Download Immunology: The Basics and Beyond

What is Immunology?
• Immunology is the study of the body’s
defenses against pathogens (viruses,
bacteria, fungi, parasites)
• Not a well-funded science in the US in the
1970’s and then came AIDS and other
diseases that compromise the immune
system plus the advent of work on
vaccines to treat cancer
Immunology: The Basics
and Beyond
Cindy Wilkening May 20, 2009
Adapted from WWW outline by Dr. J.A.
Wise, Dept of Biology, Hampton
University
2
Types of Immunity
History and Overview
• In the late 1700’s, Edward Jenner
administered the first vaccination against
smallpox
• Innate Immunity is present from birth. It is
fast but nonspecific.
– Macrophages are part of innate immunity and
are phagocytic cells that can kill pathogens.
Discovered by Elie Metchnikoff in the late
1880’s.
– A vaccine is a dead or weakened pathogen
used to induce immunity without causing any
disease
• Adaptive Immunity is acquired during an
individual’s lifetime. It is slow but specific.
• In the late 1800’s, Louis Pasteur
developed cholera and rabies vaccines
3
4
Adaptive Immunity
• Antibodies (Ab) or Immunoglobulins (Ig)
are substances in the serum of infected
individuals that bind pathogen.
Discovered in the late 1800’s.
• Antigens are generally foreign substances
(but can be self) that bind to specific
antibodies or cell receptors
Macrophages
Dendritic Cells
5
6
1
Adaptive Immunity
CD molecules
• Lymphocytes are required for all forms of
adaptive immunity.
• Each lymphocyte is specific for one
antigen (monospecificity).
• A clone is a group of genetically identical
lymphocytes specific to a particular
antigen. There can be more than 1 clone
per antigen. Discovered in the 1960’s.
• CD or “cluster of differentiation” are cell
membrane molecules that are used to
classify cells into subsets
• A monoclonal antibody (Mab) can be
manufactured from one clone of cells
specific to one cell membrane molecule
• Mabs are used to define the cluster in vitro
• Examples are CD3 on T-cells, CD19 on Bcells, CD34 on stem cells
7
8
Features of Adaptive Immunity
Clonal Selection Theory
• Clonal Selection and Tolerance
• Explains how adaptive immunity works.
• Specific lymphocyte clones exist in small
numbers before exposure to pathogen.
• After exposure, clones specific to the
pathogen proliferate, increase in number
and differentiate into effector cells,
• After fighting off pathogen, there are more
cells of these specific clones in the body
– Receptor repertoire are the receptors on all
lymphocytes that recognize all antigens
except self-antigens
– Central tolerance is the elimination of
lymphocytes with receptors for self antigens
early in development of the lymphocytes
9
• Immunological memory happens when the
body is re-exposed to pathogen. The
adaptive immunity is faster and stronger
because of the increased number of
pathogen-specific lymphocytes
10
11
12
Lymphocyte Differentiation
• B cells (as plasma cells) secrete antibody
• T cells develop into effector cells that
either kill pathogen-infected cells or
activate other parts of the immune system
• How do these cells originate? From
hematopoietic stem cells in the bone
marrow
2
13
14
15
16
Adult Normal White Cell Counts in
Blood and 5-Part Differential
• B-CELLS MATURE IN BONE
MARROW
• T-CELLS START IN BONE
MARROW AND FINISH MATURING
IN THYMUS.
• White blood cells: 5000 to 9000 cells per
mm3
•
•
•
•
•
Neutrophils: 55.0%
Lymphocytes: 35.0%
Monocytes:
6.5%
Eosinophils:
3.0%
Basophils:
0.5%
• T-CELLS AND B-CELLS THAT
HAVE NOT YET BEEN EXPOSED
TO ANTIGEN ARE CALLED NAÏVE
LYMPHOCYTES
(3000 to 7000/mm3)
(1000 to 4000/mm3)
(100 to 600/mm3)
(50 to 250/mm3)
(25 to 100/mm3)
• ANTIGEN TRAVELS IN BLOOD TO
THE SPLEEN AND LYMPH TO THE
LYMPH NODES WHERE IT IS
FILTERED OUT AND ACTED UPON
BY LYMPHOCYTES
• DISTINCT AREAS FOR B-CELLS
AND T-CELLS IN THE LYMPHOID
TISSUES
17
18
3
Antigen Receptors on Lymphocytes
• Membrane-bound Immunoglobulin (Ig) on
B-cells
• T-cell receptor on T-cells
B-CELL RECEPTOR
19
T-CELL
RECEPTOR
20
Immune Response to Pathogens
• Lymphocyte activation
21
– B-cells: first signal is binding of antigen to
surface Ig, second signal provided by T-cells
via cytokines which are like localized
hormones
– T-cells: first signal is binding of antigen to Tcell receptor, second signal by antigenpresenting cells which are macrophages or
dendritic cells
– Activated lymphocytes proliferate (clonal
expansion) then differentiate into effector cells
22
Major Histocompatibility Complex
(MHC)
• T-lymphocyte cells recognize antigen
bound to MHC molecules
• MHC Class I bearing cells (most nucleated
cells) interact with CD8 positive cytotoxic
T-lymphocyte cells that act by direct cell
killling
• MHC Class II bearing cells (antigen
presenting cells) interact with CD4 positive
helper T-lymphocyte cells which activate
other T-lymphocyte and B-lymphocyte
responses
23
24
4
HLA are Human MHC Molecules
• HLA are human leukocyte antigens
• Each person has six class I MHC-alleles
(1 of HLA-A, -B, and -C from each parent)
• Each person has at least 6 class II MHCalleles (1 of HLA-DQ and -DP, and 1 or 2
of -DR)
• Many different alleles in population for
each HLA type
25
26
Humoral Immunity
Cell-Mediated Immunity
• Mediated by Ig antibodies (IgM, IgA, IgD, IgE,
and IgG)
• Antibodies released into blood from plasma cells
(an effector B-cell)
• Good against extracellular pathogens and toxins
(eg from bacteria)
• Neutralization is physical binding by antibody
• Opsonization is binding of antibody to pathogen
which causes antigen to be ingested more
rapidly by macrophages
• Activation of complement can lyse and kill
bacteria
27
• Carried out by T-cells, macrophages, and
other cells and is good against cells with
intracellular pathogens
• Helper T cells activate other cells of the
immune system; the ‘Generals’
• Cytotoxic T cells can destroy cells infected
with viruses by direct killing with perforin
and granzymes
• Phagocytes digest cell fragments from
killed cells
28
MEMORY CELLS PRIME IMMUNE SYSTEM
29
30
5
Tumor Cell Escape
Types of tumor antigens
• Mutated self protein
• Product of oncogene or mutated tumor
suppressor gene
• Overexpressed or aberrantly expressed
self protein
• Oncogenic virus
• Failure to produce or loss of tumor antigen
• Mutations in MHC genes or genes needed
for antigen processing
• Production of immunosuppressive proteins
or expression of inhibitory cell surface
proteins
• Since many tumors arise from self cells,
the tumor may be ignored as self tissue
31
Types of Cancer Vaccines
32
Types of Cancer Vaccines
• Viral vectors and DNA vaccines
• Antigen/adjuvant vaccines
– Cancer cell antigen plus adjuvant (a substance that
causes an immune response). Expected that immune
system will respond to tumor cells that express the
vaccine antigen
• Whole cell tumor vaccines
– Cells taken from patients own tumor (autologous) or
from other patients (allogeneic). Expect to stimulate
an immune response
– DNA with gene for cancer antigen is manipulated in
the lab so it is taken up by antigen-presenting cells
(APC). The antigen is displayed on APC cell surface.
Expected that immune system will attack tumor cells
with this antigen (Not all DNA vaccines use viral vectors)
• Idiotype vaccines
• Dendritic cell (DC) vaccines
– DC removed from patient’s blood and then stimulated
with patient’s own cancer antigens, grown in petri
dishes then re-injected into the patient. Expected to
activate T-lymphocytes to multipy and attack tumor
cells with DC antigen
– Antibodies contain proteins and carbohydrates which
can be antigens. Cancer antibodies (called idiotype
antibodies) are unique for each person and can be
used to trigger an immune response in a way similar
to antigen vaccines
33
Treatment Vaccines Under Investigation
•
•
•
•
•
•
•
•
•
Patient-specific vaccines
Prostate Specific Antigen (PSA)
Sialyl Tn (STn)
Heat Shock Proteins (HSPs)
Ganglioside molecules
Carcinoembryonic antigen (CEA)
MART-1 (or Melan-A)
Tyrosinase
General DNA vaccines to modify genetic
structure of cancer cells
34
Approved Preventative Vaccines
• Hepatitis B
– Prevents infection with hepatitis B virus
associated with some forms of liver cancer
• Gardasil
– Prevents infection from 2 types of human
papillomavirus (HVP) which causes 70
percent of cervical cancer worldwide
35
36
6
HLA/MHC and Transplantation
HLA/MHC and Transplantation
• During maturation in thymus, CD8+ and
CD4+ T cells selected to recognize
peptides displayed by self MHC molecules
• Transplantation can include blood, cells,
tissues from source other than self
(allogeneic)
• T-cells from self cross-react with non-self
MHC molecules and attack the non-self
cells
• Need for tissue typing to match HLA
molecules from donor to those of
transplant host
• Need to use immunosuppressant drugs
such as cyclosporine or steroids to
suppress immune reaction in host
• If mature T-cells of donor are transplanted,
they can attack recipient’s tissues leading
to serious clinical reaction called Graft vs
Host disease. This may happen especially
in bone marrow or stem cell transplants.
37
38
Some Methods of Assessing the
Immune System
HLA/MHC and Transplantation
• There is also Graft vs Leukemia where it is
beneficial if the donor cells kill off the
receipient bad immune cells. There is a
slight mismatch made in the HLA typing
for this to happen.
• Flow cytometry
–
–
–
–
Lymphocyte subsets
Tetramer Assay
Cytokine Assay
CFSE Flow-based Cell Proliferation Assay
• Serial dilution assays
– Natural Killer Cell (NK) assay as an example
• Delayed type hypersensitivity (DTH) test
• ELISA
• ELISPOT
39
40
Flow Cytometry Sample Prep for
Subsets
Flow cytometry
“A flow cytometer is an instrument that
illuminates cells as they flow individually in
front of a light source and then detects and
correlates the signals from these cells that
result from the illumination.” Givan, 2004
A flow cytometer can also deflect cells
based on the laser illumination and sort
them on size or fluorescence
41
• Whole blood collected from patients into tubes
with anticoagulant
• Red cells are lysed with a lysing agent
• A monoclonal antibody (Mab) is connected to a
fluorochrome and this complex is added to the
tube. Fluorochromes emit fluorscence upon
excitation at a specific wavelength
• 1 to as many as 17 Mab- fluorochromes can be
added to tube depending on number of
collectors on machine
42
7
43
44
DNA CONTENT
45
S-PHASE FRACTION
G0/G1 is
resting/active
cell phase
S-phase is DNA
synthesis
G2/M 2x DNA
content for
mitosis
46
Tetramer Assay
• Advantages
– Specific, sensitive, can assess complex cell
phenotypes by flow
– Can detect dysfunctional cells which arise
during chronic exposure to antigens such as
tumors or HIV virus
• Disadvantages
– Only single specificities can be analyzed
– Measure intensity but not breadth of response
47
48
8
What is a tetramer?
• Complex of 4 MHC molecules bound with
peptide of interest and labeled with
fluorochromes
• Purchase tetramers from a company or
from NIH for custom tetramers
• Use as a stain in a flow cytometric assay
• Often a “rare event” (much less than 1%),
need 105 to 106 lymphocytes (events) to
detect
49
TETRAMER RESULTS
50
Tetramer Assay
• Results are frequency of tetramer positive
cells reported as a fraction of a major
lineage subset used to define them such
as CD3+CD8+
• Can also be reported as number of
tetramer positive cells per volume of blood
if beads are used on the flow cytometer
51
52
Cytokine Flow Cytometry
Cytokine Flow Cytometry
• Antigen-stimulated CD4+ and CD8+ Tcells can be detected by directly
measuring internal cytoplasmic levels of
cytokines such as IFN-gamma and IL2 by
flow cytometry
• PBMCs or cell suspensions are stimulated
in vitro for a short time with antigens that
cause cytokines to be made in the cell
cytoplasm.
• Drugs that block cytokine synthesis are
then added to stop cytokine production
• Cell membranes are permeabilized with
mild detergent to allow fluorescent tagged
cytokine-specific Mabs to get into cell
• Cells can be stained with Mabs for lineage
markers such as CD3, CD4, CD8 and for
activation markers such as CD69
• Results are “rare events” and require up to
106 events to detect
• Results can be expressed as percents
53
54
9
CFSE Flow-based Proliferation Assay
CFSE Flow-based Proliferation Assay
• CFSE is carboxyfluorescein diacetate
succinimidyl ester
• Labels long-lived intracellular molecules with
highly fluorescent dye
• After labeling with CFSE, cells are
stimulated with antigen or mitogen of choice
• Following each cell division, progeny have
half of the fluorescence as parent cell
• Can detect up to 8 to 10 cell divisions
• Track cells from Day 2 to Day 14 after CFSE
55
• In addition to CFSE, Mabs to surface
molecules on the lymphocytes can
determine how phenotypic properties
change with cell division
• CFSE-labeled cells can be sorted on a
flow cytometer and analyzed in functional
assays such as secretion of cytokines or
antibodies
• With use of beads, final data are number
of cells per total cells within a cell culture
for each division number
56
NK Cell Assay
•
•
•
•
NK cells part of innate immune system
Not antigen dependent
Can kill target cells such as K562 cell line
Radioactive Chromium (51Cr) is added to
K562 medium and is taken up by cells
• K562 cells are washed to remove excess
51Cr
57
58
NK Cell Assay
NK cell assay
• 96-well plate is prepared with either whole
blood or PBMCs of patient
• Add dilutions of labeled K562 cells to wells
containing PBMCs to yield specific
effector: target ratios from 50:1 to 6.25:1
• Also have wells with labeled K562 cells
but no PBMCs for spontaneous release
59
• Also have wells with K562 cells only to
which a detergent is added to disrupt cell
membranes and get maximum release of
51Cr
• Incubate for set time, stop incubation with
cold medium, remove supernatant and
transfer to counting vials
• Count on a gamma counter to get counts
per minute (CPM) which is used to
calculate % specific lysis
60
10
Delayed Type Hypersensitivity Test
NK Cell Assay
• % specific lysis=100*
(mean experimental cpm-mean spontaneous release cpm)
(mean max release cpm-mean spontaneous release cpm)
• #Lytic Units (LU) per 107 effector cells=
#Effector cells
(E:T)(#Target cells)
166 Lytic Units per 107 effector cells. 105 or
106 effector cells can also be used
• Measure of intact cell-mediated immunity
• Use antigens that patient has been
exposed to such as Candida or Tetanus
• Inject small amount of antigen suspended
in fluid just under skin of lower arm
• After 48 to 72 hours, check for induration
at site of injection
• Measure largest diameter
• Diameter >=5mm indicates intact CMI
61
DTH Test
• If it is known that the individual has been
exposed to the antigen but there is no
induration, called anergy
• Abnormal DTH can be a sign of frequent
infections, autoimmunity, and malignancy
• DTH also used to help diagnose infections
such as TB
• DTH used to aid in cell-mediated immune
response to vaccines
62
Elisa Assay
(Enzyme-linked immunosorbant assay)
Antigen on Solid Phase
63
Elisa Assay: Antibody on Solid Phase
64
Elisa Assay
• Color of fluid in wells is measured in a
reader and expressed as relative light
units
• A standard curve is generated with known
amounts of antibody and its relative light
units
• Final results expressed as pg/ml
concentration of cytokine, for example
65
66
11
ELISPOT Assay
• ELISPOT Assay is Enzyme-Linked
Immunospot Assay
• Detection of secreted molecule at site of
secreting cells
• 100 to 400 fold more sensitive than ELISA
• Final results expressed as Spot Forming
Cells (SFC) per 106 cells
67
68
69
70
71
72
12
Book Resources
Journal Resources
• Basic Immunology (3rd edition) by AK Abbas and AH Lichtman, 2008
• Flow Cytometry, Clinics in Laboratory Medicine, 2007
• Manual of Molecular and Clinical Laboratory Immunology (7th
edition) by B Detrick et al,2006
• Cellular and Molecular Immunology (3rd edtion) by AK Abbas et al,
1997
• Immunobiology (5th edition) by Janeway et al, 2001
• Flow Cytometry: An Introduction by AL Givan in Flow Cytometry
Protocols (2nd edition) by TS Hawley and RG Hawley, 2004
• Flow Cytometry: First Principles (2nd edition) by AL Givan, 2001
• Schaum’s Outline of Theory and Problems of Immunology by G
Pinchuk, 2002
• A Manual of Laboratory and Diagnostic Tests (5th edition) by F
Fischbach, 1996
• Immunology at a Glance (8th edition) by JHL Playfair and BM Chain,
2005
• How the Immune System Works (3rd edition) by L Sompayrac, 2008
• Guinn, B-A et al, Recent advances and current challenges in tumor
immunology and immunotherapy. Molecular Therapy, 2007, vol 15,
p 1065-1071
• Altman, JD, Flow cytometry applications of MHC tetramers, Methods
in Cell Biology, 2004, vol 75, p 433-452
• Lyons, AB et al. Flow cytometric analysis of cell division history
using dilution of carboxyfluorescein diacetate succinimidyl ester, a
stably integrated fluorescent probe. Methods in Cell Biology, 2001,
vol 63, p 375-398
• Klenerman, P et al, Tracking T-cells with tetramers: new tales from
new tools. Nature Reviews Immunology, 2002, vol 2, p 263-272
• Walker, EB et al. Monitoring immune responses in cancer patients
receiving tumor vaccines. Intern. Rev. Immunol, 2003, vol 22, p 283319
• Quah, BJC et al. Monitoring lymphocyte proliferation in vitro and in
vivo with the intracellular fluorescent dye CFSE, Nature Protocols,
2007, vol 2, p 2049-2056
73
74
WWW Resources
• www.utpa.edu/faculty/materon/3403/immunopaper.html (now
defunct; was JA Wise outline)
• www.cancer.gov/cancertopics/factsheet/cancervaccine
• www.cancer.gov/clinicaltrials/learning/cancervaccines
• www.elispot-analyzers.de/english/elispot-animation.html
• www.ask.com/pictures?q=immune+system&qsrc=0&o=0&l=dir
• www.freewebs.com/immunology/elispot-wells.html
75
13