Antibodies Flashcards
Antibody classes
Determined by isotype of heavy chain
mu -> IgM delta -> IgD gamma -> IgG epsilon -> IgE alpha -> IgA
General antibody structure
2 heavy chains and 2 light chains
H=H and L=L due to allelic exclusion
L-H and H-H di-sulfide bonds hold together
Fab = antigen binding (variable) - H + L Fc = crystallizable (constant) - only heavy chains
Membrane antibodies
Co-expressed with adhesion molecules
Ig-beta, Ig-alpha (similar to CD3 + TCR) - transduce signals
Membrane antibody + Ig-beta + Ig-alpha = BCR!
Antigen binding site
At N-terminus of light and heavy chains
“VL” = variable light loop
“VH” = variable heavy loop
Each loop (VL and VH) contains 3 hypervariable regions aka "CDR" aka "complementarity determining regions" - amino acid residues determine specificity of antigen binding
Antigenic determinants
Antigenic determinant = epitope = region on antigen that binds to antibody
- may be protein, carb, lipid, etc
- many antigens have multiple different determinants -> multiple Ab matches
Binding
- conformational (no affinity when denatured)
- linear -> either accessible when folded or inaccessible (MUST be denatured for Ab binding)
Fc region
Fraction crystallizable
C-terminal of light (1/2) and heavy (3/4) chain
Constant across Ig class and subclass
Determines effector function of antibody!
- CH2 binds complement
- Fc receptors (FcR) bind Fc region of a subclass
Antibody valence
Number of Fab regions = number of antigenic determinants that can be bound
“Normal” and membrane = divalent
Secreted IgM and IgA = multivalent
Affinity vs avidity
Affinity = strength of binding of antigenic determinant:Fab
Avidity = functional strength of antigen:antibody
- avidity is exponentially increased by valence (ie multivalent, multiple Fab’s can hold antigen exponentially longer)
IgG characteristics
Most abundant in blood Crosses placenta -> passive immunity Four subclasses (IgG1, IgG2, IgG3, IgG4) - different H chains -> different functions Simple dimer (2 H, 2 L) -> 2 Fab's
IgM characteristics
First antibody produced in immune reaction
Expressed on membrane of naive, mature cells as BCR (dimer form) - along with IgD
Released into blood - 5 dimers -> 10 Fab’s
- held together with J chain (not from “J” exon of DNA)
- low affinity but 10 Fab’s -> high avidity -> highly efficient
IgA characteristics
Blood as simple divalent
Secreted (sIgA) = dimer = 4 H + 4 L
- 4 Fab’s -> higher avidity
- J chain + “secretory component” hold together
- made by local plasma cells -> secreted across epithelium
IgA secretion
Produced and secreted in local plasma cells
- secreted as dimer with J chain
Binds to poly-Ig receptor on basal surface of epithelial cell
- dimer + poly-Ig receptor endocytosed
- poly-Ig receptor partially cleaved in endosome ->
- secreted with dimer + J chain + poly-Ig receptor fragment
“Secretory component” = fragment of poly-Ig receptor from epithelial cell
- helps hold together in secretion
IgD characteristics
Very low levels in blood
Primarily acts as BCR
- expressed with IgM in mature, naive cells (same Fab specificity)
- antigen binding -> internalized -> processed -> present to Th
Important for respiratory, identifying B cell development and type (ie follicular vs marginal zone)
IgE characteristics
Very low levels in serum
Important for inflammation and allergies/asthma
Binds to FcER1 receptors on mast cells (via Fc domain)
- antigen -> crosslinking of mast cells -> signal -> histamine release
“Isotype”
Differences between Ig classes
- sequence in Fc of heavy chain
- determines function of antibody (ie complement, FcR’s)
- use in lab to find specific antibody class
“Allotype”
Genetic polymorphisms in antibodies
- ex different alleles for H chain of IgM
- study in Fc region (not affected by recombination)
- use for paternity
“Idiotype”
Changes to variable domains
- directly determines antigen binding site specificity
- use for targeting therapies to destinations or pathogens
Overview of antibody diversity
Multiple exons in different gene segments
- V = variable, D = diversity, J = joining, C = constant
->rearrange DNA so VDJ (or VJ) is encoded in a single uninterrupted exon
- unique DNA rearrangement -> unique Fab
Similar process at each of three gene loci (H chain, lambda, kappa)
DNA recombination process
Requires combined expression/activity of “recombination-activating genes” - RAG1, RAG2 - limited to lymphocyte dev’t
H chain first
- DJ join
- V joins -> VDJ
- C region not rearranged (spliced in mRNA)
Either kappa or lambda (not both in same cell)
- VJ join
DNA - antibody structure
V = variable exon
- determines general shape of antigen binding site
- contains complementarity determining region (CDR) 1, 2
VDJ junction -> CD3 (most variability!)
Light chain components
Either kappa or lambda expressed in each cell
VJ exons splice -> determine variable region
Each allele also has constant region at C-terminal end
Constant region DNA
Located at 3’ (C-terminus) of allele
Not recombined!
Isotype and class switching determined by splicing
- “switch”/S sequences between C-regions
C-region exons: alpha (1,2), delta, epsilon, mu, gamma (1-4)
Membrane vs secreted antibodies
Determined by splicing and poly’A of constant DNA region of H chain
Membrane - includes transmembrane (hydrophobic) and cytoplasmic exons - at 3’ end of mu and delta C regions
Secreted - transmembrane and cytoplasmic exons spliced off
Mechanisms of antibody diversity
Most are antigen-independent -> inc likelihood of antigen match
- variation in germ line alleles (V, D, J, V, J)
- variation in exon combination (which V, which D…)
- imprecise joining -> junctional diversity
- CD3 -> extra nucleotides added -> frameshift
- terminal deoxynucleotidyl transferase (TDT) addes “N” nucleotides to 5’ end of exons
- “P” nucleotides added during recombination
Also have antigen-dependent affinity maturation during clonal expansion
Affinity maturation
Within germinal center (ie during clonal expansion)
Somatic point mutations develop in V region
Mutations accumulate if they increase binding affinity (compete for antigen:BCR binding)
-> decrease in binding coefficient/increased affinity of final plasma and memory cells
Allelic exclusion
Each mature B cell only expressed one H chain and one L (either kappa or lambda)
If first is successful -> second is silenced and never recombines
Stages of B cell maturation
Does NOT require foreign antigen
Heavy chain rearrangement -> mu expressed + surrogate light chain -signal> stop heavy rearrangement ->
Start light chain rearrangement -(if successful)>
IgM expressed -> “immature” -> tolerance induction ->
IgM signals survival, expansion -> IgD expressed -> “mature naive”
BCR co-receptors
Most active when stimulated by same complex as BCR (ex antigen + complement)
Stimulatory (-> activation) - CD21 = complement receptor 2 - CD19 - CD81 Inhibitory (slows activation or requires more antigen) - CD32 = receptor for Fc region of IgG
CD19 deficiency
Form of “common variable immunodeficiency”
CD19 is co-stimulator for B-cell activation
If Cd19 missing -> no co-stimulation -> more bacterial infections
Treat with IV-Ig -> passive immunity
Isotype switching
Induced by T-cell cytokines: IL-4, TGF-beta, INF-gamma
Via differential splicing of VDJ to different C regions
B cell tolerance
Multiple mechanisms
Apoptosis - either central or peripheral
Anergy
Receptor editing - “tries” a second time to make light chain -> new antigen specificity
T cells - don’t activate self or produce Treg (T cell tolerance is more robust)
