Innate Immunity I

Innate Immunity = rapid, general response, evolved earlier

 Cells  macrophages, neutrophils, NK cells - interact with a wide range of structure
 Receptors inherited in genome
 Lack of clonality
Adaptive Immunity = slower, more specific
 B & T lymphocytes  clonal distribution
 Receptor genes rearranged in development
 Discriminate between closely related molecular structures
 Disadvantages = time required for differentiation, not heritable (somatically mutated), random
specificity (some anti-self)

Pluripotent Cells of the Innate Immune System

hematopoietic Erythroid progenitor RBCs
stem cell
Megakaryocyte progenitor Platelets
PHSC Myeloid progenitor Macrophages, neutrophils, basophils, eosinophils
Lymphoid progenitor T & B lymphocytes

WBCs = 55-70% neutrophils, 20-35% lymphocytes, 3-7% macrophages, 1-3% eosinophils, 1% basophils

Neutrophils  Phagocytose and kill harmful targets, activated by IL-1, G-CSF, TNFα, GM-CSF
 First responders, found during acute inflammation
 2 weeks to mature from bands to polys (PMN/polymorphonuclear), live 1 day in tissue
 Recognize targets via Fc and complement receptors

Macrophages  Phagocytose, also capable of antigen presentation to T cells

 Promonocytes differentiate in marrow (2-5 days)  enter circulation as monocyte  after1 day,
enter tissue and differentiate into tissue-specific macrophage
o Tissue macrophages have more cytoplasm, more granules, and a ruffled plasma memb.
 Present in chronic inflammation, also in parasitic infections
 Activated by IL-3, M-CSF, GM-CSF

NK Cells  Kill infected host cells via release of cytotoxic granules

 Activated by cytokines, especially IL-12 and IFNγ
 Target recognition through activating and inhibitory receptors (for MHC Class I)

Functional Components of Innate Immune System

Barrier Functions  Act as the primary defense
 Epithelial cell layer: joined by tight junctions, able to secret β-defensins (antimicrobial peptides
that disrupt bacterial membrane)
 Mucosal surfaces: Mucus decreases pathogen adhesion and creates constant flow
 Low pH: In gut, lysozymes in saliva

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Recognition of Pathogenic Structures
PAMPs (Pathogen Associated Molecular Patterns)
 Expressed by pathogen and not by host
 Structurally invariant, required for pathogen's survival, often a repeating motif
 Usually a polysaccharide (e.g. LPS in gram neg bacteria) or nucleotides, not proteins
PRRs (Pattern Recognition Receptors)
 Receptors that recognize PAMPs, are encoded in genome/germline
 3 types exist:
o Extracellular  function in opsonization or complement activation
 CRP
 MBL (Mannan binding lectin)  binds mannose residues, differentiating b/n
host and pathogen by the spacing of binding domains
o Endocytic  Function in phagocytosis
 MMR (binds like MBL, but internalizes pathogen and fuses with lysosome)
o Signaling
 Cell Surface Signaling (Toll-like receptors, TLRs)
 TLRs are a family of membrane-bound receptors that recognize a variety
of PAMPs (depending on different LRR/leucine-rich repeats)
 Intracellular Signaling (PRR within cell  TLR4, NOD1 and NOD2)
 NOD proteins in cytoplasm recognize peptidoglycans (gram + bacteria)
 Regulate inflammatory and apoptotic response

Innate Immune Signaling (Ex. using TLR4, which binds LPS and ultimately activates NFκB)
LPS binds to carrier LPS-binding protein (LBP)  Carried to receptor on macrophage (CD14)  LPS/CD14
binding activates TLR4  Cytoplasmic portion of TLR4 binds MyD88  Phosphorylation cascade 
Phosphorylation of IκB (NFκB inhibitor)  Releases NFκB  NFκB enters nucleus  gene activation

Inflammasome = Large signaling compounds the detect pathogen and stressors

 Activate caspase-1 cascade, leading to formation of highly pro-inflammatory cytokines IL-IB and
IL-18
 Both extra- and intra-cellular controls

Innate Immunity II

Macrophage differentiation from resting to active depends on local stimuli

 Activated macrophages are larger (more membrane for attachment, surface proteins)
 Mediate phagocytosis more efficiently and are also cytotoxic to intracellular pathogens
 CD4+ cells activate macrophages via IFNγ

Recruitment to Site of Infection

1. Rolling Adhesion
a. Mediated by selectins (loose, rapid attachment and detachment b/n neutrophils and
endothelial cells)  L selectin on Leukocytes, E & P selectin on vascular endothelium
2. Attachment
a. Mediated by integrin (firm attachment)  Ex. of interaction = LFA-1 binding ICAM-1
b. Inflammatory stimuli can increase integrin expression
3. Diapedesis
a. Migration between endothelial cells toward chemotactic factors of an infection
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 i. Factors like PAMPs, chemokines (IL-8), and lipid-derived factors (PAF/platelet
activating factor, leukotrienes)
b. In absence of a stimulus, neutrophils follow a "random walk"

Leukocyte Recognition of Pathogens

 Neutrophils/macrophages have receptors for Fc portion of IgG antibodies (FcγR)
o 3 types of FcγR with different affinities for IgG subclasses
 Mediate attachment/uptake of antibody-coated particles by phagocytes
 Aggregation of Ig on bacterial surface allows cross-linking of multiple FcγRs on 1 antigen
o Phagocytes are only activated if cross-linking occurs
 Cross-linking of IgE molecules bound to FcεR on mast cells/basophils triggers degranulation
 Fc receptors trigger phagocytosis and killing via ROS, while complement receptors do not

Phagocytosis: A 2-step process mediated by actin filaments

1. Attachment (no energy required)
a. Opsonin dependent  Mediated by FcR/complement receptors on phagocyte reacting
with and IgG/C3b
b. Opsonin independent  Depends on surface properties of particle (hydrophobicity)
2. Internalization (energy is required)  Fuse with lysosome (& ROS only if Fc-triggered)

Intracellular Killing: Reactive Oxygen and Nitrogen Species

 NADPH Oxidase system  an electron transport chain to reduce molecular oxygen
o Activated during Fc-mediated phagocytosis  respiratory burst (neutrophil's O2
consumption increases x30)
o Initial product = superoxide (02-), which can then form other toxic product, like H2O2
 Both neutrophils and macrophages make reactive nitrogen intermediates (wrong in class notes)
o Activated by IFNγ or LPS, upregulates NO synthase, which creates NO-
o This reaction inhibited by L-NMMA (not sure if relevant)

Cytokine Production by Macrophages

 Activated macrophages release cytokines IL-1, IL-6, and TNFα  acute phase response
o IL-1 is a pyrogen (induces fever), TNFα and IL-1 activate vascular endothelial cells (for
neutrophil/monocyte attachment/diapedesis)
 Also important  IL-8 (major chemotactic factor), IL-12 (activates NK cells, CD4+ differentiation)

Response to Infection
1. Inflammation  Pain, redness, heat, swelling
2. Leukocyte recruitment
3. Acute phase response (cytokine/chemokine secretion  endogenous pyrogens)
4. TNFα-mediated containment of infection (by causing local clotting of blood vessels)
5. Complement cascade and adaptive immune response

Innate Immunity and Disease

 Chronic Ganulomatous Disease  neutrophils unable to generate superoxide, O2-
o X-linked recessive, sx's = chronic infections, abscesses, granulomas, unusual infections
 Chrohn's Disease  Subset of pts with Crohn's have a NOD2 mutation, unable to activate NFκB
o Results in hyperactive immune-mediated inflammation in the gut
 Septic shock can be triggered by LPS trigger systemic release of TNFα
o Drop in BP d/t vasodilation and edema in tissues, widespread clotting  organ ischemia
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 Immunoglobulin Structure and Function I

The recognition element of B cells is antibody/immunoglobulin/Ig

 Membrane-bound  Triggers lymphocyte activation, mediates uptake of antigen for
presentation to T cells
 Soluble  Mediates host defense via neutralization, opsonization, and complement activation
o Antibodies are concentrated in the serum, but also found in other bodily fluids

The recognition element of T cells is the T cell receptor (TCR)  only exists in membrane-bound form

**Functional difference = Ig recognizes antigen outside of cell, while TCR recognizes inside cell

Clonal Selection has 4 characteristics:

1. Each new lymphocyte contains 1 type of receptor, unique and randomly-generated
2. Antigen-binding triggers proliferation and differentiation
3. Cells produced through clonal expansion have identical antigen specificity
4. Self-reactive cells are deleted

Antibody response
1. Primary Response  Antibodies appear after 2-4 weeks (time lag for differentiation/expansion)
2. Secondary Response  Faster, specific response due to antigen-specific B cells (immunological
memory)

Structure of Antibodies
 5 classes (IgM, IgD, IgA, IgG, IgE)  4 polypeptide unit of 2 heavy (HC) and 2 light chains (LC)
o Each class has its own HC; there are 2 types of LC (κ and λ)
 Each chain has a variable region which recognizes an antigen, and a constant region which
controls effector function
 IgG is most abundant
o 4 domains on HC (VH, CH1,CH2,CH3), 2 on LC (VL,CL)
o 3 hypervariable regions in variable region that aren't adjacent in primary AA structure,
but come together in tertiary structure to form Ag-binding site
o 2 Fab and 1 Fc fragments
o Flexible  has hinge (b/n CH1 and CH2) and elbow (b/n VH and CH1)
 Hinge = 180° flexion/axial rotation / Elbow =
50° flexion

IgG  Y-shaped, can activate complement, but is most important

for opsonization, can cross placenta, γ HC

IgM  Pentamer, activates complement cascade, 1st Ig activated

in immune response, μ HC, contains J (joining) chain b/n Fab
monomers

IgA  Monomer in serum, dimer in secretions, J chain in dimer, α

HC, main Ig in mucosa/secretions
Fc
IgE  Allergic reactions, likely evolutionarily for helminth
infection ε HC

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IgD  On some B cells, unknown function, δ HC
Monoclonal Antibodies  Derived from a single B cell, are all homogenous
 Occurs in myeloma (cancers of B cells and plasma cells)  used in experiments to generate
identical, specific antibodies

To generate a monoclonal antibody:

Inject Ag into mouse  Remove spleen  Extract B cells  Fuse with myeloma cells  Allow
hybridomas (fused cells) to grow  Select for antibody in new cell populations

Antigen = Something recognized by the immune system

 Antigenic determinant = the specific part of an antigen the antibody binds  most proteins
have multiple antigenic determinants (linear/in AA sequence and conformational/in folding)
Immunogen = Something that elicits an immune response
 Immunogenicity is determined by foreignness, size (>1000 daltons), complexity, genetic factors,
and mode of administration
 Adjuvant = vehicle that increases response to an antigen
 Hapten = a small molecule that is not by itself immunogenic, but does elicit an immune
response when coupled to larger molecules

Ig molecules are antigenic:

Isotype: Encoded by different loci (e.g. IgG versus IgA)  isotypic antigenic determinants are shared by
all molecules of a given HC or LC type
Allotype: Encoded by different alleles (e.g. 2 different IgG's)  allotypic antigenic determinants are
encoded by a particular allele of a given Ig gene
Idiotype: Encoded in ways not explained by Mendelian genetics  idiotypic antigenic determinants are
localized to the variable portion of the antibody molecule and are unique to a given antibody

Antibody Binding  Saturable process similar to prior enzyme kinetics

 Small changes in binding energy = large changes in binding affinity (1.42 kcal/mol = 10-fold
change in affinity)
 Equilibrium dialysis is a method to measure affinity and valence (# binding sites per molecule)
o Valence of IgG = 2, of IgM = 10

Affinity in a Scatchard plot:

 Concave Scatchard plot indicates heterogeneous antibody
response, all with different affinities
o Can find "average" affinity by slope of tangent line
at point where 1/2 binding sites are occupied (K0)
 A linear curve indicates homogenous antibody response
 The average affinity increases over time (affinity
maturation)

Assays for Antigen Binding

1. Direct (ELISA or RIA, like ELISA with radioactive isotope)
2. Indirect/Competitive-Binding (allows calculation of amount antigen)

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 Immunoglobulin Structure and Function II

Antibody genes are clustered in 3 parts of the genome  1 for HC and 2 for the 2 LCs
 Each section has a variable and constant region, and within each variable region, there are 3
hypervariable regions
 There is no functional difference between the two LCs, λ and κ

Antibody sequence variability is clustered in 3 hypervariable regions (the CDR or complementarity

determining region/Ag-binding region)
 Framework regions (parts between hypervariable regions within variable region) have low
variability, but more than the constant region
 The surface of the antigen that bind the CDR = epitome
o Interacting surfaces are large, complementary with many hydrophobic stabilizing H-
bonds and Van de waals interactions  no change in conformation occurs with binding

Generation of Variable Region Diversity

 Genome is too small to encode each antibody separately
 Variable domains of HC and LC are encoded in genome/germline, then brought together in
unique ways
 Southern blot and PCR can be used to detect antibody rearrangements

Light chains are encoded by 2 gene segments: V and J

Heavy chains are encoded by 3 gene segments, V, D, and J

Gene Assembly/VDJ Recombination (occurring in bone marrow or thymus)

Mediated by conserved recombination signal sequences (RSS), which flank all un-rearranged gene
segments  each RSS is 7-9 bp’s, separated by 12 or 23 bp spacers
 RSS is the recognition site for recombinase proteins RAG1 & 2
 12-23 rule = only segments with a 12 spacer can recombine with 23 spacer segments
RAG 1 & 2 check spacers, then cleave DNA at junction of RSS and gene segment  the DNA is then
repaired, but in the process can lose or gain random nucleotides, adding to diversity
 One type of addition adds the N region, in which TdT randomly adds nucleotides w/o a template
 A second type = P insertion/P nucleotides, which result from opening of the hairpin (that forms
on the cut coding end), filling with DNA pol I, and re-joining with other end

VDJ diversity only affects the 3rd hypervariable region  the 1st & 2nd regions are diverse through
evolution and are encoded in the germline
 1 rearrangement for LCs
 2 rearrangements for HCS  1ST is D-J recombination, 2ND is V-DJ recombination
 Continue to rearrange on pro-B cell until a functional HC is formed (tested with surrogate LC) 
Functional HC makes the cell a pre-B cell, and signals to stop arranging HC, start for LC
o Signal to stop HC rearrangement = allelic exclusion (ensures only 1 HC is expressed)

↑ diversity via junctional variability (P/N additions, random loss) as well as somatic hypermutation
 Somatic mutations introduced into rearranged genes occurs in secondary lymphoid tissues 
mutations tend to accumulate in hypervariable regions because mutations that increase binding
affinity are selected for (mechanism for affinity maturation)

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Class Switching
 IgM is the first Ig expressed in developing B cells, then IgM and IgD, which then undergo further
differentiation to other Ig classes
o Co-expression of μ and δ HCs (expressing both IgM and IgD) occurs due to different
polyA and mRNA splicing
o Expression of HC other than μ or δ is done by recombination
 AID is an essential protein for class switch recombination
 Activated by CD40L (on T cell) and CD40 (B cell), IL-4 and stimuli like LPS can
induce switching
o Commitment to switch = transcription of the new H chain locus
 Binding specificity is maintained despite changes in expressed Ig type

Creation of membrane-bound versus soluble Ig depends on differential mRNA processing

Ig Gene Rearrangement and Disease

Agammaglobulinemia = complete lack of circulating antibodies
 Unable to eliminate extracellular bacteria or to neutralize viruses
 Scid mutation leads to immunodeficiency due to defective VDJ recombination (defect in joining
coding sequences together)  very few functional B and T cells
 If deficient in RAG1 or RAG2 proteins, complete lack of B and T cell development
VDJ Rearrangement and Cancer
 Translocations via somatic recombination between an oncogene and an Ig gene cause lymphoid
cancers (lymphomas)

B Cell Development

Hematopoietic stem cells (HSC)  all blood cell lineages

 Characterized by long term reconstitution potential (can’t run out of stem cells)

B cell differentiation occurs in two parts:

1. Generation of a large pool of B cells with unique Ag receptors (pre-immune)
2. Clonal selection (selection of B cells that bind to Ag from pre-immune pool)

BONE MARROW 2° LYMPHOID

Common lymphoid progenitor  pre-pro B  pro-B  pre-B  Immature B  Mature B

Requires IL-7 to progress HC rearrangement Naïve B cell in 2° lymph tissue

with functional HC & LC
Prior to B cell commitment LC rearrangement

Accessibility hypothesis: All rearrangeable genes cannot be acted on by recombinase machinery (RSS
and RAG) due to chromatin restraints  genes are rearranged in a certain order depending on access
 HC gene segments are made accessible before LC segments, so HC recombination occurs first
 For HC  D and J segments are available first, so DJ recombination occurs, then V-DJ
o IL-7 controls access to/recombination of V segments in pro-B cells

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A large portion (70-80%) of VDJ recombined pro-B cells do not produce a function HC
 HCs are tested for functionality with the pre-B cell receptor (BCR), acts like a surrogate LC
 pre-BCR transmits 3 signals if HC is functional:
1. Rescues cell from apoptosis and induces proliferation
2. Stops HC rearrangement
3. Activates LC rearrangement
 Pre-BCR ensures allelic exclusion by stopping all HC rearrangement and IL-7 signals

Light chain type (κ versus λ) depends on LC constant region

LC variable region generated by 1 recombination between V and J segments
 The 1-step recombination allows multiple recombination attempts to form functional LC
 If multiple recombinations are still non-functional, can switch isotypes, κ ↔ λ

B cells reactive against self-antigens must be eliminated by one of three ways:

1. Deletion  extensive cross-linking of BCR induces apoptosis
2. Editing  process through which a functionally rearranged LC undergoes secondary
recombination, which is possible at LC loci (random, no guarantee for function LC, may still be
autoreactive and trigger deletion via apaptosis)
3. Anergy  if Ag doesn't cause extensive cross-linking, cell becomes resistant to BCR signals

Only 10% of generated B cells leave the marrow as immature B cells  develop to mature B cells in
secondary/peripheral lymphoid organs (mostly spleen)
 Cells entering the spleen are called T1, develop to T2 in 2 days (different cell markers)
 T2 cells respond to BCR and BAFF/Blys (B cell activating factor) signals in order to survive and
proliferate
3 main types of mature B cells:
1. Follicular mature (FM)  Naïve B cells in follicles, participate in germinal center reactions,
present Ag to T cells, proliferate in response to BCR signals
2. Marginal Zone (MZ)  Do not participate in germinal center reactions, rapidly produce IgM to
blood borne pathogens, proliferate in response to TCR signals
3. B1 Cells  mucosal immunity, T-independent

Germinal centers are structures generated by cell-cell/chemokine interactions between FDC, B & T cells
 Location where B cell somatic hypermutation and class switch recombination occur
 GC Reaction = selection of highest affinity B cells and apoptosis of unselected cells
o Dependent on CD40 (on B cell) and CD40L (T cell) reaction

Effector Functions of Antibody

Effector function is determined by the constant regions of the heavy chain

Antibodies of the 5 Ig classes are produced in different amounts and

different times in first and second responses
 Also varies with body location (serum vs. secretion)
 IgM is 1st produced in serum, IgG is most important in 2nd
response, IgA most important in secretions **

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There are 3 general groups of antibody activities:
1. Transport (across mucosal surfaces, placenta)
2. Non-inflammatory activities (virus neutralization, blocking bacteria adherence)
3. Inflammatory activities (opsonization, complement activation, mast cell activation, cytotoxicity)

TRANSPORT FUNCTIONS
GALT & BALT = gut and bronchial associated lymph tissue local production of Ig
 Ig is produced by plasma cells in the lamina propia and transported across mucosa
 IgA acts as a barrier for pathogens and toxins, but also environmental Ags (e.g. food)
o IgA is a dimer in secretions held together by a J chain and intrachain disulfide bonds
Secretory component of IgA contains part of the polymeric immunoglobulin receptor
 Has Ig-like domains, allows IgA to bind and transcytose through mucosa epithelial cells
 Important for survival of secreted IgA

IgG transport across the placenta is mediated by the Brambell receptor

 Carries IgG across endothelium into extracellular spaces, and recycles endocytosed IgG from
endosomes back to extracellular fluid (which greatly prolongs IgG half-life)
 Baby born with 110% of mom's IgG due to active transport and recycling of IgG

NON-INFLAMMATORY FUNCTIONS
Antibodies can neutralize viruses, toxins, and bacteria in several ways:
1. Inhibit binding to target cells  via physically coating surface of the virus (IgM)
a. Influenza is recognized by Ig binding to HA and NA surface glycoproteins
b. Blocking of attachment doesn't entirely explain the anti-viral effect of antibody
2. Inhibit fusion with endocytic vessicles, which is necessary for viral uncoating and translocation
of viral nucleic acid into the infected cell's nucleus (IgA)
3. Preventing toxins from binding/entering host cells (IgA and IgG)

INFLAMMATORY ACTIVITIES
Opsonization  Coating a "slippery" polysaccharide capsule to improve uptake by phagocytes
 Phagocytes bind strongly when there are interactions between multiple Fc receptors and Ig
molecules with 1 Ag molecule (cross-linking)
 Binding of particle-bound Ig to Fc receptors provides specificity to the effector cell (phagocyte)
 For particle too large for ingestion, phagocytic vacuoles fuse with membrane and release toxic
enzymes

Complement Activation  Classical pathway is activated by Ig-Ag complexes (via C1q activation)
 Leads to opsonization directly (IgG and Fc receptor binding) and indirectly (via C3b)

Activation of Mast Cells  IgE does nothing as a soluble protein, must be bound to mast cell/basophils
 IgE concentration in serum is usually low, synthesis is stimulated by IL-4
 IgE binds to mast cells/basophils via high affinity receptor for Fc portion of IgE (Fab portion binds
Ag)  in this way, IgE provides mast cells and basophils with specificity
o Cross-linking of IgE bound to Ag triggers degranulation and activation of phopholipase
A2 (results in increased vascular permeability, tissue edema, mucus production)

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Antibody-dependent cell-mediated cytotoxicity (ADCC)
 NK cells can bind to IgG fixed on surface of infected host cell  cross-linking of Fc receptors
trigger NK release of cytoplasmic granules containing perforin and proteolytic enzymes
 Important in new B cell cancer therapies  use NK cells to lyse tumor cells

The Complement System

Complement = serum and membrane-bound proteins that are activated either directly (exposure to
foreign cell) or indirectly (via Ig) and trigger a cascade of proteolytic events

COMPLEMENT ACTIVATION
Classical Pathway  C1 (a globular protein made up of C1q, C1r, and C1s) is activated by Ig-Ag complex
 A single C1q must interact simultaneously with two or more Fc regions to activate
o A singly bound IgM is sufficient, where as you would need 2 or more IgG
o C1q does not bind free antibodies
o C1q provides the critical physical link between antibody and complement
C1q binds multiple Fc  Activates C1r/C1s  C1s cleaves C4 into C4a and C4b  C4b undergoes change
exposing a thioester group  C4b reacts with 1st nucleophile it encounters  Bound C4b binds C2 
C1s cleaves C2 into C2a and C2b  C2a remains associated with C4b  C4bC2a = C3 convertase

Lectin Pathway  bypasses Ig requirement by using a lectin similar in structure to C1q, like mannose-
binding lecting (MBL) or ficolin
MBL binds mannose  Cleaves C2 and C4 using MASPs (MBL-associated serine proteases) that are
homologous to C1r and C1s  C4b and C2a from cleavage form the C4bC2a = C3 convertase

Alternative Pathway  C3 can spontaneously hydrolyze (+ H2O) and binds Factor B  C3(H2O)-bound
Factor B can be cleaved by Factor D into Ba and Bb Ba leaves and C3(H2O)Bb = soluble C3 convertase

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When C3 binds to a surface, it binds Factor B  Factor D cleaves Factor B  Ba leaves and C3bBb that is
left binds Factor P (properdin)  Stabilized C3Bb = Alternative C3 convertase
 Produced C3b can be inactivated by Factor H, or path continues with Factor B
 Amplification via alternative pathway  C3b is part of the C3 convertase, which continues to
make more C3b and C3 convertase in an amplification loop

*The lectin and alternative pathways provide an immediate defense against pathogens
**All paths converge at the production of C3 convertase
C3a is an anaphylotoxin and leads to inflammation  diffuses away to cause nearby mast cells and
basophils to release histamine and other inflammatory mediators

Bound C3b leads to opsonization  phagocytes express complement receptor CR1 specific for C3b
 If an Ag is coated with C3b AND antibody, activates both phagocytosis and killing mechanisms of
macrophages and granulocytes (via activation of CR1 and FcR)

C3b binds to either of the C3 convertases, C4b2a or C3bBb, it forms C5 convertases (C4bC2aC3b or
C3bBbC3b)

Once C5 is cleaved, C5a diffuses away as an anaphylotoxin and a chemoattractant

 Macrophages and granulocytes migrate into tissues via chemotaxis following C5a

C5b remains associated with C3b and binds C6 and C7  C5b,6,7 inserts into cells lipid bilayer  Binds
C8 and multiple C9 molecules to form the MAC (membrane attack complex)/ C3b5b6789
 MAC forms a large pore in the membrane, resulting in death of the cell

COMPLEMENT REGULATION
Regulation of classical pathway
 C-1 inhibitor binds C1r/s, causing dissociation from C1q  if C1r/s becomes activated, C-1
inhibitor acts as a suicide substrate
 Controlling C3 convertase  multiple proteins can bind C4b, inactivating it (DAF - decay
accelerating factor, CR1, and C4BP/C4 binding protein)

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Regulation of alternative pathway
 C5 convertase (C3bBb3b) is inhibited by Factor H, CR1 and DAF
 Factor H competes with factor B for binding to cell-bound C3  depends on sialic acid content
(high sialic acid favors Factor H  host cells high in sialic acid, while pathogens are low and
favor Factor B/functional C5 convertase)

Factor I inhibits in all pathways  cleaves and inactivates C3b or iC3b with a cofactor

Regulation of the MAC  Insertion into "bystander" cells is limited by:

 Protectin (CD59), which stops C8 and 9 addition
 Vitronectin, a serum protein, which binds soluble C5b,6,7,8 and prevents completion of MAC

Complement also regulates B cell activation  cross-linking of sIgM (B cell Ag receptor) and CD19
decreases the number of receptors on B cell that must bind to Ag to activate proliferation

Disadvantage to complement = slow to evolve, easily avoided by pathogens (examples):

 Vaccinia/cowpox virus inhibits formation of classical path C3 convertase
 Trypanasomes destabilize alternative path C3 convertase
 Epstein-Barr uses complement receptors to facilitate infection

Deficiencies in complement proteins can leads to many diseases (e.g. no C1-inhibitor  unregulated
C2b and hereditary angioedema)
Common clinical tests for complement:
 C3,C4 serum level  C3 involved in all pathways, C4 only in classical and lectin
 CH50  tests overall complement function

Example questions from lecture:

1. If a patient lacks C6, but all other C's are normal, CH50 will be_____? Low
2. A child with glomerulonephritis has a kidney biopsy that shows dense deposits of C3. Serum
levels of C3 are diminished, but C4 is normal. This suggests activation of what pathway?
Alternative (because you have used up C3, but not C4  not classical/lectin because C4 isn't
involved)

Viral Dynamics and Immune Responses

Viruses = small, with RNA/DNA genome surrounded by a protein or lipid/protein coat

 Are obligate intracellular parasites
 Enveloped viruses have an out coating lipid bilayer derived from the host cell by budding

Viral Life Cycle

1. Attach to host (via capsid/envelope and host surface proteins)
a. Pattern of expression of virus receptors determine
the tropism of the virus (the cells and tissues it can
infect)
b. Example - T cell surface CD4 usually for recognition of
HLA/MHC Class II, but HIV-1 has high affinity binding
site for CD4
2. Enter host and begin intracellular phase of life cycle
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 a. Intracellular viral antigens recognition are mediated by cytolytic T cells (CTL)
3. Eclipse Phase (time between virus entry into cell and release and of new virus particles)
4. Productive Infection

Viral genomes vary in size  large ones can devote genes to immune evasion, others only encode genes
for viral genome replication
 All viral proteins are made on host ribosomes (and envelope lipids are host-derived)
 Unlike other pathogens that have unique features not found in host cells, viruses have few
unique chemical differences from host (no PAMPs for PRRs to recognize)

Viral Dynamics
Average lifetime of a cell or virus = 1/ decay rate
constant

Virus distribution in the body

 Free virus, v (viremia) can be measured
clinically
 The lifetime of free virus in circulation is only minutes, so detectable viremia indicates ongoing
virus production
 Rate of change of v = production rate - clearance rate

When a virus is first introduced to a cell, the infection dies out or grows according to the reproductive
ratio, R0
 If R0 > 1, the infection will spread
o R0 of HIV = 20!  results in explosive initial multiplication of virus
o In contrast, bacteria R0 = 2  each cell yields 2 daughters, but generation time is very
short, so they can still produce a large number of offspring in a short time
 Viruses have longer generation time (1-2 days)
 If R0 < 1, the infection will die out
o Pre-existing immune responses (e.g. vaccines) reduce R0 to < 1 to prevent infection

Following an initial period of exponential growth, viremia levels off because either 1.) the system runs
out of uninfected cells or 2.) immune system starts to control infection
 CTLs have been implicated in the control of chronic infection  decreases in the number or
activity or CTLs allow more viral replication

Antiviral Therapy  Examples in work with HIV

 Antiretroviral drugs stop new infection of susceptible cells, but do not inhibit release of virions by
cells already producing infection
o Virus in the plasma must come from the cell infected prior to therapy
o Decay rate in plasma is very fast, and is determined by the decay rate of the cells
producing the virus (or half life, which is ~1 day for most of the cells)
 HIV is highly dynamic  every possible mutation in HIV genome arises once a day statistically in
the average patient
 HAART (highly active antiretroviral therapy) = combination of 3 antiretroviral drugs, which stop
viral replication almost completely

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The failure of HAART to reduce viremia to 0 reflects cells in a state of latent infection
 Latency = reversible non-productive state of infection, a major mechanism of immune evasion
 HIV's main mechanism of evasion is mutation, but the minor use of latency prevent HAART from
ever fully curing the infection
o Arises as a consequence of normal CD4 T cells  Some activated T cells become
infected as they are in the process of reverting back to resting G0 state
o Results in a stably integrated, but transcriptionally silent form of virus in a memory cell

Restriction Factors are host proteins that interfere with viral replication (considered innate response)
Unlike PAMPs and PRRs, with restriction factors, the recognition and effector functions are mediated by
one protein
 "Classic" example from notes  Cytidine deaminase ABOBEC3G causes G  A hypermutation in
retroviral genomes
o Only acts on single stranded DNA, which is only generated when retroviral genomes are
copied with reverse transcriptase, RNA  DNA
 ABOBEC3G causes a C  U on the DNA minus strand, so when copied to a plus strand, A is
inserted instead of G  All Trp (TGG) become stop codons
o Lethal to virus because introduces stop codons in nearly every reading frame
 1 of HIV's 10 genes is devoted to counteracting ABOBEC3G with Vif (accelerates its degradation)

Antigen Recognition by T Cells I and II

TCR sees foreign Ag as a complex of "processed" foreign peptide associated with a "self" protein
encoded by the MHC  MHC gene products are polymorphic and influence whether an individual
generates an effective response to foreign Ag

Overview of T cells
 Required to assist B cells in making antibody
 Follow same clonal selection as B cells  encounter with Ig is critical
 Immature T cells develop in the thymus, mature cells go to secondary lymph tissues

T Cell Recognition of Antigen

 Unlike Ig, which recognize conformational antigen determinants (folded), T cells recognize short,
linear sequences of ~10 AAs  can recognize denatured protein
 T cells only recognize Ag on the surface of other cells, will not recognize free Ag
o Macrophages can serve as APCs, but the most important ones are DCs
 Genetic origin of the APC influences T cell ability to recognize Ag
o Referred to as MHC restriction, is the result of different MHC alleles expressed

The Major Histocompatibility Complex was initially implicated in acceptance or rejection of transplants
In humans, MHC is called HLA (Human Leukocyte Ag) and encodes 3 protein classes
 Class I and II proteins are involved in T cell function
 Class III protein are soluble serum proteins, many in the complement cascade

Class I molecules are made up of 2 chains  a heavy chain encoded by HLA A, B, or C loci (3 possibilities)
and a β microglobulin
 HLA A, B, and C are expressed (co-dominantly) in every tissue/cell except RBCs and neurons

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  There are also Class Ib molecules (E, F, and G) that are non-classical Class I (b/c they have few
alleles and are expressed in a tissue-specific fashion)

Class II molecules are cell surface heterodimers with an α and β subunit, each of which has 2
extracellular domains (α1/α2, β1/β2)  α1 and β1 form the T cell recognition site
 There are 3 types of Class II molecules: DP, DQ, DR  each w/ unique α and β subunits
 Class II are expressed in a tissue-specific fashion  constitutively expressed on B cells,
macrophages and DCs

MHC products are highly polymorphic  many allele variants at each locus, no single wild-type
**In a cell that expresses both MHC I and II, there will be 12 different MHC molecules expressed (3 loci
encoding each type, 2 alleles at each loci that are co-dominantly expressed)

Naming of MHC allelic variants = HLA "locus" * "allele" (e.g. HLA A*0301)

MHC restriction = T cells will become activated only when contacting APCs expressing the correct "self"
MHC molecules
 Experiments proved that Ag and MHC specificity depend on one receptor
o Disproved theory that T cells recognize MHC and Ag separately via 2 receptors

The TCR is made up of an α and β polypeptide chains linked via disulfide bond  like Ig, has variable N
terminal and constant C terminal domains & Ag binding site is formed from the hypervariable regions
 TCR gene rearrangement occurs identical to that for Ig on B cells (RAG1 and 2-mediated, 12-23
spacer rule)  difference is that it occurs in the thymus
 Similar ways to generate diversity (VJ combinations, junctional diversity) but somatic mutation
not used

Differences between TCR and Ig:

 TCR exists only in membrane-bound form
 TCRs are non-covalently associated with a complex of proteins, CD3
o CD3 is the same on all T cells and doesn't influence specificity
o CD3 is required for expression of the α/β heterodimer on plasma membrane and for
signal transduction once ligand is bound to TCR
o Surface expression of CD3 is used as a marker for T cells, Ig against CD3 can be used to
deplete T cells clinically (to prevent transplant rejection, to treat MS)
 Ags must be processed prior to T cell recognition to expose specific linear epitopes

Binding to the MHC molecule on APC is the first Ag-specific reaction that occurs  doesn't involve the T
cell or TCR at all
 MHC molecules only have 1 peptide binding site  a "groove" into which many Ag fit
 For a given MHC molecule, only certain amino acids are tolerated at 1 or 2 key "anchor"
positions, but at all other positions, there is no specificity for AA
o Class II grooves are open on both ends, thus more easily bind longer Ag peptides

T Cell Ag Recognition Summary

Protein Ag taken up by APC  Ag undergoes partial proteolysis and is re-expressed on surface of APC
bound to MHC groove  T cell recognizes the MHC-Ag complex
 T cells won't bind to incorrect MHC because 1.) Different/incorrect peptide fragment will be
presented and 2.) T cell contacts with MHC will be incorrect
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  MHC do present self proteins in absence of pathogen  self v non-self discrimination occurs on
level of T cell, not MHC

T Cells can be functionally divided in subsets that express either CD4 or CD8 surface glycoprotein
 CD4 and CD8 both have no variability and no allelic polymorphism (are all identical)
 Both use the αβ TCR and recognize processed Ags presented by self MHC molecules
CD4+ T cells release cytokines that activate other cells  involved in antibody formation by promoting B
cell proliferation and differentiation via cytokines (aka helper T cells, Th)
 Recognize Ags on MHC Class II (HLA DR, DQ, DP)  extracellular Ag
CD8+ T cells mediate the destruction of virally infected host cells
 Recognize Ags on MHC Class I (HLA A, B, C)  intracellular Ag (ideal for virus inside cell )

Processing pathway for extracellular Ag:

MHC Class II protein synthesized in ER  Invariant chain prevents binding to self protein  Class II
leaves ER in vesicle
Extracellular fluid and Ag enter APC via phagocytosis/pinocytosis  Ag degraded in low-pH endosomal
compartment  Fuses with vesicle containing Class II MHC  Invariant chain removed by proteolytic
enzymes  Ag binds MHC Class II  Vesicle fuses with membrane, MHC-Ag present on APC

Processing Pathway for intracellular Ag:

MHC Class I molecules fold into their 3-d conformation in the lumen of the ER, but don't bind because
they have no access to cytoplasmic peptides
Some synthesized viral protein is broken down within cell  peptide fragments free in cytoplasm 
fragments transported into ER via TAP1/TAP2 complex  Fragments bind to MHC Class I  Bound
MHC-Ag transported to cell membrane for presentation

T Cell Subset Primary Function APC Ag Origin MHC Class

CD4+ Cytokine release Macrophage, B cell, DC Exogenous II
CD8+ Lysis Any nucleated cell Endogenous (viral) I

Once activated with correct MHC-Ag, CD4+ helper cells must find B cell specific for same Ag
 B cells bind Ag with surface Ig, then internalize and process them for association with Class II 
CD4+ cells can then recognize the MHC-Ag complex (more in later lecture notes)

An important note  Ag recognition by T cells involves many costimulatory signals between T cell
surface proteins in addition to TCR and CD4/8 (but these don't influence specificity)
 LFA-1 binds with ICAM-1 and ICAM-2 (intercellular adhesion molecule 1/2)
 CD2 (on T cell) interacts with LFA-3
 CD28 (on T cell) interacts with B7 (on APC)

Superantigens are bacterial and viral proteins that can activate large numbers of T cells
 Do not require processing  bind intact to class II MHC, not on the Ag-binding cleft
 Bind MHC II AND region of the TCR (specifically the β chain - shared by many T cells)
o Able to activate ~ 1/20 of all T cells

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 o Cause toxic shock syndrome and food poisoning

Ag Recognition by Natural Killer T Cells (a small subset of T cells that express some surface protein of NK
cells as well as the αβ TCR)
 TCR is relatively invariant on NKT cells  recognizes class IB molecule CD1, which presents
bacterial lipids
 Sort of innate (recognize unique bacterial characteristic), but also adaptive (recognition
mediated via TCR)
 NKT cells secrete large amounts of cytokines when activated

Antigen Presenting Cells

Ag capturing and processing occurs in peripheral tissues, while initiation of primary immune response is
localized in secondary lymphoid organs

Dendritic Cells (DCs) are professional APCs that initiate both innate and adaptive immune responses

There are 2 major DC developmental pathway:

1. Myeloid pathway  Langerhans cells in epithelia (LC) and interstitial DC in all other tissues
(intDC)  both secrete IL-12 **main focus of the lecture
2. Lymphoid pathway  plasmacytoid DC (pDC)  secrete type I interferon (IFN)

Myeloid Lineage Development

1. DC progenitors (in marrow) enter circulation en route to peripheral non-lymphoid sites
2. The immature/processing DCs differentiate for Ag uptake/processing, MHC production, etc.
3. inflammatory mediators promote maturation of tissue DCs
4. Mature/presenting DCs move to secondary lymph tissues, located mainly in T cell areas of
lymph nodes, where they stimulate T cells with Ag presentation

The capacity to internalize and process whole protein Ags varies with the state of maturation of the DC
Freshly isolated DC from tissues have unique features from more mature cells:
 High intracellular concentration and synthesis of MHC Class II
 Very active macropinocytosis (sample a volume equivalent to their cell volume every hour)
o Macropinocytosis is turned on by GM-CSF and IL-4, turned off by LPS, TNF, and IL-1
 Use "absorptive endocytosis", a high affinity path that uses PRRs, because regular
macropinocytosis is inefficient

Mature DCs are poor at processing and presenting intact Ag, but are very efficient at presenting already
processed peptides  mature DCs give a "snapshot" of Ags that were present in tissue prior to DC
migration to lymph nodes

In the lymph node, formation of stable DC-T cell clusters occur in 2 phases:
1. Initial Ag-independent clustering, which is short-lived
2. Stable clustering with Ag-specific T cells

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 Comparison of different DC developmental stages:
Immature DC in Periphery
- High intracelluluar MHC Class II
- Active MHC Class II synthesis
- Very active macropinocytosis
- Adsorptive endocytosis using
high affinity PRRs
DC in Periphery Exposed to
Inflammatory Stimuli
- MHC Class II synthesis shut
off
- Prominent stable surface
expression of peptide-
loaded MHC Class II
- Less macropinocytosis
- Migration to T cell area of
lymph node via chemotaxis
Mature DC in Lymphoid Organ
- High levels of peptide-loaded MHC II
- Highly dynamic membrane processes that
extend and retract to sample passing T cells
- Secretion of DC-CK which specifically
attracts naïve T cells
- Stable interactions with Ag-specific T cells
- Upregulate co-stimulatory molecule
expression

Mature DC cells can prime CD4+ T cells with additional interactions (beside Ag-specificity):
 CD40 on "superactivated" DCs can bind CD40L on CD8+ to activate w/o the need for CD4+
 DC produce IL-12  differentiation of CD4+ into Th1 OR IL-4  Differentiate to Th2 cells
 Can interact with B cells aiding with Ig formation

DC can capture and present self-antigens  When this presentation occurs (signal 1) in absence of
costimulatory signals (signal 2), tolerance to the self-antigen is induced

Some viruses evade immune response by not directly infecting DC (or APC in general)  in this case you
need cross priming to activate CD8+ cells, otherwise APC's cannot
present
 When infected cells die, their fragments are taken up by
DCs and presented on MHC Class I (which is usually
reserved only for intracellular Ag) to activate CD8+ T cells

Although DCs are the major APCs in immune response, there are
others:
1. Macrophages have a large scavenging function (clear
substrates, usually by complete digestion)
a. Are also capable of adsorptive endocytosis
b. Unlike DC, macrophages have many lysosomes
which result in complete degradation
i. Some Class II presentation occurs, but
most Ags are completely digested
2. B cells also have a lot of lysosomes
a. Dependent on surface Ig-mediated adsorptive
endocytosis to ingest Ag their presentation is Ag-
specific

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 T Cell Selection and Tolerance

Tolerance = State of immune system characterized by unresponsiveness to a specific Ag

Early hypotheses and research showed:

 If the receptor on an immature lymphocyte was reactive with self-Ag, it would be deleted
 The immune system will become tolerant to any Ag present during fetal development
o Freemartin twins study (dizygotic twins sharing a single placenta)  recognizes sibling's
genotype as self-Ag, even if self-Ag is actually different
 Ability to become tolerant decreases rapidly after birth

Central tolerance = Tolerance generated during lymphocyte development

Peripheral tolerance = Induction of tolerance in mature lymphocytes after development is complete

*More is known about tolerance in T cells than B cells - although it is known self-reactive B cells/ Ig
induce deletion or anergy  These notes/lecture focus on T cells

3 stages of T cell development

1. Early genetic events leading to unique TCR expression
2. Cellular selection
a. Negative selection first eliminates thymocytes that express self-reactive TCRs, leading
to self-tolerance
b. Positive selection favors the development of thymocytes expressing TCR's that
preferentially recognize foreign Ag and self MHC
3. Acquisition of mature effector function

Rearrangement and Selection of TCR Genes

Although the α/β TCR is most common, there is also a γ/δ TCR
 The δ locus is within the α locus, ensuring during rearrangement that only α/β OR γ/δ TCR will
be expressed
 α and β loci have many more V and J segments than γ and δ = way more combinatorial diversity
Hematopoietic cells enter thymus from the bone marrow w/o CD4 or CD8 (-/-) and begin rearrangement
 γ and δ chains are rearranged first, so the γ/δ TCR is the first expressed in a small #
 Expression of complete γ/δ TCR inhibits further recombination of α and β loci  γ/δ TCR cells
are committed to one lineage and leave for peripheral lymph organs (remain CD4-8-)
 α and β genes rearrange if γ/δ TCR is non-functional or if α rearrangement occurs before δ,
deleting it
CD4-8-  CD4+8+ (coexpressed) once a functional VDJ rearranged β gene is formed  TCR α gene
continues to rearrange (if no functional α chain forms, CD4+8+ cell dies)

Creation of CD4+8+ with a functional TCRαβ is critical for thymic selection (most CD4+8+ cell never
mature and die in thymus, small % survive and turn off either CD4 or CD8 gene)
 Negative selection is the major mechanism of central tolerance

Thymocytes have 3 possible fates depending on TCR:

1. If good fit with both self-MHC and self-antigen are deleted (negative selection)
2. If no good fit with self-MHC are useless and die after failing to positively selected for
(nonselection)
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 3. If good fit with self-MHC, but not with any other self-peptide they are (positively) selected to
mature

Research indicates these 3 decision are made via detection of quantitative differences in TCR occupancy
by peptide/MHC complexes on stromal cells that are "sampled" during thymocyte development
 Poor fit = no/weak signal, while a too strong fit (self reactive) = very strong signal

The thymus has an important role in development of peripheral tolerance  Thymus epithelial cells
express (to a small degree) mRNA of many self-compounds (e.g. insulin)

No thymus = no T cells = immunodeficiency

Abnormally functioning T cells = autoimmunity

T Cell Effector Function I: CD4+ T cells

Cytokine = An active molecule produced by a cell (named for cells that produce them  lymphokine,
monokine)
Chemokine = A cytokine with chemo-attractive properties

CD4+ T cells differentiate into effector cells via clonal expansion once they are activated by foreign
antigen in either the spleen or lymph nodes
 Once T cells contact an APC with the correct Ag-MHC, the synthesis of various cytokines is
induced, especially interleukins (cytokines that are produced by and effect WBCs)

The clonal expansion of CD4 cells is driven by IL-2 (aka T cell growth factor)
 The receptor for IL-2 is a heterotrimer (α, β, and γ chains) that is assembled when T cell
recognizes MHC/Ag
o If defective in IL-2, can still mount an immune response, though not normal
 Severe combined immunodeficiency (XSCID) is a disease resulting in absent or greatly reduced T
cells  mutation in the γ chain of the IL-2 receptor
o Defective IL-2 can still mount immune response because there are other cytokines
o Mutation in γ chain is a bigger problem because many IL receptors use that chain
 Once IL-2 has bound IL-2R, follows a Jak-STAT pathway/intracellular cascade
o SCID can result from defects in the Jak STAT pathway as well

CD4+ Cells can be further divided by function:

 Th1 cells synthesize IL-2, TNF-α, and γ-IFN  involved in macrophage activation
 Th2 cells make IL-4, IL-5, and IL-13  associated with B cell activation, growth, differentiation
 Tfh (T follicular cells)  Role in antigen-specific B cell help
 Th17 secrete IL-6 and IL-17  Role in inflammation

T-Cell dependent macrophage activation requires 2 signals from Th1:

1. Release of IFNγ
2. Interaction of CD40L with CD40 on macrophage
> Some research indicates TNFα may also be able to deliver the 2nd signal via macrophage TNF receptor
> Activation of macrophages by T cells is Ag-specific, but once active, macrophages are not Ag-specific,
and so can clear co-infections even if only 1 Ag is recognized by T cell

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Th2 and Thf recognize Ag/MHC II on B cells and release cytokines (IL-4,5,13), effecting B cell
development, activation, growth, differentiation
 Thf cells are located in B cell follicles where they produce IL-4 and IFNγ
 Lymphokines secreted by Th2 and Thf also influence class switching and somatic mutation
(switching factors)

X-linked Hyper IgM Syndrome  Patients fail to express a functional CD40L, which is essential to
stimulate class switching
 CD40L isn't constitutively expressed - it is induced by TCR binding
 Disease characterized by no germinal center, LOTS of IgM, but no other Ig type

Th17 cells  recently discovered, produce IL-17, IL-6 and TNF to induce neutrophil activation and
promote inflammation

Th1, Th2, and Th17 T cells aren't pre-

programmed, but all come from CD4+
Th0 following Ag stimulation by APCS
depending on the cytokines present
during differentiation

Some diseases are effectively

controlled if Th1 is formed, but not Th2
 clinical relevance

Once differentiated, the cells produce

cytokines that actually inhibit the
proliferation of the other cell types

T Cell Effector Function II: CD8+ T cells

Cytotoxic T Cells (CTL) function to eliminate virally infected cells (major function), bacterial-infected
cells, tumor cells, and are important in graft/transplant rejection
 Triggered by recognition of MHC Class I and Ag by CD8+ T cell  undergo clonal expansion and
differentiation, expressing important lytic mediators

CD8+ activation can occur in the absence of CHD4+, but the response is short and ineffective to clear
virus  Need CD4+ to prime CD8+ cells to improve effector cell function and memory cell generation

If viruses don't infect APCs, will need to use cross priming to present viral Ag to CD8+ cells

Differentiation of a resting CD8+ T cell into functional CTL takes several days
 Once active, CTLs initiate contact with infected cell (not with Ag) through cell adhesion
molecules, LFA1 (on T cell) and ICAM (on target cell) are particularly important
 Additional interactions can take place if Ag receptors encounter MHC-Ag complex
o Surface glycoprotein CD8 binds MHC Class I and facilitate binding
 A unidirectional signal cascade is initiated  cytolytic process is very specific (no innocent
bystanders killed by accident)
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  When T cell binds target cell, cytoskeleton rearranges to bring cytotoxic granules toward target
o Granules fuse with membrane
 Killing by CTLs occurs (not with ROS) via cytoplasmic granules containing:
o Perforin punches holes in the target cell membrane
o Granzymes are proteases that enter the cell through the pores and initiate apoptosis
o Granulysin can associate with lipids, potent cytolytic activity

An additional signaling pathway for CTL activation is the Fas/Fas-L pathway:

 Fas is a cell surface protein with a "death" domain on cytoplasmic tail
 Ligand for Fas (Fas-L) is also a cell surface protein expressed on some CD8+ cells
o Provides a second mechanism to kill for CTLs, but only on cells the express Fas

Viruses can evade the host immune system in many ways: (ultimately prevent presentation)
 Latent infection
 Replicate in cells that lack MHC Class I (neurons) or in "privileged" sites not normally accessed
by CTL
 Downregulation of Class I expression to prevent presentation of viral proteins to CD8+
 Physical association with TAP 1/2 to prevent transport of peptides into ER
 Move newly synthesized class I molecules from ER to cytosol
 Mutation can escape recognition by CTL ("CTL escape mutants")

NK Cells are another type of cytotoxic lymphocyte  part of the innate immune response
 Derived from CD34+ cell precursors and mature primarily in bone marrow
o Although they are lymphocytes, don't express Ig or TCR
 Present at low concentration in the circulation
 NK Activating Receptors are a group of receptors that initiate production of cytokines and
cytotoxicity on binding to NK cells
o Best known example = NKG2D  triggers NK lysis of tumor cells using ligands MICA and
MICB (upregulated w/ cellular stress and expressed on most epithelial tumors)

NK killing is primarily mediated by NK inhibitory receptors - there are 2 main families:

 Immunoglobulin superfamily and C-type lectin-like superfamily
 Structurally different, but both contain 1 or 2 ITIM motifs (inhibitory signal) AND the ligands for
both receptor families are largely MHC Class I molecules
o Normal cells display MHC Class I on their surface, so the presence of MHC I inhibits
killing of normal cells (inhibitory signal trumps activating ones)
o Viruses often downregulate MHC I to escape CTLs, but NK cell can detect and kill the
infected host cell due to lack of MHC I
 NK important in tumor killing because tumors lose expression of MHC Class I

T Cell Tolerance

Horror autotoxicus = body's immune system attacking itself; autoimmunity

Immune response is dictated by the context in which the Ag is encountered
 Ag in presence of PAMPs/ "danger" signals will activate immune response, while Ag in the
absence of danger will lead to tolerance
 PAMPs activate APCs, and once activated, presents any Ag that are present (b/c of "danger")

22
Central tolerance is induced in the thymus  developing T cells that react with self Ag are negatively
selected; however, the thymus cannot represent/test all self-Ag, some self-reactive cells aren't deleted

Peripheral tolerance can be induced several ways:

 Ignorance  autoreactive T cells are present, but Ag is low or hidden, no response is triggered
o If T cells gain access to Ag later in life, will get a reaction and autoimmunity
 Clonal Deletion  When self Ag is present in high levels, autoreactive T cells are deleted
 T Cell Anergy  T cells that receive Signal 1 w/o Signal 2 (costimulation) fail to produce IL-2 and
proliferate, become anergic

T Regulatory cells promote tolerance and prevent autoimmunity

 Most prominent Treg is CD4+CD25+ T cells  a subset of these cells express transcription factor
Foxp3, which inhibits T cell activation
o When deleted from mice, develop organ-specific autoimmunity
 In addition to CD4+25+, TR1 cells promote tolerance via secretion of IL-10 and TGF-b (inhibits
CD4+ cells)

Immunoregulation  an active ongoing process regulating the immune response, possibly involved in
tolerance
 IL-10 negatively regulates active immune responses, but it can also inhibit auto-immune
responses, thus promoting tolerance
 Cells that have an effector role in one situation can induce peripheral tolerance in others
 Inhaled or ingested antigens can induce tolerance

Ex. Mucosal immunity and peripheral tolerance  gut immunity depends on IgA and Peyer's patches
contain T cells that produce IL-10 and TGF-b to help B cells and plasma cells make IgA (effector)
If fed antigens, the T cells (gut effector cells) mediate oral tolerance
 Ag-specific CD4+ T cells (Th3) secrete TGF-b - when they encounter Ag at target organ,
mediate bystander suppression (stop activation of naïve T cells)

Autoimmunity occurs when there is a breakdown in the mechanisms that promote tolerance

Clinical Implications for Tregs

 Tregs are protective against diabetes (autoimmune attack on the pancreas)
 Can induce tolerance for transplant (rather than induce life-long immunosuppression)
 Tumors are good at inducing tolerance  potential research for treatment

Lymphocyte Activation

Review of previously learned concepts in this lecture:

 A relatively small number of T cells respond to a given Ag
 CD8 cells respond to intracellular Ag via MHC Class I; CD4 cells respond to extracellular Ag via
MHC Class II (CD4 are further differentiated into subsets)
 Requiring signal 1 and 2 ensures only "professional APCs" activate T cells
o DCs constitutively express MHC Class I & II, B7 costimulator, major response to virus
o Macrophages scavenge, MHC Class II & B7 is inducible, major response to bacteria
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 o B Cells low constitutive expression MHC CLass II, inducible to higher, B7 inducible,
present bacterial, viral, and protein Ags
 Effector T cells are activated by Signal 1 alone

Stages of T Cell Activation:

1. T cell encounters MHC-Ag complex on APC surface
2. Induction of IL-2 production and IL-2R α chain
3. T cell proliferates in response to autocrine signaling via IL-2

1. Generation of Signal 1 by APC

 As T cells circulate through secondary lymph tissue, adhere to endothelial surfaces of vessels,
mediated by L-selectin
 Diapedesis through blood vessel wall is mediated by LFA-1 on cell and ICAM-1/2/3 on vessel
 Early phase of T cell binding to APC mediated by CD2/LFA-3 binding, LFA-1/ICAM
 MHC II binds TCR and CDR; MHC I binds TCR and CD8

2. Two main paths of T cell activation, both ultimately upregulate IL-2 and IL-2Rα:
1. Ras-Raf Pathway
2. Phospholipase C Pathway

The invariant chain of the TCR-CD3 complex couples the TCR to intracellular tyrosine kinases and other
signal transduction molecules (tyrosine kinase is important in TCR-mediated signaling)
 CD3 and ζ chain associate with transmembrane of the TCR
 The TCR ζ chain is capable of triggering signaling by itself
o Because the ζ chain contains 2 SH2 domains, which bind specific and with high affinity
o The SH2 domain = YXXL and binds phosphorylated tyrosine residues
 The active signaling component of invariant chain is an ITAM, a conserved motif
o ITAMs couple the TCR complex to the intracellular tyrosine kinase ZAP-70
o An inactivating mutation in ZAP-70 causes inherited T cell immunodeficiency
 ZAP-70 is essential for TCR-mediated signaling
o LAT is an important substrate for ZAP-70  required for activation of Ras and PLC paths

Binding of CD4 or CD8 along with the TCR is required for generating Signal 1
 Cytoplasmic portions of CD4 and CD8 are bound to a protein, Lck and association with Lck is
necessary for T cell activation
 Unknown how CD4 and CD8 interact with TCR, but likely that Lck activates ZAP-70

Costimulatory signals are Ag-independent  primary function is enhanced IL-2 production

 Best known = CD28 on T cells and B7-1/2 on APCs

When Signals 1 and 2 are provided:

TCR binding  PIP2 hydrolysis  IP3 generation  Ca++ release from intracellular store  calcineurin
is activated  dephosphorylation of NFAT  NFAT moves to nucleus  binds IL-2 promoter  Activate
transcription of IL-2
Drugs used to suppress transplant rejection (cyclosporin A) inhibit the clonal expansion of T cells by
indirectly target calcineurin, preventing NFAT entering the nucleus and IL-2 production

Another mechanism to inhibit T cell activation = interaction of B7 with CTLA-4

 On naïve T cells, only receptor for B7 is CD28  after T cell activation CTLA-4 is expressed
24
 o Expressed transiently for ~2 days after activation to limit IL=2 production and T cell
proliferation
 CTLA-4 is homologous to CD28, but binding of CTLA-4 and B7 stops T cell proliferative response

*Differentiated effector cells lose their dependence on costimulation  triggered by Signal 1 only

**Did not cover "signaling through the B cell antigen receptor complex" in lecture, though in notes

Immunological Memory

Immunological memory can be detected up to 70 years after initial vaccination.

Receiving more than one vaccination didn’t make a significant difference in the percentage of volunteers
who had detectable CD4 or CD8 responses.
The relative half-life of memory  10 years for both CD4 cells, 15 years for CD8 T cells
 Implies that a simple way to increase the persistence of T cell memory might be to simply start
out with more cells in the memory pool

2 Types of Memory Cells  Both mostly in resting stage, both make effector cytokines (IFN gamma,
TNF) in response to antigen

EFFECTOR memory cells

 Reside in the tissues, provide a more rapid first line of defense against re-infection
o More poised to mount attack in resting stage, have mRNA to quickly make perforin and
granzyme B
 Potent lytic activity, negative for CCR7

CENTRAL memory cells

 Reside in the lymphoid organs, act more like a reservoir to resupply the effector pool
 CD62L (L selectin) and CCR7 facilitate adhesion and migration to lymphoid organs
 Autocrine signaling via IL-2

Why do you need both types?

1. Resident effector memory cells provide the first response
2. This pool is supplemented from the circulation, T cells attracted to the pro-inflammatory milieu
3. By about 24 hours after infection, DCs have presented Ag to central memory cells in lymph
nodes  initiates generation of additional effector memory cells to resupply tissues

There are two models for why we get two different types of memory cell:
 Linear differentiation model: Naïve  Effector cell  Memory cell
 Bifurcative differentiation model (more data to support)  division of T cells is asymmetrical
(one is bigger), one population becomes effector, the other central (memory cells arise earlier)

**IL-7 and IL-15 are both important cytokines in memory (clinical implication = might give IL7/15 with
vaccine to create more/better memory cells)

Persisting antigen is NOT required for memory to persist  shown with mouse study, in mice without
MCH I (couldn’t present antigen), transplanted memory cells still persisted
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Persistent antigen is problematic
 Continued exposure of specific T cells to antigen in a pro-inflammatory setting leads to either
loss of memory and effector function over time (can’t produce cytokines)
o First lose the ability to make IL-2, then TNF-alpha, and finally IFN

CD4 T cell help is REQUIRED for long term memory (no Class II  No CD4 T cells  NO help)

Huge exponential expansion of infection/ immune response is not necessary to generate memory cells
 Memory cell NUMBERS were programmed early on in the CD8 T cell response.
 Even with treatment, will generate a robust and FUNCTIONAL memory CD8 T cell response.

Secondary memory response

 Increased prevalence of antigen-specific memory CD8 T cells
 Memory responses happen faster (2-3 days instead of 7-10), and stronger
 Include higher affinity T cells, because those were the cells most likely to have expanded in
response to the original antigen challenge (overall better response)
o Mostly IgG’s (and IgA), unlike first response in which IgM > IgG
o Affinity of antibody and somatic hypermutation both higher in second response

Secondary response and “booster” after vaccination

 After an initial vaccination, naïve T cells proliferate and differentiate into effector cells
o Data suggest that the optimal time for boosting is 2-3 months after the initial priming
vaccine
 No half-life can be calculated for antibodies  no appreciable decay of B cell memory
 Magnitude of the B cell response appears to be slightly dependent on the NUMBER of
vaccinations received, with 2 or more vaccinations resulting in a stronger response

Formation of B Cell Memory

 On first exposure to antigen, all B cells have similar chance of recognizing antigen
 On subsequent exposures, previously stimulated B cells more likely to recognize and bind
antigen  Darwinian process in which highest affinity B cells continue to proliferate with each
exposure
o This process occurs in germinal centers (in lymph nodes where somatic hypermutation
and affinity maturation occur)
 The germinal center reaction gives rise to TWO distinct sets of long-lived B cell populations
o Memory B Cells – stop proliferation and Ig secretion, but still express their BCR so can
response to a second exposure  do not require persistent Ag
o Plasma cells (induced via BLIMP-1), which move to the bone marrow, and a subset
survive for years in the marrow
 Too many plasma cells in bone marrow = multiple myeloma

Anatomy of the Immune System: Cellular Interactions

Microenvironments in the body must support 3 critical immune functions:

1. Enable the production of clonally diverse lymphocytes with a broad Ag recognition (1° tissue)
2. Co-localization of Ag and mature lymphocytes (2° lymph tissue)
3. Provide niches to promote long-term survival or key effector and memory cells (1,2,&3°)
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Primary Lymphoid Tissues are sites of lymphogenesis that support production of mature T and B cells
from non-functional progenitors
 At these sites, must develop Ag-recognition ability, select cells with appropriate specificities,
develop signling to response to Ag stimulation, and home to proper microenvironment
Bone Marrow
 In long bones and spongy bone, contain stroma (many different cells) that prpvide support for
marrow and produce factors to regulate hematopoiesis
 Earliest precursors are more distal while the mature elements are central in the cavity

B-lymphopoiesis in Bone Marrow

 Development from hematopoietic cell to mature Ig+ B cell during an Ag-independent process
 IL-7 and contact with stromal cells are required for B cell development
 There is extensive deletion of B cell precursors, cleared by macrophages (either nonfunctional B
receptor/Ig OR autoreactive)

The Thymus
 Has 3 major functions:
o Receive T cell progenitors from bone marrow
o Support maturation, clonal expansion, and deletion of developing T cells
o Coordinate the release of competent T cells to the periphery
 Embryologically distinct  combination of endoderm (3rd pharyngeal pouch), ectoderm, and
mesenchyme
 Mature thymus contains several types of epithelia that serve different functions
o Thymic epithelial cells express high levels of MHC Class I and II antigens, and produce
cytokines involved in T cell development (IL-1,3,6,7)
 Bone-marrow derived cells (monocytes, DC precursors) also enter thymus to form macrophages
and interdigitating cells
o Interdigitating cells  Effective APCs, dominant cell in driving clonal deletion (negative
selection) of developing T cells
 There is some T-lineage differentiation in the marrow (in absence of a thymus, it is possible for
limited T cell development)

T Cell Development in the Thymus

Prothymocytes enter thymus via large venules at CMJ  Localize to subscapular region
 Cells do not exhibit surface CD3, CD4, or CD8 at this point (CD3 might be present in cytoplasm)
 TCR rearrangement begins while all cells are double negative, DN (CD4-/CD8-)
DN cells begin rearrangement  50-fold expansion in cells  Functional β gene is rearranged  Cells
become DP (85-90% of cells in thymus are DP)  Another 50-fold expansion  DP cells mature and lose
expression of CD4 or CD8  (SP) CD4 and CD8 cells increase expression of TCR/CD3
During transition from DP  SP, cells move from thymus cortex  medulla (Final maturation in the
medulla take 12-16 days, acquire full TCR signaling capabilities)
The CMJ has a high density of DCs and macrophages, forming a negative selection barrier  cells that
survive the barrier upregulate anti-apoptotic molecules like BCL-2

Secondary Lymphoid Tissues are where naïve lymphocytes and exogenous antigen are brought together,
including the lymph nodes, spleen, and mucosa-associated lymphatic tissue (MALT)
Compartmentalization in secondary tissues
27
  T and B cells enter secondary tissues via HEV (high endothelial venules)
o HEVs have homing receptors and secrete chemoattractants to extract lymphocytes from
circulation
 B cells migrate toward B cell follicles following B lymphocyte chemoattractant (BLC, CXCL13)
 T cells express a receptor CCR7 for chemokines SLC/CCL19 and ELC/CCL21 and home to T cell
zones

To activate T cells in lymph nodes, DCs also express CCR7, responding to CCL19  move to T cell zone
 DCs have a high concentration of MHC, express T cell costimulation signals, recruit T cells with
cytokines/chemokines, and maximize surface area for T-cell/APC interaction
 Initial encounter between DC and T cell activates T cell but also determines which cytokines it
will produce and where it will go after activation
 New T cells leave secondary lymph tissues via efferent lymph, enter blood, migrate to tissues
for which they have specific receptors

Production of Memory-Effector Lymphocytes

 For T cells, naive  memory/effector transition takes place in T zones of 2° lymph tissues
 For B cells, this transition involves differentiation into distinct plasma cell populations and also
genetic modifications of Ig to increase affinity (affinity maturation) and effector capability
(class switching)

B Cell Differentiation in Secondary Lymphoid Tissues

 Unlike T cells, B cells recognize bound OR free/soluble Ag in lymph
 B cells are modified in secondary lymph  somatic hypermutation, selection based on affinity

B Cell Activation - 2 stages:

1. Rapid conversion of naïve B cells into plasma cells making genetically unmodified Ig
2. T-B cell interactions gives rise to long-lived plasma cells producing high affinity, non-IgM Igs

Germinal centers function to increase antibody affinity through somatic hypermutation of Ig genes and
selection of clones bearing receptors with increased Ig affinity

GC Reaction: B-cell blasts  centroblasts  clonal expansion and hypermutation  centrocytes 

plasma cells & memory B cells

In a follice, B cells will see Ag presented by FDC  form a secondary follicle, which becomes the GC and
the original follicle become the mantle zone
 Mature GCs are oligoclonal (only contain progeny of 1-3 B-cell blasts)
 Only bound-Ag promotes the GC reaction - free Ag in lymph will not
 All centrocytes are destined for apoptosis unless they receive positive signal via their Ig

Follicular T helper cells (TFH) regulate B cell activation and survival in the GC
 Move from T zone to B follicle b/c stop expressing CCR7 and begin making CXCR5
 CD4+ TFH binds to Ag-presented by B cell MCH II at border b/n B and T areas
o Critical part of binding is between CD40 (B) and CD40L (T)  promotes B cell survival
and allows for class switching

Mucosa-associated lymphoid tissue (MALT) = epithelial/mucosal layer barrier immune function

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  Contain many immune cell types, T and B areas, GCs, HEVs
o HEVs allow naïve cells to enter MALT for 1st encounter with Ig
 include tonsils and peyer's patches in small intestine  contain multiple GCs and are rich in IgA-
producing plasma cells
Tertiary Lymphoid Tissue = Accumulations of lymphoid tissue in non-lymphoid tissues during chronic
inflammation  can even resemble second lymph tissue with T/B areas and GCs
 Can arise in almost any tissue and is transient

Autoimmunity

Autoimmunity = An immune response to a self-antigen

 Can be organ-specific if targeted Ag is specific to an organ/tissue (e.g. Ig to insulin = DM)
 Can be systemic if targeted Ag is more ubiquitous (e.g. Ig for lupus/SLE)

Characteristics of autoimmunity:
 Immune response is highly restricted and associated strongly with phenotype
 Responses are driven by autoreactive CD4+ T cells
 Autoimmune diseases often begin with autoimmune reactions 2-15 years before onset of sx's
 2 types of auto-antibodies  those that precede diagnosis by several years (initiation) and
those that occur around time of sx onset (propagation)

4 phases of autoimmunity
1. Susceptibility  pre-disease, preconditions for later initiation are satisfied
2. Initiation  presence of autoimmune response, before onset of symptoms
3. Propagation  onset of clinical disease
4. Regulation/Resolution  activation of immunoregulatory pathways

Example: APECED  Rare disease encompassing multiple autoimmune diseases

 Numerous autoantigens, specifically enzymes in endocrine tissues
 Defect in AIRE, which normally regulates expression of various peripheral endocrine-tissue
autoantigens in thymic epithelial cells
o In order to prevent autoimmune response, thymus must express self-Ag to developing T
cells  No AIRE = unable to present endocrine auto antigens, no tolerance

IPEX Syndrome  rare, X-linked disorder w/ multiple autoimmune issues, caused by mutation in FoxP3
 FoxP3 is essential for Treg development  no regulation to stop immune response
 The outcome of immune signaling is a balance between stimulatory and inhibitory signaling

Theory why autoreactive T cells exist:

 Only a small fraction of potential antigenic determinants on a molecule are presented by MHC
Class ii (self or pathogen)
 The epitopes that are presented are immunodominant  dominance is influenced by protein
structure/folding
 Dominant epitopes are presented, cryptic epitopes are not  tolerance is only induced to
dominant epitopes and T cells that recognize cryptic self-epitopes are not deleted
o Altering a molecule's processing and presentation may then later reveal previously
cryptic epitopes and generate an autoimmune reaction
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In order to generate an adaptive response cell needs  non-tolerized Ag, above-threshold
concentrations of Ag, a pro-immune context (infection, damage, malignancy)
3 major events associated with onset/worsening of autoimmune disease:
1. Injury  Sympathetic ophthalmitis
a. Eye is normally an "immune privileged site" (T cells can't see Ag there)
b. Penetrating injury  immune response drives damage and inflammation even
spreading to other eye
2. Infection  Coxsackie virus
a. Cardiac myosin heavy chain modified during immune response to infection  T cells
continue to recognize new myosin heavy chain after virus is gone  chronic
inflammation and dilated cardiomyopathy
3. Malignancy  Paraneoplastic Neurological Degeneration (PND)
a. Tumor expresses Ag also found in CNS  when immune system responds to tumor, also
attacks CNS/cerebellum
b. Cancers can cause autoimmunity because there is over-expression, mutation, and
modified structure in tumors
c. Scleroderma (fibrosis of skin and internal organs) results from Ig to RNA pol III mutated
in cancer cells (patients have onset of cancer and scleroderma at same time)

**Initiation of autoimmune disease involves exposure to self-Ag not previously seen and tolerized due
to being in an immune privileged site, being mutated in cancer, or structurally modified during infection
 Immune response subsequently spreads to wild-type Ag, causing ongoing damage to tissues
expressing the autoantigen

E-Lecture: Evaluation of Immune Function

Clinical Features of Immunodeficiency

1. Increased Susceptibility to Infection

a. Chronic/recurrent infections without other explanation
b. Infection with Organism of Low Virulence
c. Infection of Unusual Severity

2. Autoimmune or Inflammatory Disease

a. Target Cells (e.g. hemolytic anemia, immune thrombocytopenia, thyroiditis)
b. Target Tissues (e.g. vasculitis, systemic lupus erythematosus, rheumatoid arthritis)

3. Syndrome Complex
Syndrome
DiGeorge Syndrome
Wiskott-Aldrich Syndrome
Ataxia-Telangiectasia
Ivemark Syndrome
Clinical Presentation Immunologic Abnormality
Congenital heart disease Thymic hypoplasia
Hypoparathryroidism
Abnormal facies
Thrombocytopenia Variable B- and T-
Eczema lymphocyte dysfunction
Ataxia Variable B- and T-
Telangiectasia lymphocyte dysfunction
Congenital heart disease Asplenia
Bilateral 3-lobed lungs
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Polyendocrinopathy Syndrome Endocrine organ dysfunction Chronic mucocutaneous
candidiasis

Patterns of Illness Associated with Immunodeficiency

Illnesses
Disorder
Infection Type Other
Antibody Sinopulmonary Also susceptible to autoimmune
Gastrointestinal disease (autoantibodies)
Cell-mediated Pneumonia Do not have autoimmune diseases
Immunity (present as a Gastrointestinal
classic Skin, Mucous Membranes
immunodeficient pt)
Complement Sepsis and Other Blood-borne - Likely to develop autoimmune
stereotypical for complement defects disease (SLE,
Glomerulonephritis)
Phagocytosis Skin, Reticuloendothelial System  areas Not associated with response to
w/ phagocytes viruses

Screening Tests for Immunodeficiency

Suspected Abnormality Diagnostic Tests
Antibody Quantitative immunoglobulin levels (IgG, IgA, IgM)
Antibody response to immunization
Cell-mediated Immunity Lymphocyte count
Delayed type hypersensitivity tests
T-lymphocyte (CD4, CD8)
HIV serology
Complement Total hemolytic complement (CH50)
Phagocytosis Neutrophil count
Nitroblue tetrazolium (NBT) dye test or
dihydrorhodamine flow cytometry assay

Assessment of Immunoglobulins and Antibody

- Specificity for Ag is determined by the Fab portion of the antibody molecule
- Effector function is determined by the Fc portion of the antibody molecule
- Antibody level is not the same as immunoglobulin level

Property IgG IgA IgM IgE

__________________________________________________________________________
First detectable antibody – – + –
Major part of secondary response + – – –
Major serum immunoglobulin + – – –
Major secretory immunoglobulin – + – –
Complement activation + – ++ –
Opsonic ++ –  –
Placental transport + – – –
Anaphylactic activity – – – +
___________________________________________________________________________

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Primary and secondary antibody responses differ in terms of
kinetics and relative amounts of IgG and IgM antibodies
 Primary response = More IgM than IgG
 Second response = IgM response is same, IgG
response is much greater, so IgG is Greater in 2nd

Immunoglobulin levels vary by age  no single normal

Ig, can only evaluated by age

Assessment of T lymphocyte function

Delayed type hypersensitivity responses – Small

amounts of recall antigens are injected intradermally
(e.g. TB test). Subjects who are able to react (have
had the disease) will mobilize monocytes and T
lymphocytes to the area, creating a red
(erythematous), hard (indurated) lesion. Lack of
response is anergy.

Lymphocyte proliferation assay

 Mix peripheral mononuclear cells (monocytes and macrophages), and B & T lymphocytes with
antigens in solution
 Analyze how much thymidine was incorporated into DNA as an indication of proliferation
o Proliferation = indication of normal T cell function

Example questions from lecture - how to use these tests clinically:

1.) A baby is born to an HIV-infected mother. The baby appears healthy at birth, but it is important to
determine if this child has been infected with the AIDS virus. Which tests might be useful to answer that
question?

A. Measurement of IgG antibody to HIV in the mother

B. Measurement of IgG antibody to HIV in the baby
C. Measurement of baby's serum immunoglobulin levels
D. Measurement of IgA and/or IgM antibodies to HIV in the mother
E. Measurement of IgA and/or IgM antibodies to HIV in the baby
F. Detection of viral antigen in the baby

32
Mother only has 1/3 chance of passing it on, testing must be done in baby, not in mother. IgG test in
baby would be useless because mom's IgG is passed on, even to uninfected baby. Over time, could
diagnose if baby's anti-HIV IgG increased, but this requires several titers. Could use IgA or IgM at any
time  since they aren't passed through placenta, if they are present, the baby must be making them
him/herself and thus be infected. Could also use PCR to look for evidence of virus. Serum Ig would be
useless and only indicates the baby is making Ig, nothing about its specificity.

Problem with serum tests  A window period (lag between virus appearing and antibody production)
will yield a falsely negative Ig test in someone who is actually infected. The only way to test for HIV early
on is to directly look for virus via PCR.

2.) A teenager has fever, swollen lymph nodes and an enlarged spleen. He may have infectious
mononucleosis? It is expensive and slow to culture the Epstein-Barr virus that causes infectious
mononucleosis. How can immunologic tests help to make the diagnosis?
Other members of the family are tested for antibody to EBV with the following results:
IgG IgM
Patient + +
Brother – –
Mother + –
Father – +

Which family members are currently infected with EBV,

which have had a previous infection, and which are
susceptible to EBV infection?

IgM to viral capsid Ag(VCA) is the first produced (lasts

months), then IgG to VCA (lasts years), then IgM to
nuclear antigen (persists for lifetime). IgG and IgM seen
together indicates someone who is currently infected.
IgG only indicates a prior infection. IgM without IgG
indicates a very early active infection. So in this
scenario, the patient is infected, his father is infected
(early), his mother has previously been infected.

3. Gamma globulin is used to treat patients with antibody deficiency diseases. A number of lots of
gamma globulin were contaminated with the hepatitis C virus, an organism that is non-cultivatable.
What tests could be used to detect Hepatitis C infection in this group of antibody-deficient patients?

Patients with antibody deficiency don't make antibodies, so cannot test for IgM, IgG, or IgA. The only
way to make a diagnosis in a patient who has no antibodies is a direct PCR test for the antigen.

4. An infant has severe thrush (oral yeast infection) and Pneumocystis carinii pneumonia. Both of these
are opportunistic infections (i.e., infections that do not occur in immunocompetent hosts). What
defects in host defense could account for susceptibility to these infections, and what immunologic tests
could be performed to investigate the possibilities?
33
The likely defect is cell-mediated immunity. The first test would be lymphocyte count (for total #),
then look at types of lymphocytes present. Results show very low levels of CD2, CD3, CD4, no CD8. No
increase in lymphocyte proliferation with exposure to several mitogens. Normal level of B
lymphocytes, but no CD4 to help produce humoral immunity. No T cell function at all  need bone
marrow transplant or will be fatal

Rule of 2/3
Parameter < 5 years old > 5 years old
WBC 9000 9000
Lymphocytes 2/3 (6000) 1/3 (3000)
T lymphocytes 2/3 (4000) 2/3 (2000)
CD4+ T lymphocytes 2/3 (2666) 2/3 (1333)

5. A ten-year old boy has a history of two bloodstream infections caused by encapsulated bacteria
(pneumococci) and has now developed systemic lupus erythematous (an auto-immune disease
characterized by the development of many types of auto-antibodies) with deposition of immune
complexes in the glomeruli. You suspect a deficiency of complement. What is the best screening test to
use?

CH50  determines what dilution of serum will still yield 50% maximum hemolytic activity

Vaccines

History of Vaccination
 Edward Jenner creates first vaccine when he discovers inoculation with cow pox confers
protection against small pox
 Most infection diseases have been eliminated/nearly eliminated in the US
o Indigenously transmitted measles was declared eliminated in the US in 2000
o Fear of vaccine causing autism has led to an increase in incidence of measles among kids
 Incidence of vaccine-preventable diseases in adults remains high (influenza is #1)
 Still many diseases for which vaccines don't exist or are inaccessible (e.g. measles vaccine must be
refrigerated, not accessible to warm climates w/o electricity)

Prime/Boost Method = Administering multiple doses of vaccine, which "boosts" the avidity and number of
B and T cells "primed" with the first dose; works via 3 mechanisms:
1. Generate antibodies that neutralize bacterial toxins or viral surface proteins
2. Induce antibodies to opsonize bacteria
3. Induce virus specific CD8+ cytolytic cells

Active versus Passive Immunity

Type of Immunity Active Passive
Forms Vaccines, Toxoids Immunoglobulin
Goal Long-lasting immunity Short term protection
Means of Protection B cell proliferation, Ig responses, T Ig from immune individual
cell stimulation
Boosters Given? Yes, sometimes No
Immediate Protection? No Yes
34
HBIG are HBV (Hepatitis B) antibodies given after exposure to prevent infection (passive), while the HBV
vaccine is given before exposure to prevent infection (active)
Polio  was endemic in the US prior to 1900
 Most infants were exposed to polio under 6 months, when mom's IgG provided protection and
they only displayed very mild form of disease  widespread passive immunity
 With improved sanitation, fewer babies were exposed to polio and later exposure resulted in
severe disease  vaccination to induce active immunity was required to prevent polio

Effective vaccines should be:

 Safe, with few side effects
 Protect against illness for a long period of time
 Induce neutralizing Ig and/or protective T cells
 Be practical (low cost, biologically stable, easily administered, few side effects)
**Ideally vaccination results in activation of APCs and B/T cells and induces T cell response to several
epitopes (to deal with MHC polymorphism in the population)

Types of Vaccines:
1. Whole killed vaccine  Intact pathogen is treated and incapable of replication
a. Excessive treatment can impair immunogenicity, but insufficiently treated cells may still
be virulent
b. Do not replicate, so do not enter cells  no MHC Class I signaling, limited CD8 response
2. Live attenuated vaccine  Live, avirulent strain of pathogen that cannot cause disease
a. Produced by passing virulent strain through another species/tissue until enough
mutations make it avirulent  unlikely, but possible to revert back to WT/virulent
b. Generally most potent b/c activate CD4 and CD8 responses
3. Subunit vaccine  Individual components of the relevant microorganism
a. Lack PAMPs (safer, but less immunogenic)
b. Work well for bacterial toxins and viral envelope proteins, but not bacterial polysaccharide
capsules**
4. Antigen-bearing vector vaccine  Plasmid containing cDNA that encodes a protein Ag
a. Used for highly variable pathogens (HCV & HIV) where a common Ag is hard to find
b. Current research into better PAMPs than DNA  express pathogen Ag and then have
vector activate immune response to inserted Ag
i. STEP trial 3 vectors each expressing a different HIV gene
ii. Thai trial  multiple vectors expressing same gene (heterologous prime boost)
c. Thai trial had better outcomes

**H. Influenza (especially serotype Hib) causes severe invasive infections  initial subunit vaccine
targeting its polysaccharide was ineffective (no T cell activation, no response in children < 18 months)
 Immunogenicity of vaccine was increased by conjugating it to a carrier protein that would induce T cell
response (and thus class switching, somatic mutation, etc.)
Another way to increase immunogenicity is through the addition of adjuvants
 Aluminum salts (alum) are the most common (in HAV, HBV, Tdap, Hib, HPV and others)
o Alum preferentially activates a Th2 response
 Newer adjuvants have been developed to enhance Th1 and CD8+ responses
o ASO4 is a combination of alum and MPL, an LPS-derivative that activates TLR4 pathway,
leading to increased cytokines and APC activation
o Cervarix vaccine (ASO4) might provide better response than Gardasil (alum only) for HPV
35
Type of Vaccine Example Advantages Disadvantages
Whole Killed IM influenza Inexpensive, more More side effects than
IM polio immunogenic than subunit, less
subunit immunogenic than live
Live Attenuated Polio and BGC (for TB)  Inexpensive, high Risk of reversion to
both not used in US immunogenic virulence, can cause
disease in immuno-
compromised patient
Subunit HBV (Hep B) Few side effects, can Less immunogenic,
H. influenza target critical part of expensive
pathogen, very safe
Antigen-Bearing Vector ? future HIV and HCV As immunogenic as live Expensive, unproven
vaccines attenuated, but safer

Tumor Immunology

The immune system can promote carcinogenesis:

 Chronic inflammation (and chronic diseases that cause inflammation) are associated with cancer
o Inflammatory bowel disease ↑ risk of colorectal CA, GERD ↑ risk of esophageal CA
o Chronic Hepatitis B and C increase risk of hepatocellular carcinoma
 NFκB is involved in chronic inflammation pathway (NSAIDs are inhibitors of NFκB)
o Nurses' Health Study  NSAID use correlated w/ ↓ risk of colon CA (not other types)

Tumor infiltrating lymphocytes (TIL) are T cells present within tumors  presence of TILs associated with
better patient outcome/survival
 Patient with late stage cancer and many TILs had better outcomes than early stages with less TILs

The Immune Editing Hypothesis (3Es - Elimination, Equilibrium, Escape)

1. Small tumors are eliminated by the adaptive immune system before they are detectable
a. NK cells recognize loss of MHC I in tumor cells AND sometimes tumor cells express
activating ligands (or lose inhibitory ligands)
b. CD8+ Cells can also recognize non-tolerized new, mutated or over-expressed tumor Ag
2. At some point the tumor finds way to evade the immune response and continues to exist in
equilibrium (still somewhat restricted by immune system,)
3. Eventually, tumor cell evolve to escape the immune system

Cancer Vaccines
 Nearly all approaches to infection vaccines have been applied to cancer w/o much success
 Vaccinating against cancer is limited by presence of persistent Ag
o Ag is usually tolerogenic (capable of producing tolerance)
o Most vaccines are administered in an Ag-free host

One attempt to target Prostate Specific Antigen (PSA) uses strain of virus in smallpox vaccine  increased
immunogenicity by adding 3 costimulatory surface molecules (B7, ICAM1, and LFA3)
 In Phase III trials currently  Phase 2 showed prolonged survival
 No tumor shrinkage  probably reverts back to equilibrium stage for a short time

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Dentritic cells have decreased number and function in cancer patients  2nd vaccine type targets DCs
 Remove patient's peripheral monocytes, culture with GM-CSF to induce DC differentiation, and
reintroduce DCs into body  increases survival by 4 months
 This is the one FDA-approved cancer vaccine for treatment of prostate cancer - Sipuleucel-T

Adoptive Cellular Therapy (ACT) focuses cancer treatment on T cells

Remove T cells from tumor and activate/expand with IL-2, then reintroduce into body
 To work, must deplete endogenous T cells and T regs by inducing immunodeficiency
 Only works 20% of the time, but has had 80% response rate in metastatic melanoma
Another option is to re-engineer a patient's own T cells to be tumor-specific
 T cells have existing TCRs, so end up have 2 TCRs and specificity for 2 different Ags
 New α/β chains can rearrange with endogenous α/β chains  potential to create an autoreactive
TCR
Recent research has focused on chimeric antigen receptors (CARs)
 Combine specificity and high affinity binding of Ig with killing machinery of CD8 T cells
 The antibody part of the molecule is engineered so Ag-binding part is a single chain made up of
variable parts of HC and LC (single chain fragment variable = scFv)
o CARs are only useful for Ag expressed on cell surfaces
 NEJM paper about one patient who had chronic lymphoid leukemia  had CAR made to target
CD19 on B cancerous cells in marrow
o 3 weeks after CAR treatment, "cytokine storm" from CAR-modified T cells begin to lyse
tumor  after, NO cancerous B cells located in bone marrow
o CD19 expressed on all B cells, so this patient can never make own B cells again (can be
given Ig for passive immunity)
Immune checkpoints are series of molecules on T cells that serve to restrain T cell function
CTLA-4 is a molecule that binds tighter to B7 than CD28  prevents signal 1 inducing anergy in T cell
 In tumors, upregulation of CTLA-4 induces anergy instead of T cell activation to tumor Ag
 Antibodies against CTLA-4 have been developed to allow T cell activation of tumor cells
o CTLA-4 has an organ-specific expression  CDLA-4 can cause specific organ inflammation
o Life threatening inflammation in bowel, brain, etc.
 Ipilimumab is the only FDA-approved immune checkpoint-blocking treatment for cancer
o Only 15-20% patients with metastatic melanoma have tumor shrinkage, BUT it is the only
drug that has shown long-term survival benefit
PD-1 (Programmed Death-1)  promising research in CA treatment
 PD1 on T cells bind ligand on DC and tumor cells to inhibit T cell response
 Without CTLA-4, mice die  without PD-1, no real effect (can potentially safely block all PD-1)
 When PD-1 antibody is combined with vaccine  dramatic improvement in Phase I trial
o One patient's tumors disappeared, and when he developed tumor in brain, there were no
cancer cells (only immune cells that presumably had responded)
o Four different response patterns, 30% tumor shrinkage (best current treatment = 15%)\
 Anti-PD-1 treatment is currently in Phase III trials

Summary
 Only FDA-approved cancer vaccine = Sipuleucel-T for prostate cancer
 Only FDA-approved immune checkpoint blocking antibody = Ipilimumab (anti-CTLA-4)
 It is easier to turn off a negative signal (checkpoint blocking) than to induce a positive signal
(vaccine) in tumor cells
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 Primary Immunodeficiency Diseases

Primary immunodeficiency diseases refers to the fact that the defect is intrinsic
 Most are genetically determined and inherited as single gene defects
 Are more common than previously expected
 Patients may vary in severity of the disease
 Onset can occur in adults as well as in children (81% affected are male)

Chronic Granulomatous Disease (CGD)  Deficiency in neutrophil/macrophage oxidative burst

Genetics
 CGD is the consequence of a number of different defects that all result reduced or absent
production of peroxide
 Most common defect = X-linked mutation resulting in absence of one of the subunits of
Cytochrome b-558
o Cytochrome b-558 has a critical role in the ETC reactions in reducing molecular O2
 3 other forms for CGD are inherited as autosomal recessive traits causing deficiencies in other
proteins that are part of the chain reaction results in O2-
Clinical Presentation
 Increased susceptibility to infection in organs rich in phagocytic cells (lymph nodes, spleen, liver,
lungs),
 Only infected by catalase-positive bacteria and fungi, NOT VIRUSES or catalase-negative
o Catalase is an enzyme that protects pathogens from hydrogen peroxide
 Cells are only able to kill bacteria that produce peroxide (pneumococci and streptococci),
essentially these bacteria provide the substance that kills them
Pathophysiology
 Common defect is in the metabolic machinery used to produce peroxide in phagocytes
 Unable to generate superoxide (O2-) and peroxide (H2O2)
Diagnosis  Add NBT dye to WBCs and an Ag that will activate oxidative burst by macrophages  in a
normal oxidative burst response, yellow stain turns blacks
Therapy for CGD
 Prophylactic antibiotics (against bacteria and fungi)
 Interferon-gamma (to upregulate killing by monocytes)
 WBC transfusion during severe infection (little benefit, only lasts a few hours)
 Bone marrow transplantation to repopulate body with functional phagocytes

Severe Combined Immunodeficiency (SCID)  severe deficiency in both B and T cells

Genetics  Multiple different genetic forms resulting same phenotype (don't need to memorize)
1. X-linked recessive deficiency in the γ chain of the IL-2 receptor on T-cells
2. Autosomal recessive deficiency in the purine salvage pathway enzyme ADA
3. Autosomal recessive deficiency in ZAP-70 (involved in signal transduction of the TCR)
4. Deficiency of JAK 3 (cytosolic protein that interacts with ILS receptor)
5. Deficiency of RAG 1, RAG2, or recombinase activating gene involved in VDJ recombination
Clinical Presentation
 Susceptibility to infection by virtually any microbe  fatal within first year of life if untreated
o Characterized by opportunistic pathogen infections that most people don't get
o Onset of symptoms at 2.7 months (after mom's IgG wears off), death usually by 7 months

38
  Severe lymphopenia (T cell count = 0)  always involves T cells, usually involves B cells
o In ADA deficiency, defect affects both T and B cells
o IL2R and JAK3 deficiencies only affect T cells (indirectly affect B cells though)
Pathophysiology
 Basis for each of the different genetic causes  focus on ADA deficiency
 The absence of adenosine deaminase (ADA) allows accumulation of deoxy ATP, a "metabolic
poison" which affects T-cells and interferes with their function
 Important to determine which genetic mutation causes SCID  specific therapy can be initiated
Diagnosis  Severe lymphopenia (low lymphocyte)
Treatment
 Bone marrow transplant (if match is found) before life-threatening disease is contracted cures
>80% children (>90% with early diagnosis that prevents an organ damage)
o Alternatively, if transplant isn't an option, can give ADA directly (PEG-ADA)
 Gene therapy for X-linked SCID  In patients who cannot get a bone marrow transplant, can
create a vector expressing common γ chain, transfect patient's stem cells with vector and replace
into body
o Worked initially, but 6 of 8 patients developed malignancy (leukemia/lymphoma) after
several years
o Research on vectors inserted into more stable genes have treated w/o developing CA

Allergy I: Allergy and Hypersensitivity

Prevalence of allergic disease  very common

Sequence of an Allergic Immune Response (Priming)
Ag presentation  Th2 Response (create IL-4,5,13)  IL-4 induces IgE production  IgE binds mast cells

The differentiation of a CD4+ cell into Th1 or Th2 determines whether response will be an allergic one
 IL-4 drives differentiation toward Th2, while IL-12 and IFNγ favor differentiation to Th1
 One model explain implicates NK T cells in producing the IL-4 to initially stimulate Th2 (make more
IL-4)

Th2 differentiation can also be influenced by many stimuli (e.g. common allergens)
 Many cell mechanisms for innate immune system to sense Th2-inducing stimuli (protease activity,
PRRs, tissue damage, certain amino acids)
 Some (but not all) allergens are enzymes

lL-4 and IL-13 drives accessibility to ε locus on HC, driving class switching to IgE
 IgE is the rarest Ig in blood circulation, is short-lived in serum  IgE activity occurs locally binding
to high affinity receptors on basophils and mast cells
 IgE seemed to evolve to fight helminth infections, based on research with mice

IgE Mediated Reactions:

 Skin  acute urticaria (hives) Mast cells are present in
 Inhalation  acute asthma, allergic rhinitis (in upper airways) skin, around blood vessels,
 Oral  food allergy (vomiting, diarrhea) and in mucosa of GI tract and
 Systemic  Anaphylaxis (a medical emergency) airway

39
Mast cells are critical for IgE-mediated inflammatory responses
 Contain FcεRI receptor for IgE  cross-linking of IgE's (that are already bound to FcεRI) by allergen
leads to degranulation and histamine release
o Unlike other Ig classes, in which Ag first binds/cross-links Ig, then binds target
o In addition to histamine, proteases, leukotrienes, and prostaglandins are important
products
 Mast cells will contain many different IgE's (bound to Fc) all with different specificities

The location of sensing mast cells determines the severity of immune response:
 Subcutaneous low-dose Ag triggers only a relatively small response
 Ag rapidly accessing systemic circulation can be sensed by large number of mast cells in the
connective tissue surrounding capillaries  large, systemic response
An acute asthma attack occurs with Ag reaction in the respiratory tract  smooth muscle (airway)
constriction and mucus secretion results, putting the patient in danger of hypoxia

Eosinophis (<5% of blood) are activated/expansion is triggered by IL-5 secreted by Th2 cells
 Contain complex granules filled with very toxic substances to kill parasites/helminths
o Major Basic Protein (trigger histamine release from mast cells), Eosinophil Cationic
Protein, and Eosinophil-derived Neurotoxin
 Synthesize leukotrienes and prostaglandins on activation
 Elevated eosinophil levels indicate 5 diseases  NAACP (neoplasm, asthma/allergy, Addison's
disease, collagen-vascular disease, parasites)
 Also contain Fcε receptors and bind IgE (unlike mast cells, binds to Ag that has already been
coated with IgE) to then kill Ag
o Anti-IL-5 Antibody will deplete eosinophils, but doesn't reduce allergic symptoms

Basophils (rarest cell in circulation)

 Produce IL-4 and IL-13 to perpetuate immune response, produce some histamine and tryptase
 Unknown importance of role of allergic reaction (circulating version of mast cell?)

Omalizumab (Xolair) blocks binding of IgE mast cells by binding to the constant portion of IgE normally
binding the Fc receptor  treatment in severe allergic reactions

Allergic Reaction:
1. Early phase response  Initiated by mast cell
degranulation when Ag binds IgE, resulting in immediate
changes in vascular permeability, smooth muscle
contraction, and initiation of inflammation
2. Late phase response  hours to days later, a 2nd round of
the same changes due to eosinophil attraction to site via
chemokines

The Hygiene Hypothesis  Homeostasis of mucosal immunity requires exposure to microbial

organisms/products, so basically being exposed to many antigens is protective against immune disorders
(e.g. living with pets, multiple siblings, on a farm, near animals, etc. protects against asthma/allergy)
 Th1 expression is favored by many of these antigens
 Th2 expression is favored by widespread antibiotic use, Western lifestyle, urban environment, etc.

Genetics is implicated in allergic reactions/immune disorders in research with families and twin studies
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 Types of Hypersensitivity
Type I  immediate hypersensitivity mediated by IgE reactivity to soluble Ag
 Involves mast cell degranulation, histamine release

Type II  mediated by IgG, which coats self-Ag bound on cell surface or matrix  Ag is pre-formed
 Receptors are FcRs on phagocytes or NK cells
 Caused by common drugs like penicillin and cephalosporins, which coat self-Ag (both self-Ag and
drug are cleared)
 Also induced by pathogens (Rheumatic Fever) and Ig-mediated autoimmune disorders (Graves
DELAYED RESPONSE

Disease, Myasthenia Gravis)

Type III  mediated by IgG binding to a soluble Ag to form immune complexes

 These complexes are usually cleared by macrophages in the spleen, but are not cleared in Type III
reaction (possibly due to Ag excess, persistent Ag, or the complex is too small )
 Serum sickness is a type III hypersensitivity reaction (prior to antibiotics when horse serum was
use to help clear infection  Type III response would occur with immune response to foreign
proteins in serum)
 Seen in autoimmune diseases (Lupus nephritis and Rhematoid arthritis)
Type IV  T cell-mediated reaction (any type - Th1, Th2, CD8+, Th17)
Th2  chronic asthmas CD8+  Poison ivy/contact dermatitis
Th1  Tuberculin reaction Autoimmune  IDDM

The Tuberculin reaction is a delayed hypersensitivity reaction to test for CD4 T cell memory
 Ag is injected into subcutaneous tissue Th1 recognizes Ag  Th1 releases cytokines  Recruit
phagocytes and plasma to site of Ag injection  local inflammation

Summary of hypersensitivity responses

Type I Type II Type III Type IV
Ig/Reactant IgE IgG IgG Th1 Th2 CD8+
Antigen Soluble Cell- bound Soluble Soluble Soluble Cell-bound
Mechanism Mast cells Phagocytes, Complement Macrophage Eosinophil Cytotoxicity
NK cells activation activation
Example of Allergic PCN and Serum Contact Chronic Contact
reaction rhinitis, cephalosporin sickness, dermatitis, asthma/ dermatitis
asthma, allergies lupus, RA Tuberculin allergic
anaphylaxis rhinitis
Timing Immediate Delayed

Allergy II: Origins of Allergic Disease

Quick review of Th2 mediation of allergic diseases:

Ag presentation to DC cell induces Th2 differentiation  IL-4 and IL-13 released  B cell class switching
to form IgE  IgE cross-links receptors on mast cells when bound to Ag Release of mediators like
histamine and leukotrienes  immediate changes that cause early allergic response

Differentiation into Th2 induced by DC signals 1 & 2, and ALSO a signal 3 (cytokines from epithelium)
*A sensitized patient is one in which IgE already exists for an allergen
41
Review of T cell subtypes:
 Th1 produces IFNγ, involved in clearance of intracellular organisms (macrophage killing)
 Th2 produces IL-4,-5,-13 is important in clearing extracellular organisms like helminths
o Protects fetus developing in mother, prevents inflammation
 Treg produce IL-10 and TGF-β, suppress immune response - important for homeostasis with
bacteria and parasites in the gut
 Th17 produce IL-17, IL-21 drive neutrophil development/response - involved in autoimmune
disease

Epithelial cells regulate Th2 responses through the production of IL-33, TSLP and IL-25
 All 3 are from different families and act through different pathways, but all 3 activate NFκB and
induce production of Th2-stimulating cytokines
 These 3 cytokines activate non-B, non-T cells (innate lymphoid cells/ILCs)
o These cells have a function similar to CD4+ w/o the cell surface markers/receptors
 Biggest difference is unlike T cells, ILCs don’t make IL-4
 Arise from a common lymphoid progenitor
o Directly activated by mucosal surfaces, not interaction with antigen (no memory)
o Provides quick, but short-lasting response
Production of the cytokines by epithelial cells is induced through PRR stimulation
 Many allergens are enzymes
o High exposure to LPS directly activates DC, driving Th1 response instead of Th2
 If more PRRs are activated (e.g. fungus on dust mites) a stronger allergic response is triggered

Susceptibility for atopic diseases:

Twin studies indicate a large genetic component, with some environmental factors
 26 different chromosomal regions associated with atopy/asthma (exist in many combinations)
 Some specific genes have been associated with disease (17q12-21 and childhood-onset asthma-
remodeling of epithelium; FLG/filaggrin gene and atopic dermatitis)

If a mother has atopic disease, offspring at higher risk  seem to be influenced in utero
 IFNγ production at 9 months inversely correlated with atopy at 6 years
 Cord blood from atopic mothers has impaired Treg cell # and function, lower IFNγ and higher IL-
13 levels

There are many environmental factors that influence development of these diseases:
 Exposure to allergens (cigarette smoke exposure, air pollution in cities  asthma)
 Certain infectious diseases (children with severe RSV  asthma)
 Diet (high fat diet) and lack of sunlight (Vit D) can also ↑ risk of atopic disease

Epidemiological Data:
 Prevalence of asthma symptoms among children is higher in more developed countries (but
increasing asthma prevalence globally)
 Prevalence of immune disorders to greater in countries with higher GNP
 1950-2000  Incidence of infectious diseases has decreased while incidence of immune disorders
has increased

42
Hygiene Hypothesis (revisited)  Lack of microbial exposure early in life drives disease
 Microbial exposures occurring after the first year of life had no/much weaker protective effects
What is the cellular mechanism explaining the hygiene hypothesis?
 The presence of some T cell pathways inhibits others (for example, stimulation of Tregs
prohibits Th2, thus Tregs early in life prevent Th2 reactions)
 Gut flora is different depending on geography  more stable flora in developed countries
o When normal commensal bacteria is not allowed to populate the GI tract, pathogenic
microorganisms take over and dominant/remain in gut
 There are particular risk factors for allergic responses that alter gut microbiome
o Caesarian delivery  prevents protective exposure to vaginal microbiome
o Higher antibiotic use & not breastfeeding are both risk factors

The microbiome is the ecological community of commensal, symbiotic and pathogenic organisms that
share the body's space
 Each mucosal surface has a unique combination of microorganisms within each person
o Each person has >10x more microorganisms than cells in their body
o Many of these organisms are not culture-able, so little is known about them
 Have evolved, likely beneficial  Tend to be host-specific

In patients with allergies, there are predominant shifts in the microbiome (↑ in Clostridia and ↓ in
commensal bacterial)
 Early in life one particular organism can confer an allergic response by regulating bone marrow
cell hematopoiesis  sets tone of response for rest of life
Environmental exposures may be making epigenetic changes that occur after conception/during
gestation
 Epigenetic changes are modification to DNA (structure, not sequence)  inherited
o DNA methylation suppresses gene transcription
o Histone modification can activate or silence
 Mothers exposed to air pollution confer risks to child (turn off INFγ/protective Th2 response,
alter protective Treg function)  seen to persist for generations
 Folic acid (B6) currently given to pregnant women  is a strong epigenetic regulator that may
increase risk of allergic disease in offspring

Allergy III: Clinical Manifestations and Managements of Human Allergic Diseases and Reactions

IgE doesn't cross the placenta  babies are not born with allergies
 Allergic reactions require prior Ag exposure and expansion of T cells as well as plasma cells that
secrete allergen-specific IgE
 Allergic sensitization = Production of IgE and arming of cells with FcεRI  doesn't imply disease
 Atopy = familial disposition toward allergic disease

Allergic diseases are common  affect 30-40% of world's population, 3rd/5th leading chronic disease in
children/adults
 Prevalence peaks in early age (80% cases develop < 20 years old)

43
Atopic march = phenomenon that refers to the typical progression of allergic disease
 Usually starts as eczema and food allergy in infancy/early childhood  outgrown in late
childhood, when asthma and allergic rhinitis increase

Diagnostic features of Atopic Dermatitis (AD)  characterized by chronic itchy, flaky skin
 Rash must be pruritic and follow a chronic relapsing course for diagnosis
o May also have xerosis (extreme drying of the skin)
 Onset usually in infancy  most patients have personal allergies or family history of atopy
 Treatment is 2-pronged: Moisturizing skin to prevent dryness and treat inflammation
o Also educate to avoid allergens

Asthma = Reversible airway obstruction (including cough, SOB, chest tightness, wheezing)
 Increased risk of developing asthma with smoking, air pollution, obesity, allergen exposure, and
viral infections

Allergic rhinitis = "hay fever" or "rose fever", seasonal allergies

 Presentation = sneezing, runny nose, nasal congestion, itchy eyes/nose
 Affects almost 1/3 population, most people with asthma also have allergic rhinitis
o Can affect quality of life of children with symptoms
 Prevalence and cost have both been increasing in US
 2 main types = seasonal (present <4 months/year) or perennial (>9 months/year)
o For seasonal, spring (trees), summer (grasses), fall (weeds) have different antigens
o For perennial/indoor symptoms, allergen most likely mold, dander, roaches, dust mites

Venom Allergy  venom hypersensitivity from 3 families of insects, there is cross-reactivity among some
of these venoms, so patients with allergy to one would likely have reaction to another
 Reactions vary from mild to life-threatening (patients with anaphylactic reactions may have
underlying mast cell disorder)

Diagnosis and Management of Allergic Diseases

 Evaluation includes history and physical exam, and next step is usually testing
 Recommend patients avoid allergen, especially after specific allergen is identified

2 primary ways to detect IgE in patients:

1. Skin testing  allergen is introduced under the skin, if IgE is present, will cross-link and activate
mast cell histamine release, causing a raised, pale bump (wheal) with surrounding redness (flare)
o Advantage = fast, relatively inexpensive / Disadvantage = can lead to false positives
2. Blood testing ("RAST")  an ELISA test for recognizing human IgE
o IgE in serum will bind to allergen  secondary anti-IgE antibody labeled with enzyme
binds to IgE  substrate for the enzyme is added and create a quantifiable fluorescence
 Higher fluorescence = more allergen-specific IgE in sample
Treatment for Allergies
 Can treat with anti-histamines or leukotriene-modifiers for symptom relief/control
 Corticosteroids are used for more persistent/severe disease (steroid reduce mast cell and
eosinophil number, do not change IgE levels or mast cell function)
 Immunotherapy = administration of gradually increasing quantities of an allergen over weeks,
months, years in hope of inducing immunological tolerance

44
Food Allergy = immunological response to proteins in food (in contrast to food intolerance, a response to
carbohydrate/sugar content, like lactose intolerance)
 Prevalence also increasing in food (>90% to milk, eggs, peanuts, soy, wheat, tree nuts, and fish)
 Vary from very mild to life-threatening symptoms (anaphylaxis/respiratory symptoms)
o Hives/urticaria usually occur within 2 hours of ingestion, often accompanied by
angioedema  Considered a risk for future anaphylactic reaction
 Comorbid with atopic dermatitis  40-50% patients with severe AD have food allergy as trigger

Mechanism for GI symptoms in food allergies

 IgE-mediated  vomiting directly after eating specific food
 Non IgE-mediated  T-cell mediated disorders like celiac disease
 Eosinophlic GI diseases may have component of both IgE- and T-cell mediated reaction

Oral Allergy Syndrome  localized reaction with pruritis and swelling to mouth, lips, throat
 Presents in adolescence or late childhood
 Due to fresh (non-cooked) fruits and vegetables cross-reacting with specific pollens

Eosinophilic GI Diseases  abnormal accumulation of eosinophils in any part of the GI tract

 Symptoms depend on which part of GI tract is affected
 Cause is not well understood, symptoms vary by age (may simply be poor growth in kids)

Diagnosis of Food Allergy

 Detailed history including food suspected, specific symptoms, timing of symptoms, reproducibility
of reaction
 30-40% diagnosis requires testing  gold standard = oral food challenge (which is time-
consuming and carries risk to patient)
 Skin and ImmunoCAP testing for food allergies is not reliable due to many false positives (though
the more positive the test, the more likely there is a true allergy)
 Some children outgrow their food allergies, but not as many or as fast as previously thought
o Milk, egg, soy and wheat allergies are often outgrown, whereas shellfish, nuts are not
o Patients with food allergies are likely to have a co-occurring allergic disease
 There is no treatment for food allergies, so avoidance is key
o Worst outcomes often in adolescents who refuse to carry Epipen

Anaphylaxis is diagnosed clinically, defined by either:

 Sudden onset (minutes/hours) reaction involving skin, mucosa, or both (hives, itching, lip swelling)
 Two or more of the following: sudden skin/mucosal symptoms, respiratory involvement,
hypotension, GI symptoms
 Hypotension (30% drop in BP) after exposure to a known allergen

Symptoms = urticaria/angioedema (hives/swelling), upper airway edema, wheezing, hypotension, GI sx's

Tryptase is released by mast cells and can be measured as an indicator of degranulation  has a short
half-life so must be measured within 3 hours (+ test can diagnose anaphylaxis, but - test cannot rule out)
 However, there are anaphylactic reactions for which IgE doesn't seem to be involved

Usually anaphylaxis is a uniphasic reaction (sx's shortly after allergen exposure w/ quick resolution) 
However some patients experience a biphasic reaction (sx's initially improve, but then recur hours later)
 Patients in ER are observed for 4 hours after reaction d/t risk of biphasic reaction
45
Treatment of Anaphylaxis
 Epinephrine = drug of choice (different dose for adults/kids, pts may require >1 dose)
o Is an α/β adrenergic agonist (↑HR, dilate airway) that inhibits mast cells/basophil action
 For severe reactions, airway should be protected (intubation), O2 given and IVF to maintain BP
 Antihistamines, corticosteroids can help treat the symptoms, though won't prevent the life-
threatening manifestations
 Once patient has improved, testing 2-4 weeks later is critical to determine cause of anaphylaxis

Anti-Inflammatory Drugs

Three major classes of anti-inflammatory drugs:

1. NSAIDs (Non-steroidal inflammatory drugs); 3 sub-classes
a. Aspirin (ASA)
b. Traditional NSAIDs
c. Coxibs (selective COX-2 inhibitors)
2. Steroids
3. Biologics (Antibodies against inflammatory cytokines)

Key Historical Events

 ASA use dates back to Eastern and Western medicine (Hippocrates documented in 5 B.C.)
 1829  salicin ID’d as active ingredient; 1830s  Salicylic acid synthesized for first time
 1890s  Acidity of salicylic acid was reduced; acetylsalicylic acid first called Aspirin
 1970s  Research demonstrated ASA works by inhibiting prostaglandin biosynthesis
 Further research yielded development of traditional NSAIDs and selective COX-2 inhibitors

NSAIDs Overview
 Desired effects:
o Analgesic (relief of low-moderate
intensity pain, lacking addictive effects of
opioids)
o Anti-pyretic (reduces body temperature)
o Anti-inflammatory (systemic relief)
 Side effects: GI (ulcers, bleeding, perforation),
renal and cardiovascular symptoms
 Mechanism of action = blocking prostaglandin and
prostanoids
o Prostaglandins contributes to
development of pain and can trigger fever
o Prostanoids are involved in inflammation
(↑vasodilation, vascular permeability,
promotes leukocyte migration)

Biosynthesis of prostaglandins
 Common precursor of all prostaglandins = arachidonic acid (AA, released upon activation of
phospholipase 2)
 Pharmacological management of prostaglandin = reduce production of intermediates
o NSAIDs target Cyclooxygenase
o Steroids target Phospholipase A2
46
  Prostaglandin H2 synthase (PGSH)/ Cyclooxygenase (COX) = Key enzyme in converting arachidonic
acid to prostaglandin H2 (a 2-step reaction requiring 2 active sites)
o 1st active site  hydrophobic, binds low fatty acid side chain of AA
 AA is converted to a peroxidase, PGG2
o 2nd active site  includes a heme group, binds peroxidase and converts to PGH2 (alcohol)

Aspirin inhibition of COX is irreversible  covalent modification prevents AA binding at 1st active site
 This can occur with salicylate, but the addition of acetyl group (ASA = acetylsalicylate) makes it a
much more potent inhibitor (acetylates binding site, instead of competitively binding to it)
o Acetylation of COX-1 creates a steric block preventing binding of AA
o Acetylation of COX-2 retains COX activity, but creates a different product

NSAID COX inhibition is reversible

 Substrates that mimic AA all have 1 or more free carboxylic acids

COX-1 and COX-2 are different isoforms of COX enzyme encoded by different genes
 COX-1 is constitutively active in response to hormones
o Has protective features (to platelet, kidney, GI function); “housekeeping”
 COX-2 is induced during inflammatory conditions, causing pain and fever
o 60% identical to COX-1, but in the active sites there is only 1 difference out 24 AAs (Ile in
COX-1  Val in COX-2, a difference of only one carbon)
o A COX-2 specific inhibitor would prevent inflammation w/o COX-1 side effects

Development of COX-2 Specific Inhibitors

 Despite similarities in binding site, the 3-d structure of the enzymes is different
o COX-1 binding site is more shallow, while COX-2 binding site is within a deeper V-shaped
pocket  V-shaped molecule can fit the structure of the COX-2 enzyme to access site
 Highly specific drugs have been developed to bind COX-2
o Vioxx, Celebrex, Bextra
 In clinical trials, COX-2 inhibitors have been as effective as traditional NSAIDs
o COX-2 inhibitor are better than traditional NSAIDs in preventing GI side effects
o However COX-2 increases risk for cardiac disease/MI (Vioxx taken off market)
 Celebrex continues to be used with box warning for CV event risk

Acetaminophen  structurally similar, but mechanistically different from traditional NSAIDs

 Weak COX inhibition with poor anti-inflammatory activity (reduced GI effects)
 Good analgesic and anti-pyretic activities

Steroids
 Derived from cholesterol  are hydrophobic, enter cells through membrane diffusion
 Modulate gene expression via binding to intracellular receptor proteins and steroid receptors
o All steroid receptors have a ligand-binding domain and a DNA binding domain which
allows them to regulate gene expression

Steroid/Hormone Receptors have 2 modes of regulation of transcription:

1. Constitutively expressed receptor in the nucleus, where it binds elements in chromatin and
recruits co-repressors to repress transcription (normally OFF)
a. Upon binding, dissociation of co-repressors and recruitment of activators turns gene
transcription ON (e.g. thyroid hormone receptor)
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 2. Receptor is anchored in cytosol by a chaperone complex
a. Upon binding, receptor dissociates from chaperone and translocates into nucleus to bind
target promoter sequence and turn gene transcription ON (e.g. glucocorticoid receptor)

Different steroid receptors bind DNA with high sequence specificity; receptors bind as dimers
 Response elements for receptors are either inverted repeats or direct repeats of 6 bp’s 3-5
nucleotides apart

Physiological activity of cortical steroids

 Regulate metabolism of lipids, protein and carbohydrates
 Balance of fluid and electrolytes
 Normal function of CV, immune, kidney, skeletal muscle, endocrine and nervous systems
 Responsible for resistance to stress

Agonists of glucocorticoids include hydrocortisone, dexamethasone, and prednisolone  formulated for

topical, injectable, inhalable, or oral use

Mechanism of glucocorticoids in reducing inflammatory responses:

 Inhibits prostaglandin and leukotriene biosynthesis through inhibiting expression of COX-2 and the
activity of Phospholipase A
 Inhibits the production of key inflammatory cytokines (IL-1, IL-6, TNF-α)

Clinical use of glucocorticoids  Replacement for adrenal insufficiency, lupus, allergy, asthma,
leukemia/lymphoma, organ transplantation (related anti-inflammatory and immunosuppressive effects)

Transplantation

Syngenic  genetically compatible for transplant purposes (b/n inbred mice, homozygotic twins)
Allogenic taken from 2 different individuals from same specifies (genes at 1 or more loci vary)
Alloreactivity  immune responses against foreign tissue/ “allo” antigens (allogenic peptide/MHC)
Xenogenic  derived or obtained from an organism of a different species

Historically  research into grafts rejection in mice (Medawar) led to important concepts  alloreactivity,
immunological memory, immunological specificity, memory resides in lymphocyte

Laws of Transplantation:
1. Transplants within inbred strains will succeed
2. Transplants between inbred strains will fair
3. Transplants from an inbred parental strain to an F1 (PF1) will succeed, but F1P will not
4. Transplants from F2 and all subsequent generations to F1 will succeed
5. Transplant from inbred parental strains to F2 will fail, but not always
a. Can be used to estimate the number of MHC genes (% survival = 0.75n, where n= # MHC
genes  because ¼ loci would cause rejection for each gene involved)

Identifying genes that control transplant rejection  Histocompatibility genes, mapped into 2 classes:
1. Major Histocompatibility genes (HLA) – fast rejection (7-10 days)
2. Minor Histocompatibility genes (H-Y genes) – slower rejection (30-60 days)
a. Multiple minor histocompatibility genes can create similar reaction to major
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Mixed lymphocyte reaction (MLR)  assay in which lymphocytes from 2 different individuals are cultured
together
 Cells from ONE donor are irradiated (unable to replicate), and observe “responders” from normal,
non-irradiated cell from other donor (CD4, CD8 cells)
o Can look for killing through assays for CD8
o Can analyze proliferation via measuring uptake of 3H thymidine in DNA
 One-way MLR = only T cells from one individual proliferate
 Two-way MLR = Cells from both donors respond to one another

Proliferative responses to alloantigens are orders of magnitude stronger than to normal antigens
 An unprimed response looks like a memory response
 Allo Paradox  If T cells don’t recognize presentation by non-self MHC molecule, how do they
recognize an alloantigen? (Only 1 in 105 – 106 T cells are specific for a particular Ag/MHC complex)
 A very high frequency of unprimed cells respond to alloantigens (1-10% of all cells)

The MHC Class I molecules recognized in alloresponses are the same as those in Ag-specific responses
 Most supported theory to explain this  T cells recognize alloantigen by crossreactivity,
recognizing some residues on the foreign MHC and some on the bound Ag peptide
o The structure of the allo-MHC molecule is necessary and sufficient to interact with TCR
o T cells are peptide-specific, so can still be recognized to an extent on allo-MHC
 Alternate theory  binding is driven by high density and weak affinity between TCR and
alloantigenic MHC molecule

There are no specific “allo” proteins, but are often peptides derived from house-keeping proteins
 Can be polymorphisms in the proteins themselves (HY response)
 Allo-MHC is likely to bind a different subset of peptides from same protein as self-MHC
o Even if same peptide binds, the parts exposed to TCR are likely different

Little is known about minor histocompatibility antigens

 Source of minor histocompatibility antigens  polymorphic self-proteins that differ in AA
sequence between individuals give rise to minor H Ag differences between donor and recipient
 H-Y is a specific minor histocompatibility Ag  male recipients reject grafts from females, but
females can accept grafts from males

Bone marrow transplant = blood/marrow transplant = stem cell transplant = hematopoietic cell transplant
Sources:
 Syngenic  from identical twin
 Autologous  from self
 Allogenic  from another member of same species
 HLA-matched sibling  25% siblings are a match on average
 Matched unrelated donor (MUD)
BMT used to treat/cure many blood cancers/diseases with improved outcomes over chemo

Donor cells undergo selection on MHC in recipient thymus  recipient T cells can be activated by donor
APCs and recognize infected MHC cells in recipient’s own body

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Graft-versus-leukemia effect is the positive/curative effect due to a minor histocompatibility antigen
 T cells are critical, CD8 cytotoxic cells that recognize minor histocompatibility antigens are the
major effector cells in the GVL effect

Graft-versus-host disease  mature T cells in graft attack host cells through similar alloreactive process
that results in LVK effect
 Has distinct phases, with positive feedback loops that perpetuate process
o Chemo and other procedures to prime recipient for graft cause tissue damage (target and
kill rapidly dividing cells in the body)  APC and innate immune cell get activated 
thought that this might cause GVHD
 Treatment approaches include direct targeting of alloreactive T cells and their subsets
o T cell depletion can eliminate GVHD, but increases the risk of leukemia relapse
* Want to minimize GVH disease and maximize GVL effect

Solid Organ Transplantation

General Transplantation Info

 ~8000 deceased donors and 6300 living donors each year  has remained stable over time
o Success of transplants  people on waitlist for organs continues to rise
o Sickest patients on liver/heart waitlist treated first to prevent death while on waitlist,
while kidney transplant patients wait in order of when they were put on list
 Top 3 most common transplants: Kidney > liver > heart  these 3 also have the best outcomes
 Survival of different organ transplants varies: live donor kidney has best success
 Live donations
o Increase of live kidney donors in 1990s due to development of laparoscopic technique
o Live liver donations peaked in 2001 due to death of donor (unrelated to surgery)

MHC genes and matching is essential to prevent organ rejection

 Due to class I MHC on an allogeneic cell presenting allogeneic peptide to T cell, and T cell
triggering response as if it were recognizing a pathogen
 Alloantigen presentation can occur through 2 pathways:
o Direct  donor APC stimulates T cells in lymph node
o Indirect  recipient APC processes and presents allogenic donor peptide with self-MHC
 3 most important MHC gene loci important for organ rejection are HLA A, B, and DR
o Because expression is co-dominant at each locus siblings can be an exact match, half-
match, or totally unmatched  best response = sibling > parent > stranger
o If matched at all 3 loci (6 alleles matched) have statistically improved outcome

Calcineurin is the target of cyclosporine and Prograff

 Is downstream from T cell activation of MHC-Ag complex
 Allows NFAT to move to nucleus and increase transcription of genes for IL-2 production (which
then trigger T cell proliferation via autocrine signaling)
 Blocking calcineurin, NFAT, and IL-2 production induces significant immunosuppression
 Alternative immunosuppressive strategy with MTOR-inhibitors, which blocks IL-2 signaling

Pathology in allograft biopsies

 Only heart transplants are regularly checked using biopsies to look for infiltrating lymphocytes

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  Biopsies of other organs are done when there are problems with the transplant

Overview of important immunosuppressive medications:

 Steroids are non-specific in their action  are lots of side effects if only steroids are used for
immune suppression (transplant patients kept on low-dose to avoid effects)
 Purine anti-metabolites (Imuran, Cellcept, Prograf)  non-specific, interfere with rapidly dividing
cells (can decrease cell development in bone marrow, can alter GI turnover/function)

Most common transplantation = blood transfusion (B cells/Ig matching instead of T cell/MHC)

 Blood-type (Ig) matching also important in organ transplantation  hyperacute rejection occurs if
organ is from non-blood-matched donor
 Ig would bind to Ag on organ capillaries  attract complement proteins  neutrophil recruitment
 neutrophil lytic enzymes destroy endothelial capillary walls  platelets adhere leading to
vascular blockage/thrombus (organ clots off from body)
o Occurs within minutes
o Some centers (including Hopkins) have techniques to overcome non-matched donors

Immune response to organ is only triggered if sensitized by a previous event to non-self MHC (transfusion,
prior transplant, pregnancy)  Testing prior to transplant ensures a match:
 Crossmatching assesses donor and recipient to determine if recipient has pre-existing Ig against
donor MHC
o Mix donor cells, complement, and recipient serum  if lysis of donor cells occurs, it is
considered a positive cross-match and will result in acute rejection
 Estimation of degree of sensitization PRA (panel of reactive antibody)
o ELISA-based test to estimate presence and breadth of Ig against non-self MHC
o Scale 0 (no sensitization) to 100 (have pre-formed Ig), measuring Ig against a broad
variety of MHC
o Predicts the likelihood of rejection
 If a patient knows their live donor, can do plasmaphoresis (minimize pre-formed Ig to donor MHC)
o Can do this for ABO-incompatible patients

Immunopharmacology of Transplantation

Molecular and cellular bases of organ rejection  Transplant rejection is mediated by T-cell response

Sites of action of immunosuppressants  Focused on preventing CD4+ T cell activity/proliferation

 As stated in last lecture, can focus on inhibiting calcineurin or MTOR

Different classes of immunosuppressive agents:

1. Inhibitor of MHC/peptide-TCR interaction

 Anti-thymocyte globulin  used to prevent acute rejection
o Produced in animals, administered via IV (half-life 3-9 days)
o Major side effect = serum sickness and nephritis
 Anti-CD3 antibodies (Orthoclone OKT3)
o Blocks binding of T cells and also leads to rapid depletion of circulating T cells
o Can act as TCR agonist, leading to cytokine secretion and side effect/toxicity (“cytokine
release syndrome”) which vary from mild flu-like sx’s to lethel shock-like reaction
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2. Inhibitors of TCR-mediated intracellular signaling (calcineurin)
 Cyclosporine A (CsA)  a fungal metabolite used as a front-line therapy to prevent rejection
o Changed the practice of organ transplantation
o Given IV or PO – peak plasma level reached within a few hours
 50% drug is sequestered in RBC’s, which acts as a reservoir
o Metabolized by liver into 30+ metabolites, excreted in bile/feces and urine
o Main toxicity = nephrotoxicity (via same mechanism that mediates immunosuppression)
 FK506 (Tacrolimus, Prograf)  More potent than CsA, very similar drug profile
o Given IV and PO, peak plasma level within a few hours, metabolized by liver, similar side
effect of nephrotoxicity
o Used an alternative or complementary option to Csa  used widely in liver transplant
because it stimulates hepatocyte growth

CsA and FK506 have also played important roles in understand intracellular signaling cascades and Ca-
dependent gene transcription activation
 Both have an unprecedented mode of action  Don’t bind to target enzyme’s active site, but
recruit immnunophilin  the drug-immunophilin complex inhibit target enzyme (calcineurin)
o CsA’s immunophilin receptor = cyclophilin, FK506 receptor = FKBP

3. Inhibitors of cytokine/receptor interaction  Block IL-2 dependent T cell proliferation

 Daclizumab (Zenapax)  a humanized monoclonal antibody to the IL-2Rα receptor on T cells
o FDA-approved in 1997, used primarily for kidney transplant to prevent nephrotoxic
effects of CsA or FK506
o Given in multiple doses (1st 1 hour before transplant, 5 later doses at 2 week intervals
after transplant)
 Basiliximab (Simulect)  similar antibody and action, only difference is shorter half life

4. Inhibitor of cytokine-mediated signal transduction  After binding of IL-2 to its receptor on the T cell,
this class inhibits the intracellular signaling that usually results in cell proliferation
 Rapamycin is structurally similar to FK506 (binds to same receptor, FK506), but instead of
affecting calcineurin, it inhibits MTOR (mammalian target or rapamycin)
o MTOR involved in transcription/translation of many genes required for cell proliferation,
also promising research into using it as a cancer treatment
 It is synergistic with CsA/FK506, its use reduces their associated toxicity
 Whereas CsA/FK506 are required to tolerate transplant, rapamycin seems to induce tolerance,
and patients can be taken off the drug for short periods of time

5. Inhibitors of T cell proliferation

 Mycophenolate Mofetil (Cellcept) is a prodrug for mycophenolic acid, which inhibits an
enzyme required for de novo purine biosynthesis
o T and B cells rely on de novo synthesis and are selectively sensitive, but this can also
affect the synthesis of new epithelial cells
o Primarily used in kidney transplant with other immunosuppressants

6. Other inhibitors
 Steroids  glucocorticoids are still being used to inhibit T cell activation
 Cytotoxic drugs  non-specific inhibition of cell proliferation, had severe side effects and
have been replaced by CsA and FK506

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Current research is focusing on inducing donor-specific tolerance in transplant recipients (long-term
immunosuppression is not ideal)
 Potentially through ignorance, intra-thymic tolerance, micro-chimerism, Tregs, BMT
 No successful tolerance induction thus far

Main Points
 Inhibition of CD4 T cell activation is the major strategy to block organ rejection
 Calcineurin is the major signal transducer of calcium signaling in peripheral T cells
 CsA, FK506 and rapamycin represent novel type of drugs that work by bringing 2 proteins together
to block the function of the target protein

Immunosuppression Drugs Overview Chart

Inhibition Target Examples
MHC-TCR interaction Anti-thymocyte globulin
Anti-CD3 antibodies
TCR-mediated Cyclosporine (CsA)
intracellular signaling FK506
Cytokine signaling Daclizumab
Basiliximab
Cytokine-mediated Rapamycin
intracellular signaling
T cell proliferation Mycophenolate motefil
Mode of Action Notes
Block binding of T cell to Anti-CD3 Ig can act as
MHC molecule TCR agonist
Block calcineurin Important in understand-
ing intracellular signaling
Antibody to IL-2 receptor
blocks binding of IL-2
Block MTOR May also be useful in CA
treatment
Inhibition of de novo
purine synthesis

Secondary Immunodeficiency

Causes of secondary immunodeficiency

 Newborns, especially those born premature (most IgG transport across placenta in last 6 weeks)
 Hereditary and metabolic diseases
o Ex. In sickle cell, clotting off the spleen due to sickled RBCs = decreased immune function
 Can be caused by immunosuppressive agents (treat CA, autoimmune disease, transplant)
 Infectious diseases
 Infiltrative and hematologic disease
 Surgery and trauma (burns  lose serum protein, and splenectomy  lose splenic filter)

Infections that activate a Th1 response inhibit a Th2 response (B cell

activation) via IFN-γ, no Ig is produced
 This is why TB is tested by using skin test  TB infection prohibits Ig
production against it
Infections that activate a Th2 response inhibit Th1 (T-cell help) via IL-10 
puts patient at higher risk of viral and parasitic infections
 This explains the high mortality with measles  measles isn’t lethal
but inactivates Th1 response and patients die from other diseases

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Epstein-Barr Virus (EBV)  a double stranded DNA virus of herpes family
 Most people contract before age 6/7  causes vague symptoms; if contracted later in life, patient
develops mononucleosis
 Infects pharyngeal epithelial cells and resting B lymphocytes  transforms B cells so they have the
capacity to indefinitely proliferate
o T cells control/kill the transformed cells  in a blood smear of a patient with EBV, will see
“atypical lymphocytes”, the T cells reacting to the transformed B cells
 Patients with EBV are immunodeficient 1-4 months following onset of illness
o Immunodeficiency can cause a false negative Delayed Type Hypersensitivity Skin Test
(PPD, Candida) because T-cell unable to react in tissue
 The EBV genome encodes a protein, BCRF1, which is homologous to IL-10
o IL-10 is part of the Th2 response, inhibits the Th1 response, causing anergy

Rebuck skin test  scrape part of skin, measure how many leukocytes migrate into that area over time
(measures white cell locomotion)

Immunosuppression by HIV-1  Kills CD4 cells and disables T cell helper functions
 As CD4 count ↓, susceptibility to infection ↑

Cancers that infiltrate the bone marrow can cause immunodeficiency by displacing normal immune cells
o Leukemia/lymphoma, metastatic disease of bone marrow, histiocytosis

Immunosuppression can also be caused by cancer cells secreting cytokines which help the tumor
proliferate, but may also induce systemic immunodeficiency
 TGF-β (tumor growth factor) is a cytokine normally produced in the gut that controls a lot of
immunosuppression activity (though is stimulatory in that it promotes class switching to IgA)
o Creates non-inflammatory environment for tumor, also hides tumor from IgG or IgM
o Tumors can create enough TGF-β to induce systemic immunosuppression
 Suppresses IL-1 (prohibiting signals for T cell proliferation, IL-2, etc.)
o TGF-β suppresses intracellular killing via macrophages (opposite IFN-γ activity)
 VEGF (vascular endothelial growth factor)  Induces growth of blood vessels, supplying tumor
with blood
o Causes ineffective Ag presentation by:
 Preventing maturation of DC
 Preventing MHC molecule expression

Immunodeficiency caused by drugs

 Direct destruction of injury to immune cell (cytotoxic drugs, radiation)
o Chemotherapy targeted at rapidly-dividing cells will damage bone marrow, leukocytes
numbers will decrease, and patient will be more susceptible to infection
 Indirect destruction or injury to immune cell (anti-convulsants)
o Anti-convulsants (phenytoin/dilantin) have been found to induce IgA-deficiency in some
people (sometimes irreversibly)
 Immunomodulation of immune cell function (CsA, glucocorticoids)
o Interfere with function of macrophages and T cells

Complications of Transplantation
 There is a relatively high incidence of cancer in post-transplant patients (4-18%), occurring in a
relatively young group of patients
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 o The types of cancers these patients develop are not the more common (breast, prostate,
lung, colon CA)  skin CA and lymphoma are the most common
 Infections in heart transplant patients  Pneumonia and CNS infections caused by opportunistic
infections (similar to HIV patients), recurring problems with shingles (varicella-zoster virus)

Monoclonal Antibodies as Drugs

Production of a monoclonal antibody:

Mouse is immunized with Ag  Spleen removed Spleen cells producing Ig mixed with immortal
myeloma cells  Allow cells to fuse  Transfer to HAT medium  Select hybridoma that makes Ag-
specific Ig  clone selected hybridoma

4 Types of Monoclonal Antibodies

1. Mouse (“o” in name)  can only be given a few times until immune system attacks it
2. Chimeric (“xi” in name)
3. Humanized (“zu” in name)
4. Human (“u” in name)  created via transgenic mice

4 Mechanisms of Action for Monoclonal Antibodies

1. ADCC (Antibody-Dependent Cellular Cytotoxicity)  Ab binds to target cell and Fc receptor,
effector cell mediates killing
a. Mediated by NK cells, macrophages, or neutrophils
b. Killing requires Ab binding to target cell AND FcγRIII on phagocytes
i. Can increase binding by glycosylation of Fc
2. CDCC (Complement-Dependent Cellular Cytotoxicity)  Antibodies bind to C1q receptor
a. Requires Ab cross-linking/proximity to bind to C1q receptors
b. Can have different effects in people with different C1q polymorphisms
3. Antagonist  block growth factor signaling to prevent tumor cell survival
a. Can block a receptor or a ligand
4. Agonist Ab signaling tumor cells as treatment (e.g. via death domain receptor)
a. Examples include anti-CD40 and anti-41BB (activate CD8 T cells)

4 Different IgG Types in Humans  4 Fc Gamma Receptors (FcγR) with different functions
* We only need to know FCγRIII
 IgG 1, 2, and 4 have half lives of 21 days; IgG3 short half life of 7 days (difficult to use)

4 Modified Antibody Technologies

 TRAP Molecules (Aflibercept)  Designed to trap VEGF to prevent induction of blood vessel
growth in the eye causing macular degeneration
 Single-Chain Dual Specificity (BiTe)  HC and LCs from CD3 and CD19 engineered to stick
together  drag T cell (CD3) to target Ag (CD19) on tumor cell
 Chimeric Antigen receptors  Combine killing of CD8 cells with specificity/affinity of Ab
 Antibody Drug Conjugates (ADC)  Ab very specific for target combines with a cytotoxic agent
that is highly potent in small quantities and non-immunogenic
o A linker keeps ADC intact until it reaches target, doesn't alter Ab characteristics, and
ensures the cytotoxic agent is functional at target site

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4 Examples of Monoclonal Antibodies with Clinical Relevance
1. Rituximab/Anti-CD20 = chimeric, first mAb approved for lymphoma (1997) and RA (2006)
a. Is an IgG1  works through ADCC
2. Trastuzumab = humanized mAb, IgG1  prevents dimerization (ADCC), unknown for sure
3. Urelumab(Anti-41BB) = fully humanized, IgG4 agonist to activate CD8 cells (in Phase I trials)
4. Nivolumab (Anti-PD-1) = fully humanized, IgG4 (with modified hinge region)
a. Is a PD-1 antagonist  in Phase III kidney CA, lung CA, and melanoma studies

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Cytokine Reference Chart
Cytokine Produced By Target Cell Action
IL-1 Macrophages Many cells* Pro-inflammatory (pyrogen)
IL-2 Th0/Th1 T cells Proliferation
IL-3 PHSC Differentiation of myeloid and
lymphoid progenitors
IL-4 Th2 B cells Class switching (incl. IgE)
Th0 + Th2 response
IL-5 Th2 Eosinophils Activation
IL-6 Macrophages, Th2, Th17 Many cells* Pro-inflammatory
IL-7 Stromal cells (marrow) & Immature lymphocytes Development (VDJ recomb.)
others T & B memory cells Memory persistence
IL-8 Macrophages Phagocytes Chemotaxis, phagocytosis
IL-10 Th2 & others Th0 - Th1 formation
Many cells/cytokines Inhibit immune response
IL-12 APCs Th0 + Th1 response
NK cells NK cell activation
IL-13 Th2 B cells Class switching to IgE
IL-15 APCs T cells T cell memory
NK cells NK cell development
IL-17 Th17 Neutrophils
TNF-α Macrophages, Th1, Th17 Neutrophils Pro-inflammatory, shock response
IFN-γ Th1 Macrophage, NK cell Activation
IFN-α/β Many General anti-viral effects
TGF-β Tregs & others CD4+ cells Inhibit immune response
B/plasma cells Class switching to IgA
GM-CSF Macrophages, T cells, Mast PHSC Progenitor differentiation to
cell, NK cells monocytes & granulocytes
G-CSF Lots of immune cells Neutrophils Neutrophil development
M-CSF Various Macrophages Macrophage development
CCL19/SLC Stromal cells in T zones Naïve T cells Migration to T areas of secondary
DC cells lymphoid tissue
CCL21/ELC Stromal cells in T zones Naïve T cells "
CXCL13/BLC Stromal cells in B follicles Naïve B cells Migration to B cell areas of
Follicular DCs secondary lymphoid tissues
VEGF Cancer cells (among others) Various Angiogenesis
Immunosuppression

Important Functions to Know:

IL-4 and IL-13 important in production of IgE for allergic responses
IL-7 and IL-15 important for the persistence of memory T and B cells
IL-10 and TGF-β are important in inducing tolerance (as well as B cell survival/class switching in the gut)
IL-4 drives differentiation to Th2; IL-12 and IFN-γ drive differentiation to Th1
VEGF and TGF-β important in cancer cells, ↓immune response and ↑tumor survival
*Pro-inflammatory cytokines IL-1, IL-6 and TNF-α act on many targets, including vascular endothelial
cells (↑permeability), hypothalamus (cause fever), neutrophil/macrophage recruitment
 Anti-inflammatory drugs inhibit production of IL-1, IL-6, and TNF-α
57