Download Immunology Review – Quiz 1

Immunology Review – Quiz 1
CONCEPTS IN IMMUNITY
o Background
o Goal of immune system: battle infection
o Leukocytes: interact as network to attack microorganisms
o Microbes can be intracellular (invade cells) or extracellular (grow on tissues)
o Host defense mechanisms
 External defenses (e.g. physical barriers)
 Innate immunity: non-specific molecules/cells
 Adaptive immunity: specific molecules/cells
o Components of the immune system
o Leukocytes:
 3 types of lymphocytes (B, T, large granular lymphocyte)
 myeloid derived cells (monocytes/macrophages, granulocytes)
o Hematopoietic stem cells
o Hematopoietic cells derived from common stem cell in bone marrow
o Self-renewing
o Differentiate in response to cytokines, other signals
o Important definitions
o Antigen: reacts with antibodies (foreign pathogen/particle)
o Immunogen: antigen that induces immune response (e.g. protein, na, carb, lipid)
o Hapten: small molecule that can’t induce Ig alone (e.g. penicillin); can if attached to larger protein
o Passive immunity: established by transfer of Ig or lymphocytes (provided to patients)
o Active immunity: result of host cells’ response to antigen (e.g. vaccination)
o Humoral immunity: mediated by Ig
o Cellular immunity: mediated by cells
o Specific immunity: protects against specific antigen that elicits response
o Non-specific immunity: protects against many types of antigens (usually related)
o Immunological memory/secondary response: specific response to previously-encountered antigen
o Cytokines: small proteins that signal between cells
 Membrane-bound or soluble
 Autocrine or paracrine
 Bind to specific receptors
 Know: IL-1,2,4,5,10,12; TNF-alpha, beta; IFN-alpha/beta, gamma; chemokines
o CD: cluster designation for cell suface molecules (see CD chart)
o Immune receptors: recognizing danger
o Pattern recognition receptors
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 Bind to patterns on microorganisms and their products
 Macrophages, DCs, epithelial cells, etc.
 Nonspecific (bind to similar molecules on diff organisms)
 Innate
o Immunoglobulin (Ig)
 One specific antigen
 B cells
o T cell receptors (TCR)
 One specific peptide and MHC
 T cells
o Fc receptors (FcR)
 Bind to Ig
 Allow cells to use specificity of Ig to direct function
 NK, macrophage, PMN, Eos, mast cells
o Innate immunity: first cells to respond
o Mechanisms present prior to infection
o Sentinels that find infection near site of entry
o Acute phase proteins/complement, macrophages,
PMNs, NK cells, epithelial/endothelial cells
o Phases of immune response -----------------------------------
o Recognition of antigen
o Activation of immune cells
o Clonal expansion
o Effector phase
o Decline after antigen is cleared
o Memory cells
o Antigen presentation: recognizing pathogen presence
o Antigen-presenting cells (APCs): macrophages, B cell, dendritic cell
o Small peptides presented with MHCs on APC surface
o T cells recognize specific MHC-peptide complex with TCR
o Major histocompatibility complex (MHC)
o Bind small proteins and present them to TCR
o Class I: all nucleated cells; present internal proteins
o Class II: on APCs; present external proteins
o Adaptive immunity: specific response
o Lymphocytes with highly specific receptors (Ig, TCR)
o Specificity
o Memory
o Clonal selection
o Lymphocytes exist in pools of clones
 Each clone specific for single antigen
o Clone activates when bound to antigen  divides a lot
 Differentiation to effector cells, memory cells
o Receptor cross-linking
o Must trigger several antigen-specific receptors at once to get cell
response
o Receptors localize near each other to bind to antigen on cell surface,
bringing signaling molecules close together  response
o Ig can be use to mimic this process
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o Two signals for immune cell activation
o Prevents inappropriate activation
o 1: via immune receptor (Ig, TCR)
o 2: via costimulatory signal (general signal induced by
presence of pathogen)
o Antibodies: response to remove pathogen; humoral
o Made by plasma cells (from B cells)
o Multiple sites for same antigen
o 2 heavy chains, 2 light chains
o Fab portion: antigen binding
o Fc portion: effector region
o 5 classes: Ig-M,D,G,A,E
o T cells: response to remove pathogen; cell-mediated
o Lymphocytes, develop in thymus
o Specific TCR
o Activate humoral response
o 3 types
 cytotoxic (CTL): CD8, cytokines
 helper (Th): CD4, lots of cytokines
 Th1: interact with macrophages, inflammation, development of CTLs
 Th2: interact with B cells, promote Ig making
 Th17: autoimmunity, inflammation
 regulatory (Treg): suppress T cell activity in periphery, prevent autoimmunity
o Immunological memory
o On second exposure to antigen, response is faster and more effective
o Antigen-specific memory lymphocytes in higher numbers, easier to activate
o Lymphoid tissue and trafficking: linking antigens and lymphocytes
o Primary lymphoid tissue: where lymphocytes develop (marrow, thymus)
o Secondary lymphoid tissue: where mature lymphocytes reside (spleen, nodes, tonsils, etc.)
o DCs pick up antigens in periphery and bring to node via lymphatic drainage
o DCs present antigen to lymphocyte in node
o Tolerance: avoiding immune response to “self” antigens
o Removes/inhibits lymphocyte receptors for normal proteins
o What to know:
o Cells of immune system
o Antigen
o Key cytokines and CDs
o Adaptive versus innate immunity
o Clonal selection and expansion
o Antigen presentation
o 2-signal system
o Immunological memory
o Lymphoid organs
INNATE IMMUNITY
o External defenses
o Physical barriers: microbes must pass skin/epithelial cells actively or passively
o Secretions: sweat, tears, saliva, gastric fluid have antimicrobial substances
o Microbial products/competition: non-pathogenic bacteria (commensals) on epithelial surfaces
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o Soluble molecules: mediate protection against microbes before adaptive immunity develops
o Complement
 20 interdependent proteins
 sequential activation  inflammation, attract neutrophils (chemotaxis), help attach microbes to
phagocytes (opsonization), kill microbe
 activation is direct by microbes (alternative path) or by Ig bound to antigen (classical path)
o Acute phase proteins
 Plasma proteins
 Innate defense against microbes (esp bacteria)
 Limit tissue damage cause by disease/infections
 Maximize activation of complement, opsonization
 Produced in liver in response to microbe or cytokines (IL-1/6, TNF-alpha/gamma)
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Interferons (IFN)
 Protect against viral infections (interfere with replication)
 Signaling molecules btwn cells
 Type I (alpha, beta): made by lots of cells; inhibit protein synth in infected cells
 Type II (gamma): leukocytes (Th1, NK); regulates Th1 response, increases phagocytosis and
antigen presentation
o Collectins: carb binding proteins; act as opsonins (help macrophage destroy microbe)
o Peptide antibiotics: produced by lots of cells (e.g. epithelial, phagocytic)
o Innate immunity cellular receptors/pattern recognition receptors
o Innate immune cells have receptors to recognize pathogens
o Germ-line encoded  no gene rearrangements for expression
o No memory response
o Activation of receptors  costimulation (signal 2) of lymphocytes
o Types (one cell can have many types)
 Mannose receptors
 On macrophages, DCs, endothelial
 Bind to mannosyl/fucosyl carbs  eat microbe  peptides on MHC  T and B response
 CD14
 On macrophages
 Binds LPS on gram neg bacteria
 Helps destroy microbes, induce secretion of cytokines that trigger adaptive immunity
 Scavenger receptors
 On macrophages
 Recognize carbs, lipids in bacteria and yeast cell walls
 Toll-like receptors (TLR)
 Cell surface or vesicles
 Recognize patterns on multiple pathogens  signal presence of pathogen  expression
of costimulatory molecules and cytokines needed for adaptive response
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NODs are similar to TLR but in cytoplasm (involved in Crohn’s disease)
Innate versus adaptive immune receptors -----------------------
o Macrophages
o Aka phagocytes; eat particles and dying cells
o Derived from monocytes in blood
o Differentiate in different tissues (mononuclear phag system)
 Kupffer—liver
 Mesangial—kidney
 Alveolar—lungs
 Microglial—brain
o Secrete cytokines to activate immune cells
o Can be activated to kill bacteria
o Phagocytosis -------------------------------------------------------------------
 Attraction to site of infection via chemotaxic signals MDP, C
 Interaction with microbe for easier ingestion (opsonization)
 Mannose, C, Fc receptors
 Ingestion/endocytosis (invagination of membrane)
 Fusion of phagosome and lysosome (microtubules)
 Killing ingested material (O2 dependent/independent)
 Reactive O2 intermediates (ROI)
 Nitric oxide (NO)
 Lysosomal proteases
 Upregulated by IFN-gamma
 Digested pieces released  inflammation, recruit PMNs
o Activation of macrophages to (can interchange based on stimuli):
 Pro-inflammatory (classical): kill microbes through phag
 Wound-healing: produce ECM, alter cytokine production, suppress lymphocyte expansion
 Regulatory (anti-inflammatory)
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o Dendritic cells
o Immature DCs sit in tissues and phagocytose particles to sample environment
o Mature when encounter microbe signal (via TLR or TNF-alpha)  upregulate MHCI/II on surface  can’t
phag anymore, move to nodes to present to T cells, activate antigen-specific T cells
o Subtypes for different T cell types
o Different names for DCs
 Langerhans (LH)—skin
 Interdigitating (IDC)—lymph node T cell areas
 Follicular dendritic cells (FDC)—B cell follicles of lymph tissues
o Possible immunotherapy
o Neutrophils/polymorphonuclear cells (PMN)
o Major leukocyte in the blood
o Granules with enzymes that kill bacteria
o Short life span (hours/days)
o Have Fc receptors
o Move through blood till they get signal to move to tissue:
selectin on endothelial cells
 Inflammation induces selectin expression
 E.g due to TNF-alpha from macrophage
 PMNs slow down and roll ----------------------------
 Sensitive to inflammation signals (C5a, LPS)
 causes PMN to upregulate integrins on surface  bind to ICAMS on endothelium 
move into tissue by chemotaxis due to chemokine gradients  move to infection and
kill microbe
o Natural killer (NK, LGL) cells
o In many tissues and blood
o Make and release cytokines (inc IFN-gamma, TNF-alpha, GM-CSF) for virus and tumor killing, activating
macrophages
o Can kill cells
 Perforin granules
 Fas-Fas ligand
 TNF (cytokine) release
o No TCR or Ig
o Express Fc receptors
o Help with antibody dependent cellular cytotoxicity (ADCC)
o NK activating receptors bind to molecules on stressed/tumor/virus-infected cells
 Turns on NK killing and cytokine production
o NK inhibitory receptors bind to MHCI and shut of NK to protect normal cells
 Tumor/virus-infected cells sometimes dec MHCI to avoid T cells, but then NK can kill them
o Mast cells (CT) and basophils (circulation)
o Fc receptors for IgE
o Triggered by specific antigens
o Degranulate when activated  release pharmacological mediators
 Histamine: vasodilation, vascular permeability
 Cytokines
 TNF-alpha, IL-8/5: attract neutrophils, eosinophils
 platelet activating factor: attract basophils
o Eosinophils
o Production induced by IL-5 in marrow
o Granules involved in inflammation
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o Attack parasites that can’t be phagocytosed with major basic protein
o Related to allergies
Platelets
o Release mediators that activate complement  attraction of leukocytes
o Granules with chemokines, growth factors
Interaction between innate immune cells: network
o Macrophages make cytokines (TNFalpha) 
activates its own IL-12 production, recruits
PMNs
o IL-12  NK cells make IFN-gamma  makes
macrophages better killers, upregulates IL-2
receptors on NK so they proliferate
o TNF-alpha, IFN-gamma, IL-12 also activate T cell immunity!!
Resistance to innate immunity
o Organisms can become resistant to phag, ROS, complement, antimicrobial peptide
Innate versus adaptive immunity
o Innate cells/molecules often present at infection
site when it occurs
o Innate works rapidly acute inflammation
o Adaptive takes longer and is highly specific for the
microbe’s antigens
o Adaptive system shows memory so next response
is more rapid
o Adaptive is specific, but doesn’t know “bad” from “good/benign” – innate system decides whether to
attack
What to know
o Know the different levels of innate immune defense: physical, soluble molecules, cells
o Know the characteristics of the different cells of the innate immune system
o Characteristics and function of innate immune receptors
o Functions of immature and mature dendritic cells.
o Role of class I in NK cell recognition
o Understand the differences between innate and adaptive immunity
o Understand the role of innate defenses in activating adaptive immune defenses
ANTIBODIES/IMMUNOGLOBULINS
o Basics
o Antigen due to vaccination/infection  inc Ig specific to it
o Plasma, extravascular spaces, secretions
o Glycoproteins
o Bind antigens with high specificity, affinity
o Made by B cells
o 5 classes: G, A, M, D, E
o Basic structure
o 4 polypeptide chain unit covalently bonded
o 2 identical light (L) chains
 2 kinds: kappa κ, lambda λ
o 2 identical heavy (H) chains
 5 kinds (define Ig classes): M--µ. D--δ. G--γ. E--ε. A--α
o L bound to H via disulfide and non-covalent hydrophobic/hydrophilic interactions
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Symmetrical: allelic exclusion – inhibition of the other Ig genes in the B cell making a specific Ig
L and H have disulfide loop every 90 aa’s
o  polypeptide loop domains of 110 aa’s
 VH, VL, CH1, CH2, CH3 domains each with diff
function
 VH + VL: binding site for antigen
 CH2-CH2: complement fixation
o Characteristic of Ig superfamily
Fab fragment: N-term half of H chain and all of L chain
o Antigen binding site: N quarter of H and N half of L
 Binds to antigenic determinant
 Variable region of Ig
o Constant regions CH1
 All Ig of same class/subclass have same aa
sequence in constant regions
o Fc region: C-term half of H chain
o Constant regions CH2, CH3
o Determines effector function of Ig
 Combines Ig with complement
 Binds to certain types of cells at FcR
o Fc receptors (FcR)
 Many types of cells
o IgG FcR: monocytes, granulocytes, lymphocytes (not RBC)
o IgA FcR: monocytes, granulocytes (esp mucosal)
o IgE FcR: mast cells, basophils (involved in degranulation)
o Antibody valence, affinity, avidity
o Valence: max number antigenic determinants Ig can interact with
 E.g. 2 Fab sites can bind to 2 molecules antigen or 2 identical site on same antigen
o Affinity: tightness of bond between Ig binding aite and antigenic determinant
 Kd, dissociation constant (higher = more likely to dissociate)
o Avidity: combined effect of valence and affinity
 Higher valence  higher affinity due to more bond sites
o Antigens: fits in Ig pocket via non-covalent bonding
o Innate system recognizes molecule patterns common to microbes
o Adaptive system recognizes specific antigens on a microbe
o Protein, lipid, carb, na
o Ig binds to native antigen (how it is in nature)
o Must be unique enough to induce immune response
o Often several diff antigenic determinants (epitopes) per antigen  multideterminant
 Ig binds to this (can have multiple diff Ig’s if determinant is non-identical)
 Protein: 3-6 aa’s; Carb: 5-6 sugar molecules
o Carbs tend to have repeating sugar units  several identical determinants
o Antibody classes (isotypes)
o IgG
o 150kD (small, so gets into tissues)
o vascular, extravascular, and secretions
o most abundant Ig in blood
o most immunity for blood borne microbes
o crosses placenta  passive immunity to fetus
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o 4 subclasses with diff H chain sequences (1-4)
 bind to diff FcR
o long half-life  good protection
 FcRn binds to IgG at low pH when IgG
gets to endosome and recycles it
o Requires help to activate complement
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IgM
o 900kD (big!)  mostly in vascular space/serum,
not tissue
o First Ig expressed on B cell
 Normal 4-chain unit
o Also soluble in blood
 5 5-chain units held together
 J chain causes polymerization
o Activates complement well (on its own)
o 10 binding sites per molecule  high overall
avidity
 important since IgM is first in immune
response; must act until IgG is ready
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IgA
o Vascular
 170kD
 normal 4-chain unit
o Also major Ig in secretions (colostrum, milk, saliva)
 also has secretory component (SC) and joining chain (J chain)
o SC: transepithelial transport, protection from degradation
o J chain: holds units together via disulfides
 420kD dimer
 made locally by plasma cells in mammary and salivary glands and resp, GI, GU tracts 
transported through epithelial cells to lumen
o Defense against microbes at mucosal surfaces
o 2 subclasses
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IgD
o Low quantities vascular
o Antigen receptor on B cells
 Naïve B cells have IgM and IgD for the same antigen
 Antigens internalized when bound  presented on surface to T cell  T cell signals B to
proliferate and become plasma cell  plasma makes lots of Ig
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IgE
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Very low quantities vascular
Acute inflammation
Protection from worm infections
Allergies, hay fever, asthma (hypersensitivity)
Mast cells with IgE
 Antigen binds  mast cells releases histamine
o Allotypes and idiotypes
o Allotypes
o Genetic markers on Ig’s that differ between individuals
o Often single aa differences on L or H chain
o  immunogenic when injected into individual that doesn’t have the allotype
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o don’t affect function of Ig
Idotypes
o Unique antigenic determinants in the antigen binding site
o Ig’s can be made against them when injected into other animals
o Each B cell clone expresses same idotype (produces single type of Ig)
o IgM versus IgG
o Neutralizing virus/toxins: IgM is better since it has 10 binding sites
o Wider distribution: IgG, since it’s smaller and can get into tissues
o Long-term protection: IgG due to long half-life
o Interaction with innate components: IgM can activate complement on its own, IgG requires 2 molecules
o What to know
o Know the different antibody isotypes and subclasses.
o Be able to describe antibody structure and know how this relates to different functions
o Know where different antibody isotypes are found
o Understand the basic nature of an antigen and its epitope (what is recognized)
o Understand the unique features of each isotype that relate to its specific function
o Know the differences between isotypes, allotypes and idiotypes
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ANTIBODY-ANTIGEN REACTIONS
o Basics
o Interaction is non-covalent: H-bonding, electrostatic, Van Der Waals, hydrophobic
o Better fit  tighter bonding
o Interaction is reversible
 Mass action: K = [Ab—Ag]/[Ab][Ag]
o Types of antigen-Ig reactions
o Agglutination: antigen particle plus specific Ig  aggregation of particle
o Precipitation: soluble antigen plus specific Ig  insoluble lattice formation
o Complement activation: antigen in solution/on cell plus specific IgG or IgM  complement inactivation
o Cytolysis: cell plus anti-cell Ig plus complement  lysis of cell
o Opsonization: antigenic particle plus Ig plus complement  enhanced phag
o Neutralization: toxins/viruses/hormones plus specific Ig  inactivation
o Antigens
o Microbes have lots of surface molecules each with many antigenic determinants
 Some elicit stronger responses than others (based on health, age, genetics)
o Haptens (e.g. Penicillin): small molecules that can’t elicit Ig response unless attached to larger molecules
(carriers)
o Cross-reactivity
o Similar/identical antigenic determinant on multiple molecules or cells (shared epitope)
o Cross-reactive antigen may not bind to Ig quite as well
o E.g. people have Ig’s to blood type antigens other than their own bc they’re similar to carb antigens on
some microbes
o May explain natural/innate Ig’s to a variety of molecules
o Autoimmune (e.g. group A strep infection  rheumatic fever; strep antigens similar to heart muscle)
o Precipitation: antigenic particles initially soluble
o Anti-hapten Ig plus hapten  soluble complex
o Anti-hapten Ig plus protein with many haptens attached 
insoluble complex
o Lattice formation: if protein has several hapten determinants, Ig
can bind to more than one antigen (hapten) at a time
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 Leads to precipitation
 Adding hapten to anti-hapten Ig before adding hapten-protein conjugate inhibits precipitation
o Level of precipitation
 Antibody excess zone: lattice doesn’t develop, soluble
 Equivalence zone: maximum precipitation; no antigen or Ig detected in supernatant
 Antigen excess zone: lattice doesn’t develop, soluble
o Protein antigens don’t usually have repeating sequences  1 determinant of a particular kind per
protein antigen
 So, there are usually multiple Ig’s formed for each protein antigen (specific to its diff
determinants)
 So, lattice formation doesn’t occur for most soluble proteins (unless it has repeats)
o Agglutination: linking insoluble antigenic particles
o Interaction of surface antigens on insoluble particles (e.g. cells) with antigen-specific Ig
o Need much less Ig than for precipitation
 Clinical uses:
 Blood type
 If bacteria is present in blood
o IgM is more efficient at it than IgG bc it has 10 binding sites
o Sometimes Ig binding to cell doesn’t cause agglutination
 Use second Ig reactive to first Ig
 Coombs test
 Identifying patients with hemolytic anemia
 Use Ig reactive to human Ig  causes agglutination of RBCs if human Ig is bound to RBCs
 Or indirectly: add patient serum to cells and then add mouse or rabbit anti-human Ig to
detect circulating Ig reactive to cell surface Ig
o Results in bigger circle on plates
o Monoclonal antibodies
o Making monoclonal antibodies (mAbs)
 Fuse immortal cell (myeloma tumor cell) with specific
Ig-producing B cell from immunized animal/human
 hybridoma cell
 Hybridoma cell is immortal and makes specific mAbs
 Used for clinical applications
o Fv libraries to make mAbs
 Get mRNA for Vh and Vl regions from lots of B cells
 Use the mRNA to make cDNA for each H chain V
region and randomly join it with cDNA for each L
chain V region  produces genes with lots of diff
antigen combining sites (Fv regions)
 Clone the Fv’s into cells  source for specific mAbs
o Antigen-Antibody assays used clinically
o Background
 Antigen on cell can be detected via labeled Ig
 Radioisotopes, fluorescein, enzymes
o Radioimmunoassay (RIA)
 Measure serum level of Ig to a specific antigen
 Add antigen to wells  wash  add test Ig  wash
 detect with radiolabeled ligand that binds to Ig
o Enzyme-linked immunoabsorbent assays (ELISA)
 Like RIA, but ligand is coupled to enzyme (e.g. peroxidase)
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 Bind ligand to test Ig  wash  detect by adding substrate that reacts with enzyme  color
RadioAllergoSorbent Test (RAST)
 Like RIA, but measures antigen-specific IgE with radiolabeled anti-IgE
o Antigen detection
 Competitive assay: Ig on plate  add test antigen (serum) and labeled antigen  test antigen
will out-compete labeled if it’s present
 Two-site capture assay (more common): capturing Ig on plate  add test antigen (serum) 
binds to capturing Ig  add labeled Ig  binds to other site on test antigen
o ELISPOT: for cells that produce cytokines
 Cytokine-specific Ig on plate  add activated T cells  T cells secrete cytokines  cytokines
bind to Ig  add second cytokine-specific Ig that’s labeled with enzyme  colored precipitate
o Mistakes during antigen detection
 Caused by heterophillic Ig (HA) – anti-animal human Ig
 False positives: HA bridges test Ig and labeled Ig via Fc or Fb regions
 False negatives:
 Anti-idiotype HA steric hindrance: HA blocks test-Ig binding sites by binding to Fab
 Anti-isotype HA steric hindrance: HA blocks test-Ig binding sites by binding to Fc
 Antigen binding HA steric hindrance: HA binds to antigen so it can’t bind to test Ig
o Flow cytometry
 Bind labeled Ig to cells in suspension  run through flow cytometer  cytometer measures
fluorescence of each cell
 If labeled Ig cells are run through fluorescence-activated cells sorter (FACS), cells get separated
into diff tubes based on their brightness
 Use to test for cancer types with blood cancer
o Immunohistochemistry (IHC)
 Detect antigens on histological specimens
 Bind labeled Ig to tissue section  wash  detect expression via fluorescence or enzymes
 Advantage: detect antigen within complex tissue
 Counterstain all/adjacent cells and tissues to get location of antigen
 Use to test for cancer types with solid tumors
o What to know
o Understand the basic nature of antigen-antibody reactions
o Know the difference between a hapten and an immunogen
o Understand cross-reactivity and the consequences of it
o Know requirements for lattice formation and precipitation
o Know what a Coombs test is
o Know what monoclonal antibodies are and their uses
o Be familiar with the basic concepts involved in antigen-antibody assays, including RIA, RAST, ELISA, and
flow cytometry
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ANTIGEN PROCESSING AND PRESENTATION
o Immune system must monitor for pathogens inside and outside cells
o Outside: bacteria, protozoa, worms, fungi
o Inside: viruses, bacteria, protozoa, tumors
o Monitor outside via Ig
o Hard to get at the inside ones  small pieces of proteins inside cells (peptides) are presented on surface
via MHC
o MHC-peptide complex scanned by TcR
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If TcR binds MHC-peptide complex AND gets costimulatory signals, T cell
responds  proliferation, cytokine production, differentiation
MHC Class I pathway: presentation to CD8 T cells (CTL) – kill virus-infected cells ----
o Occurs in all nucleated cells
o Proteosome: protein complex that degrades defective cellular proteins in
cytoplasm for reuse
 During immune response, proteosome changes shape better  makes
peptides that have better fit with MHC I
o Transporters associated with antigen processing (TAP): Brings peptides into ER
o In the ER: peptides associate with MHC I exported to cell surface
o Cell surface: MHC-peptide complex recognized by CD8 T cells
MHC Class II pathway: present to CD4 helper T cells – interact with DC, macro, B cell
o Occurs in antigen presenting cells (APC) – macrophage, B cell, dendritic cell
o APC phagocytoses extracellular proteins
 Macrophage and DC take up lots of antigens via nonspecific receptors
 B cell takes up specific antigen via specific Ig  B cell activation
o Endocytic/lysosomal vesicles: pathogen proteins broken into peptides
o MHC II fuses with endocytic vesicle and gets loaded with peptide
 MHC II is assembled in ER but doesn’t take up peptides there (MHC I does)
 Invariant chain blocks binding pocket of MHC II when in ER
 Invariant chain degraded in endosome, leaving CLIP peptide
 HLA-DM removes CLIP, so MHC II can bind to peptides in endosome
o MHC-peptide complex goes to cell surface  recognition by CD4
 TcR binds to specific MHCII (recognizes antigen)
 CD4 binds to invariant region of MHC II
Features of MHCs
o One peptide displayed at a time  each T cell responds to single MHC-peptide
o Peptides acquired during MHC complex assembly intracellularly (endosome for
MHC II, ER for MHC I)
o Low affinity, broad specificity  different peptides can bind to the same MHC
o Slow off-rate  peptide displayed long enough to get specific T cell
o Stable expression requires peptide  empty MHCs recycled
o MHCs only bind peptides  MHC-restricted T cells only respond to
protein antigens (not carb, lipid, na!)
Cross-presentation
o Costimulation is only provided well by APCs (especially DCs)
o So, CD8 cells need a way to respond to virus-infected cells, which
don’t provide costimulation
o DCs ingest virus-infected cells and present viral peptide on MHC I
 = cross-presentation
 antigens move from endosome to cytoplasm!
 Then they follow MHC I pathway and are presented to CD8
 They’re also present to CD4 via MHC II like usual
MHC molecules and peptide binding
o MHC’s have polymorphic peptide binding domains
o Peptides that bind are 9-12 aa
 Have anchor residues (Y) which bind to MHC
o Diff MHC’s bind to diff peptides from same antigen bc they have diff aa’s
o MHC must be able to bind to peptides of an antigen to have T cell response to that
antigen
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o MHC structure
o MHC I and II genes linked on chromosome 6
o MHC I
 HLA- A, B, C genes
 10-70 alleles for each
 Complex: MHC I alpha chain, beta-2-microglobulin, peptide
 CD8 binds to alpha 3 domain
o MHC II
 HLA- DR, DQ, DP genes
 7-70 alleles for each
 Complex: MHC II alpha chain, MHC II beta chain, peptide
 Alpha and beta are polymorphic, mostly in alpha 1
and beta 1 domains, which interact with TcR
 CD4 binds to beta 2 domain
o Codominant expression; complete set of genes inherited from each parent  increases diversity of MHC
o Polymorphic genes  diff individuals present and respond to diff microbial peptides
Examples:
1. influenza virus
2. mycobacterium
tuberculosis
3. strept pneumoniae
o Tissue distribution of MHC I and II antigens
o B cells, DC, macrophages express lots of MHC II 
proficient at presenting to CD4 T cells
o All nucleated cells express MHC I  proficient at presenting to CD8 T cells
o Non-classical MHC molecules
o HLA-G
 MHC I
 Nonpolymorphic, no HLA-A, B, or C
 In extravillous trophoectoderm (fetal tissue contacting maternal circulation)
 May prevent maternal immune response (T and NK cells) against paternal antigens in the fetus
o CD1d
 Nonpolymorphic
 Not encoded in MHC region
 On B cells, DC, and some non-APCs
 Presents non-peptide antigens (lipid and glycolipid) to T cells
 Restricts T cell response to foreign microbial antigens
o Uptake and presentation of foreign antigens (MHC II, or MHC I via cross-presentation)
o Immature DCs take in foreign antigens at epithelium/CT
o DCs get signals that there’s a pathogen
 Pathogen-associated molecular signals (lipopolysaccharide, double stranded RNA)
 Inflammatory cytokines
o DCs migrate to lymph nodes via lymphatics to present to T lymphocytes
 Migrate to T cell areas using cytokine gradients
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
DCs mature and upregulate expression of costimulatory molecules  activate antigen-specific T
cells by presenting antigen and giving costimulation
o Antigens that enter blood stream can be captured by APCs in spleen
o What to know
o Understand MHC class I pathway
o Understand MHC class II pathway
o Know the fundamental differences in the Class I and II MHC processing pathways, and with which
subsets of T cells they interact.
o Know the general properties of class I and class II MHC molecules
o Know the restrictions for peptide binding to MHC molecules.
o Know the significance of MHC gene polymorphisms
o Understand the role of dendritic cells in antigen presentation to naïve T cells
T CELL RECOGNITION OF ANTIGEN
o Combatting intracellular infections is primary role of T lymphocyte
o Microbes in phagosome can resist proteolysis
o Viruses in cytoplasm
o T lymphocytes
o Scan for intracellular pathogens via TcR
o TcR recognizes antigenic peptides on MHC
o Once recognized, T cells become effectors  attack, recruit other cells, increase killing efficiency
o CD8 cells (CTL)
o CD4 cells (Th)
 Can differentiate to effectors, secrete cytokines
 Target of AIDS virus
o Induce adaptive immune response and immunological memory
o Antigen recognition
o DO NOT recognize native antigen
 Recognize peptides from antigens (bacteria, virus) on MHC
o TcR --------------------------------------------------------------------------------
 Alpha and beta chains created from gene recombination
 Each T cell expresses TcR with diff sequence that recognizes
unique peptide-MHC complex
 Rearrangement/expression in thymus during T cell devel.
 TcR complex
 TcR: unique alpha and beta for each MHC-peptide
 CD3 proteins: activate signaling pathways in T
cell; identical on all T cells
o Coreceptors needed for T cell activation
o CD4: binds to MHC II invariant residues in beta 2 domain
o CD8: binds to MHC I invariant residues in alpha 3 domain
o Immunological synapse
o Naïve T cells circulate through nodes and spleen looking for
APCs with MHC-peptide that matches their TcRs
o T cell stimulation requires:
 TcR binding to MHC-peptide
 CD4/8 coreceptor activation
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

o
o
o
o
o
CD3 signal conduction
Adhesion molecule (integrin) engagement
 ICAM-1 on T cell, LFA-1 on APC
 Surround TcR-MHC proteins and hold cells
together
o = immunological synapse
 Chemokines and antigen-TcR binding make
integrins become high-affinity  strong adhesion
between cells  T cell response
 Costimulation (see below)
Costimulation
o Activates specific pathways, ESSENTIAL for naïve T cell activation
o CD80 (aka B7): on APCs, binds to CD28 on T cells
o CD86: on APCs
o Expression on APC increases in response to microbe  signal 2
o Only on DC, mac, B cell  ONLY THES PROFESSIONAL APCs can
activate naïve T cells!!
o Two-signal model to prevent response to self antigens
 Signal 1: TcR binds to MHC-peptide
 If CD8 T cell only gets signal 1  tolerance (T cell is unresponsive/inactived – anergy)
 Signal 2: CD28 binds to costimulator, which upregulates at APC surface in presence of microbe
 Signal 1 and 2  growth and cytokine production of T cell
o Cross-presentation of extracellular peptides by MHC I is needed so that CD8 T cells can get activated!
 ??????
Early T cell activation, growth, cytokine production
o Clonal expansion
 Activated T cell begins to divide A LOT
 Daughter cells (clones) have same TcR and same MHC-peptide specificity
o T cells make growth factors to support their growth
 Interleukin 2 (IL-2)
 Interleukin/lymphokine: soluble proteins
make by immune cells
 Production stimulated by TcR stim
 T cell increases high-affinity Il-2 receptor as well
  autocrine signaling loop
 Also paracrine function of IL-2 on other cells
 B cell differentiation
 Growth factor for NK cells
Turning off immune response
o Activated T cells begin expressing CTLA-4
o CTLA-4 binds strongly to CD80 and 86, replacing CD28  inhibitory signal
o Lack of CTLA-4 function can cause inflammatory disorders, tissue injury
Superantigens
o Some bacteria/viruses make proteins that are superantigens bc they
stimulate lost of diff T cells
o Bind to sides of MHC II and Vb region of TcR, gluing APC and TcR together
o Tons of cytokine production  vascular leakage, shock
o Staphylococcal enterotoxins (food poisoning, toxic shock syndrome)
What to know
o How the TCR recognizes antigen
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o
o
o
o
o
o
Coreceptors involved in T cell activation
What is costimulation?
Clonal expansion of antigen specific T cells
Role of cytokines in T cell activation
Inhibition of T cell activation by CTLA-4
What are superantigens?
T CELL RESPONSE AND T CELL MEDIATED IMMUNITY
o T cell selection
o T cell precursors migrate to thymus (progenitor cells committed to T cell lineage)
o Most die within thymus
o T cells undergo gene rearrangement in thymus  TcR expressed on thymocytes
 Have CD4 and CD8 = double positive thymocytes
 Many can’t express usable TcR
o Positive selection: cells with TcR that weakly binds to MHC I or II survive
 Thymic epithelial cells in thymic cortex are the presenting cells, present
lots of peptides that would normally be expressed elsewhere
 Then express only CD4 or 8 depending on if they bind MHC I or II
o Negative selection: cells with TcR that strongly binds to MHC I or II die
 Autoreactive, so may cause problems in periphery
 DC, mac, medullary epithelium are the presenting cells
o Select T cells that recognize self MHC and foreign peptide, not self peptide!
o Regulatory T cells: possibly come from cells with very high affinity for self
 CD4 cells that suppress immune responses when exported to periphery
 Inhibit via cytokine production (active) and interaction with other cells (passive)
o Lymphocyte trafficking and recirculation: speed dating btwn lymphocytes and DC’s
o Naïve B and T cells (have not found their antigen yet) recirculate through secondary lymph organs
o Adhesion molecules on surface let them attach to endothelial cells to exit blood stream via HEV
o Homing adhesion molecules attach to specific molecules on
endothelial cells (addressins) in certain sites ---------------------
 Allows lymphocytes to target mucosal or peripheral
lymph node areas
 MALT: lymphocytes stimulated in one MALT site will
migrate to other MALT sites via homing
o Once activated, lymphocytes alter chemokine receptors so
they can go into blood stream and infection
o Tolerance
o Antigen-specific T or B cells reactive to self tissues may be:
 Clonally deleted
 Made functionally unresponsive
 Prevented from responding (suppressed)
 Lacking antigen-specific receptor or MHC element
(CD4/8)
o Tolerance is specific
 Can still respond to other antigens
 May be partial (e.g. weak/altered immune response)
 Can be humoral or cellular
o Central tolerance
 Clonally deleting T and B self-reactives in the thymus
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and marrow (i.e. negative selection)
Antigen exposure during development
 Virus in neonate may be seen as self  deletion of lymphocytes responsive to it
o Peripheral tolerance
 Some autoreactive T cells make it to the periphery
 If self-antigen is on an APC when the APC also happens to
have antigen particles, autoreactive T cell can get
costimulation and respond; otherwise, they won’t
respond  anergy (expression of proapopotic proteins or
of death receptor and death receptor ligands Fas/Fas-L)
 CD4 T regulatory cells can keep T cells from responding
o Effector cells in T cell immunity -----------------------------------------------
o Activation requires:
 Specific signal via TcR
 General costimulatory signal

Activation  autocrine growth factors (IL-2), alters cells surface molecules
(for trafficking), inc membrane proteins to trigger other cells (e.g. CD154),
production of cytokines
o CD8 cytotoxic T cells (CTL): recognize MHC I
o Host resistance to pathogens that live in cytosol (e.g. viruses)
o Recognize pathogens via MHC I
o Killing mechanisms
 Lyse cells they bind to with perforin and granzymes  pores in cell
membrane  apoptosis
 Release factors (TNF-alpha, IFN-gamma) onto cells they bind to or
express molecules on surface (Fas-L) that trigger apoptosis
o CD4 helper T cells (Th): recognize MHC II
o Early mature cells: Th0
 Broad spectrum of cytokines
o Later mature cells: Th1, Th2, Th17
 Th1: inflammatory responses (activate mac and CTL) via cytokines
IFN-gamma, IL-2, TNF-alpha
 Intracellular microbes – help macrophages become
completely activated (IFN, TNF)
 Makes cytokines that recruit macrophages and inc release
from marrow (GM-CSF, IL-3)
 TNF promotes adhesion of macrophages to
endothelial cells at infection site
 Induce B cells to make IgG
 IFN-gamma: prevents induction of Th2; increase
production of cytokines that inc Th1 differentiation
(pos feedback)
 Th2: help B cells grow/differentiate via cytokines IL-4, IL-5, IL13
 Induce B cells to make Ig, isotype switching, affinity
maturation!! Via:
 Cytokines
o IL-4  IgE  IgE binds to mast cells  mast
cells make IL-4  Th2 induction (pos fdbck)
o
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o Allergic rxns
 Surface molecules that engage B cells
 IL-5  eosinophil production in marrow (worms, large parasites)
 Th17: inflammation, autoimmunity, inflammatory disorders via IL-17
o CD4 regulatory T cells (Treg)
o Usually derived from strongly self-reacting CD4 cells in the thymus; become T reg with IL-2 and Foxp3
o Suppress T cell immune responses in thymus (inhibit activation) and periphery (inhibit effector function)
o Maintain tolerance to self-proteins
o Contact-dependent mechanism that reduces inflammatory response via IL-10, TGF-beta
o Cytokines
o Act like hormones and NTs for cellular communication
o Proteins, peptides, or glycoproteins
o Interleukins, interferons, etc
o Have lots of functions; related to allergies, cancer, inflammatory disease, immune reconstitution
o Cytokines enhance or suppress cell-mediated immunity  treatment of autoimmunity, cancer, etc.
o What to know
o Thymic structure
o T cell development in the thymus
o What is MHC restriction? Why is this important?
o Mechanisms of central vs peripheral tolerance
o T cell trafficking in the periphery and into tissue
o Role of cytokines in T cell effector cell development
o Mechanisms of CD8+ T cell function
o CD4+ T cell subsets and their function in immunity
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ANTIBODY DIVERSITY
o Nomenclature for Ig gene segments
o Variable (V): exons encoded as a family of related sequences
o Diversity (D): small exons that contribute to sequence variability; 3’ to V
o Joining (J): small exons that contribute to sequence variability; 3’ to J
o Constant (C): exons that define class and effector function of Ig; 3’ to VDJ
o Antibody genes
o 3 unlinked gene groups on diff chromosomes encode Ig
 one for kappa L chains
 one for lamba L chains
 one for H chains
o Recombination occurs within each group
 Multiple coding exons recombine
o Variable region (V)
 Mature B cell or plasma cell: Vregion of H chain is
continuous
 Germ-line or non B cell: V region of H chain is
interrupted by introns
 V region of H chain has 3 segments
 V segment
 D segment
 J segment
 V region of L chains
 V segment
 J segment
o Constant region (C)
 In both L and H chains
 3’ to V genes but separated from them by unused J segments and non-coding DNA
 1 functional gene segment for each class and subclass
 C regions of H chains
o 1 segment each for M, D, G1-4, E, A1-2
o also encodes transmembrane domain
 C regions of L chains
o 1 segment for kappa group, 7 segments for lambda group
o Gene rearrangement/translocation
o In development, B cell selects:
 one V,D, J combo for H chain
 one V, J combo for L chain
o H chain group rearranges first
 Selected D moved next to selected J
 Selected V moved upstream of DJ  heavy chain V region
 C genes are downstream, the closest one is M
 Primary transcript has VDJ, intervening DNA, M
 That is spliced to mRNA with VDJC  complete IgM heavy chain
o L chain rearrangement next
 Selected V from either kappa or lambda moves next to selected J  light chain V region
 C region
 Kappa chains: C region 3’ to V region with unused J between
 Lambda chains: one of 7 lambda C genes associated with the J segment
 Primary transcript has VJ, intervening DNA, C
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o
 That is spliced to mRNA with VJC  complete IgM light chain
Rearrangement requires recombination activating genes RAG-1, RAG-2
 Only expressed in developing lymphocytes
 Break and rejoin DNA during translocation
o Synthesis and assembly of chains
o L and H polypeptides combine in the ER to form Ig
o Antigen-specific Ig transported to plasma membrane
o Heavy chain has C terminal sequence that anchors Ig to plasma membrane
 This sequence is removed in plasma cells so Ig can be secreted
o Ways to create diversity
o Antigen independent mechanisms
 Combinational diversity
 Gene segments chosen at random in each B cell (number of possible VDJ, VJ combos)
 Random selection and pairing of L and H chains in the ER
 Junctional diversity
 Imprecise joining of VDJ of H chain or VJ of L chain (variability in position of joining)
o Last nt of V could be replace by first nt of J  change in antigen binding area
 Addition or subtraction of nt during recombination
o TdT adds nt’s to ends of gene segments, called N nucleotides
o Overhangs during recombination may be filled in with P nucleotides
o Antigen-dependent mechanisms
 B cells stimulated by antigen and Th undergoes somatic mutation: changes in V regions of L and
H chain that increase/decrease affinity
 Affinity maturation: B cells that increase affinity of their Ig will become plasma cells with
a higher affinity Ig
o Lots of B cells are wasted, but excess diversity ensures response to wide array of antigens
o Allelic exclusion
o After successful rearrangement of VH and VL, other gene segments are suppressed  1 specificity
 If rearrangement doesn’t work, another rearrangement will occur
o Unique to B and T cell antigen receptors
o Differential splicing and Ig receptor expression -------------
o IgM expressed first on B cell
o Then IgM and IgD
 Have same VH and VL  same specificity
 Mature naïve B cell
o Primary RNA transcript is differentially spliced to yield
two mRNA’s (IgM and IgD)
 Primary has both M and D constant regions
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o Stages of B cell differentiation ---------------------------
o In bone marrow
o Signals: marrow stromal cells, cytokines induce
o H chain rearranged first
o Pre-BCR complex: M protein expressed on cell
surface with 2 invariant chains (L-like)
 Signal to stop H chain rearrangement and
work on L chain
o Successful H and L chain  functional IgM
 = immature B cell
 susceptible to tolerance induction
 IgM is signal for survival and expansion
o Then get coexpression of IgM and IgD
 = mature naïve B cell
 responds to antigen; required for diff to plasma cell
o B cell receptor (BCR) complex ---------------------------------------------------------------------------
o Ig alpha and beta are 2 TM polypeptides associated with Ig that signal for BCR
 Ig are TM molecules but cytoplasmic domain is only 3 aa long  can’t signal
 Signal prepares B cell to interact with Th cell (effects on transcription factors
like Myc, AP-1)
 Identical signaling for all B cells
o B cell coreceptor complex: CD21/CR2, CD32, CD19, CD81
 Enhances or inhibits BCR complex
 If BCR and coreceptor both bind to antigen/complement, CD21 and 32 are
engaged  CD19 and 81 influence signaling via Ig-alpha/beta complex
o B cell tolerance
o During B cell development
 Negative selection: deletion or anergy (can’t respond) of self-reactive B cells which express
receptors with high affinity for self or bind to membrane antigens
 Receptor editing: recombination of L chain genes, making B cell with new specificity
 Chance to change from self-reactivity and survive
o After secondary stimulation of memory B cells
 Susceptible to tolerance by epitopes presented multivalently if they don’t get T cell help
o Self-reactive B cells can get activated if they express both self and non-self peptides at the same time
(will get T cell help)
o What to know
o Know how antibody diversity is generated (recombination)
o Understand how differential splicing is used to create immunoglobulin
o Know what allelic exclusion is and why it is important (get 1 specificity!)
o Understand the role of antigen in the generation of antibody diversity (no much of a role!)
o Know stages of B cell development and when self-tolerance can occur (2 occurrences)
o Know the different components of the B cell receptor
o Understand B cell tolerance to self antigens
HUMORAL IMMUNITY
o Antigen origin impacts response
o Introduced in blood  spleen  splenic macs and CDs  presentation of antigenic determinants 
T cells
o Mucosal areas  B cells, macs, DCs below mucosa  presentation of antigenic determinants  T
cells  B cell has antigen plus T cell help  humoral response  IgA released to mucosa
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Tissues  travels through lymphatics to lymph tissue/node  B cells, macs, DCs  presentation of
antigenic determinants to T cells  T cells in paracortical region, B cells in follicle and divide in
germinal center
Primary response
o Occurs within 5-8 days of exposure
o Production of IgM followed by IgG or IgA by plasma cells
o Durations depends on quantity of antigen and mode of entry
o Ig reacts with antigen, making complexes or precipitates eliminated by phagocytosis
o Ig production reaches peak after antigen is gone
o One B cell makes one specific Ig, but lots of B cells get activated by diff antigenic determinants
B cell activation
o Need to avoid activating non-specific B cells
o B cells recognize antigenic determinant via Ig on membrane (affiliated with Ig-alpha-beta = BCR)
o Antigen coated with complement interacts with CD21/CR2, CD19, CD81
o Results in signaling pathway activation, transcription factor activation
o Consequences of antigen binding
 Proliferation
 Upregulation of CD80/B7
 Upregulation of cytokine receptors
 Processing and presentation of antigen on MHC II
 Downregulation of chemokine receptors so it can leave follicle
 Low-level secretion of IgM
 Now can encounter antigen-specific T cell
o B-T interaction for activation of naïve B cells when they get to parafollicular zone (T cell zone)
 B cell presents antigen to activated Th
 Th has CD154 (CD40 ligand, reacts with CD40 on
B) and cytokines
 CD154-CD40 interaction required 
ensures antigen-specific activation
 B cells proliferate and become:
 Memory cells
 Plasma cells that secrete specific Ig
B cell differentiation after activation: occurs in germinal center
o Ig isotype switching ------------------------------------------
 B cells with IgM and IgD can switch to other H
chain classes
 E.g. to IgA or IgE in mucosal region
 Requires stimulation via CD154-CD40 and
cytokines (IFN-gamma, IL-4, IL-5)
 Get translocation of VDJ 5’ to another H
constant (C) region
 Guided by repetitive DNA
 Intervening C DNA cut out
 If a plasma cell, heavy chain C regions
are removed to allow secretion
o Affinity maturation
 Affinity of Ig for antigen increases with time
 B cells increase in number, start competing 
those w/ high affinity expand
o
o
o
o
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o
o
o
 Also, somatic mutation of VH and VL in B cells stimulated by antigen and T cells
 Decrease in dissociation constant
Secondary (memory) response -------------------------------------
o Reintroduction of antigen  faster, better response
o Dec lag period for Ig presence due to memory cells
o More plasma cells develop
o Higher conc Ig in serum
o More antigen-binding cells  those with highest
affinity for follicular DC’s or Th win  results in
plasma cells with high affinity
 Competition = need higher affinity!!
o Vaccination
T cell independent (TI) B cell response
o Some antigens can activate B without Th = TI antigens
 Resistant to degradation
 Often repeated epitopes, so they cluster around the B cell and activate it
 E.g. bacterial cell wall, poly-aa, dextran
o Primary IgM response peaks a little early
o But poor induction of memory B cells (secondary response is like primary)
o No IgG response, prob bc they don’t induce cytokines/signaling that lead to isotype switching
Immunity in newborn: immature and takes a bit longer
o Lymphocytes in newborn
 Higher thank normal numbers of T and B (and NK)
 But, limited ability to respond to some antigens
 Age of exposure to antigen is important
 Sequential expression of genes encoding for receptors for antigens
 Immaturity of B and T cells
 Immaturity of APCs
o Ig in newborn
 Maternal IgG crosses placenta (using Fc receptors),
compensates for lack of IgG until infant makes it
 IgM made during fetal development
 IgA is made by 1-2 months age
 Maternal IgA obtained via colostrum, milk gives
mucosal immunity (passive)
 Passive immunity from mother may interfere with development
of active immunity  don’t respond to some antigens
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