Immunology Quiz 2!!

All antibody structure

"Immunoglobulins" in the g-globulin fraction Identical H and L chains, each chain has a variable (V) region and a constant (C) region Ag-binding site made of H and L at NH2 termini Effector functions reside in the COOH terminal regions of only the H-chains(L-chain COOH regions have no effector function

Use of FcR per antibody

- IgG1 and IgG3 dominate this because of their high affinity interaction with FcR8a) (aka CD64) although the other forms of IgG bind to some extent as well - IgA utilizes a different form of the receptor (FcgammaRIaka CD89) to accomplish the same thing on M and PMN - FcalphaRI's Ka for the Fc of IgA is about 1 X 107 (10X less than FcR-I for IgG) but this is probably compensated for to some extent by IgA's multimeric nature) - IgA and IgM utilize yet another form, Fcalpha/muR at 3 X 109, probably for lung defense and Ag uptake in LN especially the gut

Operational forms of humoral immunity

1) Active Immunity - natural infection, vaccination and boosting Passive Immunity Artificial - g-globulin fractions Naturally occurring - transplacental IgG, oral IgA Do not confuse Passive Humoral Adaptive Immunity with Barriers (Passive Innate Immunity) Adoptive Immunity - adoptive transfer of already differentiated immune cells into a (hopefully) genetically compatible animal the irradiated mouse as a living test tube

Sequential formation of antibodies

1° structure within each Ig domain is what creates the 2° structure of b-pleated sheets. The "sidedness" of the 2° structure of the β−pleated sheets in each Ig-domain is what forms the 3° structures that are the Ig-domains. But these 3° structures have another layer of information that results in important 4° structures: Monomorphic associations between opposing 3° Ig domains spontaneously assemble the four chains into the 4° Ig monomer (charged and uncharged interactions) 4° Oligomophic structures in the Fc regions that specify a limited number of effector function interactions. Highly polymorphic 4° structures that are the epitope binding sites toward the amino termini - literally the N-terminal loops between strands of the b-pleated sheets of both V-regions (H and L). Again, ALL are encoded within the 1° sequence. ****

IgM structure

950 kD (19S not 7S) "M" = macroglobulin 5 X 190 kD + 15 kD "J" (joining) chain (mild reduction) Heavily glycosylated J-chain is made by the same B-cell 10 H chains (72 kD) (µ-chain is larger than g-chain)(strong reduction and dissociation) No hinge - stiff but the pentamer compensates for this 10 L chains (23 kD) - all identical in class (k or l) Always pentameric in serum (with 10 identical binding sites) but cell surface BcR is a monomer (with an additional membrane-spanning region)

antibody made to antigen

A spectrum of different epitopes stimulates a spectrum of different B-cell clones Each unique antibody has a unique "intrinsic affinity" for a given epitope Each epitope stimulates a spectrum of related but unique clones = "multiple binding solutions" As a population, the spectrum of antibodies made against the spectrum of different epitopes on a given antigen displays an "average binding affinity" of the antibody population for that antigen.

class switching

All B-cells start by making IgM 2) IgG is usually the majority of the 2° response measured as Ab in the blood 3) IgG has different properties than IgM 4) The IgG has the same binding specificity as the IgM 5) Class Switching depends upon helper T-cells (Th) - most Ab responses are Th-dependent and are, therefore, class-switched 6) Switching is a process independent of affinity maturation (although this is not obvious)

hapten and carrier

An initial simplification of the problem was to limit the structural diversity of the epitope to a small chemical group whose structure was known = a hapten In order to provoke an antibody response the hapten had to be part of a larger molecule = a carrier (usually a protein) Together the immunogen was a hapten-carrier conjugate

Affinity maturation

As immune stimulation proceeds, the average affinity of the antibody being produced becomes higher The sum of antibody produced in a 2° response has a greater affinity than the sum of antibody produced during a 1° response Affinity Maturation is due to B-cells competing for Ag when free Ag concentrations become limiting 1) The surface Ab of the B-cell has the same intrinsic affinity and epitope specificity as the soluble Ab it will ultimately secrete 2) Physical binding of the Ag to the surface Ab is required for stimulating cell division and soluble Ab production 3) Cell division and differentiation toward soluble Ab production continue only as long as stimulation of surface Ab continues

IgD

At first believed not to be secreted at all, ever! However, recently . . . only secreted by some B-cells in specific locals, upper aerodigestive MALT (tonsils, adenoids, salivary, lachrymal glands) 30 µg/ml (with widest range of any Ig class between different individuals), half life 2.8 days the IgD Paradox - secreted form has λ-light chains while cell-surface form has k-light chains - no precedent, no good explanation IgD is mainly a cell-surface antibody on virgin, mature, immunocompetent B-cells but not on memory B-cells probably important to the initial response of virgin B-cells Largest (64 aa), most flexible hinge region of any class, heavily glycosylated coexpression with IgM may facilitate optimal recognition of Ags at different concentrations with IgM dominating high [Ag] (inflexible) and IgD dominating at low [Ag] (flexible) Signaling functions may be distinct from IgM but have eluded firm characterization gene knock out seems to have no phenotype in mice !!

BcR

BcR is the surface form of Ab that, ultimately, is secreted in a soluble form that has the same specificity The BcR tends to recognize Ag in "native" conformations 1°, 2°, 3°, 4° structure of proteins hydrophillics outside on the surface, hydrophobics inside versus" denaturation" which is 1° only whole microbes have carbohydrates and proteins on their outsides - again, quite hydrophillic and in "native" configuration Microbes and proteins enter the system in native configuration so that is the kind of spatial configuration the BcRs normally see and also what the Ab that is ultimately made binds to Ab against 1° denatured structure of a protein is perfectly possible but is not what usually enters the immune system (and not what is tested for) Consequently: MOST antibody responses are directed at hydrophillic epitopes that are found in their native configurations on the surface structures of antigen molecules and microbes.

Structure of Phosphoryl Choline

Bound by the T15 idiotype found in all "Balb/c" mice against gm(+) gut bacteria found in McPC 603 ("methylchoanthrene-induced plasmacytoma 603), an IgAk anti-phosphorylcholine plasmacytoma with a low affinity Ka = 2 X 105

Class switching and secreted/bound antibodies

Class switching will change the entire Fc region and its effector function(s). In membrane immunoglobulin, ONLY the COOH terminus of the soluble molecule is replaced by a short, hydrophobic, transmembrane segment. This is not a replacement of the entire CH3 (IgG, IgD, IgA) or CH4 (IgM, IgE) but simply a swap of the last 20 odd amino acids for a different, hydrophobic transmembrane set.

Allotype expression in the individual

Co-dominance in the Serum of a Whole Animal we are diploid for a given allele in our species population we can, as individuals be homozygous or heterozygous in the serum you express all the immunoglobulin allotype alleles that you have inherited - they are "co-dominant" in expression

Monoclonal antibody theory

Combine the clonal restriction of a B-cell with the immortality of a cancer cell by fusing them ***** use a myeloma cell for the cancer cell - Ab production ) Fusogens - polyethylene glycol (PEG), some enveloped viruses Must have some means of selection amidst thousands generated - two different forms of selection: selection of successful hybrids ("hybridomas") - requires a mutant myeloma that can be selectively inhibited without inhibiting the hybrid cell (a lack of Ab from the myeloma is nice too) of Ag-binding clones among the successful hybrids (biased at the start by immunization and boosting)

Isotypes and Allotypes vs Idiotypes - isotopes and allotypes

Constant Region Shared between many different Ig molecules

IgM

Contains m-heavy chains (no subclasses) Characteristic of a primary response About 10% of normal serum Ig Is the first Ig produced by any B-cell before any class switching occurs First Ig to appear in the serum against a new antigen Relatively low "intrinsic" affinity Reflects the unbiased B-cell population before affinity maturation Its structure of 10 binding sites compensates for this

Immunization tends to create some antibodies of all H-chain classes

Depending upon kind of Ag, route of administration, other factors, one kind usually predominates unless the antigen in question is one which gets little or no T-help notable example is the anti-ABO antibodies (the "isoagglutinins") which are exclusively IgM probably due to the actual Ag's being soluble crossreactive bacterial CHO's sampled from the gut without bacterial proteins to stimulate Th cells this results in Abs that are mainly IgM Abs due to poor T-help and, therefore, no class switching in order to help, T-cells need to see a peptide, not a carbohydrate (CHO)

Opsonization by Fc region of antibody

Depends upon Fc interacting with Fc-receptors (FcR) on phagocytes - multimerization There is an heirarchy: IgG1 > IgG3 > IgG4, IgA, IgM(IgG2 can opsonize in 50% of Caucasians due to allelic variation in FcR structure) - the hierarchy is determined by the nature of the Fc region on Ab and the FcR itself signals phagocytosis, activation of respiratory burst and killing - with some encapsulated bacteria, opsonization by Ab is critical for clearance

Selection of different consequences of FcR engagement

Depends upon a complex interplay between The Kind(s) of Ig opsonizing a target The physical size of the target and the density of the Ig opsonizing it (i.e. the local density of Fc regions). The affinity of the FcRs in question on a given host cell. The relative expression of different of FcRs on a given host cell. The relative presence of Inhibitory FcR - the ratio of these to stimulatory FcR sets a threshold.

Uses of monoclonal antibodies

Diagnostic reagents - Enzyme-linked or color (chromogen) linked or radioisotopic assays Tumor imaging and therapy Disease prevention / therapy Research reagents - detection, stimulation Affinity purification Ab-zymes Exceptions - precipitation, cost of production when Ag is cheap

Inhibitory FcR consequences

Different cells can vary the level of expression of different activating and inhibiting FcRs This allows adjustment of the "threshold of activation" Examples: Dendritic cell activation is inhibited by low levels of immune complexes but with additional PRR signaling, activation is achieved Mice deficient in FcRIIB(inhib) have DCs activated by lower amounts of Ag and become autoimmune spontaneously

Template theory

Does every cell have a separate receptor for each and every epitope ? if so, what is the means of information storage ? how can you create a sufficiently large signal if there are only a very few of any one kind of receptor molecules in each cell ? how can such a signal be unambiguous and result in the formation only of antibodies specific for the given antigen ?

Experimental Techniques

Electrophoresis - native state, charge, size, shape Proteolysis using purified proteases Many proteases are non-specific and simply degrade proteins entirely BUT some are "specific" protease specificity - some cut at or near specific amino acids - physical accessibility of cut site Reduction and Alkylation Mild reduction (with a "reducing agent" e.g. HOCH2CH2SH) breaks only the superficial -S-S- cystine bonds to -SH + HS- (two cysteine amino acid side chains in this "reduced" form) but this is reversible Strong reduction breaks all -S-S- cystine bonds Alkylating agents covalently react with -SH (and example would be -S-CH3 ) and thus prevent reformation of -S-S- bonds when the reducing agent is removed Reduction and Alkylation = permanent breakage dissociating agents - can remove 2°, 3° and 4° protein structure - "weak" - charge repulsion by lowering pHe.g. propionic acid - mainly 4° - "strong" - chaotropic action on water structuree.g. 8 M urea - denaturing - 2°, 3°, 4° size exclusion chromatography - protein size and shape under "native" conditions - size only under denaturing conditions(usually with reduction first)

Microcomplexity

Energetic map of an Ag:Ab Interface assembled from "scanning mutagenesis where alanine is substituted for different amino acids and the resultant effects on Ag:Ab binding are measured by isothermal calorimetry. It shows that most contact residues contribute little individually but are important collectively and that there are "hot spots" that can have a much greater individual contribution

ELISA

Enzyme Linked Immunosorbant (ELISA) Assay Microwells are coated with the antigen of interest Samples of the supernatants of 1000s of individual hybrid clones are added After washing, the wells are screened for those that have retained antibody bound to the immobilized antigen By means of an anti-antibody covalently coupled to an enzyme When this 2°antibody is bound and a colorless substrate is introduced, a colored product is produced by the coupled enzyme IF there was an antibody in the test supernatant that bound the Ag on the plate's well

Opsonization - antibody clearance

Epitopes are repeated on the surfaces of pathogens Many PRRs (soluble and/or cell surface) can bind Many Ab molecules can bind Many C' fragments can be deposited Phagocytes have low affinity receptors for each of these and are actively engaged when they encounter repeating arrays (high avidity) of them on a surface (and not by the monomers of those molecules that are in solution) also opsonization by Fc region of antibody - depends upon Fc interacting with FcR on phagocytes

Antibody structure experiment

Fab ("antigen binding") binds Ag but does not precipitate it is electrophoreticly diffuse both Fab have same Ag binding specificity (size exclusion chromatography under native conditions) Fc ("crystalizable") does not bind Ag and is electrophoreticly uniform divalent crosslinks Ab univalent does not

Quaternary structure of antibodies

For the 4° structure of an immunoglobulin this means the stable interaction of paired domainsbetween Light and Heavy chains(VL:VH, CL:CH1) and between Heavy Chains(CH2:CH2, CH3:CH3) Again, driven by a combination of H-bonds, ionic bonds, hydrophobicity and Van der Waals forces Note that these are spontaneous associations resulting from the matching of charges and hydrophobic regions between domains and that even this 4° level of structure is contained within the evolved primary sequences. Additional covalent stabilizing bonds (like -S-S-) can be formed as post translational modifications in 3° (intra-chain) or 4° (inter-chain) structures. carbohydrates also associate with antibodies

H-chain classes

H-chain CLASS (aa sequences differ by >10%) - IgM, IgG, IgA, IgE and IgD Class = English capital letter (M, G, A, E, D) Polypeptide chain = Greek lower case letter Amino acid sequences (epitopes) that define "class" are part of the whole H-chain, very antigenically distinct in another species. This is why class-specific (isotype-specific) antibody is always raised in another species e.g. goat anti-mouse IgM. In a given species, H-chain Fc differences are responsible for the profound differences in effector function attributed to each class. H-chain SUBCLASS - IgG and IgA only Subclass (differ from each other ~10%) Subclass is denoted by a subscript number or number/letter i.e. IgA1, IgA2, IgG1, IgG2, IgG3, IgG4 (human) IgA1, IgA2, IgG1, IgG2a, IgG2b, IgG3 (mouse) "Class" and "sub-class" have no effect upon antigen binding by the amino termini Sub-class amino acid differences tend to be clustered in the Fc region Sub-class can have subtle differences in effector function in IgG (but not IgA)

L chain nomenclature

H-chain SUBCLASS - IgG and IgA only Subclass (differ from each other ~10%) Subclass is denoted by a subscript number or number/letter i.e. IgA1, IgA2, IgG1, IgG2, IgG3, IgG4 (human) IgA1, IgA2, IgG1, IgG2a, IgG2b, IgG3 (mouse) "Class" and "sub-class" have no effect upon antigen binding by the amino termini Sub-class amino acid differences tend to be clustered in the Fc region Sub-class can have subtle differences in effector function in IgG (but not IgA) Humans have 5 subclasses of L-chain - 1, 2, 3, 4 and 5 Mice only 1, 2 and 3 Humans have only one subclass of (mice and rats?)

Binding to Ag

H-chain always contributes > 50% of the binding capacity However without any L-chain, there is no binding at all. This probably represent a need for overall structural stabilization of the binding site shape as well as the contribution of the light chain's CDRs H-chains from a mAb against the gp120 of HIV were combined with different irrelevant L-chains and all the resulting hybrids exhibited some affinity for the Ag

IgA

Has a-heavy chains * Predominant Ig in secretions * - most of it is produced in the MALT, Peyer's Patches Initially secreted into the serum (15%) * Characteristic of 2° responses (like IgG) * Displays both monomeric and multimeric forms Tends to be high affinity * Multimeric forms give higher avidity Most abundantly produced because it is the most rapidly lost secondary response characteristic

IgE

Has e-heavy chains Last to be discovered because it is normally at very low levels in the serum discovered in a multiple myeloma patient Responsible for mediating "anaphylaxis" (allergic hypersensitivity) Anti-helminthic is probably its true role like IgM, IgG and IgA, it starts out as an Ag-sensing surface molecule on resting B-cell and becomes a secreted molecule, but passes through the blood only briefly - because it rapidly reattaches itself to the surface of other cells via FceR specific for the e-heavy chain Fc region

IgG

Has g-heavy chains (abundant in the g-globulin fraction hence the "G") - subclasses have different functions Is the serum Ig characteristic of 2° (and later) responses Is the most abundant in serum (75%) ***** Is generally high affinity (= high quality) But this is not direct consequence of class switching (both are signaled by Th with a similar mechanism) Class switching usually requires time and boosting which allows many rounds of B-cell competition for Ag, selection and "affinity maturation" to occur IgG1 > IgG2 > IgG3 > IgG4 (with g1, g2, g3 and g4 H-chains)

In general w/ classes

Heavy and Light chain together determine epitope binding specificity Only H-chain determines effector function by means of the Fc region All H-chain classes expressed as sIg (BcR) All except IgD are subsequently expressed as major secreted, soluble forms

High affinity antibody opposes (either/and/or)

High intrinsic affinity binding interactions of pathogen molecules at low concentration - e.g. a single soluble toxin molecule : binding to a single toxin receptor on the surface of a host cell High avidity binding interactions of pathogen molecules at high (local) concentration - e.g. multiple adhesin molecules on the surface of a microbe : binding multiple attachment receptors on the surface of a host cell - bacterial/viral adhesion requires multiple interactions (avidity)

IgM structure consequences

Higher "avidity" = "operational" affinity (most microbes have multiple copies of the same epitope on their surfaces) - note that most IgMs are rather low affinity even with this feature. This could be due to truly low intrinsic affinity Or inappropriate spacing Or simply the high entropy price paid for 10-fold valency But it is still an improvement ) Pentamer is better at crosslinking (therefore "clumping" microbes) and clearance Very efficient at firing Complement (C') Opsonization by C' (and by Fcm/a) Confined by its physical size to blood (enters lymph and tissue mainly during inflammation) - in combating sepsis, low affinity is compensated for by rapid production, high avidity and very efficient firing of complement Thus even low levels of IgM can be protective at the start of an immune response

Isotypes

Ig classes that are present in all members of a given species

IgA location significance

IgA is specialized to neutralize (high affinity, high avidity), specialized to enter the gut and specialized to remain intact in the gut inflammation due to C' fixation is not as crucial in the mucosae to accelerate delivery of Ab, C' and phagocytes to the site of infection to mediate clearance and disposal - those processes are already part of the function of the mucosal surfaces - what is mainly needed in the mucosal sites is to prevent attachment and colonization and let the mucosae wash themselves into the gut and expel

Transport across placenta

IgG1 - high, IgG2 - mild, IgG3 - moderate, IgG4 - sometimes

Sensitization of mast cells

IgG1 - mild, IgG3 - mild, IgE - high, rest none

Activates complement system

IgM - high, IgD - none, IgG1 - moderate, 2 - mild, 3 - high, 4 - none, IgA - mild, IgE - none

Transport across epithelium

IgM - mild, IgA - high (dimer)

Effector functions of Ig Classes: neutralization

IgM - mild, IgD - none, IgG - moderate, IgA - moderate, IgE - none high intrinsic affinity is important for neutralization (IgG, IgA better than IgM)

Opsonization

IgM - mild, IgD - none, IgG1 - high, 2 - none, 3 - moderate, 4 - mild, IgA - mild, IgE - none

Sensitization for killing by NK cells

IgM - none, IgD - none, IgG1 - moderate, 2 - none, 3 - moderate, 4 - none, IgA - none, IgE - none

Fixation of Complement

IgM = IgG3 > IgG1 > IgG2 = IgAwhile IgG4, IgD and IgE do NOT fix C'

IgE and IgD differences from other isotypes

IgM, IgA and IgG function in soluble form to seek out Ag-bearing pathogens and trigger effector functions which neutralize them or bring them into phagocytes. In contrast, IgD and IgE mainly function only on cell surfaces to sense Ag binding and pass that information directly to the cell that bears them.

clonal selection hypothesis

In an immunologically virgin, mature animal, all antigenic specificities exist in advance of antigen exposure. * 2) The antigenic binding specificities are "clonally distributed". * The specificity of the interaction between an antigen and a cell is mediated by and resides in a receptor * which is: a) On the cell surface (where it is accessible to Ag) b) Highly specific (discriminatory in epitope binding) Expressed in a "clonally distributed" manner - * BcR (aka sIg, mIg) TcR

Induced fit

Induced fit of a binding site - left, the crystal structure from the antibody alone (influenza hemagglutinin peptide epitope is electronically overlaid); right, the crystal structure of antibody with peptide bound in place

Size exclusion isolation experiments

Isolated H or L did not bind Ag when separately "renatured" (meaning that the dissociating and reducing agent were removed by "dialysis") BUT if H and L were renatured together, binding of Ag was restored! THEREFORE both chains contribute to binding the binding structure forms spontaneously and is quite stable

Hybrid selection

Myeloma cell line - Ig-, HGPRT-, TK-, immortal Normal B-cell - Ig+, HGPRT+, TK+, mortal HAT Medium Aminopterin Inhibitor of de novo pathway - no cells can use this pathway Hypoxanthine and Thymidine sole nucleotide sources - therefore all cells MUST use the salvage pathway . . . And ONLY the salvage pathway Ig only made with B cell, survives HAT only with B cell, immoral only with myeloma

IgA in secretions

Neutralizes (high affinity) without inflammation The secretory component confers resistance to proteases in the gut (crucial to newborns)

IgA in serum

Neutralizes (high intrinsic affinity, high avidity) without inflammation (does not "fix" C') Promotes adherence and phagocytosis as an opsonin (FcRm/a) Serum monomer (along with IgG) can enter uninflamed tissues (but it does not cross the placenta) Dimeric form recognized by poly-Ig receptor on mucosal endothelial cells

About allotypes

No functional consequences for Ag binding or effector function Usually single amino acid point mutations which are conservative in nature e.g Km(1), Km(1,2) & Km(3) are different alleles caused by amino acid changes at positions 153 and 191 in the mouse k-chain Defined by ability to provoke Ab responses specific to them in members of the same species which to not bear that allotype literally a single epitope ("natural hapten") that can be tracked ****** generally clinically safe to transfuse antibody across allotype boundaries ****** in the lab, generating anti-allotype antibody requires LOTS of boosting with adjuvant present

Usefulness of monoclonal antibody producing hybridomas

Polyclonal antisera made by conventional immunization - expensive, in limited quantity, limited shelf life, each batch required extensive and repeated characterization, specificities directed at many epitopes mAbs are produced in a stable transformed cell line (a "hybridoma") which can be easily propagated, frozen and shared in any quantity, providing every one with the same antibody reagent in unlimited quantity, also sold for high profit ($300 - 600 / mg) Antigen specificity of the resultant antibody is very restricted, one epitope only rather than the heterogeneous mixture of epitopes represented in a polyclonal antiserum mAbs can be easily purified so that they provide homogenous material uncontaminated by other irrelevant antibodies which might confuse analyses of Ab structure or function

Pepsin digestion of IgG

Products of Pepsin Digestion of IgG followed by treatment with a reducing agent (Fab')2 still precipitates Ag (the ' means "Pepsin") After mild reduction both Fab' fragments bind the same Ag but no longer precipitate it. No Fc fragment formed here - pepsin degrades it

Anti-antibodies

Proteins are superb immunogens. Antibodies are proteins whose homologues differ significantly (i.e. have a number of unique epitopes) between vertebrate species - due to drift of non-critical structures. Antibodies raised in one species can be used to discriminate the antibodies or fragments of antibodies of another species. ******* Using papain, make Fab and Fc preparations from Rabbit IgG.****** Rabbit Fab injected into a goat = goat anti-rabbit Fab Rabbit Fc injected into another goat = goat anti-rabbit Fc Make separate H and L chain preparations from reduction and dissociation of Rabbit IgG and, having removed the dissociating agents and allowed the separate, purified H and L preparations to renature into their native conformations, test their reactions with region-specific antisera: H chain react with ab and c, L chain only ab

Anti-idiotype

Raised in same strain of same species.Still difficult without a monoclonal Ab as the immunogen. Only then are the "foreign" epitopes identically represented on a large enough fraction of the immunogen's molecules.

Ag:Ab binding

Reversible and Non-covalent Changing the pH, [salt] or the addition or organic solvents can modify or nullify binding Four types of weak interactions ("bonds") H-bonds Ionic bonds Hydrophobic interactions Van der Waals forces (attractive and repulsive)

Idiotypes

Serological definition polyclonal antisera to a human myeloma, extensively absorbed with purified parts of OTHER myeloma proteins - WHAT's LEFT? anti-idiotype antibodies Idiotopes, idiotypic determinants - collectively, an idiotype Always in variable region, on H, L or both in combination A Unique idiotype is the minimal definition of a unique clonal specificity

IgG structure

Small size (~150 kd) allowed into tissues Crosses placenta and enters fetal circulation - by specific transport (FcRn) Present in breast milk too (not dominant) and is pumped by the baby's gut into the baby's bloodstream (FcRn) Longest half-life of the serum Abs due a specific rescue mechanism (FcRn) - t1/2 = ~ 3 weeks Intrinsic Affinity - inappropriate epitope spacing for multivalent binding Avidity - for the IgG to fall off, both interactions have to break simultaneously, a lower probability event.

Consequences of Clonal selection

Specificity Of stimulation what is stimulated - selects only those cells that bind a given epitope * what is NOT stimulated - the vast majority of cells with binding specificities irrelevant to a given epitope Of the response - which Antibody effectors selectively appear in the circulation (refer to model immune response) * An unarticulated assumption that BcR = secreted Ab: true! Bottom Line: The "clonal" nature of the specificity dictates the clonal expression of the receptor (membrane form of Ab): each cell is pre-committed for life to respond to 1 epitope by making 1 kind of antibody that specifically binds that epitope The collective "clonal" nature of the stimulation dictates the specific nature of the collective soluble antibodies produced in response to the epitopes of a given antigen. A Proposal that employs "Selectivity" to explain "Memory" It was known that "bulk" lymphocyte stimulation (using "lectins") causes "activation", cell division and multiplication of cell numbers. If selective activation leads to selective division and multiplication, the result is a selective increase in the numbers of cells responding to the selecting epitope(s), i.e. a selective increase in the "clonal representation" of those specific cells as a proportion of all cell specificities in the system. A Possible Mechanism! An animal's specificity universe starts out unbiased (because it is randomly constructed) Memory is (at least in part) the accumulation of representational bias in the frequency of clones that have been selected in the past Note: Self Tolerance was explained as selective "clonal deletion" by antigen overload during some early, critical stage of cell life - this turned out to be true to a large extent

TcR

TcR is NOT Ab and is never found in soluble form TcR recognizes epitopes found only in proteins TcR recognizes protein epitopes as short, linear, denatured fragments (1° sequence structure only) which are NOT in their native configuration TcR usually recognizes epitopes derived from interior protein structures which tend to be (more) hydrophobic (than what the BcR sees). Always sees its epitopes in association with a specialized antigen-presentation molecule (Class I or Class II) on the surface of an antigen presenting cell (NOT in solution like Ab)

FcR affinity

The Ka of the various mouse and human FcRs spans over at least five logs. High affinity FcRs have a Ka ranging from 107 to 1010 can bind monomeric immunoglobulins. Low-affinity FcRs have a Ka ranging from 105 to 107 are unable to bind monomeric immunoglobulins but are able to bind immune complexes (avidity again).

temporal nature of response

The innate immune response acts early in minutes to hours, first line of defense it is also how we achieve "clearance" or "functional" removal of a microbe to an acceptably low level. eliminates (or simply controls) > 95% of all the infections we face (opportunistic, commensal) and, in so doing, "buys time" for the adaptive system to make a response. The adaptive system (T and B) acts late, taking days to weeks to deal with the other < 5% of infections, a last line of defense against "pathogens". these infections reveal themselves as "disease" in normal vertebrates. once adaptive is working it functions mainly to temporarily increase the effectiveness of the innate system to a level that will achieve "clearance" for that remaining < 5% of all infections.

Hydrophobic interactions of immunoglobulin domains

The same is true of the TWO b-pleated sheets of a typical immunoglobulin domain. The protruding side chains are nowhere near as uniform as silk. Instead each forms a (relatively) hydrophobic face on one side of the sheet and a (relatively) hydrophillic face on the other. The two hydrophobic sides bury themselves in each other, the hydrophillic sides face outward, and the reduced disulfide bond spans and "locks" this molecular "sandwich" of two 2° structures into the 3° structure we call the Immunoglobulin Fold of an Immunoglobulin Domain. Note how each b-pleated sheet has loops the protrude from each turn of the strands at both ends of each Ig domain. In most cases these loops are not functional. But in the loops at the amino-terminal end of the VL and VH regions, these loops form the CDRs and, collectively, the six loops form the epitope binding site.

Difference between primary and secondary immune response

There are four major differences between the primary (1°) and secondary (2°) responses to a given antigen the first two have to do with the rate of delivery of bulk antibody to the animal's circulation and are visible within the model the second two are more subtle and have to do with the quality of the antibody that is delivered; only visible upon closer analysis of the delivered antibody itself 2° is Larger higher peak titre longer duration of the response peak X duration = amount of antibody (area under the curve) 2° comes Faster after re-adminstration of Ag less "lag time" before Ab can be observed higher rate of production ("explosive") and 2) can be explained by Clonal Selection clonal expansion in the 1° response produces memory cells that are more numerous the system acquires a representation bias 10 - 20% of the expanded B-cells become plasma cells plasma cells eventually die, antibody eventually "turns over" "protective levels" are an operational term that can be long (polio) or short (tetanus) "memory cells" can live a very long time (much debate) 2° displays "Affinity Maturation" relative to the 1° 2° response antibody has acquired different effector functions ("class switching") - the B-cells have differentiated

Bence-Jones K chains first subjected to aa sequencing

There is a repeating unit of about 107 - 110 amino acids with an intra-chain disulfide

priming

This refers to the initial exposure to an antigen. In classic immunology experiments, priming was used to expand the numbers of responding cells so that a large, very observable response could be measured upon the second application of the antigen. This was mainly because the methods of that time made measuring primary responses much more difficult than measuring the larger secondary responses. This showed that Receptors for specific, distinct antigens were present upon the exterior surface of living cells. The receptors were on non-overlapping cell populations - they were (probably) clonally distributed.

Antibody against allotype

Usually made in same species in a recipient (host) strain that does not bear that allotype. The allotypic epitope is present as the common "foreign" element on each molecule of the immunogen. Anti-idiotype still very unlikely to be made - the immunogen is still too polyclonal

Antibody against isotype

Usually polyclonal made in another species (e.g. goat anti-mouse g-chain) Anti-allotype may be also made but is a minor component of all "foreign" epitopes Anti-idiotype not detectable - the immunogen is too polyclonal for each idiotope to stand out as enough mass

Idiotypes comparison

Variable Region Shared only by identical clones Often in Ag-combining site Often made of residues that directly contact Ag But not always; some can be framework region structures

Bubonic plague virulence factors

Virulence Factors - MANY, a complex host parasite interaction It is the flea bite that traverses the barrier of the skin yadBC - an adhesin that permits attachment to and invasion of epithelial cells in the skin A separate component acts as activator of plasminogen - degrades clots, allows access to blood and septicemia (all the better to infect the next flea). Septicemia provides sudden access to resident phagocytes populations Stimulation by exotoxins and endotoxins (e.g. gm(-) LPS) produces a "cytokine storm" TNFa (in particular) disseminated intravascular coagulation, peripheral necrosis, accelerating transfer to LNs, and "shock". Peripheral necrosis liberated mitochondrial f-Met proteins, more edema, more "shock". YOPS - see next slide

Domain term

What we call "Domains" are local 3° structures within the overall 3° structure consisting of domains and non-structured stretches. Driven by H-bonds, ionic bonds, hydrophobicity and Van der Waals forces of the aminoacid R-groups ("side chains")(still, water and entropy)

Inhibitory/activating FcR

While activating FcRs use "immuno-tyrosine activation motifs" (ITAMs)(often on g-chains)that engage tyrosine kinases (e.g. syk) causing their phosphorylation and activation, Inhibitory FcRs use "immuno-tyrosine inhibition motifs" (ITIMs)(often part of the cytoplasmic portion of the a chain)that engage phosphatases that de-phosphorylate active tyrosine kinases and inactivate them(e.g. "SH2-containing inositol phosphatase 1", SHIP)

Adaptive immune advantage

a nearly infinite repertoire of recognition(an initially small but pre-differentiated part). a large potential for further differentiation, a process which is highly adaptable and results in the generation of effectors which can be "tailored" both in size and kind to the particular threat. displays memory because this adaptable differentiation leaves a bias in the system which causes bigger, quicker, tailored responses to a second exposure to the same pathogen. exquisite recognition specificity that permits: the creation of effectors which can distinguish fine differences between pathogens while tolerating closely related commensals; specific targeting of virulence factors (i.e. the important problems that the innate system cannot address); allows effectors to distinguish self and non-self (no collateral damage).

Diffusion into extravascular sites

all IgG high, IgA (moderate) monomer

Adaptive humoral immune system is designed to be unambiguous

allelic exclusion - of the 4 inherited, only one pair of Ig loci (i.e. one H and one L) is expressed in each B-cell to prevent mixing of H and L chains. clonal restriction - each B-cell is allowed to build only one binding site from each pair (H+L) of chosen loci Binding events must be unambiguously connected to a chosen effector function Physical (covalent) connection of binding site (in the Fab) to the effector region (Fc)

Allotypes

allelic variations in the amino acid sequence of the only the constant regions of H and L-chains of Ig's that are present in only some members of a given species H-chains have allotypes only in g and a (only a2 subclass) L-chains have allotypes only in k, not in l Found in C-regions of H and L chains "allotopes", "allotypic markers"

Immunoglobulin domain

antiparallel B sheets The constant and variable regions of an immunoglobulin EACH possess TWO b-sheet 2° structures with slightly different numbers of strands in each sheet. Note the unstructured "loops" connecting the strands of the b-pleated sheets (which have been distorted and pressed flat here).

Lower Affinity FcR

degranulation of the phagocytes internal granules on to objects too big for ingestion (e.g. FcgRII-A on eosinophils reacting to helminths) (e.g.FcgRIII on NK cells reacting to antibody coated, virus-infected or cancerous self cells) important to distinguish bound or unbound antibodies

Disadvantage

each specific element of its highly diverse repertoire has only a small initial mass which takes time to become large enough to defend effectively - energetically costly to enlarge the mass. parts are mobile but there is also a requirement for highly organized "organs" - energetically costly. only partially pre-differentiated; in particular, the "effector" end is NOT pre-differentiated and time must be spent to create an effector response to the threat - strategically costly. the price of a nearly infinite recognition repertoire means self / non-self discrimination is not built in and must be enforced. Receptor specificity is generated at random In T-cells degeneracy of self / non-self discrimination is actually required. Elaborate control mechanisms are needed to distinguish and enforce self and non-self discrimination. This control is energetically costly to create, strategically costly if the regulation fails (autoimmunity).

serum levels

highest IgG1, then IgG2, then IgA, then IgM, then IgG3, then IgG4, then IgD, then, way low, IgE

adoptive transfer

irradiate immune system and add new immune cells Adoptive Transfer" means the transfer of living cells that will colonize the recipient and install their properties and capacities (such as "memory") for immune response. This particular adoptive transfer experiment depended upon the ability of surface Ig to physically bind to Ag in its native configuration as it passes by on the outside of the cell in the design of these particular experiments this binding leads to preferential removal of the cells that bind a specific antigenPRIOR to adoptive transfer of this cell population (now depleted of specific antigen responsive cells) into an irradiated recipient animal

BcR complex

mIg (secreted antibody) + Ig-B. Ig-a Schematic of IgM in its membrane form (and associated with Iga:Igb signaling dimer). When cell switches to secretion, the soluble form without the trans-membrane segment is produced as a pentamer + J chain. But surface and soluble forms have the same Ag binding site

Absorption

means removal of Ag and Ab from serum by precipitation and centrifugation. More than one binding site per Ab molecule + more than one epitope (or copy of an epitope) per antigen molecule allows lattice formation and precipitation.

Precipitation

of Ag and Ab when mixed in the right proportions.

Clearance

phagocytes Achieved by phagocytes whose efficiency depends upon access - inflammation and phagocyte exit from the circulation - very dependent upon complement (C') fixation by Antibody ability to adhere to the pathogen (Soluble, non-antibody PRRs also opsonize in concert with phagocyte receptors for them) (PRRs on the phagocyte surface) Opsonization of the pathogen by Antibody binding(FcR's on phagocytes) Opsonization by C' fragments due to Antibody firing C'(C' fragment Receptors on phagocytes)

Neutralization in primary and secondary response

prevent initial entry/attachment - we make Ab with high intrinsic affinity (IgG, IgA) - very important AND we multimerize the binding sites on these immunoglobulin molecules so that they work even better (high avidity) IgG - 2 binding sites, IgM - 10 binding sites, IgA - 2 - 4 binding sites (4 mainly) In primary response want to prevent immediate re-infection with same bug in secondary response want to prevent re-infection - high affinity even more important

Neutralization - effector function of antibodies

preventing initial damage or colonization High Intrinsic Affinity is critical - IgG, IgA >> IgM - in order to oppose the binding of critical molecules (i.e. the virulence factors) of a pathogen to the surface receptors the pathogen uses on the host cell. - generally, these critical pathogen molecules have been selected by the pathogen to possess high affinity for their receptor molecules on host cells Attachment proteins Toxins

C-terminal Ch regions

reside the amino acids responsible for the biological effector functions peculiar to each heavy chain isotype (µ, g, d etc.) - specific stereoelectrochemical surfaces formed by the 4° structure. polymerization of monomers into IgM or IgA - interaction with C' first component (C1q) of C' cascade (IgM, IgG) - specific binding of structures in Fc leading to: - opsonization and phagocytosis (FcgR, Fcm/aR) - transport across placenta (IgG - FcRn) and mucosal epithelia (IgA - poly Ig receptor) and rescue of IgG from endosomes and lysosomes (FcRn) - mast cell binding (FceR)

Interactive adaptive/innate

the Adaptive system amplifies the capabilities of the Innate system but the Innate System is still required for "Clearance" Antibody "neutralization" (prevents binding or interferes with microbial biology) - if they don't attach, they don't infect; if they cannot function biologically, no infection forms "immune complexes" (ICs) antibody can crosslink microbes into aggregates (ICs) a lower number of infectious units means the disposal system of phagocytes is more efficient "opsonizes", therefore phagocytosis is more efficient amplifies inflammation by complement (C') activation, thereby delivering more phagocytes and soluble defensive molecules to the site of infection Cell Mediated Immunity (CMI) kills microbes hiding inside cells (mycobacteria, viruses) kills cells serving as reservoirs (bacteria) or factories (viruses, cancers) liberates microbes hiding inside cells so that the cellular and soluble effectors of the innate system can get at them to produce clearance The Innate system prepares the way for the Adaptive inflammation routes microbes, microbial antigens and the phagocytes that bear them away from the circulation and into the lymphatic system and the anatomical structures designed to allow the lymphocytes of the adoptive immune system interact productively with antigens to produce antibodies and CMI "clearance" (i.e. capture, phagocytosis and killing) of microbes by phagocytes leads to "antigen processing" and "antigen presentation" required for helper T-cells (Th) to respond to Ag most of the adaptive immune response depends upon Th in order to function at their best PAMP recognition by phagocytes (particularly dendritic cells) leads to "costimulation" (aka "Signal #2") = "permission" to the adaptive system to respond to Antigen (Ag) (aka "Signal #1) required for a naive Th to respond to Ag (aka "Signal #1) most of the adaptive immune response depends upon helper T-cells by requiring costimulation to activate a Th, the adaptive system relies upon the innate system to tell it which antigenic stimuli are "dangerous" (i.e. are of probable microbial origin because they have PAMPs associated with them) otherwise the adaptive system would respond to self Ags as easily as it responds to microbial Ags

Active innate defense advantage

they are large pre-existing masses, rapid and often self-amplifying, no time wasted generating the effectors - opposes things that multiply much more rapidly than we do. they are mobile and this large mass can be focused upon a (relatively small) threat. the defense mechanisms are pre-differentiated characteristics ("hard-wired") - opposes things that reproduce much more rapidly than we do. evolutionary selection of a few (and therefore cost effective) germ line receptors for the detection of microbial chemistry allows for highly reliable self / non-self discrimination at the level of sensing a danger.

disadvantage

they have a very limited repertoire of recognition (low diversity) although of proven value due to recognition of nearly immutable pathogen components. they have broad specificity due to the recognition of shared critical pathogen components but are not very selective. they have little or no adaptability (i.e. for further differentiation); either they work or they don't. they have no memory; the response is always the same size. inflammatory effector mechanisms are NOT particularly good at self / non-self discrimination and there is often a lot of collateral damage to bystander self.

Antibody against isotope, allotype or idiotype

to cause antibody to be made, an epitope has to be foreign to the reacting individual. to cause detectable antibody to be made, that epitope must be present on a significant fraction of the molecules used to immunize an individual - a foreign epitope can be there but present on so few molecules of the immunogen dose that it is below a threshold for causing detectable antibody formation

FcRn

transporter It is "Class I-like" and associates with b2M FcRn ("n" for neonate) is found in the placenta and transfers IgG from mother's circulation into the fetus' In the maternal breast it pumps IgG from blood into milk (as well as into the mother's own GI and respiratory mucosae) minor protective function compared to IgA Found in fetal intestinal epithelia where it takes IgG found in milk and pumps it into the blood (the opposite of the pIgR and IgA) It is also found in the membranes of phagolysosomes where it rescues IgG that was ingested along with the object it opsonized and returns it to the circulation It is also found in endothelial cells where it rescues IgG and albumin from endocytic vessicles Lengthens t1/2 of IgG to ~ 3 weeks Prevents waste of the effort of making high affinity IgG But there is still turnover and thus passive immunization does not last forever

Single cell allotype expression

we are diploid of the two copies of each locus we inherit (one from each parent) individual B-cells choose only one of each pair of loci for expression the choice is random being random, all the inherited loci get expressed by some of the cells = co-dominance in the serum But at the level of the individual cell there is absolute exclusion. Individual Ab molecules produced by a single B-cell are, by definition, "monoclonal" in origin and have: the same L-chain the same H-chain (until any class switching occurs) the same allotypes In a population of antibody molecules, H and L chains can be different (and ARE in natural Ab populations) UNLESS from a "monoclonal" cell population Mixed Ab molecules (2 different H's, and/or 2 different L's in the same molecule) are only possible in the lab