no cross linking occurs. As the concentration of
antibodies is lowered by dilution in succeeding tubes, the numbers of cellular epitopes
and antibodies then reach a ratio where effective agglutination occurs.
ZETA POTENTIAL-An electrical potential between two like charged particles
prevents them from physically associating. The short distance between Fab arms of IgG
molecules may not overcome this repulsion, but the larger IgM molecule might be
sufficiently large to overcome zeta potential. The high sialic acid density on the surface
of red cells is difficult to overcome and the size, coupled with the multivalency, of IgM
makes it more efficient as an agglutinator of red cells.
COOMBS’ TEST-The Coombs’ test can overcome zeta potential by using a
second layer of antibodies to bridge cells. If the red cell is coated with IgG antibodies, an
antiglobulin antiserum can be added (Definition: a serum containing antibodies specific
for the Fc region of IgG) and it can then cross-link the IgG antibodies previously bound
to the cell thereby agglutinating the red cells. This assay is described in Figure 5.2 in the
textbook.
Direct Coombs’ Test
In this assay, patient blood that is suspected of having antibodies already bound
to the red cell (i.e. blood from a baby at risk for Erythroblastosis fetalis) is mixed
with the antiglobulin serum and positive agglutination is diagnostic for the
presence of anti-Rh antibodies bound to the red cells.
Indirect Coombs’ Test
This is to detect the presence in serum of a non-agglutinating antibody. For
example, serum from a pregnant patient suspected of having circulating IgG antiRh antibodies is mixed with Rh+ red cells, then the antiglobulin is added.
Positive agglutination is then diagnostic for the presence of anti-Rh in patient
serum, indicating that the fetus is at risk for erythroblastosis fetalis.
148
Clinical Vignette
Review Case 46—Hemolytic Disease of the Newborn. Indirect Coomb’s titers were used as a principal
diagnostic tool in this case.
PASSIVE AGGLUTINATION-Passive agglutination is a way to use the
extraordinary sensitivity of agglutination assays to detect antibodies specific for soluble
antigens such as thyroglobulin to help diagnose Hashimoto’s disease, for example. In
this assay, purified soluble thyroglobulin is attached to something particulate such as
micro-latex beads or red cells. Then sera containing suspected antibodies specific for
thyroglobulin can be titered in a standard agglutination format. If red blood cells are
used as the particle, the assay is usually called passive hemagglutination to
acknowledge the red cell as the carrier of the antigen.
PRECIPITATION REACTIONS
Precipitation Reaction in Solution (Fluid Phase Reactions)
Antigen-Antibody reactions that result in the formation of visible precipitation of
the reactants are classed as secondary manifestations of Ag-Ab reactions. This reaction
provided the first quantitative assay (NOT Qualitative, as written on page 63 of your
149
textbook) for antibody, but is rarely used today. However, understanding the Ab-Ag
interactions that lead to this reaction is important, as the immune complexes formed are
also found in vivo.
In this reaction, various amounts of soluble antigen are added to a fixed amount of
serum containing antibody. As illustrated in the figure below, when small amounts of Ag
are added, Ab-Ag complexes are formed with excess Ab, and each molecule of Ag is
bound by Ab and cross-linked to other Ab molecules. When enough Ag is added, ALL
of the antigen and antibody complex and fall out as precipitate (the zone of equivalence).
When an excess of Ag is added only small Ag-Ab complexes form (no crosslinking) and
the precipitate is reduced. This reaction is affected by the number of binding stes that
each Ab has for antigen, and the maximum number of Abs that can be bound by an
antigen or particle at one time. This is defined as the valence of the antigen or antibody
(see figure below) and valence of Ab and Ag has to be > 2 or precipitation will not occur.
It is important to note that the valence of an Ag is almost always less than the number of
epitopes on an Ag, since steric considerations limit the number of distinct antibody
molecules that can bind to a single antigen at any one time.
PRECIPITATION REACTIONS IN GELS
Often, precipitation reactions are used for analysis in situations where
quantitation is important but in other situations only a qualitative answer is necessary.
These latter reactions can be done in a gel matrix that slows down the rate of diffusion of
reactants and holds the precipitate in the gel web so that it is effectively immobilized for
visualization either directly or with the aid of various staining methods. Several
qualitative and quantitative methods are in wide use in medicine today for analysis of
numerous hormones, enzymes, toxins, and for analysis of the products of the immune
system itself. All methods described below will be designated as qualitative or
quantitative analysis methods.
OUCHTERLONY DOUBLE DIFFUSION ASSAY
The Ouchterlony Assay was developed by Orjan Ouchterlony in the 1950’s and is
still in widespread use. It has two important features.
a) it is inexpensive to use.
150
b) it can be used to compare the relatedness of two antigens (Antigenically, are
they totally different, are they the same, or only similar?).
The assay is called a Double Diffusion assay because both the antigen and antibodies are
diffusing. It is a qualitative assay.
Format: Reagents are put in wells made in a thin layer of agar or agarose made
up in physiological buffer. The molecules in each well then diffuse slowly into the agar
in a radial fashion (diffusion in a circular fashion with an ever-increasing radius). Thus,
antigen and antibody slowly diffuse toward one another. A positive result will be that a
thin opaque precipitate line or band will form in the agar at right angles to a line
connecting the centers of the two wells and it will usually be symmetrical, extending the
same distance either side of the line connecting the well centers. The presence of a line is
a qualitative assay for the presence of either antibody in the antiserum (using a standard
antigen solution) or for the presence of antigen (using a standard antiserum). See Figure
5.5 in the Textbook.
The most widespread use of the Ouchterlony technique is for comparison of
antigens. It has also been used in forensic medicine and in a variety of diagnostic assays.
Study note: The three patterns of reactions (identity, non-identity, and partial
identity) described in Fig. 5.5 on pg. 64 are important to understand
RADIAL IMMUNODIFFUSION
Radial immunodiffusion is a type of agar gel precipitation technique that is
quantitative. Once again, it relies on having a standardized antiserum or standard antigen
on hand in order to analyze the unknown sample.
Technique: The technique of doing radial immunodiffusion and some typical results are
described in Fig. 5.6, pg. 65 in the textbook.
IMMUNOELECTROPHORESIS
Immunoelectrophoresis is a variation of the Ouchterlony double diffusion in gel
technique. It is designed to analyze complex protein mixtures containing many different
antigens. The method and typical results are shown in Fig. 5.7, pg. 65 in the textbook.
Clinical Relevance: The medical diagnostic use of immunoelectrophoresis is
for diagnosis of conditions where certain proteins are suspected of being
absent (e.g. hypogammaglobulinemia) or of being overproduced (e.g. Multiple
Myeloma). It is usually used as a first screening test, followed by quantitative
tests.
151
Immunoelectrophoresis is a qualitative assay. It is also used in medical research for
following the different steps of a purification protocol to show the disappearance of
unwanted proteins when purification of one component from a mixture is desired.
NEPHELOMETRY
Nephelometry is a widely used methodology for accurately measuring quantities
of the Ig classes in serum. Obviously, dramatic increases or decreases in quantities of
these could contribute to diagnosis of numerous diseases.
In this assay, proteins in the sample react with specific antibody (ie an anti-IgE
antibody). The mixture is placed in a tube and inserted into the Nephelometer. When
light passes through the suspension that contains aggregated particles, a portion of the
light is scattered. The scattered light is measured and compared with stored standards.
Thus, this is a quantitative method using liquid-phase precipitation principles. It
can be applied to measuring any soluble substance provided specific antisera are
available.
WESTERN BLOTS
Also called immunoblotting-a mixture of antigens is usually separated by
electrophoresis on a gel, transferred onto a medium such as nitrocellulose that binds
proteins tightly and then antibodies that have an enzyme covalently attached are poured
on the nitrocellulose. Substrate for the enzyme is added, turns colors when enzyme is
present, and the colored line shows that the antigen was present. See textbook, Fig. 5.8.
Clinical Correlation—HIV infections are frequently diagnosed by doing Western Blots of patient’s
serum for content of antibodies specific for various HIV antigens. See Figure 5.8 in the Coico book. See
Case 10—Acquired Immune Deficiency Syndrome (AIDS).
IMMUNOASSAYS
DIRECT BINDING IMMUNOASSAYS
The principle of radioimmunoassays is diagrammed in Figures 5.9 & 5.10 in the
textbook. In Fig. 5.9 radioactive antigen is reacted with a limited amount of antibody. In
the second step, Figure 5.10, unlabelled test antigen is mixed with the labeled antigen
prior to addition of the antibody. The amount of labeled antigen bound to antibody is
then reduced by a factor related to the ratio of labeled to unlabeled antigen in the mixture.
The unlabeled antigen effectively competes for available antibody since it is identical
immunologically, but unlabeled. A standard curve of inhibition can be generated using
precisely known amounts of unlabeled antigen and then test samples containing unknown
concentrations of antigen can be analyzed and simply read off the standard curve to find
the concentration of antigen in the unknown.
152
SOLID-PHASE IMMUNOASSAYS
There are a group of assays in which the antigen or the antibody is coated on the
surface of a plastic microplate and sensitive indicators such as radioactivity or enzymatic
action are used to detect the presence of Ag or of Ab. There are 5 of these assays
classified in two groups according to the types of antigens being analyzed: soluble or
cellular.
A. SOLUBLE ANTIGENS
RADIOIMMUNOASSAY--There are many different formats for doing
radioimmunoassay (RIA). Only one will be described here, that is to detect antigen. In
this format the following steps are done:
a) free antigen is first coated onto the surface of plastic plates and the excess is
removed by rinsing out the wells with buffer solutions.
b) the remaining plastic surface is then blocked by adding an irrelevant protein
solution and washing
c) antiserum is added to the plate, incubated and then washed out. This leaves
the plate with Ab bound to the Ag that is, in turn, bound (noncovalently) to
the plastic.
d) a radioactive indicator is added which recognizes the Ab but not the Ag.
(There is a protein called Protein A derived from Staphylococcus aureus that
reacts specifically with the Fc region of most vertebrate IgG molecules. The
Protein A can be radiolabeled. The overall procedure is shown in the
following diagram:
Clinical Relevance:
Current Laboratory RIA Assays
The specific assays are to quantitate the amounts of specified antigens in
body fluids such as blood or urine.
Assays are available for measuring Renin, Gastrin, Parathyroid Hormone,
Growth Hormone, Urine Microalbumin, Vitamin B12 and Folate. MemorialHermann Hospital currently is phasing out the radioimmunoassay laboratory.
153
ENZYME LINKED IMMUNOADSORBENT ASSAY (ELISA)
The ELISA assay is quite similar to the RIA except that the indicator reagent used
in ELISA is not radioactive. Instead, the indicator (Protein A) is coupled to an enzyme
molecule that converts added substrates to a colored product that can be detected
spectrophotometrically due to the color change. The assay is done as diagrammed below:
Clinical Relevance: There are a large number of commercial ELISA kits available
for diagnostic purposes. Currently the Memorial-Hermann Hospital Laboratories
offer ELISA assays for Hepatitis antigen, HIV and HTLV antigens. Specific assays
are also available for detection of antibodies in patient’s serum for Hepatitis A virus,
Hepatitis B surface antigen, Hepatitis B core antigen, Hepatitis C virus,
cardiolipin, H. pylori, and for HIV types 1 and 2. Another ELISA assay is
available for detection of antibodies to Human T-lymphotropic virus type I.
Clinical Vignette
This is the case of a woman who contracted the AIDS virus from a blood transfusion
and transmitted it to her fetus later. Antibodies specific for the gp120 HIV antigen
were measured in the infant using an ELISA. The mother and father were also
tested and were found to have anti-gp120 antibodies by ELISA.
154
ELISPOT assays
Variation of the ELISA method. Incubate with cells instead of soluble antibody. The #
of spots after addition of detection antibody and precipitable substrate = the number of
cells secreting a specific antibody, thus can be used to determine the frequency of antigen
specific B cells. Also used for T cell assays (e.g. the number of T cells producing a
cytokine, as illustrated below). Used in biomedical research.
B. CELLULAR ANTIGENS
IMMUNOFLUORESCENCE
It is sometimes of diagnostic value to determine if a particular antigen is
found on or in the cells of a particular tissue. In this case, assays are needed that can be
performed directly on biopsies of tissue and seen using a microscope. The method
originally developed by Albert Coons and his colleagues at Harvard involves covalent
attachment of fluorescent organic compounds to specific antibodies that then can be used
to detect the antigen in question. The fluorescent compounds excite at different
wavelengths. This is a highly sensitive and specific assay, and cells individual cells can
be stained with up to 12 different compounds.
155
1. Direct Immunofluorescence-The antibody specific for the antigen in
question is directly labeled with the fluorophor and used to identify the
antigen.
2. Indirect Immunofluorescence-This is similar to the Coombs’ reaction
discussed earlier (review that if necessary). It is a two step method in which
the unlabeled antibody specific for the antigen in question is reacted first with
the tissue and the excess antibody is washed away. Then the slide is flooded
with a fluorescent anti-Ig (preferably Fc specific). This method has the
advantage that it is significantly more sensitive than the Direct method.
Clinical Relevance: Immunofluorescence, using the indirect format, is used in
clinical laboratories for screening patient’s sera for anti-DNA antibodies in
suspected cases of systemic lupus erythematosus.
IMMUNOHISTOCHEMISTRY is a similar technique. Instead of fluorescent labels,
the detection antibodies are labeled with enzymes such as horseradish peroxidase or
alkaline phosphatase (these are also used in ELISA). Addition of substrate then colors
the membranes of the cells expressing the antigen of interest.
FLUORESCENCE ACTIVATED CELL SORTING (FACS) ANALYSIS
FACS analysis is used to identify, and sometimes purify, one cell subset from a
mixture of cells. The technique and a diagram of the instrument are on page 69, Fig.
5.12. This is an extremely effective tool to identify and/or isolate specific cell subsets.
The organic fluorescent compounds attached to the detection antibodies are excited by
different fluorescent wavelengths, and all emit at different wavelengths as well, allowing
for specific detection of the markers. Current instrumentation can detect up to
15different antigens on one cell (though most investigators use 4 colors at most).
Sorting of cells can also be accomplished using antibodies coupled to magnetic
beads (magnetic activated cell sorting, or MACS). The cells are then placed over a
magnetized column, and any cells with labeled antibody bound to them can be isolated
from the unbound population.
156
Clinical Relevance: FACS can measure CD4+ cell numbers in AIDS patients to
follow disease progression. FACS was used in Case 5—MHC Class I Deficiency to
measure peripheral blood lymphocytes
LYMPHOCYTE FUNCTION ASSAYS
Lymphocyte function can be compromised in certain diseases or can occur as a
result of a genetic abnormality. A diagnosis can be confirmed in many cases if it is
known whether or not the B or T cells are normal, if the existing B cells can make
antibodies, or if the T cells can produce the correct cytokines.
Mitogen Activation—Lipopolysaccharides can cause polyclonal stimulation of B
cells in vitro. This activation is accurately measured by incorporation of radioactive
nucleosides. Several lectins, including concanavalin A and phytohemagglutinin are
effective T cell mitogens. Pokeweed mitogen stimulates polyclonal activation of both B
and T cells.
Numerous assays can measure antibody production by stimulated B cells (ie
ELISA).
Cytotoxicity assays measure the ability of cytotoxic T cells or NK cells to kill
radioactive target cells that express a specific antigen for which the cytotoxic T cells may
be sensitive.
MONOCLONAL ANTIBODIES AND T CELL HYBRIDOMAS
Due to cross reactivity of antibodies and the need for more controllable assays it
is sometimes of great advantage to have a homogeneous antibody preparation that is
specific for only a single epitope and with high affinity. Since polyclonal antibody
mixtures consist of a multitude of antibodies specific for different epitopes on even
simple antigens like tetanus toxoid, and the fact that there are an array of different
subpopulations of antibodies with different affinities even in the subset specific for a
single epitope, significant cross reactions can occur when using polyclonal antibodies for
analytical assays. This can lead to misinterpretation of results occasionally. Kohler and
Milstein developed a method for making murine antibodies that are monoclonal, that is,
all antibodies are derived from a single precursor plasma cell so that all the antibodies in
the preparation are identical and derived from the same original clone. The method is
outlined in Figure 5.13 in the textbook as well as in the following:
157
The specific details of the hybridoma technology are covered in the textbook. (See
Figure 5.13, Coico and Sunshine, 2009).
T CELL HYBRIDOMAS
The general method is also used for making T cell hybridomas.
Clinical Relevance: T cell hybridomas are valuable for the large-scale production
of several T cell-derived lymphokines that are used as antigen in diagnostic kits and
are also used therapeutically.
GENETICALLY ENGINEERED ANTIBODIES
Attempts to develop human hybridoma technology have not been very successful.
To adapt the murine system for making human antibodies, recombinant DNA
methodologies have been developed to “humanize” murine antibodies. The methods
usually use murine V-region sequences coupled to human C-region sequences. There are
many variations on this theme and the methodologies are also applicable to engineering
receptors (for cytokines, etc.) into cell lines in which they are not normally expressed.
MICROARRAYS TO ASSESS GENE EXPRESSION
Levels of expression of thousands of genes can be measured simultaneously using a
technology called gene chips or microarrays. Briefly, thousands of short cDNA
representing genes from all parts of the genome are attached to a slide. Samples of
mRNA from cells in culture are used and reverse transcribed into cDNA and by labeling
158
this cDNA from different sources (ie normal cells and tumor cells) with different
fluorochromes, the differential expression of distinct sets of genes can be measured. By
scanning with a laser, different spots can have different colors depending on the success
of binding by the two different cDNA’s.
This methodology has great potential in fields such as clinical diagnosis of lymphoid
tumors.
EXPERIMENTAL ANIMAL MODELS
Note: These will be covered very briefly in the lecture but students should review these
models for general knowledge as they will be mentioned later in the course and will
likely be mentioned in other courses.
Numerous strains of mice exist that mimic specific human diseases. These
became available due to the development of inbred strains of mice that freely accept cell
and tissue grafts from other members of the strain. Since the Major Histocompatibility
Complex antigens are identical in all members, grafts are accepted. One type of graft that
is useful is to do Adoptive Transfer of lymphoid cells from an antigen primed mouse to
one that was not immunized to study the function of various cell subsets. This is a type
of Passive Immunization.
The SCID mouse (Severe Combined Immunodeficiency Disease) mouse has no T
or B cells. It is useful for studying human hematopoietic stem cells since they do not
reject the foreign human cells.
Thymectomized and congenically athymic mice (Nude mice) are useful for
studying T cell function and T cell subsets. Neonatal thymectomy coupled with
irradiation (to destroy B cells and lymphoid precursors) coupled with reconstitution with
bone marrow (to repopulate with B cells) is widely used for T cell study. Nude mice are
a mutant strain that fail to develop a thymus. They can be reconstituted using syngeneic
adult thymic epithelial tissue.
TRANSGENIC MICE
Transgenic mice are made by injecting a cloned gene into fertilized mouse egs.
The eggs are injected into pseudopregnant mice. If the transgene is constructed with a
specific promoter region, it is possible to control the gene’s expression in certain tissues.
Care must be used in interpreting results with transgenic mice since the transgenes
frequently are grossly overexpressed.
KNOCKOUT MICE
Replacing a given gene with one that has been mutated or has been changed so it
is not expressed in the adult creates Knockout mice. These are widely used to study the
function of specific cytokines and MHC molecules.
159
SUMMARY
1. In addition to functioning in vivo, antibodies are used in numerous diagnostic
formats in the clinical laboratory.
2. The primary binding reaction of antibody with antigen follows the rules of the
Law of Mass Action and an Association Constant (antibody affinity) can be
accurately measured while functional avidity is defined as the affinity
enhancement due to multivalency.
3. Secondary Ag-Ab reactions include the agglutination assay used in blood
banking. A prozone in agglutination assays is due to a huge excess of
antibody molecules. Zeta potential is an electrical repulsion of like-charged
particles. Coombs tests utilize anti-Ig reagents.
4. Precipitation reactions between antibodies and soluble antigens occur
regularly in vivo. The degree of precipitation depends on valency of antigen,
ratio of antibody to antigen and the classes/subclasses of antibodies that
predominate. Usually IgG is the only effective antibody class mediating
precipitation.
5. Several precipitation reactions in gel media are widely used for different
purposes. The Ouchterlony double diffusion assay is a qualitative assay for
measuring antigen presence and comparing antigens. Immunoelectrophoresis
is a qualitative method for measuring the numbers of components in mixtures
and Radial immunodiffusion is a quantitative method.
6. Nephelometry is a widely used method for measuring Ig concentrations.
7. Radioimmunoassays and ELISA (Enzyme Linked ImmunoSorbent Assay)
are the two most widely used immunoassays used in US clinical laboratories
although radioimmunoassays are slowly being phased out in favor of ELISA
160
STUDY QUESTIONS - ANTIGEN-ANTIBODY REACTIONS
Study questions for Antigen-Antibody Interactions
1. What is the classic example where a Coomb’s type agglutination assay would be more
sensitive than the direct agglutination method.
2. Describe the steps in setting up a quantitative precipitation reaction. What does the
experiment tell you?
3. Make a list of all the immunoassays in this chapter and categorize them as a)
Quantitative or as b) Qualitative.
4. Describe a situation where you would order an Ig class quantitation measurement
done on a patient’s serum. What instrument would the lab use to do this?
5. Describe the steps in developing an enzyme linked immunosorbent assay. How
would you make it quantitative?
6. Write the two equations that together define antigen-antibody Affinity.
Answers to study questions may be found at:
http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/antibodies.html
161
IMMUNE EFFECTOR MECHANISMS I: ANTIBODY-MEDIATED REACTIONS
Steven J. Norris, Ph.D.
Recommended Reading: Actor, 2012, Chapters 7 and 10.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/geneab.html
I. INTRODUCTION
The immune system cannot be understood in isolation from infectious diseases. All living
organisms exist in a hostile environment and are continually used as hosts and energy sources by other
organisms. Since there are many different foreign invaders which can infect humans, we must have
many different ways to defend ourselves. To provide the versatility required, the two major effector
arms of specific immunity: antibody (humoral) and cellular, employ an incredible variety of
accessory mechanisms. These lectures will introduce you to some of these mechanisms.
In defense against infections, antibody is generally operative against extracellular bacteria or
bacterial products, whereas cell mediated immunity (CMI) primarily operates against intracellular
viral and bacterial infections, as well as fungal infections. The killing effects of immune reactions are
extremely efficient and, when specifically directed to a given infection, are able to eliminate large
number of organisms in a short period of time.
The immune response is a double-edged sword. In most cases, the immune system is
protective, providing life-saving defenses against infectious diseases and tumors. However, it can
also be destructive, causing immunopathology, defined as tissue damage resulting from the immune
response. These destructive responses result in some of the adverse effects of infections, in allergies
or hypersensitivity reactions (antibody- or T cell-mediated reactions to environmental or
administered antigens), and in distinct autoimmune disorders (antibody- or T-cell mediated reactions
to self-antigens). The seven immune mechanisms listed below are active in both immunoprotective
and immunopathologic reactions.
II. SEVEN IMMUNE MECHANISMS
Until the 1960s, immune reactions where not classified according to mechanism, but were
presented as a bewildering list of lesions with peculiar names. The first working classification of Type
I to Type IV immune mechanisms as introduced be Gell and Coombs was a major advance in
understanding immunopathologic reactions; seven mechanisms are presented in this handout. The
terms Type I - Type IV reactions, although out of date, are still used in some textbooks.
TABLE 1: Classification of Immune Mechanisms
Handout
Gell and
Coombs (1963)
General Properties
Antibody-Mediated
Inactivation or Activation
Cytotoxic or Cytolytic
Immune Complex
Atopic or Anaphylactic
-Type II
Type III
Type I
Toxin, virus inactivation
Opsonization, ADCC, C’-mediated lysis
Ag-Ab complex formation in tissue
IgE mediated allergic reactions
Cell-Mediated
T-cell Cytotoxic (TCTL)
Delayed Hypersensitivity (TDTH)
-Type IV
Lysis of virus-infected cells; contact hypersensitivity
CD4+ T cell-mediated activation of macrophages
Either
Granulomatous Reactions
--
Chronic reaction to poorly degradable antigens
Page 162
These immune mechanisms are similar in many ways to antibody- or cell-mediated reactions observed
in vitro. Primary reactions consist of the formation of Ag-Ab complexes or Ag-TCR reactions, secondary
reactions the effects of this interaction that can be demonstrated in vitro, and tertiary reactions the
corresponding in vivo manifestations (see figure).
Factors affecting the induction of different forms of immunity
•
Type of infectious agent or antigen.
•
Route of infection/exposure.
•
Activation of Th1 vs. Th2 cells.
•
Location/cell type involved in antigen presentation.
•
Cytokines expressed by antigen presenting cells and T cells.
•
Genetic factors.
•
Non-genetic factors. (e.g. age and nutritional status)
Page 163
ANTIBODY-MEDIATED IMMUNE MECHANISMS
1. INACTIVATION (NEUTRALIZATION) REACTIONS
A. Definition - binding of antibody to an epitope (toxin, virus, cell receptor, etc.) resulting in
inactivation (loss of function), neutralization (loss of infectivity), or abnormal activation.
B. Mechanisms
1. Binding of antibodies to a protein can stearically inhibit its binding to substrate, or
alter its conformation, resulting in loss of activity.
2. Antibody binding to viral receptor proteins can interfere with binding to cells, alter
viral structure, or mediate Ab- or C’-mediated opsonization and clearance
3. In some cases, antibodies against hormone or neurotransmitter receptors can either
block or activate the receptor.
C. Medical Aspects - examples
1. Protective
a. Immunization of
individuals with
diphtheria toxoid or
tetanus toxoid results in
expression of antibodies.
These preformed
antibodies do not prevent
colonization by C.
diphtheriae or C. tetani,
but bind to the toxins and
prevent them from
interacting with the
corresponding host cell
receptors, thus preventing
disease.
b. Infection or immunization with viruses (including polio, influenza, measles,
mumps or rubella) results in expression of antibodies that bind to viral receptors
and prevent infection upon subsequent exposures.
2. Immunopathologic
a. Myasthenia gravis - autoimmune antibodies bind to acetylcholine receptors at
the neuromuscular junction, causing their internalization and downregulation.
The synaptic folds also become decreased or ‘simplified’, reducing interaction
with the neurotransmitter and inhibiting skeletal muscle contraction. (Aristotle
Onassis had this disease.)
b. Graves disease - antibodies against the TSH receptor bind to thyroid cells and
result in activation and abnormally high production of thyroxines. (George and
Barbara Bush and dog Millie)
c. Pernicious anemia - antibodies against intrinsic factor interfere with its binding
of vitamin B12 in the GI tract, resulting in B12 deficiency and anemia.
Page 164
Clinical Vignette – Inactivation Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th edition)
Case 42
Myasthenia Gravis – binding of anti-AchR antibodies results in skeletal muscle weakness
2. CYTOTOXIC REACTIONS
A. Definition - reaction of antibodies with
cell surface antigens may result in
destruction of cells by opsonization,
complement activation, or AntibodyDependent Cellular Cytotoxicity (ADCC).
Also called Type II hypersensitivity.
B. Mechanisms
1. Complement activation may lyse
bacteria directly through formation of
the membrane attack complex (MAC).
A single IgM molecule or 2 or more
IgG molecules complexed to surface
antigens are sufficient to activate the
classical pathway.
2. Phagocytosis of infectious agents by
macrophages or neutrophils can be enhanced through antibody binding (interaction with
Fc receptors) or fixation of C3b
(interaction with complement receptors).
3. ADCC results from IgG-mediated binding
of null lymphocytes (and in some cases
macrophages) to target cells via Fc
receptors, and direct killing of the target
cell through cytolytic mechanisms (see
below).
4. In parasitic infections, IgE-mediated
binding of eosinophils to helminths results
in eosinophil degranulation and damage to
the worm tegument (surface).
C. Medical Aspects (Examples)
1. Protective
a. Many bacteria (particularly Gram
positive bacteria) are susceptible to C’mediated killing and/or opsonization.
This is particularly true of pyogenic
bacteria (such as Staph and Strep) that
result in massive accumulations of
neutrophils (see Immune Complex
reactions below).
b. Ab and C’-mediated MAC formation
and opsonization are active against
some protozoal infections, including
Plasmodium and Trypanosoma.
Page 165
c. ADCC may be active against virally-infected cells, tumor cells, protozoa, and
helminths.
2. Immunopathologic
a. Transfusion reactions - ABO mismatches result in rapid lysis of transfused cells
due to anti-A or anti-B isohemagglutinins, naturally occurring IgM antibodies that
bind to the transfused erythrocytes and activate complement.
b. Rh reactions - birth of an Rh+ infant to a previously sensitized Rh- mother may
result in binding of maternal anti-Rh antibodies to the infant’s erythrocytes, causing
opsonization and phagocytosis  hemolytic disease of the newborn.
c. Hemolytic anemia - autoantibodies can cause erythrocyte lysis, anemia.
d. Goodpasture’s syndrome - autoantibodies to basement membrane components and
complement are bound in an even, ribbon-like pattern to glomeruli and other
tissues. (Contrast with lumpy-bumpy appearance of immune complex disease; see
below).
Clinical Vignettes – Cytotoxic Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th ed., 2012)
Case 46
Case 41
Hemolytic Disease of Newborn– maternal anti-Rh antibodies cause hemolysis in Rh+ newborn
(Cynthia Waymarsh)
Autoimmune Hemolytic Anemia – patient Gwendolyn Fairfax develops hemolytic autoantibodies
following a mycoplasma infection
3. IMMUNE COMPLEX REACTIONS
A. Definition - formation of soluble or insoluble Ag-Ab complexes that can be deposited in tissue,
leading to attraction of PMNs,
inflammatory changes, and tissue
damage. Also called Type III
hypersensitivity.
B. Mechanisms
1. As increasing concentrations of
antigen-specific antibodies
(particularly IgM and IgG) are
expressed, any remaining antigen
will form Ag-Ab complexes or socalled immune complexes.
2. The size of immune complexes
formed in vivo will depend on the
degree of cross-linking as it relates to
antigen excess, equivalence, and
antibody excess, similar to
quantitative precipitation and agar
double diffusion assays in vitro (see
figure).
3. Depending on their size, immune
complexes can fix complement,
resulting in binding of C3b and
release of the anaphylotoxins C3a
and C5a. These cause local mast cell
degranulation and attraction of neutrophils, leading to inflammation.
Page 166
1. Large immune complexes are typically phagocytosed and destroyed by phagocytic cells
(such as resident macrophages of the reticuloendothelial system). Smaller complexes can
become lodged in the walls of venules, in joints, and in glomeruli. Deposition of immune
complexes causes complement activation,
attraction of neutrophils, and release of
lysosomal contents (“frustrated phagocytosis”),
resulting in vasculitis, reactive arthritis, and
glomerulonephritis.
2. The uneven distribution of immune complexes,
complement components, and lysosomal
contents results in the formation of lumpybumpy membrane deposits detectable by
binding the anti-Ig or anti-C3 antibodies.
3. Injection of an antigen in a previously
immunized individual can result in an Arthus
reaction due to deposition of Ag-Ab
complexes, complement activation, and
resulting erythema, edema, and attraction of
neutrophils. An Arthus reaction typically takes
2 to 6 hours to develop.
Page 167
C.
MEDICAL ASPECTS (EXAMPLES)
1. Protective
In pyogenic infections (e.g. Staphylococcus aureus), immune complexes attract
neutrophils which marginate on the endothelial cells and enter the tissue. A
predominance of neutrophils constitutes an acute inflammatory response (occurs
within a few days). Bacteria are killed through phagocytosis and release of lysosomal
contents. Accumulation of dead bacteria, neutrophils and other cells killed by bacterial
toxins or lysosomal contents, and fibrin accumulate, forming pus. This reaction may
wall off the infection.
1. Immunopathologic
a) Serum sickness - In the early
1900’s, serum from horses
immunized with rabiesvirus or
other agents was used for
passive immunization.
Administration of horse serum
elicited antibodies against horse
serum proteins in the patient, so
that subsequent injections
yielded immune complexes.
These could cause severe
muscle and joint pain and fever,
as well as glomerulonephritis.
Use of hyperimmune human serum antibodies for passive immunization has
virtually eliminated this problem.
b) Systemic lupus erythematosus (SLE) and related autoimmune diseases (e.g.
Sjogren’s syndrome and scleroderma) are caused by antibodies against DNA and
other normal cell components. The accumulation of immune complexes results in
skin rashes, glomerulonephritis, and pericarditis.
c) Rheumatic fever - infection with Streptococcus pyogenes can result in formation of
antibodies cross reactive with heart antigens (cytotoxic reaction) and circulating
immune complexes (immune complex disease). These cause heart and kidney
damage and vasculitis in other tissues.
Clinical Vignettes – Immune Complex Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th
ed. 2012)
Case 37
Case 52
Systemic Lupus Erythematosus – Nicole Chawner, age 16, butterfly rash after sun exposure. Immune
complexes due to antibodies against DNA and other nuclear components cause tissue damage
Drug-Induced Serum Sickness – Gregory Barnes, antibodies against penicillin cause vasculitis,
hemorrhage
4. ANAPHYLACTIC OR ATOPIC REACTIONS
A. Definition - IgE-mediated activation of mast cells and other cells types and its effects.
Also called allergic reactions, immediate type hypersensitivity and Type I
hypersensitivity. The term anaphylactic (literally “away from protection”) arose from the
recognition that immunization and subsequent challenge with some antigens lead to adverse
reactions rather than protective effects (prophylaxis).
Page 168
B. Mechanisms
1. Requires the production of antigen-specific IgE, also called reagin or reaginic
antibody. Isotype switching to IgE during formation of memory cells requires Th2
expression of IL-4. IL-6 further enhances the production of IgE. An individual
having significant levels of IgE against a certain antigen is said to be sensitized.
Individuals vary greatly in levels of IgE production; those expressing high levels are
called atopic patients.
2. Very little IgE is found in the circulation. Rather, most is bound to the surface of
mast cells present in tissue around blood vessels, or basophils found in the
circulation or tissue. IgE binds specifically to the FcR1 receptor, and can persist
for weeks to months on the surface of mast cells.
3. Crosslinking of antigen-specific bound IgE by antigen causes a decrease in cyclic
AMP levels and mast cell activation, resulting in rapid degranulation and de novo
synthesis of arachidonic acid, which is subsequently converted to leukotrienes,
prostaglandins, and thromboxanes.
4. Within seconds to minutes, the preformed contents of mast cell granules act locally
to produce a typical wheal and flare reaction (in cutaneous exposures) or hayfever
symptoms (in
CROSS LINKING
respiratory tract
IgE
exposures).
ALLERGEN
Histamine
+
bind
MAST
s to
CELL
tissu
LEUKOTRIENES
PROSTAGLANDINS
DEGRANULATION
e
HISTAMINE RECEPTORS
hista AND ALLERGIC REACTIONS
INFLAMMATORY EFFECTS
min H2 RECEPTORS - DILATION
SMOOTH MUSCLE DILATION
(INCREASED BLOOD FLOW)
VASCULAR = SHOCK
e
NEUTROPHIL
rece H1 RECEPTORS - CONSTRICTION
EOSINOPHIL
INFILTRATE
LUNG = ASTHMA
ptor
ENDOTHELIAL CONTRACTION
GI = DIARRHEA
(INCREASED VASCULAR
s H1
GU = URINATION
PERMEABILITY)
(ind
VASCULAR ENDO = EDEMA
uces
smooth muscle contraction, endothelial cell separation and leakiness 
vascular permeability) and H2 (mucus secretion, vasodilation).
Eosinophil chemotactic factor (ECF-A) - attracts eosinophils (present in latephase or chronic anaphylactic reactions)
Neutrophil-chemotactic factors (NCF) - attract neutrophils (late-phase)
Heparin - anti-coagulant, not directly involved in anaphylaxis
Wheal and flare - local erythema (due to vasodilation), edema (due to increased
vascular permeability
Hayfever - increased mucus secretion, mucosal swelling
Prausnitz-Kustner reaction - passive cutaneous anaphylaxis, caused by
experimental injection of IgE and antigen into skin.
5. In severe cases, systemic effects can cause shock (vascular collapse, loss of blood
pressure) and/or airway obstruction (laryngeal edema, bronchoconstriction and
mucus production resulting in suffocation).
6. Leukotrienes (formerly known as Slow-Reactive Substance A) cause long-term
smooth muscle contraction which is not alleviated by antihistamines. Cause some
Page 169
manifestations of asthma. Prostaglandins also promote bronchoconstriction,
vasodilation, and chemotaxis of granulocytes.
7. Eosinophils attracted to the area also have bound IgE which can be crosslinked to
cause release of granule contents:
Major Basic Protein - damages parasites, may provide some protection in
parasitic diseases. Also causes damage to host epithelium cells,
contributes to asthma.
Eosinophil Cationic Protein - also toxic to helminths, neurotoxin
Platelet Activating Factor - yet another bronchoconstrictor.
8. Long-acting cells and substances contribute to late-phase reactions, including
asthma.
9. Anaphylactic reactions can be reduced by
a) avoidance of allergens;
b) drugs such as cromolyn sodium (inhibits mast cell degranulation),
corticosteroids (block arachidonic acid metabolism, inflammation);
antihistamines (block binding of histamine to receptors); and epinephrine
(reverses bronchoconstriction, decreases vascular permeability);
c) hyposensitization - long-term injection of antigen to stimulate production of
blocking IgG to reduce allergy symptoms; and
d) desensitization - short-term injection of small quantities of antigen to deplete
IgE, desensitize mast cells (e.g. desensitization with penicillin prior to
administration of therapeutic doses).
Page 170
C. Medical Aspects (Examples)
1. Protective
a) Helminth (worm) infections. IgE-mediated responses are thought to aid in
the expulsion or killing of parasitic worms. In the GI tract, increased mucus
secretion, intestinal mobility, and release of inflammatory products may
result in dislodgement of intestinal worms such as Ascaris lumbridicoides.
In addition, release of MBP and other products by eosinophils and mast cells
damage schistosomes and trichinella parasites. Other parasites that also
cause chronic inflammation against nematode associated antigens
(Wuchereria bancrofti / Brugia malayi) may cause lymphatic obstruction
and elephantiasis.
2. Immunopathologic
a) Hay fever - allergic reactions to pollen and other allergens, causing
increased nasal secretions, watery eyes.
b) Asthma - a more severe respiratory reaction causing bronchoconstriction,
increased mucus secretion. May be life-threatening.
c) Cutaneous anaphylaxis - insect bites or exposure of skin to other allergens
may cause a rapid anaphylactic reaction. Distinct from contact
hypersensitivity (see next lecture).
d) Food allergies - IgE-mediated reactions to seafood, nuts and other foods
may cause severe anaphylactic reactions.
e) Systemic anaphylaxis - hypersensitive individuals may develop vascular
shock and respiratory failure as the result of exposure to an allergen (e.g. bee
stings). Can be reversed by rapid administration of epinephrine.
Clinical Vignettes – Anaphylactic Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed.,
2012)
Case 50
Case 49
Allergic Asthma –14 yo Frank Morgan rhinitis and persistent wheezing
Acute Systemic Anaphylaxis – toddler John Mason has a near-fatal allergic reaction after repeated
exposure to cookies containing peanut butter
Page 171
SUMMARY -- ANTIBODY-MEDIATED REACTIONS
1. The immune response is a double-edged sword, in that it can be both protective and destructive.
The immune mechanisms involved in both protective and destructive immune reactions are the
same.
2. Immune mechanisms can be subdivided into antibody-mediated and cell-mediated reactions.
The antibody-mediated reactions include inactivation or activation, cytotoxic or cytolytic,
immune complex, and atopic or anaphylactic reactions. The cell-mediated reactions include Tcell cytotoxicity and delayed-type hypersensitivity. Granulomatous reactions can be caused by
either humoral or cellular responses, but typically result from chronic reactions to poorly
degradable antigens.
3. The type of response that occurs is dependent on several factors, including the type of agent or
antigen, the route of infection or antigen exposure, the relative activation of Th1 or Th2
subpopulations, the cell type involved in antigen presentation, host genetic factors (such as HLA
type), and other factors such as age and nutritional status. Cytokines produced by Th1 and Th2
cells play a central role in what type of responses occur.
4. Responses to a given infectious agent or antigen are rarely, if ever, of a single type. Rather, there
is a mixture of several responses, some of which may be protective and others destructive.
5. Inactivation (or neutralization) reactions are caused by direct inactivation of toxins or
neutralization of viruses by the binding of antibody. Binding of antibodies to host receptors can
cause abnormal blocking (as in myasthenia gravis or pernicious anemia) or activation (as in Graves
disease).
6. Cytotoxic reactions result in cell damage or lysis due to antibody binding and complement
activation. Cell lysis through formation of the complement membrane attack complex or
opsonization by antibody or C3b derivatives are possible outcomes. Cytotoxic reactions are
particularly effective against many bacterial and protozoal infections, and antibody-dependent
cellular cytotoxicity can kill infected host cells or tumors. Immunopathologic effects include
transfusion reactions, Rh reactions, hemolytic anemia, and Goodpasture's syndrome.
7. Immune complex reactions result from formation of antigen-antibody complexes that can lead to
complement activation, attraction of PMNs, inflammatory changes and tissue damage. The size
and location of the complex formation determines the pattern of disease. Although immune
complex reactions can aid in the attraction of PMNs to a region of infection, we typically think of
them as being destructive, as in glomerulonephritis, serum sickness, and rheumatic fever.
8. Anaphylactic or atopic reactions occur through IgE-mediated activation of mast cells and other
cell types. Crosslinking of surface-bound IgE results in release of preformed granule contents
(such as histamine and eosinophil and neutrophil chemotactic factors) as well as the de novo
synthesis of arachidonic acid metabolites including leukotrienes and prostaglandins. Anaphylactic
reactions may participate in protection against helminth infections, but are also wide-spread causes
of hayfever, asthma, and other allergic reactions.
Page 172
IMMUNE EFFECTOR MECHANISMS II: CELL-MEDIATED REACTIONS
Steven J. Norris, Ph.D.
Recommended Reading: Actor, 2012, Chapters 7 and 10.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/geneab.html
Cell-mediated immunity (CMI) is defined
as immune reactions in which T cells play a
central role as effector cells (as opposed to
regulatory cells). CMI includes T-cell
cytotoxicity and delayed type
hypersensitivity (DTH). Granulomatous
responses usually result from DTH
reactions to poorly degradable antigens,
although antibody responses can also be
involved.
5. T-CELL CYTOTOXICITY
A. Definition - T mediated cellular
cytotoxicity involving direct
contact between the effector cell
(CTL) and a target cell, resulting
in target cell lysis or apoptosis.
B. Mechanisms
1. In general, T-cell
cytotoxicity involves
CD8+ T cells. However
CD4+ cytotoxic T cells also exist.
2. As in other effector mechanisms, naïve CD8+ cells must be activated by exposure to Ag-MHC I complexes and interleukins (e.g. IL-2) produced by helper T
cells and must undergo proliferation and differentiation before becoming active
Cytotoxic T Lymphocytes (CTLs).
3. The Ag-specific TCR of the cytotoxic T cell binds to the Ag-MHC-I complex on
the surface of a target cell. In addition, a protein called Fas on the target cell
binds to Fas ligand on the CTL. As in T-cell activation, other accessory proteins
also form bridges between the cytotoxic cell and the target cell.
4. Binding of the TCR activates the release of granules containing perforin and
granzymes by the CTL. The target cell is in close contact with the CTL, so most
of the granule contents bind to the target cell. (Note: CTLs have mechanisms
protecting themselves from self-destruction.)
5. Perforin forms a pore in the target cell, very similar to the pore formed by C9 in
the complement pathway. If a sufficient number of pores are formed, the target
cell can undergo rapid lysis.
6. Cytokines released by the CTL (including IFN-and TNF-may have cytotoxic
effects on the target cell.
Page 173
7. Target cells can also undergo
apoptosis or programmed
cell death. In this case,
killing is activated by two
signals: the binding of Fas
to the Fas ligand, and the
leakage of granzymes into
the target cell.
8. These two signals result the
activation of two endogenous
proteases in the target cell:
JUN kinase and Caspase 8.
These two enzymes act
through a series of
cytoplasmic and nuclear
signals to start the
irreversible process of
apoptosis or cell death.
Steps include nuclear
condensation and
fragmentation of nuclear
DNA by endogenous Dnases.
The process of cell death is
complete in 1-2 days.
9. Once the target cell is ‘programmed’ to die, the CTL can detach and go on to kill
many other target cells.
10. Null lymphocytes also generate lysis and apoptosis by similar mechanisms
during natural killer (NK) activity and antibody-dependent cellular cytotoxicity
(ADCC). However, T-cell receptor binding is obviously not involved in these
activities. Apoptosis is also important in the elimination of self-reactive
lymphocytes and the remodeling of tissues during development.
11. The protein Bcl-2 can block apoptosis by preventing the activation of caspases.
It may be involved in the resistance of certain tumors to killing.
A. Medical Aspects (Examples)
1. Protective
a) Viral infections - T cell-mediated cytotoxicity appears to be the principal
means of eliminating virally infected cells, although delayed type
hypersensitivity must also play a role (see below). By killing cells
expressing viral antigens on their surface, the host reduces virus
production but may also destroy essential cells (e.g. neurons).
b) Cancer - CTL along with DTH and NK activities are also thought to be
important in eliminating malignant cells before they proliferate and
become tumors. This process is called immune surveillance. Tumor
cells often express so-called tumor-specific transplantation antigens or
TSTAs. In virally-induced tumors, the TSTAs are often the same from
one patient to the next, whereas chemical- or radiation-induced tumors
usually express unique TSTAs. This complicates experimental strategies
for specific immunotherapy, in which the subjects are vaccinated with
TSTAs or given TSTA-specific T cells or antibodies to aid in tumor
elimination.
Page 174
c) Intracellular pathogens. Although less important than DTH, T cell
cytotoxicity is also active in destroying intracellular pathogens. Most
notably, CTL can destroy Plasmodium-infected hepatocytes during
malaria. Also, this mechanism can lyse infected macrophages in
tuberculosis, so that activated macrophages can then kill the released
bacteria.
2. Immunopathologic
a) Autoimmune diseases. Although it is often difficult to separate out T cell
MALARIA
ENDOGENOUS ANTIGEN PROCESSING AND T-CTL IMMUNITY
INFECTION OF HEPATOCYTES
SPOROZOITES
ENDOGENOUS
PROCESSING
CLASS I MHC
IL-1
INDUCTION
IFN-g
T-CTL
T-CTL
T-CTL
TL
T -C
CIRCUMSPORATE
ANTIGEN
EXPRESSION
TL
T -C
cytotoxicity and DTH, CTL almost certainly play a role in some autoimmune
diseases. An example is insulin-dependent diabetes mellitus, in which the  cells in
the islets of Langerhans are destroyed by autoreactive immune responses. Cytolytic
T cells specific for  cells can be found at the scene in IDDM experimental models.
Also, CTL are thought to be
A. TISSUE CULTURE MONOLAYER
responsible for thyroid cell
DYING CELLS
killing in Hashimoto’s thyroiditis
(see figure). Reactive
SPECIFIC T-CTL
lymphocytes also surround target
TISSUE CULTURE
cells and separate them from
TARGET CELLS
neighboring cells and basement
membranes, similar to what is
B. AUTOIMMUNE THYROIDITIS
THYROID
seen in cell cultures. This
DYING FOLLICULAR
FOLLICULAR
CELLS
‘disorientation’ also favors target
CELLS
cell death.
b) Contact dermatitis. Again,
T-CTL TO THYROID
FOLLICULAR CELLS
both CTL and TDTH are involved
in contact dermatitis (described
BASEMEMT MEMBRANE OF THYROID GLAND
in more detail below).
c) Viral exanthems. The eruptive lesions and fever characteristic of many viral
infections are partially due to the host immune response. Tissue damage due to
cytotoxic T cell responses may cause permanent loss of function.
d) Graft rejection. Cytotoxic T cell (and DTH) responses are involved in acute graft
rejection in transplant patients.
Page 175
Clinical Vignette –T-Cell Cytotoxicity (Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed., 2012)
Case 45
“Acute Infectious Mononucleosis” – 15 yo Emma Bovary had a severely sore throat,
lymphadenopathy, and 2 weeks of fever, but eventually improves with supportive therapy.
Chromium release assay - measure of
cytotoxic activity. Used to screen potential
donor-recipient pairs in transplant patients.
1. Incubate virus-infected cell culture and
normal cell culture with 51Cr to radiolabel
cells.
2. Wash to remove excess radioactivity.
3. Incubate cell cultures with lymphocytes
from virus-infected subject.
4. CTL activity will result in cell lysis and
release of radioactivity into culture
medium.
5. Determine radioactivity in supernatant,
compare to control. Results are typically
expressed as “percent specific killing”:
% killing = cpm releasedexp - cpm releasedcontrol
total cpm
6. What would be the percent specific killing
in this example?
7. Another control would be to perform the
same experiment with lymphocytes from an
uninfected individual. What results would
you expect?
51
Chromium Release Assay
Virus-infected cell monolayer
450 cpm
lysis, release
of 51Cr
viral Ag-MHC complex
50 cpm
Normal cell monolayer
25 cpm
No lysis, little
release of 51Cr
MHC
475 cpm
6. DELAYED TYPE HYPERSENSITIVITY (DTH)
A. Definition - an in vivo reaction involving activation of macrophages by cytokines
produced by lymphocytes (TDTH). Also called Type IV Hypersensitivity.
B. Mechanisms
1. Naïve T cells are stimulated by specific interaction of their TCR with AgMHC II complexes on the surface of antigen presenting cells. They must
undergo activation, proliferation and differentiation, as in other immune
responses.
2. Upon restimulation with antigen (typically in the ‘target’ tissue such as skin,
lung, or transplanted organs), the resulting memory TDTH cells (which have
Th1 characteristics) express large quantities of cytokines including IL-2,
macrophage chemotactic factor (MCF), IFN- and tumor necrosis factor
 (TNF-.
Page 176
IFN
IT
!
FO
R
IL-2
GO
DTH
ETC.
CTL
HELP!
ACTIVATION
AH! THANKS FOR THE GOODIES
5.
6.
7.
8.
3. IL-2 activates additional T cells,
MCF attracts macrophages to
the area, and IFN- activates
macrophages, increasing their
motility, phagocytic activity,
and ability to kill intracellular
bacteria (e.g. by oxidative
mechanisms). TNF- can be
cytotoxic.
4. Even in a sensitized individual,
it takes 1-2 days for a sufficient
number of T cells and
macrophages to accumulate to
cause a visible reaction (e.g.
erythema and induration EVOLUTION OF A DTH RESPONSE (SYPHILIS)
[hardening] in a
DAY 1
tuberculin skin test).
DAY 3
DAY 7
DAY 12
That is why the reaction
H
is called delayed type
H
hypersensitivity. In
H
DAY 14
INDUCTIVE STAGE
H
contrast, anaphylactic
reactions take minutes
H H
and immune complex
H
H
REACTIVE STAGE
reactions are maximal
within ~6 hours after
DAY 21
H
exposure of sensitized
individuals.
TDTH cells have little or
LATENT (HEALED) STAGE
no direct effect on
FIBROSIS
pathogens or tissues.
Their main activity is the recruitment and activation of macrophages.
These guys do the dirty work of phagocytosing and killing pathogens or
damaging tissue (in contact hypersensitivity, transplants, autoimmune
reactions, etc.). Nonactivated macrophages are relatively quiescent; for
example, they are incapable of killing M. tuberculosis and actually serve as
hosts for its intracellular growth.
The DTH activity of a patient can be tested by using antigens to which
everyone is exposed, such as Candida albicans extracts. Patients who give
negative skin test reactions to such antigens are considered to be anergic, i.e.
deficient in cellular responses.
DTH reactions can be inhibited by corticosteroids or blocked by
cyclosporin and other immunosuppressive agents. These agents are
commonly used to control autoimmune diseases and transplant rejection.
Recent studies have shown that basophils may play a role in certain types of
DTH reactions.
B. Medical aspects
1. Protective
Page 177
a) Destruction of intracellular bacteria and other pathogens. DTH is the
principal protection against mycobacterial infections and most parasitic
and fungal infections. The importance of DTH is underscored in AIDS
patients, who extremely susceptible to these organisms.
b) Cancer - as mentioned above, DTH most likely plays a role in the
immune surveillance for malignant cells. Unusual tumors occur at high
frequency in patients with decreased CD4+ cell function (e.g. Kaposi’s
sarcoma, lymphomas in AIDS patients).
2. Immunopathologic
a) Contact hypersensitivity - skin reactivity to certain
environmental agents, including poison oak/ivy, nickel,
rubber products (including latex exam gloves!), PABA in
suntan lotions, adhesives, and many other compounds.
Typically the sensitizing agent is a hapten that binds to
tissue proteins to form a hapten-carrier conjugate.
These are processed and presented by Langerhans cells
that are present in the skin and may migrate to lymph
nodes. TDTH cells are sensitized and will react to
subsequent exposures to the antigen. When exposed to
irritants, keratinocytes often express MHC Class II
proteins and cytokines, enhancing the hypersensitivity
response.
b) Autoimmune diseases - DTH reactions are involved in many autoimmune
diseases, including multiple sclerosis, insulin dependent diabetes mellitus,
Hashimoto’s thyroiditis, and rheumatoid arthritis. None of these appear to be
‘pure’ DTH responses, but rather involve a mixture of different effector
mechanisms.
c) Transplant rejection - DTH is active in acute allograft rejection, along
with CTL reactions. In this type of reaction, activated macrophages cause
tissue damage by release of lysosomal contents and oxygen radicals (rather
than phagocytosis). Reactive T cells apparently recognize the allograft MHC
proteins as “altered self”, and therefore are able to respond despite MHC
restriction. An unusually high proportion of T cells (up to 10%) respond
during allograft rejection.
Clinical Vignette – DTH Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed., 2012)
Case 51
Case 53
Case 48
Atopic Dermatitis – Tom Joad, 2 yo male with severe eczema
Contact Hypersensitivity to Poison Ivy – 7 yo Paul Stein develops itchy eruptions after a hiking trip
which responded to corticosteroids; the lesions ‘rebounded’ after the corticosteroids were stopped.
Lepromatous Leprosy – Ursula Iguaran has leprosy, and develops disseminated lesions with large
numbers of M. leprae due to a Th1-Th2 imbalance and a resulting poor DTH response.
Page 178
Blast transformation assays - in vitro measures of T cell reactivity.
Proliferation
Add *Thymidine
*
Activation
1 day
2-5 days
*
*
*
*
ConA Added
* Thymidine Incorporated
1. Peripheral blood lymphocytes are incubated in the
presence of:
a) Mitogens - agents that cause nonspecific
proliferation of certain populations of lymphocytes:
Measure of the overall activity of that cell
population.
Concanavalin A (ConA) and phytohemagglutinin
(PHA) - plant proteins that cause proliferation of
T cells
Lipopolysaccharide (LPS) - causes proliferation of B
cells
b) Antigens - provides information on the reactivity of
the individual to specific antigens. Example:
Mixed leukocyte culture - inactivated recipient
cells mixed with donor lymphocytes. Shows
whether CD4+ cells of recipient react to Class II
MHC of donor.
Add mitogen
or antigen
Donor lymphocytes
added
Nothing added
2. If reactive, lymphocytes begin to proliferate. 3HTime (days)
thymidine is added, and the amount of radioactivity
incorporated into DNA determined as a quantitative measure of proliferation.
3. High levels of incorporation relative to controls indicate a response. Why are responses to
mitogens typically much higher than responses to specific antigens?
7. GRANULOMATOUS REACTIONS
A. Definition - space-occupying lesion consisting of a predominantly mononuclear
infiltrate (lymphocytes and macrophages) at the site of deposition of a poorly degradable
antigen.
B. Mechanisms
1. Usually caused by DTH reactions, but sometimes brought about by nonspecific
reactions (e.g. silicosis) or antibody-mediated reactions. The archtypical
example is the granuloma characteristic of tuberculosis.
2. CD4+ lymphocytes and macrophages accumulate at the site of the antigen in a
typical DTH response. If the antigen (such as M. tuberculosis) continues to
replicate or is not easily degraded, it will persist and cause continued
accumulation of cells. The resulting granuloma can be up to several cm in
diameter, and contains epithelioid cells (enlarged macrophages expressing TNF)
and multinucleate giant cells (formed by the fusion of macrophages). In large
granulomas, the center can become necrotic, forming a cavity. The granuloma
can also displace normal tissue and cause fibrosis, decreasing tissue function
(e.g. in the lung).
3. In inactive TB, granulomas containing viable M. tuberculosis can persist for
decades without affecting health. However, breakdown of the granuloma or
changes in the immune status of the individual may allow mycobacteria to grow
out, resulting in active disease.
4. Persistence of immune complexes can also cause granuloma formation.
Page 179
GRANULOMATOUS REACTIONS
INSOLUBLE ANTIGEN
C1->C3b
OPSONIZATION
MACROPHAGE
C3a, C5a, C5-7
CHEMOTAXIS
IgG ANTIBODY
+
LYMPHOKINES
T-DTH
ACTIVATED
MACROPHAGES
SENSITIZED
CELLS
CLINICAL CONDITIONS
GRANULOMA - SPACE OCCUPYING MASS
TUBERCULOSIS
LEPROSY
PARASITIC INFECTIONS
SARCOIDOSIS
GRANULOMATOSES
C. Medical aspects
1. Mycobacterial infections - as described above, granulomas are important in
tuberculosis and leprosy. They can be detected in chest Xrays and are indicative
of past or present active TB.
2. Parasitic infections - attempts to destroy or wall off parasites (such as worms)
can result in granulomas. In extreme cases (eg. Roundworm Wuchereria
bancrofti), these can occlude lymphatic vessels and cause elephantiasis.
3. Sarcoidosis - disease of unknown etiology that causes granulomas in multiple
sites, including the lungs and skin.
4. Crohn’s disease - inflammatory disease of the bowel, in which granulomatous
reactions can cause stricture (obstruction) and fistula formation. Etiology
unknown.
Clinical Vignette – Granulomatous Disease, Geha and
Notarangelo, “Case Studies in Immunology”, 6th Ed.
Case 26
Chronic Granulomatous Disease – Randy
Johnson develops granulomas and is unable to
ward off Aspergillus and other opportunistic
pathogens due to inability of his phagocytes to
produce H2O2 and superoxide anion.
Page 180
SUMMARY -- CELL-MEDIATED REACTIONS
1. Cell-mediated reactions come about when T cells play a central role as effector cells. Another
term is cell-mediated immunity or CMI. These reactions include T-cell cytotoxicity and
delayed type hypersensitivity.
2. T-cell cytotoxicity occurs when an activated cytotoxic T cell (usually CD8+) binds directly to a
target cell via a specific interaction of the TCR with Ag-MHC complexes on the target cell
surface. Killing of the target cell occurs through two mechanisms. Release of granules
containing perforins and granzymes result in formation of a pore in the target cell membrane,
causing rapid lysis. A second major mechanism involves apoptosis, where programmed cell
death is activated through a complex cascade involving Fas-Fas ligand interaction, activation
of Jun kinase, Caspase 8, and other target cell signal transduction proteins, nucleus
fragmentation, organelle destruction, and DNA cleavage. Cell death occurs over a 1-2 day
period. T-cell cytotoxicity is protective against many viral infections, tumors, and intracellular
pathogens, but is also involved in autoimmune diseases, contact dermatitis, viral rashes, and
graft rejection. It can be quantitated through cell lysis assays, including the chromium release
assay.
3. Delayed-type hypersensitivity (DTH) (also called Type IV hypersensitivity) is the activation
of macrophages by cytokines produced by lymphocytes, typically Th1 cells. When Th1 cells
are activated by exposure to antigen, they produce macrophage chemotactic factor, interferongamma, and tumor necrosis factor which attract and activate macrophages. These activated
macrophages are much more effective in destroying intracellular pathogens and tumor cells.
DTH is protective against many intracellular bacteria and protozoa, including mycobacteria
and Pneumocystis carinii. Adverse effects include participation in contact hypersensitivity,
autoimmune diseases, and transplant rejection. DTH responses can be measured indirectly by
blast transformation assays or more directly by quantitation of cytokine production.
4. Granulomatous reactions are collections of lymphocytes and enlarged macrophages resulting
from a chronic response to an antigen that is difficult to destroy. Persistent M. tuberculosis
infection is an example of a disease process leading to granuloma formation. CD4+ Th1 cells
attract macrophages to the area, but they continue to collect due to failure to eliminate the
antigen. Granulomatous reactions are prominent in mycobacterial infections, some parasitic
infections, sarcoidosis, and Crohn's disease.
Page 181
IMMUNOLOGY OF HIV INFECTION
Steven J. Norris, Ph.D.
Required Reading: Geha and Notarangelo, 6th edition (2012). Case Studies in Immunology.
Garland Publishing, New York, NY. Case 10: AIDS.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/aids.html
I. HUMAN IMMUNODEFICIENCY VIRUS
A. AIDS Related Retroviruses
1. Human Immunodeficiency Virus-I (HIV-1) is the type most commonly
associated with HIV infection and AIDS in the United States and Europe.
HIV-2, which shares ~50% nucleotide identity to HIV-1, is associated with a
small number of cases in the U.S., but is prevalent in regions of Africa.
2. HIV-1 and HIV-2 are members of the retrovirus family, a group of viruses
that have an RNA genome but form a DNA intermediate that is incorporated
into the genome of the host cell. There are oncogenic (tumor causing) and
cytolytic (cell-killing) subfamilies of retroviruses. HIV is part of a group
called lentiviruses, slow-acting cytolytic retroviruses (lento means slow in
music). An example of an oncogenic retrovirus is Human T Lymphocyte
Virus (HTLV), which is associated with T cell lymphomas.
Lentiviruses
.
Virus
Human immunodeficiency virus
Simian immunodeficiency virus
Visna/maedi virus
Equine infectious anemia virus
Caprine arthritis/encephalitis virus
Disease
Cause of human AIDS
AIDS in monkeys
Neurologic and lung
disease in sheep
Horse anemia
Goat encephalitis
B. The HIV Genome and Structure
1. The HIV genome consists of a 9,000 bp segment of single-stranded
RNA. It encodes a series of gene products that are cleaved by the HIV
protease to form important structural and nonstructural proteins (see
figure).
2. Proteins important in the immune response to HIV include:
a) The envelope (env) glycoproteins gp120 and gp41, and their
precursor gp160
b) The group antigen (gag) proteins p24 (major core protein) and
p17 (protein that forms a scaffold during virion assembly)
c) The pol proteins p66 and p51 (form reverse transcriptase),
protease, and p32 (endonuclease)
d) Regulatory proteins including tat (transactivator), rev (regulator of expression), vif
(virion infectivity factor), and nef (negative factor). These regulate HIV virus gene
expression and assembly.
182
C. HIV Replication and Gene Expression (see figure)
1. Binding and internalization. gp120 on the surface of the virion
binds with high affinity to CD4 on the surface of CD4+ T cells and
some other cells types. Gp120 is removed, exposing gp41 underneath,
which then promotes fusion between the cell membrane and viral
membrane. As a result, the viral core is released into the cytoplasm of
the cell. Alternatively, antibody or C3b bound to the surface of the
virion can bind to Fc or complement receptors on macrophages and
other cells, resulting in internalization (antibody dependent
enhancement). Lastly, other “co-receptors” such as chemokine
receptors (e.g. CXCR4 and CCR5) and the glycolipid galactosyl
ceramide can interact with HIV, promoting infection of other cell
types, albeit at much lower efficiency than CD4 binding. A 32-bp
deletion in the CCR5 gene (ccr532) that eliminates CCR5 expression
has been linked to resistance to HIV infection.
2. Reverse transcription and incorporation. The virion RNA is
replicated by virus-associated reverse transcriptase, resulting in a
double-stranded DNA copy of the viral genome. This DNA becomes
circularized and then incorporated into host chromosome.
183
3. Transcription and translation of viral genes. Host transcription
factors along with viral factors such as tat activate transcription of the
viral genes, resulting in protein expression. Large precursor proteins
are cleaved into the final protein products by HIV protease. This step
is blocked by drugs called protease inhibitors, thus inhibiting viral
replication.
4. Assembly and budding. The viral core, including two copies of the
RNA genome, assemble and bud through the cell membrane to form
an infectious virion.
5. Latent infection vs. virus production. Resting CD4+ T cells
typically exhibit latent infection, that is they contain HIV DNA but
do not actively produce virus. Cell activation as indicated by
expression of HLA-DR and other Class II MHC proteins is required
for high level virus production. Enhanced viral production is linked to
expression and activation of nuclear factor kappa B (NF-B) and
other host cell transcription factors. During primary HIV or late
symptomatic infection, 1 out of 10 peripheral blood CD4+ T cells may
be latently infected, whereas only 1 out of 300 to 400 are actively
producing virus. Tissue macrophages are an important reservoir of
infection, in that they can become infected and produce low levels of
virus without being killed. Many other cell types, including epithelial
cells, can be infected. Macrophages and other cells can be latently
infected for long periods and then express virus when activated by
exposure to cytokines, viruses or other infectious agents, and other
factors.
184
II. CLINICAL COURSE OF HIV INFECTION
HIV infection and AIDS are not equivalent. HIV infection means, quite
literally, infection with HIV-1 or HIV-2. HIV+ patients can lead normal, healthy,
productive lives. Unfortunately, HIV infection almost inevitably progresses to the
profound immunodeficiency and opportunistic infections of Acquired Immunodeficiency
Disease Syndrome (AIDS). This process usually takes 8 to 12 years for sexually
transmitted infection, fewer years for blood transmission, and <1 year for congenital
infection. The rapidity of progression is related to the viral dose and, in the case of
congenital infection, the immature immune system of the host.
A. Primary infection
1. Nearly all cases of HIV infection arise from sexual contact, transfer of
infected blood (by used needles, transfusion, etc.), or maternal/fetal
transmission.
2. Although blood, semen, and other body fluids often contain HIV
virions, most sexual transmission is thought to be due to transfer of
infected cells. In infected individuals, the percentile of infected cells
in peripheral blood mononuclear cells is ~0.001-1%, and in semen is
0.01-5%. Virion and infected cell levels are highest during primary
infection and late stages (AIDS).
3. Sexual transmission occurs through mucus membranes and is
apparently enhanced by tissue disruption or ulcerative diseases (such
as syphilis and chancroid).
4. One to three weeks after exposure, the patient may develop primary
infection symptoms, consisting of headache, retro-orbital pain,
muscle aches, low- to high-grade fever, and lymphadenopathy
(swollen lymph nodes). A characteristic maculopapular
erythematous rash (red spots or raised bumps) may appear on the
trunk and spread to the extremities. These symptoms last for a few
weeks.
5. HIV virions are found in the blood, cerebrospinal fluid, and seminal
fluid in high numbers at this stage. The blood viral load in these
patients averages 5 x 106 (see table). Anti-HIV antibodies may be
detected in some patients within 6 to 14 days after the development of
symptoms. Over 90% of HIV-infected patients have seroconverted
(have detectable anti-HIV antibodies) within 3 months of infection. A
small percentage may remain seronegative after 6 months.
185
Viral Load at Different Stages of HIV Infection. Viral load is the total number of
virions per ml, as determined by a quantitative competitive polymerase chain reaction
(QC-PCR) technique. It is estimated that the number of infectious virions is about
60,000-fold less than the total number of virions determined by QC-PCR and other
means. The noninfectious virions may have defective RNA or be missing other required
components.
Clinical Stage
Primary (acute) infection
Asymptomatic
Early symptomatic
AIDS
Ave. Viremia (Virions/ml blood)
5 x 106
8 x 104
35 x 104
2.5 x 106
.
B. Asymptomatic Infection (Category A)
1. After primary infection, patients enter an asymptomatic phase in
which they appear healthy. Although the patients have high viral
loads, they have normal CD4+ lymphocyte levels and immune
responses.
2. Originally, it was thought that virus production was low during the
asymptomatic stage of HIV infection. However, recent studies have
shown that up to 109 virions are produced per day, and that the average
half-life of infected CD4+ cells is 2 days. Therefore asymptomatic
infection actually is a steady state involving massive turnover of both
new virions and newly produced CD4+ T cells.
C. Early symptomatic infection (Category B)
1. At this stage (previously called AIDS-related complex or ARC), the
years of viral infection and cell destruction begin to take their toll.
CD4+ cell numbers begin to decline, and other changes (e.g. chronic
lymphadenopathy) occur. CD4+ cell counts in peripheral blood
decline to 200-499 per l (between normal levels and those
characteristic of AIDS)
2. CD4+ cell function, as measured by lymphocyte proliferation assays
and other tests, declines before decreases in cell numbers are evident.
3. May be accompanied by fever, chronic diarrhea, oral or persistent
vulvovaginal candidiasis, and other manifestations.
186
Late symptomatic infection (AIDS) (Category C)
1. Acquired immunodeficiency disease syndrome (AIDS) is defined as
HIV infection together with the occurrence of unusual infections,
tumors, or other manifestations (e.g. HIV-related encephalopathy)
or blood CD4+ cell levels of <200/l (1993 AIDS Surveillance Case
Definition, Centers for Disease Control & Prevention).
2. Unusual infections include a long list, but most typically involve
Pneumocystis carinii pneumonia, chronic or disseminated fungal
infections, M. tuberculosis, M. avium complex, or M. kansasii
pulmonary or extrapulmonary infections, herpesvirus or CMV
infections, or intestinal parasites (e.g. Cryptosporidium).
3. Tumors include Kaposi’s sarcoma, a variety of lymphomas, and
invasive cervical cancer.
4. Other conditions are HIV-related encephalopathy (apparently caused
by CNS infection by HIV), progressive multifocal
leukoencephalopathy, and generalized wasting.
187
III. HIV INFECTION AND THE IMMUNE RESPONSE
The interaction between HIV and the immune system is, to say the least, complex. Most
of the manifestations of AIDS are directly related to decreased CD4+ T cell numbers
and function, which increases susceptibility to intracellular pathogens, viruses, fungi,
and certain tumors. CD4+ T cells play a central role as helper cells and mediators of
delayed type hypersensitivity. Other patients with depressed CD4+ T cell activity (e.g.
renal transplant patients on immunosuppressive therapy) have a similar pattern of
increased susceptibility (although usually not as severe).
As described below, virtually every arm of the immune response is involved in
combating HIV infection, but is also compromised by the effects of CD4+ cell
deficiency. Because the virus genomic sequence becomes incorporated into the host cell
DNA and can establish long-term latent infection, the virus eventually overwhelms the
immune response, leading to the breakout of rampant HIV infection, opportunistic
infections, and tumor growth that characterizes AIDS. Highly Active Anti-RetroViral
Therapy (HAART) prevents this progression by reducing the number of infectious
virions to extremely low levels; however, the viral load will again increase if HAART is
discontinued.
A. Antibody responses
1. Antibodies against a variety of HIV proteins are expressed following primary
infection. These can be detected in serum by an HIV ELISA test or Western
blot assay (see below). The combination of two positive ELISA tests and one
positive Western blot assay is considered to be diagnostic of HIV infection.
Negative tests obtained within 6 months of potential HIV exposure should be
repeated later, because the patient may be infected but not have expressed
sufficient antibodies for a positive result.
ELISA TEST FOR
ANTI-HIV ANTIBODIES
1. Add test serum to well coated
coated with HIV proteins
2. Incubate and wash
3. Add goat anti-human IgG
horseradish peroxidase conjugate
4. Incubate and wash
5. Add substrate for
horseradish peroxidase
6. Color change indicates
presence of anti-HIV antibodies
in test serum (positive result)
WESTERN BLOT ANALYSIS
NOTE: DESCRIPTION OF HIV ELISA AND
WESTERN BLOT PROCEDURES: Geha and Notarangelo, “CASE STUDIES IN
IMMUNOLOGY”, 2012, CASE 10.
188
2. Anti-HIV antibodies may have neutralizing activity, i.e. be able to inactivate
the virus. Such protective antibodies are generally directed against the surface
proteins gp120 and gp41. These antibodies may also mediate antibodydependent cellular cytotoxicity (ADCC) of infected cells.
3. Anti-gp120 antibodies may also increase the efficiency of infection in vitro
(so-called Antibody Dependent Enhancement or ADE). Antibody and
complement components on the surface of HIV can bind to Fc receptors and
complement receptors on macrophages, increasing the efficiency of binding
and internalization. Neutralization vs. enhancement may depend on the
particular epitopes recognized by the antibodies. As with many other in vitro
findings, the clinical significance of ADE is not known.
4. Immune complexes of virions, antibodies, and complement have been shown
to be infectious, indicating that immune complex formation may also increase
the efficiency of macrophage infection.
5. During late symptomatic infection, antibody levels (both total and anti-HIV)
decline due to the lack of functional T helper cells, further exacerbating HIV
and opportunistic infections.
6. Vaccination protocols must take into account the possibility that the resulting
antibody responses may enhance the efficiency of viral infection.
B. CD4+ T Cell responses - help and DTH
1. CD4+ T cells are the principal target of HIV infection, and as such are the
most profoundly affected cell type. CD4+ T cell levels are measured as the
total number of cells per l blood using a flow cytometer (also called a
fluorescent activated cell sorter; see pages 103-104 in book).
2. Even during the so-called asymptomatic phase, there is extensive HIVmediated killing of CD4+ T cells, so that the HIV-infected patient must
produce many-fold more cells to maintain normal levels (>500/l).
3. After several years, the ability of the host to replace the CD4+ lymphocytes
declines, resulting in reduction of CD4+ counts into the 200-499 cells/l
range.
4. When CD4+ cell counts fall below 200 cells/l, the patient becomes deficient
in DTH and helper activities, and thus much more susceptible to opportunistic
infections, tumors, and rampant HIV infection. This threshold corresponds to
the onset of AIDS symptoms.
5. Some CD4+ T cells have cytotoxic activity and may be involved in combating
HIV infection.
6. Functional activities of Th1 cells such as IL-2 and IFN-production decline
long before cell numbers decrease. Conversely, IL-4 and IL-10 expressed by
Th2 cells increase late in infection. This shift in cytokine expression may
play an important immunoregulatory role during HIV infection.
189
C. CD8+ T cell activities – cytotoxicity and antiviral activity
1. CD8+ lymphocytes from HIV+ patients can kill HIV-infected cells. This
CTL activity is high during the asymptomatic phase, and decreases with
progression of the disease.
2. CTLs may also be involved in the destruction of uninfected CD4+ cells (see
figure below). Soluble gp120 produced by HIV-infected cells can bind to
CD4 molecules on uninfected cells and result in expression of Fas. Fas
ligand on CTLs will bind to Fas and initiate apoptosis of the target cell. (see
Cell-Mediated Reactions lecture for review). Uninfected CD4+ cells could be
destroyed by this mechanism.
3. In addition, CD8+ cells express a cell antiviral factor (CAF) that inhibits
HIV replication in infected cells without killing the cells. When the factor is
removed, viral replication resumes. Recently, CAF activity was found to be
associated with expression of -defensins by CD8+ T cells. -defensins are
peptides have anti-bacterial activity and are usually produced by neutrophils.
The mechanism by which defensins inhibit HIV-1 replication is unknown
currently. Defensins are expressed in higher quantities by long-term
nonprogressors (LTNPs), i.e. HIV-infected patients who remain healthy.
IV. PROSPECTS FOR IMMUNOTHERAPY AND VACCINATION
At present, therapy for HIV infection involves treatment with nucleoside analogs
such as zidovudine (AZT) to inhibit viral reverse transcriptase and protease inhibitors,
which block the cleavage of protein precursors by HIV protease. In addition,
antimicrobial compounds can be administered prophylactically (as a preventative
measure); an example is the use of aerosolized pentamidine to prevent Pneumocystis
carinii pneumonia. Ideally, it would be best to prevent HIV infection through
vaccination or other means. Alternatively, immunotherapeutic measures could be used to
augment the immune response of HIV-infected individuals.
A. Immunotherapy
1. Immune reconstitution. The idea behind this therapy is akin to a
bone marrow transplant – replace the infected cells with those of an
HIV-negative donor. However, human donor lymphocytes would
190
2. Passive immunotherapy. Injection of “cocktails” human anti-HIV
monoclonal antibodies into HIV+ patients is being tried as a means of
decreasing viral loads. However, it is unclear how these cocktails are
in any way better than the patient’s own antibodies, particularly given
the degree of antigenic heterogeneity in HIV (see below).
B. Vaccination
1. Given that vaccines against Feline Leukemia Virus, a retrovirus, are successful
in preventing transmission of FLV in cats, it is possible that an effective vaccine
against HIV could be developed. It is also possible (although unlikely) that
vaccination of infected individuals could enhance viral clearance.
2. Stage II clinical trials have shown that a
recombinant form of gp160 or gp120
(VaxGen) is safe and induces antibodies against
HIV-1. Thus far, the antibodies neutralize
strains expressing the same gp160, but not other
primary HIV isolates (due to sequence
variation). This vaccine will is being tested for
its ability to prevent maternal/fetal transmission
of HIV.
3. Recombinant Live-Vector Vaccines consist of
a carrier virus containing HIV genes. Ones that
are being tested extensively are a recombinant
vaccinia-HIV gp160 (a modified smallpox
vaccine) and a canarypox-HIV vaccine. The
canarypox form has improved safety, because it
does not replicate in human cells. “Prime boost” strategies with a
boost of recombinant gp120 or gp160 have been more effective.
Several other live vectors are being developed.
4. Virus-like particles (VLPs) consist of the protein capsid without the
nucleic acid, and thus are non-infectious. A p17/p24 VLP of the HIV
core is being tested, and a p55 (gag) form is also being produced.
5. Vaccines using peptides corresponding to important epitopes on
gp120 (e.g. the V3 loop and the CD4 binding region) could
potentially be used. Branched and lipidated forms (to increase
immunogenicity) are being tried.
6. DNA vaccines are naked DNA encoding viral proteins that is taken
up and expressed by the vacinee’s own cells. These show promise,
particularly in combination with boosts with protein vaccines.
191
7. Attenuated viruses represent another approach. Monkeys
vaccinated with a Simian Immunodeficiency Virus variant lacking the
nef gene were resistant to infection with “wild-type” SIV. Intentional
infection of humans with an attenuated form of a virus that
incorporates into host DNA and mutates rapidly has hefty ethical
implications.
8. Immunization of chimpanzees with formalin-inactivated HIV has not
provided convincing evidence of protection.
C. Problems with vaccination - multiple
1. Intracellular location/latency - latently infected cells that have HIV
DNA incorporated into their genome can be activated at a later time,
after the effects of vaccination have waned.
2. Antigenic modulation - RNA viruses have a high mutation rate,
resulting in rapid changes in antigenic structure. In the human
population there are many different antigenic variants of HIV, called
clades. In addition, a patient infected with one genotype of HIV later
has several different genotypes, some of which differ in the structure
of important antigens (e.g. gp120). Immunization with a single
‘serotype’ of HIV may not be effective in preventing infection or
enhancing immune clearance.
3. Inappropropriate or ineffective immune responses. As mentioned
earlier, antibodies against HIV may actually enhance the infection of
macrophages. T-CTLs activated by vaccination may destroy
uninfected T cells by the mechanism involving gp120 binding
described above. Furthermore, activation of infected macrophages and
T cells through immunotherapeutic means may increase virus
production.
192
D. Animal models
Animal models of HIV infection provide a means of studying HIV pathogenesis
and immunity. However, none of these closely resemble the human disease. One of the
most effective models is the SCID mouse- human lymphoid cell chimera. In this case, a
mouse strain with severe combined immunodeficiency can be injected with human
lymphoid cells. The human cells persist, and can be infected with HIV upon subsequent
injection of the virus. This system permits the study of human cell infection in a
surrogate model.
Animal
Chimpanzee
Macaques
Virus
HIV-1
SIV
Rabbits
Mice- HIV genome
transgenic
Mice - tat transgenic
SCID-human lymphoid
cell chimera
Sheep
Goats
Horses
HIV-1
--
Result
Latent Infection
AIDS-like wasting
disease
Defective infection
AIDS-like illness
Used to study:
Vaccine efficacy
Vaccine efficacy,
therapy, pathogenesis
Latent infection
Pathogenesis
-HIV-1
Kaposi’s sarcoma
AIDS-like disease
Carcinogenesis
Pathogenesis, therapy
Visna
Chronic
neurodegeneration
arthritis, encephalitis
hemolytic anemia
Lentivirus pathogenesis
Caprine
EIAV
193
Lentivirus pathogenesis
Lentivirus pathogenesis
III. SUMMARY
1. HIV-1 and HIV-2 are lentiviruses that cause CD4+ T lymphocyte depletion and
immunodeficiency in humans.
2. Infection of CD4+ T cells, macrophages, and other cell types can lead to virus
production and cytolysis or long-term latent infection.
3. HIV infection progresses through primary infection, asymptomatic infection,
early symptomatic infection, and late symptomatic infection (AIDS).
4. Depletion of CD4+ cells leads to profound defects in DTH and T helper activity,
resulting in susceptibility to rampant HIV infection, opportunistic infections,
and tumors.
5. Antibody and cytotoxic lymphocyte activities combat virus production, but
eventually are overwhelmed by the virus’ ability to inactivate and destroy CD4+
cells, to change its antigenic structure, and to alternate between latent and active
infection.
6. In their current forms, immunotherapy and vaccination have not demonstrated
the ability to prevent or combat HIV infection. However, intensive research for
the development of an HIV vaccine is ongoing.
194
INFECTION AND IMMUNITY
Jeffrey K. Actor, Ph.D.
713-500-5344
Objectives: Understand the course of response and major immune defense mechanisms to
infectious agents.
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New
York, NY. 6th edition, 2009. Chapter 20. Geha and Notarangelo. Case Studies in Immunology.
Garland Publishing, New York, NY. 6th edition, 2012. Case 48: Lepromatous Leprosy.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/infimmunity.html
The course of response against typical acute infections can be subdivided into distinct stages.
Initially, the level of infectious agent is low, beginning with breach of a mechanical barrier (e.g.
skin, mucosal surface). Once inside the host, the pathogen encounters a microenvironment for
suitable replication. The agent replicates, releasing antigens that trigger innate immune function,
generally characterized as non-specific. This innate function assists in limiting expansion of the
organism. After 4 or 5 days, effector cells and molecules of the adaptive and specific immune
response enable control and eventual clearance of the infectious agent. Once the agent is cleared,
the host is left with residual effector cells and antibodies, as well as immunological memory to
provide lasting protection against reinfection.
The response to initial infection is therefore subdivided into 3 phases. The first is an early innate
and non-specific response. Preformed effector cells and molecules recognize microorganisms
within the first 4 hours of infection. Although this may be enough to clear the organism, typically
more help is necessary. The second phase lasts from 4 hours to 4 days. Again, this is primarily a
non-specific encounter with the organism. This phase is characterized by recruitment of effector
cells (professional phagocytes, NK cells) to the site of infection, and specific activation of these
effectors. The final phase is one where one where adaptive immunity occurs. Antigen specific
cells (B and T lymphocytes) undergo clonal expansion to become specific effectors. Some of these
effector cells remain even after clearance of the organism, and are able to provide a much more
rapid and specific memory response if the organism is re-encountered.
A wide variety of pathogenic microorganisms exist. They may be globally classified into groups:
Bacterial, Mycobacterial, Viral, Protozoal, Worms, and Fungal. The major immune defense
mechanisms are summarized in the following chart:
Type of Infection
Bacterial
Mycobacterial
Viral
Protozoal
Worms
Fungal
Major Immune Defense Mechanisms
Antibody (Immune complex and cytotoxicity)
DTH and granulomatous reactions
Antibody (Neutralization), TCTL and DTH
DTH and antibody
Antibody (Atopic, ADCC) and granulomatous reactions
DTH and granulomatous reactions
194
The host defense is based upon availability of resources to combat a localized pathogen. Virtually
all pathogens have an extracellular phase where they are vulnerable to antibody-mediated effector
mechanisms. An extracellular agent may reside on epithelial cell surfaces, where antibodies (IgA)
and non-specific inflammatory cells may be sufficient for combating infection. If the agent resides
within interstitial spaces, in blood or in lymph, then protection may also include complement
components, macrophage phagocytosis and neutralization responses. Intracellular agents require a
different response to be effective. For cytoplasmic agents, T lymphocytes and NK cells, as well as
T-cell dependent macrophage activation, are usually necessary to kill the organism.
195
Pathogens can damage host tissue by direct and indirect mechanisms. Organisms may directly
damage tissue by release of exotoxins that act on the surface of host cells, or via released
endotoxins that trigger local production of damaging cytokines. Pathogens may also directly
destroy the cells they infect. Adaptive mechanisms can cause disease; formation of
antibody:antigen complexes can lead to the release of proteins and factors that both mediate
control of disease as well as cause tissue damage. Some of the representative infectious agents and
the common names of associated diseases are listed in the figure.
196
BACTERIAL INFECTIONS
Bacterial infections begin with a breach
of a mechanical barrier. Release of
bacterial factors upon replication
initiates a cascade of events. Initially,
infection may be resisted by
antibody-mediated immune
mechanisms, including neutralization of
bacterial toxins. With the help of
complement factors direct cytotoxic
lysis can occur. Release of C3a and C5a
in the complement cascade will cause
vasodilation and vasopermeability
resulting in an influx of professional
phagocytes and acute
polymorphonuclear infiltration (Arthus
reaction). Opsonization of bacteria leads
to increased phagocytosis and acute
anaphylactic vascular events permitting
exudation of inflammatory cells and fluids. Phagocytosis may also occur via specific surface
receptors for ligands such as mannose or sialic acids. During the chronic stage of the infection cell
mediated immunity is activated. TDTH-cells that react with bacterial antigens may infiltrate the site
of infection, become activated and release lymphokines that attract and activate macrophages. The
activated macrophages phagocytose and degrade necrotic bacteria and tissue, preparing the lesion
for healing.
The role of complement in response to bacterial infection must be stressed. There are three major
biological components of the complement system. They are (1) activation of phagocytes including
macrophages and neutrophils, (2) direct cytolysis of target cells, and (3) opsonization of
microorganisms and immune complexes for cells expressing complement receptors. The
polymorphonuclear cells, especially neutrophils, are an excellent example of the first line of innate
defense. Puss is composed of dead and dying polymorphonuclear and host cells, local fluids and
exudates, and dead and dying bacteria.
Important factors released by macrophages in response to bacterial antigens include cytokines that
exert both local and systemic. Locally, IL-1, TNF-, and IL-8 cause inflammation and activate
vascular endothelial cells to increase permeability and allow more immune cells to enter infected
area. TNF- will also destroy local tissue to limit growth of bacteria. In addition, IL-6 can
stimulate an increase in B cell maturation and antibody production, and IL-12 will lead to
activation of NK cells and priming of T cells towards a TH1 response. Systemically, IL-1, IL-1,
TNF-, IL-6 and IL-8 all contribute to elevated body temperature (fever) and production of acutephase protein production.
197
MYCOBACTERIAL INFECTIONS
Mycobacterial infections such as
tuberculosis and leprosy are extremely
complex. The Mycobacteria have evolved to
inhibit normal macrophage killing
mechanisms (e.g. phagosome-lysosome
fusion) and survive within the “disarmed’
professional phagocyte. These organisms
are resisted mainly by TDTH initiated cellular
mechanisms, including granulomatous
hypersensitive responses, but only after the
infection have become established. T cells
are mainly responsible for control and
containment of the infection. They
recognize mycobacterial antigens expressed
on the surface of infected cells and release
factors that recruit additional immune
effectors. A local environment is established
to contain infection. Healing of the infected
center may occur, with limited necrosis of
the infected tissue. However, if the infection
198
persists, an active caseous granuloma results. Here, an infected nidus is comprised of infected and
active macrophages, ringed by T cells. A host mediated destructive response occurs inside a
contained area. The infection itself is surrounded by activate epithelioid cells, with the presence of
Giant cells (activated syncytial multinucleated cells,).
At one time it was thought that the tissue lesions of the disease tuberculosis required the effect of
delayed hypersensitivity. The term hypersensitivity was coined because animals with cellular
immune reactivity to tubercle bacilli developed greater tissue lesions after re-inoculation of bacilli
than did animals injected for the first time. The granulomatous lesions seen in tuberculosis do
depend upon immune mechanisms for their formation. However these lesions are not really the
cause of the disease but an unfortunate effect of the protective mechanisms; the granulomatous
inflammatory reaction to the infective mycobacterium results in destruction of normal tissue. In
the lung, for instance, extensive damage done by the formation of large granulomas in response to
a tuberculosis infection can result in respiratory failure. The granulomatous immune response
produces the lesion, but the mycobacterium causes the disease.
VIRAL INFECTIONS
Immune resistance to viral
infections is mainly mediated by
cell-mediated-immunity, but
humoral (antibody) responses also
play a role by preventing virus from
attaching to cell receptors. The
antiviral response id dictated by
availability to be seen by the
immune system. Viruses live within
the host's cells and can spread from
cell to cell. To be effective in
attacking intracellular organisms, an
immune mechanism must have the
capacity to react with cells in solid
tissue. This is a property of
cell-mediated reactions, in
particular TCTL, but not of antibody
mediated reactions. Antibodies do
play a role during the extracellular
life cycle of the virus. Antibodies
can bind to virus forming
complexes to inactivate virions, and
allow them to be cleared effectively
by profession phagocytes. Humoral antibody can prevent the entry of virus particles into cells by
interfering with the ability of the virus to attach to a host cell, and secretory IgA can prevent the
establishment of viral infections of mucous membranes. However, once the virus is within cells, it
199
is not susceptible to the effects of antibody.
Natural Killer cells are an early component of the host response to viral infection. NK cells will
non-specifically recognize and kill virally infected targets, although the mechanism of recognition
still remains unclear. Infected cells have a limited mechanism to down regulate viral replication by
the production of IFN- and IFN-. Non-specific interferon responses are not sufficient to
eliminate the virus. NK cells release IFN- (physically different from the other interferons) and
IL-12, molecules which both activate macrophages and help to prime T cells for an effective antiviral TH1 response. Non-specific response can not totally eliminate virus.
Many cells infected with a virus will, at some stage of the
infection, express viral antigens on the cell surface in
combination with Class I molecules. It is at this stage that
specifically sensitized CD8+ TCTL cells can recognize and
destroy the virus-infected cells through the release of factors
(granzymes, perforins, and/or interferons) that either kill the
infected host or limit viral replication. Adverse effects of
this reaction occur if the cell expressing the viral antigens is
important functionally, as is the case for certain viral
infections of the central nervous system. If the virus infects
macrophages, TDTH-cells can activate the macrophages to kill
their intracellular viruses through the activation of the
infected macrophages by lymphokines. Lymphokine
activated macrophages produce a variety of enzymes and
cytokines that can inactivate viruses. Patients with deficiencies in antibody production alone
usually do not have serious viral infections but develop life threatening bacterial infections.
Patients with defects in cell-mediated-immunity develop serious virus infections.
HELMINTH (WORM) INFECTIONS
Host response to helminth infections are generally more complex because the pathogen is larger,
and not able to be engulfed by phagocytes. They also typically undergo life cycle changes as they
adapt for life in the host. Worms are located in the intestinal tract and/or tissues. Tapeworms,
which exist in only the intestinal lumen, promote no protective immunologic response. On the
other hand, worms with larval forms that invade tissue do stimulate an immune response. The
tissue reaction to Ascaris and Trichinella consists of an intense infiltrate of polymorphonuclear
leukocytes, with a predominance of eosinophils. Therefore, a variety of antigens that are life cycle
stage dependent are displayed in changing tissue environments. Numerous cells play a role,
depending on the location of the organism. Antigens on surface of organisms, or antigens released
into the local environment, may stimulate T cells and macrophages to interact with B cells to
secrete specific antibodies. One T cell factor (type II) is also instrumental in stimulation of
eosinophils (IL-5). The eosinophils act by associating with specific antibody to kill worms by
antibody dependent cell cytotoxic mechanisms (ADCC), or by releasing enzymes from granules to
exert a controlling effect on mast cells.
200
Antigen reacting with IgE antibody bound to intestinal mast cells stimulates release of
inflammatory mediators, such as histamine, proteases, leukotrienes, prostaglandins and serotonin.
These agents cause an increase in the vascular permeability of the mucosa, exposing worms to
serum immune components, stimulate increased mucous production and increase peristalsis. These
activities are associated with expulsion of parasitic worms from the gastrointestinal tract through
formation of a physical barrier to adherence and interactions with the mucosal surface.
Eosinophils contain granules containing basic proteins which are toxic to worms. Eosinophils may
be directed to attack helminths by cytophilic antibodies that attach to the eosinophil through the Fc
region and to the helminth by specific Fab binding (ADCC). Anaphylactic antibodies (IgE) are
also frequently associated with helminth infections, and intradermal injection of worm extracts
elicits and wheal-and-flare reaction. Children infested with Ascaris lumbricoides have attacks of
urticaria, asthma, and other anaphylactic or atopic types of reactions presumably associated with
dissemination of Ascaris antigens.
FUNGAL INFECTIONS
A great deal is not known concerning immune response to fungal agents. Cellular immunity
appears to be the most important immunologic factor in resistance to fungal infections, although
humoral antibody certainly may play a role. The importance of cellular reactions is indicated by
the intense mononuclear infiltrate and granulomatous reactions that occur in tissues infected with
fungi and by the fact that fungal infections are frequently associated with depressed immune
reactivity of the delayed type (opportunistic infections). Chronic mucocutaneous candidiasis refers
to persistent or recurrent infection by Candida albicans of mucous membranes, nails, and skin.
Patients with this disease generally have a form of immune deviation, i.e., a depression of cellular
immune reactions, with high levels of humoral antibody; similar to lepromatous leprosy. Fungi
appear to be resistant to the effects of antibody, so that CMI is needed for effective resistance.
LEPROSY -- IMMUNE DEVIATION
ANTIBODY PRODUCTION
DELAYED HYPERSENSITIVITY
The protective function of granulomatous reactivity is exemplified by the spectrum of leprosy.
The clinical manifestations of leprosy are determined by the immune response of the patient. The
high resistance of tuberculoid leprosy is associated with delayed hypersensitivity and the
formation of granulomas, whereas the
FORMS OF LEPROSY
low resistance characteristic of
lepromatous leprosy is associated with
TUBERCULOID
LEPROMATOUS
the accumulation of "foamy"
BORDERLINE
macrophages and the presence high
levels of humoral antibodies.
201
Immune deviation or split tolerance is
defined as the dominance of one
immune response mechanism over
another for a specific antigen and has
been implicated in the tendency for certain individuals to develop IgE (allergy) antibodies rather
than IgG antibodies. In addition, for reasons that are unclear, but may be genetically determined,
some individuals tend to make strong cellular immune responses, but weak antibody response to
certain antigens, whereas other individuals will have the opposite response. The course of leprosy
depends upon the immune reaction of the patient. Leprosy is classified into three major
overlapping groups: tuberculoid, borderline and lepromatous. In tuberculoid leprosy there are
prominent well-formed granulomatous lesions, many lymphocytes and few if any organisms.
Delayed hypersensitivity skin tests are intact and there is predominant hyperplasia of the diffuse
cortex (T-cell zone) of the lymph nodes. The level of antibodies is low. In lepromatous leprosy
granulomas are not formed, there are few or no lymphocytes and lesions consist of large
macrophages filled with viable organisms. Delayed hypersensitivity skin tests are depressed and
there is marked follicular hyperplasia in the lymph nodes with little or no diffuse cortex. The
levels of antibodies are high and vascular lesions due to immune complexes are seen (erythema
nodosum leprosum). Borderline leprosy has intermediate findings. The prognosis in tuberculoid
leprosy is good and the response to chemotherapy is excellent. In borderline leprosy a good
response to therapy is associated with a conversion to the tuberculoid form. The prognosis in
lepromatous leprosy and the response to chemotherapy is poor. The example of the forms of
leprosy illustrates the role of cellular immunity (delayed hypersensitivity) in controlling the
infection, and the lack of protective response provided by humoral antibodies. This concept is also
considered valid for immunity to Candida albicans (chronic mucocutaneous candidiasis).
Depressed cellular immunity is associated with chronic mucocutaneous candidiasis, a condition in
which the infected individual is unable to clear Candida infections.
A diagram illustrating the relationship of the degree of cellular and humoral immune response to
the stages of leprosy is shown. The overlapping triangles indicate the relative strength of delayed
hypersensitivity and antibody production. The cross-hatched triangle indicates delayed
hypersensitivity; the open triangle, antibody production. High levels of delayed hypersensitivity
(DTH) are associated with cure of tuberculoid leprosy; weak DTH is associated with progressive
disease; balanced DTH and antibody production with borderline leprosy and slowly progressive
disease. The cytokine patterns in the two polar forms of the disease are different. Typically TH2
cytokines (IL-4, IL-5 and IL-10) dominate in the lepromatous form, while cytokines produced by
Th1 cells (IFN-, TNF and IL-2) predominate in tuberculoid leprosy. IFN-g would be expected to
activate macrophages to kill intracellular pathogens and control organism expansion; high IL-4
may explain hypergammaglobulinemia in lepromatous patients.
EVASION OF IMMUNE DEFENSE
In the ongoing evolution of host-parasite relationships between humans and their infections,
infectious organisms have developed "ingenious" ways to avoid immune defense mechanisms.
Organisms may locate in niches not accessible to immune effector mechanisms (protective niche)
or hide themselves by acquiring host molecules (masking). They may change their surface
antigens (antigenic modulation), hide within cells, produce factors which inhibit the immune
response (immunosuppression), or fool the immune system into responding with an ineffective
effector mechanism (immune deviation). The ultimate endpoint of co-evolution of the human
202
host and its infectious organisms results in an eventual mutual co-existence with most
environmental organisms. No better evidence of this is the loss of this coexistence when the
immune mechanisms do not function properly. Then organisms which do not normally cause
disease become virulent. The lesson of AIDS demonstrates that new infectious organisms will
become dominant when introduced into a previously unexposed population. In a fully evolved,
mature relationship host and infectious agent initially co-exist without detrimental affects. Thus
the ultimate evolution of the host parasite relationship is not "cure" of an infection by complete
elimination of the parasite, but least mutual co-existence without deleterious effects of the parasite
on the host. In fact, in many human infections, the infectious agent is never fully destroyed and the
disease enters a latent state that can be reactivated under different conditions.
Bacteria have evolved to evade different aspects of the phagocyte-mediated killing. For example,
they may (1) secrete toxins to inhibit chemotaxis, (2) contain outer capsules that block attachment,
(3) block intracellular fusion with lysosomal compartments, and (4) escape from the phagosome to
multiply in the cytoplasm. Viral entities also subvert immune responses usually through the
presence of virally encoded proteins. Some of these proteins block effector functions of antibody
binding, block complement mediated pathways, and inhibit activation of infected cells. The
Herpes virus produces a factor that inhibit inflammatory responses by blocking effects of
cytokines through receptor mimicking, and another that blocks proper antigen presentation and
processing. Finally, Epstein-Barr virus encodes a cytokine homolog of IL-10 which leads to
immuno-suppression of the host by activating Th2 rather than Th1 responses.
203
Summary: Infection and Immunity
The response to initial infection may be divided into 3 phases. The first is an early innate and nonspecific response, where preformed effector cells and molecules recognize microorganisms. The
second phase is again primarily a non-specific encounter with the organism, characterized by
recruitment of professional phagocytes and NK cells to the site of infection. The final phase
involves antigen specific cells (B and T lymphocytes) effectors which undergo clonal expansion;
these cells provide memory responses in case of reinfection.
The host defense is based upon availability of resources to combat a localized pathogen. Virtually
all pathogens have an extracellular phase where they are vulnerable to antibody-mediated effector
mechanisms and complement components, macrophage phagocytosis and neutralization
responses. Intracellular agents usually require T lymphocytes (helper and cytotoxic) and NK cells,
as well as T-cell dependent macrophage activation, to kill the organism. Pathogens can damage
host tissue by direct and indirect mechanisms.
The main immune mechanisms against pathogens are as follows: Bacterial, Antibody (Immune
complex and cytotoxicity); Mycobacterial, DTH and granulomatous reactions; Viral, Antibody
(Neutralization), TCTL and DTH; Protozoal, DTH and antibody; Worms, Antibody (Atopic,
ADCC) and granulomatous reactions; Fungal, DTH and granulomatous reactions.
Virtually all classes of infectious agents have devised ways to avoid host defenses. These
mechanisms include: non-accessibility in protective niches, antigenic modulation of surface
molecules, and release of factors to either suppress the immune response, or cause immune
deviation and ineffective response to the agent.
204
Clinical Vignette - The case of Ursula Iguaran (Case 48 in Geha and Notarangelo): Ursula Iguaran, a
native of Columbia, developed hypopigmented lesions on her hands and arms when she was 16, with
progressive lesions developing through the next two years. Blood tests revealed normal white blood
counts. Dermatological evaluation revealed numerous Virchow's cells (foamy macrophages) and few
lymphocytes within the lesions. Histological analysis of a forearm biopsy revealed clumps of acid-fact
bacilli. She was diagnosed as having Mycobacterium leprae.
Ursula was aggressively treated with a multiple drug regime (dapsone, clofazamine and rifampin). Her
skin lesions gradually flattened and improved.
The immune response in patients with Lepromatous Leprosy is skewed towards the production of T
helper 2 cytokines. On this basis, would Ursula be more susceptible to certain types of infections? Which
ones and why?
205
IMMUNE REGULATION:TOLERANCE
Shen-An Hwang, Ph.D.
MSB 2.221E, 713-500-5265
Learning Objectives
1. Understand the rationale for regulating various elements of the host immune
response
2. Appreciate mechanisms of tolerance as a basis for understanding
pathophysiology of diseases and as potential targets for therapeutic intervention
Keywords
immune tolerance, immunosuppression, apoptosis
Required Reading: Coico and Sunshine, 2009. Chapter 12.
Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York,
NY. 6th edition, 2012. Case 40 Multiple Sclerosis; Case 50 Allergic Asthma.
Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/cytokines.html
Overview: control of immune response.
The major goal of the adaptive immune response is directed towards recognition of
specific non-self antigens. Specificity allows recognition of foreign (non-self) antigens.
The intensity and duration of the response dictates sufficient protection against
invading pathogens with prompt and specific downregulation when foreign antigen is no
longer present. Recognition of self-antigens does occur, with immunological tolerance
defined as specific unresponsiveness to an antigen while allowing the rest of the host
response to remain intact and capable of response.
Tolerance of Lymphocytes. Immunological tolerance is defined as a state of
unresponsiveness to a particular antigen while leaving the rest of the response intact.
Immune tolerance usually targets activity of antigen receptor bearing T and/or B cells.
Tolerance can be induced early during T and B cell development, or peripherally upon
antigen encounter. Mechanisms of tolerance include apoptosis (programmed cell
deletion), anergy (a state of clonal inactivation due to lack of secondary signals), and
regulation.
Elimination of Self Reactive Lymphocytes (Central vs. Peripheral). Central
tolerance eliminates immature lymphocytes and prevents entry of self-reactive cells into
circulation. For T lymphocytes, negative selection (via apoptosis) in the thymus
eliminates the majority of self-reactive effector T-cells, and favored selection of selfreactive cells towards the regulatory T-cell lineage. Immature B cells in the bone
marrow undergo apoptosis upon recognition of self antigens, by activation of induced
cell death mechanisms. Alternatively, the B cell may undergo receptor editing to change
206
the binding specificity of the surface immunoglobulin, thus rendering the cell no longer
self reactive.
Peripheral tolerance controls T- and B- cell reactivity once they are released into the
circulation. There are several factors that are involved in the induction of tolerance to Tcells including: route of antigen administration (oral delivery of antigens are well
tolerated), location of antigen presentation (cancer microenvironment), extremely high
or low dose of antigen, the type of antigen presenting cell involved (dendritic cell
subtypes), the type of cells responding to antigen (naive vs. memory, memory cell
subtypes, effector vs. regulatory cells, etc), the environment (cytokine factors present),
and lack of co-stimulatory signals.
For self-antigen reactive B-cells that escape into the periphery, anergy is the major
mechanism for B-cell tolerance. B-cell anergy is induced primarily through continuous
exposure to antigen.. Level of antigen also plays a role as low dose soluble monomeric
antigens do not permit receptor cross-linking on the surface of the B cell, sending
signals to clonally inactivate B cell activation. Excessively high antigen dose can also
result in anergic response due to overwhelming recognition in the absence of sufficient
T cell co-stimulation. Finally, self reactive B cells that escape elimination or induction of
anergy may be incapable of activation due to lack of T cells available to help initiate
development of autoimmune response.
I. GOALS OF CONTROLLING THE IMMUNE RESPONSE
A.
Specificity
1. Self – recognized but not responded to
2. Nonself – recognized and response initiated
B.
Intensity
1. Sufficient to protect against invaders
2. Promptly down-regulated to minimize pathologies due to immune
system activation
C.
Duration
1. Long enough to clear infection/aberrant cells
2. Generate compensatory responses, immune modulation
3. Develop antigen specific memory
II. IMMUNOLOGICAL TOLERANCE
A. General Considerations
1. Definition: state of unresponsiveness to a particular antigenic epitope
while the rest of the response remains intact
2. Type of tolerance
207
a. central – induced during early stages of development
b. peripheral – induced in mature lymphocytes
3. Consequences of Tolerance
a. Apoptosis – deletion by programmed cell death
b. Anergy – inactivation and unresponsiveness to further stimuli
c. Regulation (ignorance) – negation of activating signals
(cytokines, chemokines, cell receptors, etc)
4. Key elements involved in tolerance mechanisms
a. Antigen presenting cells (B-cells, macrophage, dendritic cells
subtypes, etc)
b. Cytokine/chemokine environment (IL-10, TGF-1, etc)
c. Regulatory cells (T regulatory cells, B regulatory cells, and their
subtypes)
B. Induction of Tolerance in Immature T and B Lymphocytes (Central tolerance)
1.
Negative selection of self-reactive cells
a. CD4+8+ T cell in thymus binds with high affinity to self-peptides
at corticomedullary junction >>>apoptosis
b. Immature B cells (bone marrow) bind to self antigens on other
BM cells >>>apoptosis or anergy
208
2.
Directing cell development towards regulatory lineage of selfreactive cells – natural T-regulatory cells
a. Developed in the thymus
b. Usually recognizes self antigens
c. Controlled by multiple factors, including thymic DCs, Hassall’s
corpuscles (epithelial cells), and “conditioned” DCs
209
3.
Tolerance induced by foreign antigens
a. T-cell exposure of alloantigen in the thymus – similar
development as observed for natural T-regulatory cells that
recognize self antigens
b. Immature B-cell, recognizing foreign antigens, development with
regulatory functions in secondary lymph organs (e.g. spleen)
i. Naïve B-cells encountering helper CD4+ T-cells
expressing CD154
ii. Regulatory B-cells produces IL-10
c. This is part of self tolerance mechanism in utero
i. Dizygotic twins sharing common placental blood supply
ii. Neonatal tolerance induction in allogeneic animals
 apparent apoptosis of allo-MHC clones
 persistence of alloantigen needed for tolerance
210
D.
Induction of Tolerance in Mature T and B Lymphocytes
1.
Major mechanisms
a. Apoptosis
 FAS/FASL interaction
i. Autoimmune models – Lpr (Fas deficient) and Gld
(FasL deficient)
211
 Control of lymphocyte cell numbers
i. Activation induced cell death (AICD) - autonomous
vs. cell-cell contact
ii. Lack of growth/proliferation factors (e.g. IL-2)

b. Anergy – unresponsive to subsequent antigen challenge
 Lack of secondary signals for T and B cells (Ig and TNF
superfamilies)
i. CD28, ICOS/ICOSL (inducible co-stimulator)
ii. CD40, OX40
 Expression of suppressive secondary signals
i. CTLA-4 (induced upon T-cell activation)
ii. PD-1
 Lack of helper cells
i. Large antigen dose bypass T helper cell activation
of B-cells
ii. Usually CD40/40L interaction
c. Immune regulation – cell types and function
 T-cells
i. T-regulatory cells (natural vs. inducible)
ii. T-helper cells (TH1, TH2, TH9, TH17, etc)
iii. Cytokine production (e.g. IL-10, TGF-1)
iv. Cytokine sequestration (e.g. IL-2)
v. Direct cell-cell contact (e.g. CTLA-4/CD80)
 B-cells
212
i. B-regulatory cells
ii. B-effector cells (Be-1 vs. Be-2)
iii. Cytokine production (IL-10)
 Dendritic cells
i. Immature vs. mature
ii. Tissue specific DCs (e.g. mucosal DCs)
iii. Cytokine production (TGF-1, IL-10, IL-4, etc)
iv. Alteration of surface marker expression (CD80/86,
CD40, etc)
d. Antigen sequestration
 Immune privilege sites
 Exposure of privilege site antigens will result in
autoimmunity (e.g. CNS)
213
III.
PARAMETERS THAT INFLUENCE IMMUNE RESPONSES OF INDIVIDUALS
A.
Age
1. Young
a. Fetus makes IgM but not IgG until almost term
b. Term babies immunocompetent but immature at birth
c. Takes several months to develop full immunocompetence
2. Aged
a. Immune senescence
b. Deficiency vs. dysregulation
c. Good memory response, poor naïve (primary) response
d. Infectious deaths in elderly often occur with new organism strain
B.
Neurologic and Endocrine Factors
1. Psychoneuroimmunology – relationships between behavior (stress)
neuroendocrine changes and immunity
a.
hypophyseal-pituitary-adrenal axis - corticosteroids
b.
sympathetic nervous system – catecholamines
(epinephrine, norepinephrine)
2. Hypophyseal-pituitary-gonadal axis – particularly female hormones
a. may explain female preponderance of immune-based
diseases
b.
both TH1 and TH2 diseases noted
3. Nutritional Status
a.
trace mineral deficiency – zinc, selenium, etc.
b.
malnutrition – total calorie vs. protein – energy
 infection activates TNF, IL-1, etc. (pyrogens)
 fever produces anorexia, affecting appetite
 poor appetite worsens malnutrition
 cell mediated immunity affected first
 c.
leptin - associated with obesity
- reverses starvation-induced immunosuppression
d.
Retinoic acid

C.
MHC Message Expression
1. Allotypes of MHC expression affects specific responses
2. Affects repertoire/magnitude of responses to vaccines
3. may also affect susceptibility to certain autoimmune
responses
D.
Effects of Antigen
1.
Dose
a. low and high doses tolergenic, mid dose immunogenic
214
b. low dose tolerizes T cells only, high dose tolerizes both T and B
cells
2.
2.
Route of Exposure
 parenteral exposure is enhanced by adjuvant effects
 IV exposure can tolerize naïve (but not memory) T cells
a. IV antigen gets to spleen rapidly
b. Picked up by resting B cells
c. Resting B cells lack costimulatory molecules
d. Result is anergy
 Oral exposure
a. activates muscosal T cells to secrete TGF-beta
b. result is anergy
Regulation by Antibody
 Antibody feedback - antibody inhibits further specific
antibody production
 Antigen-antibody complex
i. Binds BCR, CD32 simultaneously
ii. Inhibits activation signal
iii. Result is anergy
215
Coico, et al., Fig 10.14. Antibody feedback inhibits
B-cell activation, resulting in negative signal to the
cell. 2009.
IV.
Tolerance in the clinical setting
A.
Oral tolerance – a major environment for tolerance
i.
Dose of antigen
1. Low – favors regulatory T-cells
2. High – favors anergy/apoptosis
ii. Mechanism
1. Dendritic cells – DCs that are CD103+ ( integrin) produces
mainly TGF-b1 and induce activation of T-regulatory cells
2. Retinoic acid – Enhances TGF-b1 production and induction of
T-regulatory cells
3. T-regulatory cells – Nearly all classes are induced by oral
antigens (e.g. TH3, secretes TGF-1)
216
B.
Pregnancy (tolerance of paternal antigens)
i. The paradox
1. Fetal expression of paternal (foreign) antigens
2. Shedding of trophoblast into maternal circulation
3. Cellular debris from fetal tissue
4. Presence of fetal cells in maternal circulation
ii. Mechanism
1. Immune stimulation by sperm exposure – Activation of effector
T-cells also triggers T-regulatory cell activity, and overall
consequence is expansion of T-reg populations (driven by TGF1 and prostaglandin E)
2. Extravillous trophoblast cells lack of expression of antigen
presentation molecules (Class 1 and 2) but expresses nonclassical class I to evade killing by NK cells
3. T-regulatory cells – Maternal T-reg numbers peak during the
window when implantation can occur and during pregnancy.
Fetal T-regs are similar to adult T-regs
C.
Cancer (tolerance of tumor antigens)
i. Cancer immunosurveillance hypothesis – immune response is
efficient in controlling tumor growth and clinical disease
ii. Cancer immuno-editing
1. Elimination – tumors detected and destroyed
2. Equilibrium – establishment of balance between tumor and
immune response
3. Escape – immune control falters and tumor variants grow
uncontrollably
iii.
Tumor microenvironment
1. Concomitant immunity (transient) – growth of tumor at one site
despite rejection of subsequent introduction of the same tumor
cells at a distant site
iv. Mechanism
1. Direct immune suppression by tumor cells
a. Expression of T-cell inhibitory molecules (e.g. PD-L1, B7H3, etc)
b. Production of anti-inflammatory cytokines (e.g. IL-10,
TGF-1, etc)
c. Alters recruitment of “suppressor” cell types, both APCs
and T-cells
2. T-regulatory cells – Population expands in cancer patients
3. Dendritic cells – IDO (indoleamine 2,3-dioxygenase) competent
APCs activate mature T-regs
217
Summary

Immune response must be regulated to allow sufficient response to protect host
with excessive or inappropriate responses that may create disease

Immunological tolerance is specific unresponsiveness to an antigen while
allowing the rest of the host response to remain intact and capable of response

Tolerance can be at the level of B or T lymphocytes or both and can be
accomplished by a variety of mechanisms including apoptosis, anergy, and
suppressor T cell activity

Many factors influence the nature, intensity and duration of an immune response
including age, neuroendocrine hormone levels, HLA allotypes, antigen dose,
antigen access, and cytokine milieu.
218
AUTOIMMUNITY AND AUTOIMMUNE DISEASES
Sandeep K. Agarwal, M.D., Ph.D.
Medicine-Immunology, Allergy & Rheumatology, BCM
713-798-3390
[email protected]
Objectives
1. Define and discuss autoimmunity.
2. Use autoimmune diseases to illustrate mechanisms of autoimmunity.
3. Provide you with clinical correlations and applications of the basic principles of
immunology.
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc,
New York, NY. 6th edition, 2009. Chapter 12 (p190-204).
R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6th
Ed) Garland Publishing, New York, 2012. Chapter 36. Rheumatoid Arthritis; Chapter 40.
Multiple Sclerosis; Chapter 41. Autoimmune Hemolytic Anemia.
Web Resource:
http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/autoimmunity.html
INTRODUCTION:
The regulation of immune function and overall immuno-homeostasis is under control of
multiple factors that include genetic and environmental components. HLA allotypes,
antigen dose, and existing cytokine milieu can all influence responses to both pathogenic
agents and self antigens.
It is highly recommended to review the reading materials PRIOR to
lecture, as the lecture will primarily concentrate on clinical
manifestations of autoimmune disorders.
219
Autoimmune Lecture Outline
Autoimmunity
• Specific adaptive immune response mounted against a self-antigen
– Loss of Self-tolerance to self-antigens
– Loss of central and peripheral tolerance
• Loss of central tolerance likely occurs all the time
• May have a physiological role to clear defective or denatured
molecules through the RE system
• Normally kept in check by mechanisms of peripheral tolerance
• May be triggered by infections or aging
• May or may not cause disease
Autoimmune Disease
• Termed “horror autoxicus” by Paul Ehrlich
• Tissue response and damage triggered by autoimmunity
• Results from the dysregulation of immune processes and pathways that
are involved in normal immunity
Architecture of an Autoimmune Response
• Innate and Adaptive components
Autoimmune Disease and Clinical Phenotypes:
AUTOIMMUNE DISEASE
CLINCAL PHENOTYPE
Systemic Lupus Erythematosus Rash; inflammation of joints and serosal linings;
glomerulonephritis; hemolytic anemia, systemic
symptoms
Rheumatoid Arthritis
Inflammation of synovium of diarthroidal joints,
systemic inflammation
Scleroderma
Inflammation, dermal fibrosis, internal organ
fibrosis, vasculopathy
Ankylosing Spondylitis
Inflammation of spine, joints, and tendon
insertions; uveitis
Multiple Sclerosis
Demyelination, optic neuritis, neurological deficits
Myasthenia Gravis
Skeletal muscle weakness, diplopia, dysarthria,
dysphagia
Hashimoto’s Thyroiditis
Hypothyroidism
Graves Disease
Hyperthyroidism, opthalmopathy
Celiac Disease
Diarrhea and malabsoprtion
Autoimmune hemolytic anemia Anemia through lysis of red blood cells
Type I diabetes
Failure of insulin production and glycemic control
220
Genetic Susceptibility to Autoimmune Diseases
Simple Genetic Traits Associated with Autoimmune Diseases
Autoimmune Diseases and Concordance in Twins
Common diseases: Multiple SNPs
• Common diseases are believed to result from a combination of susceptibility
alleles at multiple loci, environmental factors and stochastic events
• Non-Mendelian Inheritance Patterns
• Single nucleotide polymorphisms (SNPs)
– Individual bases that exist as either of two alleles in the population
Major Histocompatibility Complex – Association with Autoimmune Diseases
Class I MHC Associations
Ankylosing Spondylitis
HLA-B27
Grave’s Disease
HLA-B8
Class II MHC Associations
Rheumatoid Arthritis
Sjogren’s Syndrome
Systemic Lupus
Erythematosus
Type I Diabetes
Celiac Disease
Myasthenia Gravis
Multiple Sclerosis
HLA-DR4
HLA-DR3
HLA-DR3, DR2
HLA-DR3
HLA-DR3
HLA-DR3
HLA-DR2
HLA-B27and Autoimmune Disease
HLA-DR4 and Rheumatoid Arthritis
Single Nucleotide Polymorphisms in Autoimmune Diseases
Mechanisms of Autoimmune Disease
• Previous attempts to classify them as T-cell and B-cell mediated are outdated
• Involve Innate and Adaptive Components
• Classified based on the effector mechanisms that appear to be most responsible
for organ damage:
– Autoantibodies
– T-cells
221
Autoantibodies
• Antibodies against to self-antigens
• Can be found in normal, healthy individuals
• Important effectors in autoimmune disease
Autoimmune Hemolytic Anemia
• Autoantibodies against RBC antigens
– Warm autoantibodies
• IgG, react with Rh antigen on RBC at 37degC
• Result in opsonization of RBCs and macrophage phagocytosis
– Cold autoantibodies (cold agglutinins)
• IgM, react with I or i antigen on RBC when <37degC
• Activate complement and result in complement mediated lysis
– Drug induced antibodies
• Penicillin acts as a hapten, binds to RBC and form antibodies
against RBCs
Myasthenia Gravis
• Target antigen is alpha chain of the nicotinic acetylcholine receptor in the
neuromuscular junction
• Autoantibodies act as antagonist
• Symptoms of muscle weakness, diplopia, dysarthria, dysphagia
• May be associated with a thymoma
• Can be transmitted to fetus through placental transmission of autoantibodies
Graves Disease
• Symptoms of hyperthyroidism
– Heat intolerance, Increased metabolism, weight loss
– Palpitations, increased HR, Hair loss, Fatigue
– Nervousness, Opthalmopathy
• Autoantibodies against thyrotropin stimulating hormone receptor (TSHreceptor)
• Autoantibodies act as an agonist
• Symptoms of hyperthyroidism
• Maternal antibodies can be transmitted to fetus through the placenta resulting
transient neonatal hyperthyroidism
Systemic Lupus Erythematosus
• Autoimmune disease characterized by
– systemic autoimmunity
– multi-organ involvement
– production of autoantibodies against nuclear components
– immune complexes
• Autoantibodies and immune complexes deposit in tissues including skin, joints,
blood vessels, kidneys, etc.
222
Antinuclear Antibodies: Presence in multiple autoimmune diseases
SLE: ANA Associations and Immunofluorescence
Viral Triggering of Autoantibody Production
TLRs and Autoimmunity
Scleroderma Associated Autoantibodies
Multiple sclerosis
• A T-cell mediated autoimmune disease of the central nervous system
characterized by
– Demyelination in brain and spinal cord
– inflammation and dissemination of lesions in space and time
• Symptoms: visual defects, weakness, sensory deficits, diplopia, ataxia, cognitive
deficits, bowel/bladder incontinence
Pathology of MS
• An immune-mediated disease in genetically susceptible individuals
• Demyelination leads to slower nerve conduction
• Axonal injury and destruction are associated with permanent neurological
dysfunction
• Lesions occur in optic nerves, periventricular white matter, cerebral cortex,
brain stem, cerebellum, and spinal cord
Possible Mechanism of Demyelination and Axonal Loss in MS
• Activation of autoreactive CD4+ T cells in peripheral immune system against
myelin proteins
• Migration of autoreactive Th1 cells into CNS
• In situ reactivation by myelin autoantigens
• Activation of macrophages, B cells
• Secretion of proinflammatory cytokines, antibodies
• Inflammation, demyelination, axonal transection, and degeneration
Other Autoimmune Diseases
• Hashimoto’s Thyroiditis
– Autoantibodies and autoreactive T-cells to thyroglobulin and thyroid
microsomal antigens
– Th1 cells also play a role
– Destruction of thyroid gland leading to hypothyroidism
– Symptoms of hypothyroidism: fatigue, goiter, dry skin, brittle hair and
nails, cold intolerance, weight gain, depression
•
Rheumatoid Arthritis
– Antibodies to citrullinated peptides (anti-CCP antibodies)
– Antibodies to Fc portion of IgG (rheumatoid factor)
223
–
–
–
•
Immune complex formation and T-cell infiltration in synovium
Leads to activation of innate immune system components through Fc
receptors
Synovial inflammation, destruction of cartilage and bone erosions
Type I Diabetes Mellitus
– Autoreactive CD8+ T-cells to pancreatic islet cells
– Destruction of islet cells and failure of insulin production
– Autoantibodies to insulin and islet cell antigens (GAD) are also present,
might be a result and not causative
Targeted Therapeutics
• As our understanding of the pathogenesis increases, targeted therapeutic
approaches are becoming available
– TNF-alpha inhibitors for the treatment of rheumatoid arthritis,
ankylosing spondylitis, psoriasis, inflammatory bowel disaese
– CTLA-4 Ig for the treatment of rheumatoid arthritis
– antiCD20 antibody (targeting B-cell) for the treatment of rheumatoid
arthritis
– Beta interferon for the treatment of multiple sclerosis
– Anti-type I interferons for the treatment of systemic lupus
erythematosus (in development)
– Many others in development
CONCLUSION:
“…. The mechanisms underlying all autoimmune diseases are not fully elucidated;
however, genetic polymorphisms of MHC class II genes (alleles of HLA-DR and/or HLADQ) are associated with increased susceptibility to autoimmune diseases. Possible
mechanisms for a loss of tolerance leading to autoimmune reactions include (1) a lack of
Fas-Fas ligand–mediated deletion of autoreactive T cells in the thymus during
development, (2) loss of T-regulatory or T-cell suppressor function, (3) cross-reactivity
between exogenous and self-antigens (molecular mimicry), (4) excessive B-cell function
due to polyclonal activation by exogenous factors (of viral or bacterial origin), (5)
abnormal expression of MHC class II molecules by cells that normally do not express these
surface molecules, and (6) release of sequestered self-antigens from privileged sites, thus
priming for responses not previously seen by the immune system.
Autoimmune diseases can be classified as organ specific or systemic in nature …. Three major
types of autoimmune reaction mechanisms are recognized as causing different autoimmune
disorders ... Two of these mechanisms involve autoantibodies directed against self-antigens; for
both, classical complement pathway activation exacerbates local damage and inflammatory
response. In the first case, autoantibodies may be directed against a specific self-component,
224
such as a surface molecule or receptor. Examples include antibodies against the acetylcholine
receptor producing myasthenia gravis, and antithyroid- stimulating hormone receptor antibodies
producing Graves’ disease. Autoantibodies may also bind with antigens present in the blood,
forming antigen-antibody (immune) complexes that later deposit in organs, thus inciting an
inflammatory response. An example is seen in lupus glomerulonephritis in which complexes of
anti-DNA antibodies and free DNA accumulate in the kidney. The third mechanism is that of
autoreactive T cells that recognize targeted self-antigens on organs, leading to direct damage to
tissue. In many cases, autoreactive T cells coexist with autoantibody responses, leading to
exacerbation of disease and organ damage. In the case of multiple sclerosis, T cells reactive to
myelin basic protein destroy the protective layer surrounding axons, thereby eliminating
effective transfer of signals through nerves." [adapted from Actor, J.K. Elsevier’s Integrated
Immunology and Microbiology, Mosby/Elsevier, Philadelphia, 2007.]
SUMMARY (included materials from required reading):
1. Autoimmunity represents a failure of effective tolerance to self-antigens.
2. Genetic and environmental factors play a role in the etiology of disease.
3. Mechanisms of disease include autoantibodies that are directed against specific selfcomponents, deposition of circulating antibody-antigen complexes, and deleterious
responses by autoreactive T cells.
225
CLINICAL CORRELATIONS
Faculty Taught Class Correlations
Clinical Cases will be presented by faculty. Cases are taken from the Geha and Notarangelo. Case Studies in Immunology.
Garland Publishing, New York, NY. 6th edition, 2012.*
Clin. Corr.
Class
Date Time
2/14 11:00-11:50 AM
First Case
36. Rheumatoid Arthritis
Second Case
37. Systemic Lupus
Erythematosus
Clinical Correlation Cases will be presented by faculty. This is NOT an extra credit assignment, but rather a clinical
correlate that is part of the Immunology curriculum.
Review the study questions at the end of each chapter for each of the cases presented.
There is no formal requirement to complete the questions below. No extra credit points will be given for this Faculty
taught Clinical Correlate. Rather, it is recommended that you be prepared to answer the following during class discussion:
Questions:
1. Define the deficiency/hyperreactivity involved in this case, if one can be identified.
(Hyporeactivity)
Immunodeficiency
2.
(Hyperreactivity)
Health
Immunopathology
/\
How does this immune disorder directly or indirectly involve or impact each of the
following (answer all):



Innate immune system activities
B cell activities
T cell activities
3.
Describe the underlying mechanism(s) (e.g. at the organ, cellular or molecular level) in this case (brief paragraph).
4.
Give a short, succinct summary of the immunologic principle illustrated by this case.
*Note: Cases also appear in the previous edition: Geha and Rosen. Case Studies in Immunology. Garland Publishing, New York,
NY. 5th edition, 2007. Case numbers indicated below:
Cases for 2/14
42. Rheumatoid Arthritis
43. Systemic Lupus Erythematosus
226
Tumor Immunology
Priya Weerasinghe, MD.PhD
[email protected]
LEARNING OBJECTIVES:





Describe the concepts of tumor antigens.
Describe the effectors mechanisms in tumor immunity.
Describe how immunology is used in diagnoses of cancer.
Describe the concept of immunoprophylaxia.
Introduce concept of immunotherapy.
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New
York, NY. 6th edition, 2009. Chapter 19.
Web Resource:http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/cancerimm.html
Full Syllabus Chapter to be distributed via Blackboard prior to lecture presentation.
227
MODIFICATION OF THE IMMUNE RESPONSE: IMMUNOPROPHYLAXIS AND
IMMUNOTHERAPY
Semyon A. Risin, MD PhD
OBJECTIVES:
1. To understand the application of the major immunological principles and
concepts to modification of immune response, immunoprophylaxis and
immunotherapy of human diseases.
2. To define the current approaches and future strategies to
immunoprophylaxis and immunotherapy of immune-mediated and nonimmune-mediated diseases
KEYWORDS:
Immunoprohylaxis,
vaccine,
immunization, immunotherapy
active
READING:
Coico and Sunshine, 2009. Chapter 20
and
passive
WEB RESOURCE:
http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/prophylaxistherapy.html
I.
INTRODUCTION
Considering the number of saved lives, the active process of deliberate
exposure of individuals to infectious agents or their antigenic components vaccination or immunoprophylaxis - has been one of the most important
medical advances in the history of mankind. Historically it was based on early
recognition of resistance to secondary exposure to infectious challenges of
individuals recovered from infectious diseases.
Reports of immunization go back to the ancient Chinese (10th century) when
they used crusts from smallpox lesions (variolation) to provide an attenuated
episode of smallpox with subsequent protection. Jenner, in 1798, introduced
the use of vaccinia (cow) virus as a cross reactive agent for protection against
smallpox (from which the terms vaccine and vaccination are derived). In
1860-65, Pasteur introduced the concept of attenuated vs. killed vaccine to
prevent anthrax. These were examples of early active immunizations.
Later the concept and technology of passive immunization –use of sera of
convalescent individuals or sera from hyperimmunized animals for immediate
protection- was introduced. It was particularly successful in treating toxic
complications of diphtheria and tetanus.
Since then the theory and practice of immunization and the vaccinemanufacturing technology developed significantly. It incorporated the major
advances of molecular biology and biotechnology, including DNA228
recombinant technology, gene transfer, use of bacterial and viral vectors,
modification of protein structure, “humanization” of animal-derived antibodies,
use of hybridoma technology for manufacturing of monoclonal antibodies, etc.
In addition, based on understanding the complexity of the immune system,
new approaches to modification of the immune response targeting such key
elements as affector cells, cell interaction and cytokines were introduced.
These achievements also revolutionized the field of immunodiagnostics
(ELISA, immunostaining, Flow-cytometry) as well as triggered the use of
monoclonal antibodies for blocking biologically active molecules playing a key
role in non-immune pathology (herceptin, antiplatelet antibodies etc.), and
thus creating the basis for immunotherapy of non-immune diseases.
Smallpox vaccination based on cross-reactivity
between cowpox and smallpox viruses
II.
IMMUNIZATION BASICS
A. Types of Immunizations
1.
Active – exposure to antigen with the host generating
protective immunity. Objective: provide long lasting immunity
against future exposures
2.
Passive – administration of humoral and/or cellular factors
that provide immunity for the host. Objective: provide
temporary immediate protection against an imminent or
ongoing exposure
3.
“Heard” immunity in preventing spread of infection
B. Examples of Active and Passive Immunization
229
Type of immunity
Active
Natural (unintended)
How acquired
Infection
Artificial (deliberate)
Vaccination
Passive
Natural
Transfer of antibody
from mother to infant
in placental
circulation or though
breast-feeding
(colostrums)
Passive antibody
therapy (serum
therapy, immune
human globulin,
monoclonal
antibodies)
Artificial
III.
Active Immunization
1.
1a.
Current US Recommendations for active immunization
Schedule for Active Immunization of Children and Adults
Age
Birth
1–2 months
2 months
4 months
6 months
12–15 months
4–6 years
11–12 years
25–64 years
>65 years
Vaccine
Hepatitis B (Hep B)
Hep B
Diphtheria and tetanus toxoids and acellular
pertussis (DTP), Haemophilus influenzae
type b (Hib), inactivated polio (IPV)
DTP, Hib, IPV, rotavirus (Rv)
Hep B, DTP, Hib, IPV, Rv
Oral poliovirus vaccine (OPV), measles,
mumps, rubella (MMR), varicella vaccine for
susceptible children
DTP, OPV, MMR
Hep B, MMR, varicella
Measles, rubella
Influenza, pneumococcal disease
Adapted from JAMA, Vol 281:601–603, with permission.
1b.
Vaccines for Specific Populations
a. BCG – vaccine against TB in endemic populations
(Obscures PPD skin testing – not used in US)
b. PneumoVax – susceptible populations
230
i. Cancer
ii. Elderly
iii. Immunocompromised, postsplenectomy
c. Meningococcus vaccine – military recruits and institutionalized
subjects
d. Travel vaccines – typhoid, anthrax, yellow fever, plague, etc.
Depends upon endemic area for travel
2.
BASIC MECHANISMS OF PROTECTION
2A.
Primary vs. Secondary Immune Response
1.
Rapidity of response is critical in light of incubation
period of infection
2.
If incubation period short (i.e. 3-4 days), there may
not be enough time for anamnestic response to develop
before disease ensues
2B.
Age and Timing of Immunizations
Fetus
a.
IgM appears at 6 months gestation
b.
decreases to 10% adult level at birth
c.
IgG appears at 6-8 weeks gestation, maternal origin
d.
majority of IgG at birth is of maternal origin
(transplacental)
231
Infants
a. generally do not respond well to polysaccharide antigens
at less than 2 yrs of age
b. antigenicity improves when conjugated to protein or
toxoid
2C.
Mixed or multiple antigen vaccines
a.
routine vaccines often group by antigen types
 toxoids
 polysaccharides
 virus coats
b.
concern about relative competition between vaccine
responses
c.
with 1012 lymphocyte repertoire, competition not a
practical issue
2D.
Route of vaccine administration
a. parenteral vs. oral or respiratory
b. deltoid muscle vs. gluteus (e.g. Hepatitis B)
c. systemic vs. mucosal immunity
- Oral administration should stimulate mucosal immunity,
parenteral often does not (e.g. Sabine vs. Salk)
- Mucosal immunity stops infection, systemic -> stops illness
2E.
Hazards
a. Live vaccines in immunocompromised
individuals and pregnant women
b. Reversion to wild type
c. Arthralgias/myalgias
d. Hypersensitivity reactions
1. Arthus phenomenon
232
2. arthritis and arthralgia
3. anaphylaxis
3.
Vaccine Production Methods
a.
b.
c.
d.
e.
f.
g.
h.
IV.
Recombinant DNA – makes antigen-specific oligopeptide
Conjugated polysaccharides – add protein to involve T
cells
Synthetic peptides – largely covered by rDNA-induced
peptides – must be big enough to induce T and B cell
memory
Specific receptor blockade –stops pathogen entry (i.e.
virus)
Antiidiotype vaccines
Gene constructs – virus vector or naked DNA
Bacterium – carrier : bacterium acts as adjuvant
Toxoids – inactivated toxins which may produce better
immunity than natural infection due to relative amounts of
antigen exposure
PASSIVE IMMUNIZATION
A.
Natural
Placental Antibody Transfer
1.
majority of IgG in neonate’s plasma is passive from mom
2.
protection wanes by 6mo as infant makes own immunoglobulin
3.
specific immunization of mother antenatal can protect
neonate (i.e. tetanus neonatorum)
Colostrum protection
1.
contains enzymes, cells, antibodies
2.
B cells migrate to breast from intestine (enteromammary)
3.
antigen-specific T cells also transmitted but role is unclear
B.
Artificial
Passive Antibodies – Specific vs. Nonspecific
 specific antigen raised in animal sera (e.g. horse)
 result was serum sickness with repeated exposure
 now use hyperimmune Ig purified from human donors
 although peak levels may be lower, end result is an
extended duration of circulating protective antibody
 use of humanized monoclonal antibodies
233
C.
Monoclonal vs polyclonal antibody
 monoclonal highly specific for single epitope
 can make very large amounts in biologically active form
 polyclonal represents activity against larger number of
antigens
 must be purified from serum of human donors if single
antigen specificity
 more common to use IVIG
D.
Intravenous Immunoglobulin (IVIG)
 purified from pooled sera of thousands of donors
 advantage of multiple specificites
 dilutes out any adverse influences (drug, infections, etc.)
 IgG1 is major component – 25 fold higher concentration
than plasma
234
Comparison of immunoglobulin contents of Human Immune Serum
Preparations
Immunoglobulin (mg/100 ml)
Source
IgG
IgA
IgM
Whole serum
Immune serum globulin
Intravenous immunoglobulin
Placental immune serum globulin
1,200
16,500
3000–5,000
16,500
180
100–500
trace
200–700
200
25–200
trace
150–400
E.
Uses for Immune serum globulins
 Hyperimmune globulins
 Rhogam – prevent Rh immunization
 CMV-IGIV – prevent CMV in bone marrow
transplants
 Rabies Ig – prevent clinical rabies
 VZIG – leukemia patients exposed to VZV
 IVIG
 Humoral (IgG immunodeficiency) producing
chronic infection
 Idiopathic thrombocytopenia purpura
Precautions
 IM – aggregates may cause anaphylactoid reaction
 aseptic meningitis
 noninfectious hepatitis
 anaphylactoid in selective IgA deficiency
V.
IMMUNOTHERAPY
Use of immunological approaches for treatment of immune-based
and non-immune-based human diseases
A.
Immune-based diseases
1.
Mechanisms
 Deficiency
 Dysregulation
 Dysfunction
2.
Clinical Manifestations
 Infectious
 Hypersensitivity
 Cancers
 Others
235
3.
Potential Roles for Cytokine Therapy in Immune Diseases
Disease Mechanisms
Diagnosis
Prognosis
Monitoring response to therapy
B. Non-immune-based diseases (examples of immunoprophylactic
and immunotherapeutic approaches)
1. Cardio-vascular (antiplatelet Ab abciximab)
2. Tumors
a. nonspecific stimulation of innate immunity by
BCG
b. use of ex vivo propagated tumor infiltrating
lymphocytes (TIL) in melanoma
c. use of dendritic cells loaded ex vivo with
multiple tumor epitopes
d. new antitumor vaccines (melanoma, prostate
cancer, HPV vaccine for cervical cancer, H. pylori
vaccine for gastric and gastro-esophageal cancer
etc.)
e. use of bcr-abl vaccine for CML Herceptin for
breast cancer, rituximab for B-cell malignancies,
alemtuzumab for CLL)
C.
Clinical Examples with Potential for Using Cytokine Therapies (immunebased and non-immune-based diseases)
1.
Metabolic Diseases
 Osteoporosis – IL-6
 Diabetes mellitus – TH1
2.
CNS diseases
 Multiple sclerosis – TH1
 ALS –TH1
 Alzheimer’s – TH2 (?)
3.
Infectious Diseases
 Opportunistic infection – T cell deficit
 HIV disease – CD4 T cell deficit
236
4.
Inflammatory bowel disease
 Crohn’s
TH1
 Ulcerative colitis TH2
 Rheumatoid arthritis – blocking inflammatory cytokines
5.
Sepsis syndrome/ARDS –TNF, IL-1, IL-6
6.
Hypersensitivity Diseases
 Allergic/asthmatic diseases – TH2
 Autoimmune/inflammatory diseases – TH1 and TH2
D.
Rationale for immunotherapy of bronchial asthma
a.
b.
Asthma is a classic example of TH2 disease.
IL-4 serves not only as a signal for isotype switches to IgE but is,
along with IL-3 and GM-CSF, a mast cell growth factor
IL-4 can upregulate expression of VCAM-1/VLA-4, which is an
adhesion molecule pair that facilitates eosinophil-specific
inflammation.
IL-4 appears to be involved in goblet cell hypertrophy and
hyperplasia which result in increased mucus production, a hallmark
of asthma inflammation.
IL-4 may also be involved in airway remodeling. Additionally, IL-5
induces eosinophil differentiation from myeloid precursors in the
bone marrow
IL-5 in conjunction with eotaxin serves as an important chemotactic
factor for eosinophils,
IL-5 inhibits apoptosis thus prolonging survival of eosinophils in the
periphery
IL-5 activates eosinophils to release cytotoxic products such as
major basic protein, eosinophilic cationic protein and others.
c.
d.
e.
f.
g.
h.
Thus, when considering the therapeutic utility of various new biotechnology
molecules, a fundamental approach would attempt to regulate the milieu that
activates mast cells and eosinophils and recruits them to airways. If mast cell
numbers and/or activities can be regulated, asthma activity can likewise be
affected. Thus, many efforts are underway to regulate activity and/or production
of TH2 cytokines IL-4 and IL-5 as well as allergen-specific IgE.
In 2003 FDA approved a humanized monoclonal antibody against IgE – Xolair
(omalizumab) - for clinical use.
237
SUMMARY

Immunization can be by either exposing the host to an antigen preparation
that induces a protective immune response (active) or by supplying the
immune products (i.e. antibody or effector cells ) from another immune
host (passive)

Immunizations occur after a primary exposure that creates a sensitization
and a secondary “booster” challenge that provides an accelerated,
heightened response capable of protecting the host against subsequent
infection and disease

There are multiple vaccine preparation methods, each with their own
advantages and disadvantages

Passive immunization can be natural - from maternal source (placental
transfer or colostrums) or artificial – from an exogenous source of
immunoglobulins or immune cells. The exogenous antibody preparation
can be for either a specific antigen source (antiserum) or for more general
immunoglobulin replacement (i.e. intravenous Immunoglobulin). Antibody
preparations may be monoclonal or polyclonal.

Immunotherapy is used primarily as either a modulator of the immune
response based upon the notion of TH1/TH2-based immunological
diseases that can be treated by altering the underlying imbalance (such as
increasing one helper population over another), or as a cancer treatment
modality to block the expression of biologically important molecules and
suppress cancer cell proliferation.
238
PRIMARY IMMUNODEFICIENCIES
WILLIAM T. SHEARER, M.D., PH.D.
Objectives






I.
Define immunodeficiency and note its frequency and inheritance patterns.
Understand the genetic basis for primary immunodeficiency.
Describe deficiencies in B and T lymphocytes.
Describe deficiencies in NK and phagocytic cells and complement.
Learn how to diagnose immunodeficiencies.
Consider treatment options for patients with congenital immunodeficiencies.
Key Words








II.
SCID, Severe Combined Immunodeficiency
XLA, X-linked Agammaglobulinemia
CVID, Common Variable Immunodeficiency
Hyper IgM, Immunodeficiency with Elevated IgM
Wiskott-Aldrich Syndrome
DiGeorge Syndrome, Cellular Immunodeficiency with Hypoparathyrodism
CGD, Chronic Granulomatous Disease
LAD, Leukocyte Adhesion Deficiency
Definitions
The immunodeficiency disorders are a diverse group of illnesses that, as a
result of one or more abnormalities of the immune system, predispose a person
to infection. The abnormalities of the immune system can involve absence or
malfunction of blood cells (lymphocytes, granulocytes, monocytes) or soluble
molecules (antibodies, complement components) and can result from an
inherited genetic trait (primary) or from an unrelated illness or treatment
(secondary).
III.
Reading Assignments
A.
B.
C.
Coico R, Sunshine G. Chapter 17: Immunodeficiency disorders and
neoplasias of the lymphoid system. In Immunology: a short course, 6th
Edition. New York: Wiley-Liss, 2009.
[recommended reading] Shearer WT, Fischer A. Editorial. The last 80
years in primary immunodeficiency: How far have we come, how far need
we go? J Allergy Clin Immunol 2006;117:748-752.
[recommended reading] Notarangelo L, Casanova JL, Conley ME,
Chapel H, Fischer A, Puck J, Roifman C, Seger R, Geha RS; International
Union of Immunological Societies Primary Immunodeficiency Diseases
Classification Committee. Primary immunodeficiency diseases: an update
from the International Union of Immunological Societies Primary
239
D.
E.
Immunodeficiency Diseases Classification Committee Meeting in
Budapest, 2005. J Allergy Clin Immunol 2006;117:883-896.
[recommended reading] Orange JS, Hossny EM, Weiler CR, Ballow M,
Berger M, Bonilla FA, Buckley R, Chinen J, El-Gamal Y, Mazer BD,
Nelson RP Jr, Patel DD, Secord E, Sorensen RU, Wasserman RL,
Cunningham-Rundles C; Primary Immunodeficiency Committee of the
American Academy of Allergy, Asthma and Immunology. Use of
intravenous immunoglobulin in human disease: A review of evidence by
members of the Primary Immunodeficiency Committee of the American
Academy of Allergy, Asthma and Immunology. J Allergy Clin Immunol
2006;117:S525-S553.
[recommended reading] Puck JM, Malech HL. Gene therapy for
immune disorders: good news tempered by bad news. J Allergy Clin
Immunol 2006;117:865-869.
WEB RESOURCE:
HTTP://WWW.UTH.TMC.EDU/PATHOLOGY/MEDIC/IMMUNOLOGY/IMMUNO/DISORDERS.HTML
IV.
General Considerations
There are greater than 120 types of primary immunodeficiency that have
been characterized and their incidence in the general population is 1:10,000
(aside from the extremely common selective IgA deficiency, 1:500). The most
serious forms of immunodeficiency, such as severe combined immunodeficiency
and X-linked agammaglobulinemia, occur in 1:100,000 individuals. At least 58%
of cases are diagnosed in children (less than 5 yr) and 83% of these patients are
male. Autosomal recessive, X-linked recessive, and familial inheritance patterns
are observed.
V.
Etiology
It is likely that genetic abnormalities underlie all primary states of
immunodeficiency. The simplest concept to remember is the scheme of the
developing immune system. T and B lymphocytes pass through unique
development stages as they mature and differentiate from the primordial stem
cells in the bone marrow. Probably due to an underlying genetic lesion causing
absent or altered enzymes, lymphocyte maturation may stop at a certain stage,
deemed an “arrest point." Lymphocyte maturation arrest points may lead to
clinical immunodeficiency states; for example, in patients with X-linked
agammaglobulinemia (X-LA, B lymphocyte maturation is interrupted between the
pre-B and B cell stage. By virtue of their lack of mature B cells, patients with XLA have no circulation of antibody-producing plasma cells, and are very
susceptible to infection with multiple organisms. Patients with common variable
immunodeficiency possess B cells which are present but malfunctional, due to
intrinsic developmental block between B cells and plasma cells, failure to secrete
immunoglobulin, or lack of effective T helper cell function. Patients with X-linked
severe immunodeficiency lack maturation of stem cells into mature T cells.
240
VI.
Clinical Features
The principal manifestation of immunodeficiency is an increased susceptibility
to infection as documented by: a) increased frequency of infection, b) increased
severity of infection, c) prolonged duration of infection, d) development of an
unexpected complication or unusual manifestation, e) infection with organisms of low
pathogenicity. Certain types of cancer, e.g., Epstein-Barr virus-induced B-cell
lymphomas, appear in patients with certain immunodeficiencies. Autoimmune
conditions also appear in patients with immunodeficiencies.
VII.
Antibody (B Cell) Disorders
A.
X-Linked (Bruton's) Agammaglobulinemia (X-LA)
Patients with X-LA suffer from recurrent serious infections that begin in
early childhood, and are found to lack all classes of immunoglobulin and
circulating B cells. Plasma cells do not develop and antibody responses are
absent. Pre-B cells are present in the bone marrow of patients indicating a
failure of differentiation into mature B cells.
Recurrent bacterial and pyogenic (pus producing) infections begin
between 6 and 9 months due to wane of maternal IgG acquired transplacentally.
Since the half-life of IgG is 23 days, in about 5-6 months the infant's serum IgG is
virtually nil. Infections include pneumonia, sinusitis, otitis, pyoderma,
osteomyelitis, and meningitis. Sepsis may result from infection with organisms
such as pneumococci and streptococci, which have an exterior polysaccharide
capsule normally coated by host IgG (opsonin) and digested by phagocytic cell
(contain Fc receptor for IgG).
The molecular basis of X-linked agammaglobulinemia has been shown to
be due to the lack of a cytoplasmic tyrosine kinase, which prevents B cell
maturation, and production of immunoglobulins. There are also autosomal
recessive forms of agammaglobulinemia that involved mutations in the mu heavy
chain of immunoglobulins.
B.
Selective IgA Deficiency
This is the most common form of immunodeficiency, which occurs in 1/500
individuals and has significant associations with several other diseases usually of
an autoimmune nature. There is a strong familial association--possibly
autosomal dominant inheritance with incomplete penetrance, although recessive
patterns have been seen. Patients with IgA deficiency may develop CVID or
have relatives with CVID (see below.). There are low or normal numbers of
mature B cells that fail to secrete IgA due to an unknown genetic lesion (the
alpha heavy chain gene is intact). Since IgA coats mucosal surfaces, it is not
unreasonable that gastrointestinal symptoms of infection and malabsorption
should be prominent in the IgA-deficient population.
C.
Common Variable Immunodeficiency (CVID)
This name represents a heterogenous group of patients that sometime
after infancy, usually from 15-35 years of age, develop recurrent bacterial
infections, decreased immunoglobulin levels and impaired antibody responses.
Cellular immunity is usually normal or minimally defective except when patients
become debilitated. Approximately 20% of cases of CVID are now known to be
241
activator and calcium-modulator and cyclophilin interaction) located on B cells
that normally binds to B cell activating factor of the TNF family (BAFF) and a
proliferation-inducing ligand (APRIL) which regulates isotype switching.
Autosommal recessive inheritance patterns have been noted in a subset of CVID
patients who lack the normal inducible co-stimulator (ICOS), leading to inability to
make specific antibody.
Prominent clinical problems are those of bronchiectasis (persisting
infections of the periphery of the lung tissue) and intestinal giardiasis (parasite)
leading to extreme debilitation and premature demise. Immunization with an
antigen leads only to production of low levels of IgM antibody without the normal
switch to IgG upon repeated immunization. There is also a high incidence of
associated autoimmune disease and malignancies and a familial tendency for
these diseases. Both CVID and IgA deficiency may map to a susceptibility gene
on the 6th chromosome in the Class II MHC region.
D.
Hyper IgM (HIM) Disorders
This immunodeficiency is usually X-linked and appears in males (HIM-1);
but females are also affected, indicating an autosomal inheritance as well (i.e.,
defective activation-induced deaminase [AID]) or a defect in uracil DNA
glycosylase (UNG), both termed HIM-2. Another autosomal recessive defect
(abnormal CD40 molecule on B cells) has been discovered (HIM-3). In all types,
the serum IgG and IgA are usually totally absent or markedly reduced; IgM
concentration is normal to very high. Patients may have high titers of some IgM
antibodies but often no or very low specific antibody formation. The percentage
of peripheral blood lymphocytes with surface Ig is normal. There are usually
normal T cell mitogen responses but some patients develop T cell deficiency with
time. Patients often have associated neutropenia and/or autoimmune
phenomena and infections are common. A high incidence of malignancies is
also seen in these patients. In males, the abnormal gene in the X-linked type of
HIM-1 has been mapped to Xq26,27. The normal gene product is the ligand
(CD40L present on T cells) that binds to CD40 on B cells, leading to cell
activation. Thus, the B cells appear to be intrinsically normal.
E.
Wiskott-Aldrich Syndrome
The Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder with
variable clinical phenotypes that correlate with the type of mutations in the WAS
protein (WASP) gene. WASP, a key regulator of actin polymerization in
hematopoietic cells, has 5 well-defined domains that are involved in signaling,
cell locomotion, and immune synapse formation. WASP facilitates the nuclear
translocation of nuclear factor kappaB and was shown to play an important role
in lymphoid development and in the maturation and function of myeloid
monocytic cells. Mutations of are located throughout the gene and either inhibit
or dysregulate normal WASP function. Analysis of a large patient population
demonstrates a phenotype-genotype correlation: classic WAS occurs when
WASP is absent, X-linked thrombocytopenia when mutated WASP is
expressed, and X-linked neutropenia when missense mutations occur in the
Cdc42-binding site. The progress made in dissecting the function of WASP has
242
provided new diagnostic possibilities and has propelled our therapeutic
strategies from conservative symptomatic treatment to curative hematopoietic
stem cell transplantation and toward gene therapy.
VIII.
Cellular Disorders
A.
T-Cell Deficiency: DiGeorge Syndrome (DGS)
DiGeorge first described (1965) the association of infection, absent
thymus, and congenital hypoparathyroidism. There are other associated
congenital abnormalities: cardiovascular defects, abnormal facies, urinary tract
abnormalities, and orthopedic abnormalities. There is failure of the 3rd and 4th
pharyngeal pouches to develop at about 10 weeks of embryonic life. Originally,
DGS was believed to occur on a sporadic basis but familial associations have
been noted. Children with the syndrome come to the attention of the physician
because of seizures on the first day of life due to low calcium in the blood. If the
children survive the neonatal period (1 mo.) increased susceptibility to
opportunistic infection occurs with fungi, such as Candida albicans and
Pneumocystis carinii. About 95% of DGS patients have chromosomal deletions
(22q11 [also have velocardiofacial syndrome] or 10q13). A chest x-ray is
frequently helpful in making the diagnosis, since the thymic shadow is absent or
reduced in size. An animal model of DiGeorge syndrome lacks the Tbx1 gene,
but this abnormal gene is not universally found in DGS.
It is now becoming clear that, in addition to the complete DGS, where
there is a total lack of T-cell immunity, there exist partial syndromes where T-cell
numbers and T-cell responses to mitogens and antigens may be completely
absent or intermediate in value. Infants usually have normal serum
immunoglobulins and normal circulating B lymphocytes but do not mount a
specific antibody response to antigens because of lack of T cell help.
B.
T- and B-Cell Deficiency
1.
Severe Combined Immunodeficiency (SCID)
There are many types of combined (B- and T-cell) immunodeficiency
which result in recurrent life-threatening infections, severe diarrhea, and failureto-thrive. The classical form of SCID is X-linked, but there are autosomal
recessive forms, as well as sporadic forms. The usual lymphocyte analysis
reveals an absence of lymphocytes bearing mature T-cell antigens (CD3, CD4,
CD8) and inability of lymphocytes to respond to mitogenic and antigenic
stimulation. Frequently, there are small numbers of B cells with mature antigens
(CD19, CD20), but most infants with SCID do not make serum immunoglobulins.
The genetic and molecular defect in X-linked SCID is a mutation in the gene that
codes for the gamma chain of the IL-2 receptor. Without a normal IL-2 receptor,
normal T-cell maturation and proliferation cannot take place. Consequently, all
normal T-cell functions are absent. The profound nature of the SCID defect is
due to the fact that the defective gamma chain renders not only the IL-2 receptor
dysfunctional but also the IL-4, IL-7, IL-15, and IL-21 receptor.
There are several other forms of SCID involving genetic lesions (all
inherited in autosomal fashion) in the: 1) T-cell receptor complex, CD3; 2) kinase
243
enzyme, JAK-3; 3) interleukin receptor, IL-7R; 4) IL-2R; 5) T-cell receptor
recombinase enzymes, RAG1/RAG2; 6) maturity marker, CD45; 7) major
histocompatibility complexes, MHC-1 due to TAP-1, -2 (transporter associated
with antigen processing) deficiency; 8) MHC-2; 9) kinase enzyme, ZAP-70
(absence of CD8+ T cells); 10) unknown gene termed ARTEMIS; 11) nucleic acid
enzyme adenosine deaminase, ADA; and 12) nucleoside phosphorylase, NP.
These defects can also be classified according to T-cell phenotypes, e.g.,
X-linked SCID is T cell (-) B cell (+) NK cell (-); ADA deficiency is T cell (-) B cell
(-) NK cell (-); RAG1, RAG2 deficiency is T cell (-) B cell (-) NK cell (+); and IL-7
deficiency is T cell (-) B cell (+) NK cell (+).
2.
Non-SCID forms of T- and B-Cell Deficiency
These conditions, although eventually fatal, permit up to several years of
life, although the quality is poor because of recurrent infections and malignancy.
These conditions are: 1) Wiskott-Aldrich syndrome (WAS), 2) Ataxiatelangiectasia (AT), 3) Nijmegen breakage syndrome (NBS), 4) X-linked
lymphoproliferative disorder (X-LP), 5) NFkB essential modifier (NEMO), 6)
warts, hypogammaglobulinemia, infections, myelokathexis (WHIM), 7) absence
of caspase 8, and 8) hyper IgM due to X-linked (HIM-1), and an autosomal
recessive gene defect in the gene coding for CD40 on B cells (HIM-3).
C.
Other Cell-Derived Combined Immunodeficiency
Defects in non-B/T cells can cause serious immunodeficiencies as severe
as SCID. For example, defects in the IFN-R or IL-12R on antigen-presenting
monocytes/macrophages cause repetitive serious infections, most commonly
with atypical mycobacteria. Natural killer (NK) cell deficiency leads to chronic viral
infections and malignancy because of lack of immunosurveillance. Although
these other cells are not part of the adaptive immune system, their impact upon
B- and T-cell function is so profound that the result of their dysfunction is
tantamount to severe B- and T-cell deficiency. In the case of IFN-R/IL-12R
deficiency, there is so much infection with mycobacteria that B- and T-cell
resistance prove inadequate. In the case of NK-cell deficiency, the innate
function of viral clearance is missing, and again B- and T-cell resistance is
overwhelmed.
IX. Phagocytic Immunodeficiency
A.
Chronic Granulomatous Disease
The engulfment process in phagocytic cells is associated with increased
anaerobic glycolysis and ATP consumption. There is a burst of respiratory
oxidative activity, increased oxygen consumption, and a shift to glucose
metabolism via the hexose monophosphate shunt. As glucose is utilized by the
leukocytes, reduced pyridine nucleotides (NADH and NADPH) accumulate, and
cytochrome oxidase activities increase with the final production of H2O2, which
(after subsequent metabolism) is toxic to bacteria (O2-, superoxide). CGD
leukocytes, however, demonstrate no increased O2 uptake, no shift to the hexose
monophosphate shunt and no H2O2 production. There are several genetic
defects, the principal defect being the abnormal gp91 in the membrane portion of
the cytochrome B558 which is inherited in X-linked fashion (chromosome
244
). The white blood cell count (WBC) is high in an attempt to compensate
for lack of bacterial killing.
Laboratory tests used to diagnose CGD are the nitroblue tetrazolium dye test
(NBT), dihydro rhodamine test (DHR), and the chemiluminescence assay. All tests
measure H2O2 and subsequent superoxide production by the granulocyte. Gamma
interferon has been shown to augment the effectiveness of antimicrobial treatments in
CGD, but bone marrow transplantation offers the only permanent therapy.
B.
Leukocyte Adhesion Deficiency
Leukocyte adhesion deficiency-1 (LAD-1) is a rare autosomal recessive disorder
characterized by recurrent bacterial and fungal infections and impaired wound healing in
spite of leukocytosis. Classically there is a history of delayed separation of the umbilical
stump in affected individuals. In these patients, most adhesion-dependent functions of
leukocytes are abnormal. The molecular basis of the defect is absent or deficient
expression of the β2 integrins, or the CD11CD18 family of glycoproteins (chromosome
21q22.3), which includes leukocyte function-associated antigen-1 (LFA-1 or
CD11aCD18), Mac-1 (CD11bCD18), and p150,95 (CD11cCD18). These proteins
participate in the adhesion of leukocytes to other cells and in the phagocytosis of
complement-coated particles. In all patients studied so far, the defect has been
mapped to the 95 kD β chain (CD18). The gene encoding this chain may be mutated,
producing an aberrant transcript, or its transcription may be reduced.
Leukocyte adhesion deficiency-2 (LAD-2) is another disorder described in a very
small number of patients that is clinically indistinguishable from LAD-1 but is not due to
integrin defects. In contrast, LAD-2 results from an absence of sialyl-Lewis X, the
carbohydrate ligand on neutrophils that is required for binding to E-selectin and perhaps
P-selectin on cytokine-activated endothelium. It is likely that LAD-2 patients have
mutations in genes encoding enzymes involved in fucose metabolism.
Like other genetic defects affecting leukocytes, leukocyte adhesion
deficiencies are candidates for bone marrow transplantation and ultimately
specific gene therapy.
X.
Disorders of the Toll-Like Receptor and Complement Systems
A.
Toll-Like Receptors
Toll-like receptors (TLRs) are a series of 10 receptors in humans that
participate in detecting pathogens. TLRs recognize a small number of pathogenassociated molecular patterns (PAMPs). PAMPs represent molecules (LPS,
lipoproteins, flagellin, unmethylated DNA, double-stranded and single-stranded RNA)
that promote for the survival of a pathogen. Activation of TLRs leads to recruitment of
neutrophils and macrophages to sites of infection and augmentation of antimicrobial
activity. On engagement of TLRs, dendritic cells (DCs) undergo maturation and migrate
to draining lymph nodes, where they present antigen to T cells. PAMPs binding to all
known Toll-like receptors cause the production of inflammatory cytokines, including
TNF-α. IRAK-4 (interleukin receptor-associated kinase-4) is a critical effector in
signaling by TLRs and the IL-1 receptor, which share homology in their intracellular
domain. Patients with IRAK-4 deficiency are susceptible to invasive bacterial infections
and to viral infections.
245
B.
Complement
The complement system is a complex of approximately 31 innate host defense
proteins that acts in three activation patterns (classical, alternate, and lectin-binding) to
augment adaptive host defense mechanisms and to exert a bacteriolytic effect of its
own. Genetic deficiencies or mutations of any one of these proteins leads to impaired
host defense.
The prototype complement component deficiency disease is that of C3
deficiency, a very rare disorder but one that allows us to assess the crucial importance
of the total complement pathway in host-defense mechanisms. Since C3 is the pivotal
complement component through which both classical and alternative pathways act, its
absence does not permit complete activation of complement. Because of the lack of C3
chemotactic factors, C3a and C5a are not released and phagocytic cells are not drawn
to the focus of infection. Moreover C3b serves as an opsonin of bacteria and by virtue
of a chemical affinity of C3b for a receptor on phagocytic cells (polymorphonuclear
leukocytes and monocytes) the engulfment of a C3b-coated bacterium by a phagocytic
cell is facilitated. Persons with complement component deficiencies of C1, C2, or C4
can still activate the complement cascade via the alternative pathway and persons with
complement component deficiencies of C5, C6, C7, C8, or C9 can still generate
chemotactic factors and have opsonin (C3b) function.
Pyogenic infection (streptococcal, staphylococcal) and autoimmune diseases are
associated with early complement component deficiencies (i.e., C1, C2, C4) and
meningococcal and gonococcal infections are associated with late complement
component deficiencies (i.e., C5-C9).
XI.
Treatment of Immunodeficiency Diseases
A.
Antibody (B Cell) Disorders
1.
Replace IgG deficiencies with intravenous immunoglobulin
2.
General supportive care very important
3.
Treat complications with appropriate medications, e.g., autoimmune
disease with immunomodulators.
B.
Cellular Disorders (T and B cells, monocyte/macrophages)
1.
Bone marrow stem cell transplantation
2.
Intravenous IgG
3.
Gene therapy where possible
4.
General supportive care
5.
Cytokine therapy in selected defects, e.g., IL-2 for IL-2R
deficiency
Phagocyte Deficiency
1.
IFN- for CGD
2.
General supportive care
3.
Bone marrow stem cell transplants (patients bone marrow must be
ablated with chemotherapy)
C.
D.
Complement Deficiency
1.
General supportive care
246
XII.
Diagnosis of Immunodeficiency Diseases
A.
Medical History
1.
Age of patient at onset of symptoms
2.
History of live microbial immunizations
3.
Severity of illness
4.
Family history
B.
Physical Examination
1.
Tonsils present
2.
Palpate lymph nodes
3.
Organomegaly
4.
Growth - measurements
C.
Laboratory Investigation
1.
Antibody function
a.
Immunoglobulin levels
b.
Specific antibody responses
- Isohemagglutinins (anti A, anti B)
- Anti-diphtheria and tetanus antigen antibodies
- Anti X 174 antibody
2.
B and T Lymphocyte, NK cell, and monocyte/macrophage function
a.
T cell
- Delayed hypersensitivity skin testing (e.g. SK-SD, monilia
antigens)
- T cell subsets (flow cytometry)
- Mononuclear cell phenotypes (subsets-monoclonal
antibodies)
- PHA reactivity and antigen reactivity
- Specific antigen stimulation in vitro
- T cell excision circles (TREC)
- V TCR spectratyping
b.
B cell (B cell subsets by flow cytometry)
- Memory (CD27+) B cells
c.
Nucleic acid enzyme assay
- Adenosine deaminase
- Nucleotide phosphorylase
d.
NK cell surface markers and functional assay
e.
Monocyte/macrophage receptor assays
3.
White blood cell function
a.
WBC
b.
NBT, DHR tests
c.
CD11a,b,c CD18 assay by flow cytometry
d.
Chemotaxis and opsonization assays
4.
Complement function
a.
Total hemolytic complement
b.
C3
c.
C4
247
5.
XIII.
Molecular and genetic studies
Summary (Key Concepts)
A.
B.
C.
D.
E.
Inherited gene defects are causes of primary immunodeficiency.
Lack of immune effector function produces infection.
Defective function occurs in B, T, NK, PMN, or M cells.
Diagnostic tests augment medical history and physical exam.
Treatment attempts to replace what is missing.
248
XIV.
Questions
1.
A one-month-old boy develops severe respiratory syncytial virus
infection and requires ventilator support. His father is healthy, but
his 21-year-old mother has never enjoyed good health since being 15
years of age. His T and B cell phenotyping and function are normal.
His phagocyte function and complement function are normal. His
serum IgM and IgA levels are normal, but his serum IgG level is
almost zero. How would you explain this child’s illness?
A.
B.
C.
D.
E.
2.
A 16-year-old girl develops recurrent fevers and a red fixed rash over
her nose and upper cheeks that resembles a butterfly. A diagnosis of
autoimmune disease (systemic lupus erythematosus) is made by the
clinical immunologist. Which additional test is likely to be abnormal?
A.
B.
C.
D.
E.
3.
The child has selective IgG deficiency.
The father’s silent X-LA is being expressed in the child.
The child has hyper IgM syndrome.
CVID is being expressed at an early age.
The mother has an immunodeficiency.
NBT
IgA
C9
ADA
CD18
A 30-year-old woman with recurrent pneumonias is diagnosed with
hypogammaglobulinemia. A search of her family members reveals that
three of her children have low serum immunoglobulins, two of the
children being girls and one a boy. Both parents of the woman have low
serum immunoglobulin levels and two of her sisters have low serum
immunoglobulins. What is the most likely diagnosis?
A.
B.
C.
D.
IFN-R deficiency
IgA deficiency
Common variable immunodeficiency
Hyper IgM syndrome
4.
A 2-year boy was found to have repeated infections, easy bruisability,
and mucosal bleeding. His platelet count was 10,000/L (low) and his
antibody isotype failed to switch from IgM to IgG upon boosting with a
de novo polysaccharide antigen ( X-174 bacteriophage). What test
would lead to a definitive diagnosis?
A.
Lymphoproliferation to  X-174
B.
Flow cytometry test for intracellular WASP
c.
Delayed hypersensitivity test for TB
D.
Superoxide generation by neutrophils
5.
A two-month-old boy develops high fever and pan lobar pneumonia.
There is no thymic shadow on the chest x-ray. His lymphocyte
249
phenotyping is normal, except for a complete absence of CD8+ cells.
What would be your diagnosis?
A.
B.
C.
D.
E.
The child has idiopathic CD8+ T-cell deficiency.
The laboratory technician forgot to add the anti-CD8 monoclonal
antibody to the lymphocytes.
The child has the Wiskott-Aldrich syndrome.
The child has JAK-3 deficiency.
The child has ZAP-70 deficiency.
6.
A 4-year male patient, who has suffered draining lymph nodes in the
neck on 3 occasions and a liver abscess all due to Staphylococcus
aureus (4 items) infection, is diagnosed with chronic granulomatous
disease by means of stimulated production of superoxide in white blood
cells (WBC) using the dihydrorhodamine assay in a flow cytometer. His
parents are also tested. What would you expect the WBC test results to
show?
Mother
Father
Patient
A
0%
10%
100%
B
0%
0%
100%
C
0%
50%
50%
D
0%
100%
10%
7.
A one-year-old girl is brought to the pediatrician because of a deep and
painful perirectal abscess. Her white blood cell count (WBC) (mostly
neutrophils) is greater than 50,000 (normal less than 10,000), and her
spleen is greatly enlarged. Mother, father, and two siblings are healthy.
Lymphocyte function, antibody formation, NBT test, and complement
function are normal. What would you tell the anxious parents?
A.
B.
C.
D.
E.
8.
The child has an intercurrent illness that requires antibiotics.
The WBC is too high and needs reduction by splenectomy.
Another white blood cell test is necessary.
Complement tests by a reference laboratory will be necessary.
IFN- therapy will eliminate the infection.
An 18-year-old student who has been well all of his life develops
three bouts of gonococcal infections in his first year at college. How
would you respond?
A.
B.
C.
D.
E.
The exuberance of youth explains this problem.
Investigation of congenital T-cell deficiencies will be necessary for
diagnosis.
The patient needs intravenous IgG to remain infection free.
The IFN-R on his antigen-presenting cells is defective.
There is a defect in one of the C5-C9 complement pathways.
250
TRANSPLANT IMMUNOLOGY
Wasim Dar, M.D., Ph.D.
MSB 6.256
713-500-7400
[email protected]
Objectives: (1) Discuss the immunobiology of transplantation. (2) Detail the contributing cells
and factors involved in transplant acceptance vs. rejection. (3) Appreciate the importance of
innate and adaptive functions in graft recognition. (4) Define molecular aspects of hyperacute,
acute and chronic rejection. (5) Recognize clinical consequences of transplantation.
Keywords: Allorecognition; rejection, GVHD, xenograft.
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New
York, NY. 6th edition, 2009. Chapter 18; Geha and Notarangelo. Case Studies in Immunology.
Garland Publishing, New York, NY. 6th edition, 2012. Case 11. Graft-Versus-Host Disease.
Kidney Graft Complications (Blackboard file, case #46). Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/transplantation.html
Immunobiology of Transplantation
I. The Contributors: The immunobiology of transplantation should be considered as a complex
immune response to stress and injury.
A.
Innate Immunity: the contributions of innate immunity to immune reaction in organ
transplantation have only recently been more robustly explored
1. Toll Like Receptors (TLR)
a. Expressed on immune cells and epithelial tissue of organs
b. TLR signaling
c. DAMPS (damage associated molecular patterns)
d. Ischemia reperfusion injury leads to increased expression of DAMPS with
interact with TLRs to induce further direct cell injury and injury via immune
cells
e. TLR injury primes the adaptive immune system to react against allografts
f. TLR2 and TLR4
251 2. Complement
a. Three pathways
i.
Lectin
ii.
Alternative
iii.
Classical
b. The Lectin and alternative pathways appear to have greatest role in
transplantation
c. Mechanisms of tissue injury
i.
Complement activation leads to direct tissue injury via the
membrane attack complex
ii.
Alternatively, complement products, particularly C5a act on C5a
receptors in organ tissue to cause cell death.
iii.
C5a and C3a cause migration of neutrophils into transplant organs;
neutrophils mediate tissue destruction and injury
d. Complement and Adaptive immunity: Complement products C5a and C3a can
act on antigen presenting cell receptors and T-cells
B.
Adaptive Immunity: Well studied in transplantation
1. T-cells-thymic selection of T-cells results in a large repertoire of cells that have
allospecificity.
a. Allospecific T-cells react against transplanted organs when they are activated
by antigen presenting cells
b. Direct vs. indirect allorecognition
c. Many T-cell populations arise: Activation via the TCR is key-3 signal
model/costimulation
2. B-cells: B-cell contributions to transplant immunobiology have been historically less
well explored
a. B-cells are now well recognized to have key roles in the following processes
i.
Hyperacute rejection
ii.
Antibody mediated rejection
iii.
Graft fibrosis and chronic graft injury
252 iv.
C.
Sensitization
These processes are almost always a reflection of IgG antibodies in the recipients
against donor HLA
b. Thus before completing our discussion about the process above we need to put
into context how these processes were discovered. Terasaki reported that
patients with antibodies against HLA lost their grafts
3. The Crossmatch: basically the idea of detecting antibodies against donor HLA
a. Methods
i.
CDC crossmatch
ii.
Flow crossmatch
iii.
Virtual crossmatch
b. Which organs is it important for?
c. Does tissue typing matter?
II. Consequences of Transplantation
1. Rejection
2. Chronic graft loss
3. Tolerance.
253 Spring Semester, 2013
Team Based Learning Exercise
The Immunology course will have one Team Based Learning exercise where
students will be required to address a clinically based scenario and provide answers
to related questions. Students will be assigned specific reading prior to the session,
which will assist in mastering of the material so as to allow participation in the group
activities. Materials will include new material in Immunology, as well as materials
already mastered in other courses. The format will be similar to the Clinical Applications
course.
The Team Based Learning Exercise is mandatory.
The Team Based Learning Exercise encompasses a graded set of exercises related to
multiple integrated aspects of a clinical scenario. The exercise is worth a maximum of 10
points towards your overall Immunology grade.
The session will utilize clinical scenario(s) to present problem(s). Students are divided
into teams; utilizing the groups already in place for the Clinical Applications course.
Approximately 5 problem questions arising from the clinical scenario are crafted to
foster discussion within the teams; each team is required to come to a consensus as to the
solution to the problem. Written justification may be required for the team solution, to
be prepared and handed in for grading at the end of the session.
Team Based Learning Exercise: February
Immunology
28th
8:00-9:50 a.m.
Persons missing the session must provide written notice explaining circumstances
for not attending. Written approval must be obtained from the Office of
Educational/Student Affairs prior to consideration for any makeup session or
alternate assignment.
254
EVOLUTION OF THE IMMUNE SYSTEM
Adan Rios, M.D.
Department of Internal Medicine, Oncology Division
UT Medical School at Houston Texas
[email protected]
Objectives: (1) Discuss the emergence of innate and adaptive immunity on a phylogenetic scale.
(2) Examine the pressures in nature leading to maturation of immune potential. (3) Discuss the
fate of duplicated genes in evolutionary development of immune function. (4) Compare the
evolutionary co-option of ancient biological systems. (5) Recognize immunological paradoxes as
necessary for elicitation of protective responses to infectious agents.
Web Resource:http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/Evolution.html
Full Syllabus Chapter to be distributed via Blackboard prior to lecture presentation.
255
Timeline of Immunology
Sources: Wikipedia, Timeline of Immunology; Immunology History IV, History of Immunology
Time Line (Keratin.com); Stewart Sell and Scott L. Rodkey, A short history of
Immunopathology.




























3000 B.C.E. – Fever (Mesopotamia)
2000 B.C.E. - Recognition of “adaptive” protection against disease (Egypt, China)
400 B.C.E. – Anatomic identification of organs (Hippocrates)
80 B.C.E. – Acquired resistance to poinsons (Mithridate Eupator, King of Pontus)
25 – Four cardinal signs of inflammation (Celsus)
1000 – “Snuff” variolation for smallpox (Sung Dynasty, China)
1590 – Bursa of birds described (Fabricius)
1690 – Lymphoid tissue identified in small intestine (Peyer)
1718 - Lady Mary Wortley Montagu, the wife of the British ambassador to Constantinople,
observed the positive effects of variolation on the native population and had the technique
performed on her own children.
1798 - First demonstration of vaccination smallpox vaccination (Edward Jenner)
1837 - First description of the role of microbes in putrefaction and fermentation (Theodore
Schwann)
1838 - Confirmation of the role of yeast in fermentation of sugar to alcohol (Charles
Cagniard-Latour)
1840 - First "modern" proposal of the germ theory of disease (Jakob Henle)
1850 - Demonstration of the contagious nature of puerperal fever (childbed fever) (Ignaz
Semmelweis)
1855 – Tuberculous granulomas identified (Rokitansky)
1868 – Langhans Giant Cells identified (Langhans)
1857-1870 - Confirmation of the role of microbes in fermentation (Louis Pasteur)
1862 - phagocytosis (Ernst Haeckel)
1867 - First aseptic practice in surgery using carbolic acid (Joseph Lister)
1876 - First demonstration that microbes can cause disease-anthrax (Robert Koch)
1877 - Mast cells (Paul Ehrlich)
1878 - Confirmation and popularization of the germ theory of disease (Louis Pasteur)
1880 – Birth of Cellular Pathology (Virchow)
1880 - 1881 -Theory that bacterial virulence could be attenuated by culture in vitro and
used as vaccines. Proposed that live attenuated microbes produced immunity by depleting
host of vital trace nutrients. Used to make chicken cholera and anthrax "vaccines" (Louis
Pasteur)
1883 - 1905 - Cellular theory of immunity via phagocytosis by macrophages and microphages
(polymorhonuclear leukocytes) (Elie Metchnikoff)
1885 - Introduction of concept of a "therapeutic vaccination". First report of a live
"attenuated" vaccine for rabies (Louis Pasteur)
1887 – Anti-rattlesnake venom discovered (Sewall)
1888 - Identification of bacterial toxins (diphtheria bacillus) (Pierre Roux and Alexandre
Yersin)
Time Line 1










































1888 - Bactericidal action of blood (George Nuttall)
1890 - Demonstration of antibody activity against diphtheria and tetanus toxins. Beginning
of humoral theory of immunity. (Emil von Behring) and (Shibasaburo Kitasato). Attempt to
cure tetanus with passive immunotherapy (Behring)
1891 - Demonstration of cutaneous (delayed type) hypersensitivity (Robert Koch)
1893 - Use of live bacteria and bacterial lysates to treat tumors-"Coley's Toxins" (William
B. Coley)
1894 - Bacteriolysis (Richard Pfeiffer)
1896 - An antibacterial, heat-labile serum component (complement) is described (Jules
Bordet)
1900 - Antibody formation theory “side chain theory” “horror autotoxicus” (Paul Ehrlich)
1901 - blood groups (Karl Landsteiner)
1901-8 Carl Jensen & Leo Loeb, Transplantable tumors
1902 - Immediate hypersensitivity anaphylaxis (Paul Portier) and (Charles Richet)
1902 Paul Portier & Charles Richet, Anaphylaxis
1903 - Intermediate hypersensitivity, the "Arthus reaction" (Maurice Arthus)
1903 - Opsonization (Almroth Wright & Stewart Douglas)
1905 - "Serum sickness" allergy (Clemens von Pirquet and (Bela Schick)
1905 – successful organ transplantation (Correl and Guthrie)
1906 – Clemens von Priquet, coined word “allergy”
1907 - Svante Arrhenius, coined the term immunochemistry
1910 - Emil von Dungern, & Ludwik Hirszfeld, Inheritance of ABO blood groups
1910 - Peyton Rous, Viral immunology theory
1911 - 2nd demonstration of filterable agent that caused tumors (Peyton Rous)
1914 - Clarence Little, Genetics theory of tumor transplantation
1915-20 - Leonell Strong & Clarence Little, Inbred mouse strains
1917 - hapten (Karl Landsteiner)
1921 - Cutaneous allergic reactions (Carl Prausnitz and Heinz Küstner)
1922 – Fleming found lysozyme and penicillin
1924 - Reticuloendothelial system (Aschoff)
1925 – Chemical mediators of inflammation (Lewis)
1926 - Lloyd Felton & GH Bailey, Isolation of pure antibody preparation
1935 Quantitative precipitin reaction (Heidelberger)
1936 - Peter Gorer, Identification of the H-2 antigen in mice
1938 – Gammaglobulin identified (Tiselius and Kabat)
1938 - Antigen-Antibody binding hypothesis (John Marrack)
1940 - Identification of the Rh antigens (Karl Landsteiner and Alexander Weiner)
1941 – Hemolytic disease of the newborn (Rh antigens) (Levine)
1941 - Albert Coons, Immunofluorescence technique
1942 - Anaphylaxis (Karl Landsteiner and Merill Chase)
1942 - Adjuvants (Jules Freund and Katherine McDermott)
1944 - hypothesis of allograft rejection (Peter Medawar)
1945 - Passive transfer of cell mediated immunity (Chase)
1946 - identification of mouse MHC (H2) by George Snell and Peter A. Gorer
1947 – Twins do not demonstrate transplant rejection (Owen)
1948 - antibody production in plasma B cells (Astrid Fagraeus)
Time Line 2



































1949 - growth of polio virus in tissue culture, neutralization with immune sera, and
demonstration of attenuation of neurovirulence with repetitive passage (John Enders) and
(Thomas Weller) and (Frederick Robbins)
1949 - immunological tolerance hypothesis (Macfarlane Burnet & Frank Fenner)
1950 - Richard Gershon and K Kondo, Discovery of suppressor T cells
1952 - Ogden and Bruton, discovery of agammagobulinemia (antibody immunodeficiency)
1951 - vaccine against yellow fever
1953 - Graft-versus-host disease (Morton Simonsen and WJ Dempster)
1953 - immunological tolerance hypothesis (Rupert Billingham, Leslie Brent, Peter Medwar, &
Milan Hasek)
1953 - James Riley & Geoffrey West, Discovery of histamine in mast cells
1955-1959 - Niels Jerne, David Talmage, Macfarlane Burnet, Clonal selection theory
1957 - Clonal selection theory (Frank Macfarlane Burnet)
1957 - Discovery of interferon (Alick Isaacs & JeanLindermann)
1957 Ernest Witebsky et al., Induction of autoimmunity in animals
1958-1962 - Discovery of human leukocyte antigens (Jean Dausset and Snell)
1959-1962 - Discovery of antibody structure (independently elucidated by Gerald Edelman
and Rodney Porter)
1959 - Discovery of lymphocyte circulation (James Gowans)
1960 - Discovery of lymphocyte "blastogenic transformation" and proliferation in response
to mitogenic lectins-phytohemagglutinin (PHA) (Peter Nowell)
1961-1962 Discovery of thymus involvement in cellular immunity (Jacques Miller)
1961- Demonstration that glucocorticoids inhibit PHA-induced lymphocyte proliferation
(Peter Nowell)
1962 – Classification of immune mechanisms (Gell and Coombs)
1963 - Development of the plaque assay for the enumeration of antibody-forming cells in
vitro (Niels Jerne) (Albert Nordin)
1963 - Jaques Oudin et al., antibody idiotypes 1964-1968 T and B cell cooperation in immune
response (Anthony Davis)
1964 – Mixed lymphocyte reaction (Bain, Vas, et al.)
1965 - Discovery of the first lymphocyte mitogenic activity, "blastogenic factor" (Shinpei
Kamakura) and (Louis Lowenstein) (J. Gordon) and (L.D. MacLean)
1965 - Discovery of "immune interferon" (gamma interferon) (E.F. Wheelock)
1965 - Secretory immunoglobulins (Thomas Tomasi et al.)
1966 - Identification of T and B cells (Claman)
1967 - Identification of IgE as the reaginic antibody (Kimishige Ishizaka)
1968 - Passenger leukocytes identified as significant immunogens in allograft rejection
(William L. Elkins and Ronald D. Guttmann)
1968 – Accessory cell role in immune response (Mosier)
1969 - The lymphocyte cytolysis Cr51 release assay (Theodore Brunner) and (Jean-Charles
Cerottini)
1969 – Immune response genes (Benacerraf and McDevitt)
1971 - Donald Bailey, Recombinant inbred mouse strains
1971 - Peter Perlmann and Eva Engvall at Stockholm University invented ELISA
1972 - Structure of the antibody molecule
1974 – Network theory for antibody control on immune response (Niels K. Jerne)
Time Line 3
































1974 - T-cell restriction to major histocompatibility complex (Rolf Zinkernagel and (Peter
Doherty)
1975 - Generation of the first monoclonal antibodies (Georges Köhler) and (César Milstein)
1975 – Identification of natural killer cells (Kiessling, et al.)
1976 - Identification of somatic recombination of immunoglobulin genes (Susumu Tonegawa)
1979 - Generation of the first monoclonal T cells (Kendall A. Smith)
1980 – Immunoglobulin structure (Kabat)
1980-1983 - Discovery and characterization of the first interleukins, 1 and 2 IL-1 IL-2
(Kendall A. Smith)
1981 - Discovery of the IL-2 receptor IL2R (Kendall A. Smith)
1981 – Appearance of AIDS on a global scale
1983 - Discovery of the T cell antigen receptor TCR (Ellis Reinherz) (Philippa Marrack) and
(John Kappler) (James Allison)
1983 - Discovery of HIV (Luc Montagnier)
1984 - The first single cell analysis of lymphocyte proliferation (Doreen Cantrell) and
(Kendall A. Smith)
1984 - Robert Good, Failed treatment of severe combined immunodeficiency (SCID, David
the bubble boy) by bone marrow grafting
1984-1987 - Identification of genes for the T cell receptor (Leroy Hood, et al.; Hedrick
Davis, Mak)
1985 Tonegawa, Hood et al., Identification of immunoglobulin genes, somatic generation of
Ig variable regions
1985-onwards - Rapid identification of genes for immune cells, antibodies, cytokines and
other immunological structures
1987- Structure of MHC I defined (Wiley and Strominger)
1986 - Hepatitis B vaccine produced by genetic engineering
1986 - Th1 vs Th2 model of T helper cell function (Timothy Mosmann)
1988 - Discovery of biochemical initiators of T-cell activation: CD4- and CD8-p56lck
complexes (Christopher E. Rudd)
1989 – Catalytic antibody cleavage of peptide bonds (Sudhir Paul)
1990 - Yamamoto et al., Molecular differences between the genes for blood groups O and A
and between those for A and B
1990 - Gene therapy for SCID using cultured T cells
1991- Role of peptide for MHC Class II structure (Sadegh-Nasseri & Germain)
1992 – Hepatitis A vaccine developed
1993 - NIH team, Treatment of SCID using genetically altered umbilical cord cells
1994 - 'Danger' model of immunological tolerance (Polly Matzinger)
1995 - Regulatory T cells (Shimon Sakaguchi)
1996-1998 - Identification of Toll-like receptors
2001 - Discovery of FOXP3 - the gene directing regulatory T cell development
2005 - Development of human papillomavirus vaccine (Ian Frazer)
2011 – Nobel Prize awarded to Bruce A. Beutler, Jules A. Hoffmann, and Ralph M. Steinman
for landmark discoveries indicating TLRs are gatekeepers of innate immunity
Time Line 4
APPENDIX
Nomenclature of Immune System Cells.
Table 2-2. Lymphoid Leukocytes and Their Properties
Total Lymphocytes
1.3-3.5 × 109/L
B cell
Monocytic
Adaptive Humoral immunity
Plasma cell
Monocytic
Adaptive Terminally differentiated, antibody secreting B cell
T cell
Monocytic
Adaptive Cell-mediated immunity
Natural killer cell
Monocytic
Innate
Effector Function
Innate response to microbial or viral infection
Table 2-1. Myeloid Leukocytes and Their Properties
Phenotype Morphology Circulating Differential Count* Effector Function
9
Neutrophil
PMN granulocyte 2-7.5 ×10 /L
Eosinophil
PMN granulocyte 0.04-0.44 ×10 /L
Phagocytosis and digestion of microbes
Basophil
PMN granulocyte 0-0.1 ×10 /L
Immediate hypersensitivity (allergic)
reactions
Mast cell
PMN granulocyte Tissue specific
Immediate hypersensitivity (allergic)
reactions
Monocytes
Monocytic
0.2-0.8 ×10 /L
Circulating macrophage precursor
Macrophage
Monocytic
Tissue specific
Phagocytosis and digestion of microbes,
antigen presentation to T cells
Dendritic cell Monocytic
Tissue specific
Antigen presentation to naïve T cells,
initiation of adaptive responses
9
Immediate hypersensitivity (allergic)
reactions, defense against helminths
9
9
*Normal range for 95% of population, +/- 2 standard deviations.
PMN, polymorphonuclear.
Appendix I
Antibodies
Table 3-1. Classes of Antibody Isotypes and Functional Properties*
Immunoglobulin Class
IgG
IgE
Isotype
IgM
IgD
Structure
Pentamer
Monomer
Monomer
Monomer
Monomer, dimer
Heavy chain
designation
μ
δ
γ
ε
α
Molecular weight (kDa) 970
184
146-165
188
160 × 2
Serum
concentration(mg/mL)
1.5
0.03
0.5-10.0
<0.0001
0.5-3.0
Serum half-life (days)
5-10
3
7-23
2.5
6
J chain
Yes
No
No
No
Yes
Complement activation Strong
No
Yes, except
IgG4
No
No
Bacterial toxin
neutralization
Yes
No
Yes
No
Yes
Antiviral activity
No
No
Yes
No
Yes
Binding to mast cells
and basophils
No
No
No
Yes
No
Additional properties
Effective
agglutinator of
particulate antigens,
bacterial
opsonization
Found on surface
of mature B cells,
signaling via
cytoplasmic tail
AntibodyMediation of
dependent cell allergic response,
cytotoxicity
effective against
parasitic worms
Appendix II
IgA
Monomer in
secretory fluid,
active as dimer
on epithelial
surfaces
Table 3-2. Unique Biological Properties of Human IgG Subclasses
IgG1
IgG2
IgG3
IgG4
Occurrence (% of total IgG)
70
20
7
3
Half-life (days)
23
23
7
23
Complement binding
+
+
Strong
No
Placental passage
++
±
++
++
Receptor binding to monocytes
Strong
+
Strong
±
Appendix III
T Cells
Appendix IV
Appendix V
Effector Cells in Cytotoxic Cell Mediated Immunity
Effector Molecules
Effector Cell
CD markers
CTL
NK cell
NK cell
ADCC
LAK cell
Macrophage
TCR,CD3,CD8,CD2 Perforin, cytokines
(TNF-β, IFN-)
CD16,CD56, CD2
Perforin, cytokines
(TNF-β, IFN-)
CD16,CD56, CD2
Perforin, cytokines
(TNF-β, IFN-)
CD16,CD56, CD2
Perforin, cytokines
(TNF-β, IFN-)
CD14
TNF-α, enzymes,
NO, O radicals
Appendix VI
MHC
recognition
Antigen
recognition
required
Class I
no
specific TCR
no
specific IgG
no
nonspecific
no
nonspecific
nonspecific
Complement Cascade
Appendix VII
Biological Functions of Complement.
Appendix VIII
Interleukins are the cytokines that act specifically as mediators between leukocytes.
Major Cell Source
IL-1
Macrophages
IL-2
IL-3
Activated T cells
T lymphocytes
IL-4
T cells and mast cells, B cells,
stromal cells
IL-5
T cells and mast cells
Activated T cells, B cells,
IL-6
Monocytes and PMNs
thymus and bone marrow
IL-7
stromal cells
IL-8 (CXCL8) Macrophages
IL-9
Activated T cells
Activated T cells, B cells and
IL-10
monocytes
Major Functions
Stimulation of T cells and antigen-presenting cells
B-cell growth and antibody production
Promotes hematopoiesis (blood cell formation)
Proliferation of activated T cells
Growth of blood cell precursors
Promotes TH2 cell development
B-cell proliferation
IgE production
Eosinophil growth
Promotes granulomatous response
Induces fever and shock, synergistic effects with IL-1 or TNF-
Development of T cell and B cell precursors.
Chemoattractant for neutrophils
Promotes growth of T cells and mast cells
Inhibits inflammatory and immune responses
Inhibits TH1 cell responses
Synergistic effects on hematopoiesis
Promotes TH2 cell response
Promotes TH1 cells while suppressing TH2 functions
Similar to IL-4 effects, attenuates macrophage function
Similar to IL-2 effects
Chemoattractant for CD4 T cells
Promotes T cell proliferation
Induces IFN- production
Inflammatory response, induction of IL-6 and TNF-
Involved in inflammatory skin diseases
IL-11
Stromal cells
IL-12
IL-13
IL-15
IL-16
IL-17
IL-18
IL-19
IL-20
Macrophages, B cells
TH2 cells
Epithelium and monocytes
CD8 T cells
Activated memory T cells
Macrophages
Monocytes and B cells
Monocytes and Keratinocytes
NK, B, T, and dendritic cells,
macrophages, and endothelial Modulates B, T, and NK cell function
cells
T cells (CD4+) and NK cells
IL-10 homologue
Stimulate IFN- production and proliferation in blast T cells and activated
Activated dendritic cells
(memory) T cells
B cells, CD4+ (naïve) T cells,
Inhibition of endothelial cell differentiation and migration of endothelial
TH2 cells, epithelium and
cells (anti-tumor)
fibroblasts, NK cells,.
Bone marrow stromal and Mast
Possible mediator of allergic disease (TH2 responses)
cells
CD4+ (mature) T cells and NK
IL-10 homologue
cells
Activated APCs
Inhibits hyperactive T cells (CD4+)
IL-21
IL-22
IL-23
IL-24
IL-25
IL-26
IL-27
Appendix IX
Interferons were first recognized for their ability to confer resistance to viral infection.
Major Cell Source
IFN-;
24
Leukocytes
distinct species
identified
IFN-β
Fibroblasts
IFN-
T cells (TH1), macrophages
(rare)
Major Functions
Anti-viral, Anti-tumor
Regulate differentiation
Modulates lipid metabolism
Inhibits angiogenesis
Immunoregulates (monocyte/macrophage activation)
Enhances MHC expression
Class I: IFN- and IFN- β beta
Class II: IFN-
Properties of selected immune mediators, growth factors and chemokines.
Major Cell Source
CCL5 (Rantes)
T cells, Endothelium
CCL11 (Eotaxin)
Monocytes and Macrophages,
Endothelium and Epithelium
Chemoattractant for monocytes
Proliferation/activation of Chemokine-activated killer cells
Chemoattractant for neutrophilic granulocytes
Stimulates TNF secretion by macrophages
Chemoattractant for Eosinophils and Basophils, Monocytes and
Dendritic cells, and T cells
Increases monocyte adherence to endothelial cells
Activates Basophils (degranulation)
Chemoattractant for Eosinophils
Mediator of allergic response
CXCL1
CXCL2
CXCL3
Monocytes, Fibroblasts,
Epithelium
Chemoattractant for Neutrophils
Activates Neutrophils (degranulation)
CXCL8 (IL-8)
Monocytes and Macrophages,
Fibroblasts, Endothelial cells
Chemoattractant for Neutrophils
Activates Neutrophils (degranulation)
CCL2 (MCP-1)
CCL3 (MIP-1)
GranulocyteMacrophage
Colony
Stimulating
Factor (GMCSF)
Transforming
Growth Factorbeta (TGF-β); 5
isoforms
Tumor Necrosis
Factor-alpha
(TNF-);
Lymphotoxin B,
Cachectin
Tumor Necrosis
Factor-beta
(TNF-β)
Monocytes and Macrophages,
Fibroblasts
Monocytes, T cells, Fibrobalsts,
Mast cells
Major Functions
Stimulates growth of macrophages and granulocytes
T cells and macrophages,
Stimulates differentiation: Monocytes, Neutrophils, Eosinophils
endothelial cells and fibroblasts Stimulates release of arachidonic acid metabolites from granulocytes
and increased generation of reactive oxygen species, granulocytes
Growth inhibitor for Lymphocytes, epithelium, endothelium,
Platelets, Macrophages, T cells,
fibroblasts, neuronal cells, hepatocytes, keratinocytes, and
Endothelial cells, Keratinocytes hematopoietic cell types.
Inhibits MHC Class II expression
Moncytes and Macrophages
Activates vascular endothelium, increases vascular permeability
Induces fever and shock
Induces acute-phase responses
Tcells, Fibroblasts, Astrocytes,
Endothelium and Epithelium
Cytolytic or cytostatic for tumor cells
Induces reactive oxygen species from Neutrophils
Critical component of wound healing
Appendix X
Appendix XI
Appendix XII
Type I Hypersensitivity (also called immediate hypersensitivity) is due to aberrant production
and activity of IgE against normally nonpathogenic antigens (commonly called allergy). The
IgE binds to mast cells via high affinity IgE receptors. Subsequent antigen exposure results in
crosslinking of mast cell bound IgE with activation of mast cells that release preformed
mediators (eg. Histamine, leukotrienes, etc.) and synthesize new mediators (i.e. chemotaxins,
cytokines). These mediators are responsible for the signs and symptoms of allergic diseases.
Appendix XIII
Type II Hypersensitivity is due to antibody directed against cell membraneassociated antigen that results in cytolysis. The mechanism may involve complement
(cytotoxic antibody) or effector lymphocytes that bind to target cell-associated
antibody and effect cytolysis via a complement independent pathway (Antibody
dependent cellular cytotoxicity, ADCC). Cytotoxic antibodies mediate many
immunologically-based hemolytic anemias while ADCC may be involved in the
pathophysiology of certain virus-induced immunological diseases.
Coico, Sunshine, Benjamini, 2003. Fig. 15.11
Appendix XIV
Type III Hypersensitivity results from soluble antigen-antibody immune complexes that
activate complement. The antigens may be self or foreign (i.e. microbial). Such complexes are
deposited on membrane surfaces of various organs (i.e. kidney, lung, synovium, etc). The
byproducts of complement activation (C3a, C5a) are chemotaxins for acute inflammatory cells.
These result in the inflammatory injury seen in diseases such as rheumatoid arthritis, systemic
lupus erythematosus, postinfectious arthritis, etc).
Appendix XV
Type IV Hypersensitivity (also called Delayed Type Hypersensitivity, DTH) involves
macrophage-T cell-antigen interactions that cause activation, cytokine secretion and potential
granuloma formation. Diseases such as tuberculosis, leprosy and sarcoidosis as well as contact
dermatitis are all clinical examples where the tissue injury is primarily due to the vigorous
immune response rather than the inciting pathogen itself.
Appendix XVI
Type IV Hypersensitivity (continued)
Figure 16.1. DTH reaction. (A) Stage of sensitization by antigen
involves presentation of antigen to T cells by APC, leading to the
differentiation of TH0 T cells to TH1 and TH17 cells. (B) Challenge
with antigen (the elicitation stage) involves antigen presentation to
TH1cells by APC, leading to TH1 and TH17 activation, release of
cytokines, and recruitment and activation of macrophages.
Appendix XVII
Table 8-1. Autoimmunity and Disease
Autoimmune Disease
Mechanism
Pathology
Autoimmune hemolytic anemia
Autoantibodies to RBC antigens
Lysis of RBCs and anemia
Autoimmune thrombocytopenia
purpura
Autoantibodies to platelet integrin
Bleeding, abnormal platelet
function
Myasthenia gravis
Autoantibodies to acetylcholine receptor in neuromuscular junction
Blockage of neuromuscular
junction transmission and
muscle weakness
Graves' disease
Autoantibodies to receptor for thyroid- stimulating hormone (TSH)
Stimulation of increased
release of thyroid hormone
(hyperthyroidism)
Hashimoto's thyroiditis
Autoantibodies and autoreactive T cells to thyroglobulin and thyroid
microsomal antigens
Destruction of thyroid gland
(hypothyroidism)
Type I diabetes (insulindependent diabetes mellitus;
IDDM)
Autoantibodies and autoreactive
T cells to pancreatic islet cells
Destruction of islet cells
and failure of insulin
production
Goodpasture's syndrome
Autoantibodies to type IV collagen
Glomerulonephritis
Rheumatic fever
Autoantibodies to cardiac myosin (cross-reactive to streptococcal cell Myocarditis
wall component)
Pemphigus vulgaris
Autoantibodies to epidermal components (cadherin, desmoglein)
Acantholytic dermatosis,
skin blistering
Multiple sclerosis
T-cell response against myelin basic protein
Demyelination, marked by
patches of hardened tissue
in the brain or the spinal
cord; partial or complete
paralysis and jerking
muscle tremor
Systemic lupus erythematosus
(SLE)
Circulating immunocomplexes deposited in skin, kidneys, etc, formed Glomerulitis, arthritis,
by autoantibodies to nuclear antigens (antinuclear antibodies, or
vasculitis, skin rash
ANA), including anti-DNA
Rheumatoid arthritis
Autoantibodies to IgG (rheumatoid factors); deposition of
immunocomplexes in synovium of joints and elsewhere; infiltrating
autoreactive T cells in synovium
AUTOIMMUNE DISEASE
Joint inflammation,
destruction of cartilage and
bone
CLINCAL PHENOTYPE
Systemic Lupus Erythematosus Rash; inflammation of joints and serosal linings; glomerulonephritis;
hemolytic anemia, systemic symptoms
Rheumatoid Arthritis
Scleroderma
Inflammation of synovium of diarthroidal joints, systemic inflammation
Inflammation, dermal fibrosis, internal organ fibrosis, vasculopathy
Ankylosing Spondylitis
Inflammation of spine, joints, and tendon insertions; uveitis
Multiple Sclerosis
Demyelination, optic neuritis, neurological deficits
Myasthenia Gravis
Hashimoto’s Thyroiditis
Skeletal muscle weakness, diplopia, dysarthria, dysphagia
Hypothyroidism
Graves Disease
Hyperthyroidism, opthalmopathy
Celiac Disease
Diarrhea and malabsoprtion
Autoimmune hemolytic anemia Anemia through lysis of red blood cells
Type I diabetes
Failure of insulin production and glycemic control
Sjorgren’s Syndrome
Disorder of the moisture-producing glands
Appendix XVIII
Figure 8-5 Primary immunodeficiencies. Manifestation of immunodeficiency is dependent upon the etiology of response. B-cell
deficiency is marked by recurrent infections with encapsulated bacteria. T-cell deficiency manifests as recurrent viral, fungal, or
protozoal infections. Phagocytic deficiency with associated inability to engulf and destroy pathogens usually appears with recurrent
bacterial infections. Complement disorders demonstrate defects in activation patterns of the classical, alternative, and/or lectin-binding
pathways, which augment adaptive host defense mechanisms.
Appendix XIX
Tissue Rejection and Host Response to Transplantation.
Appendix XX
Mechanisms of Tolerance.
Appendix XXI
http://www.cdc.gov/vaccines/recs/schedules/downloads/child/0-6yrs-schedule-pr.pdf
Appendix XXII