Adaptive Immunity: B-Cell Mediated Immunity III PDF

11/28/2023 LECTURE OUTLINE I. Overview Adaptive Immunity: II. B Cell Receptor Diversity B-C...

11/28/2023 LECTURE OUTLINE I. Overview Adaptive Immunity: II. B Cell Receptor Diversity B-Cell Mediated Immunity III III. B Cell Development IV. B Cell Maturation MCB 11338 – Immunology V. B Cell Abnormalities LS Meadows VI. B Cell Activation VII. Antibody Structure & Function VIII. Antibody Receptors IX. Immunoassays Figure 4.1 Plasma cells secrete antibody of the same antigen specificity as that of the antigen receptor of their B-cell precursor Antibodies soluble proteins produced by plasma cells found in blood, lymph, & on mucosal surfaces bind to extracellular pathogens / antigens directly disable pathogen/antigen tag pathogen/antigen for destruction by C’ and/or phagocytes specific = each ab binds to one ag (or small # of very similar ags) Typical Antibody Structure Heavy Chain Constant Domains 4 polypeptide chains differ in location of disulfide bonds, carbs, presence/absence of hinge 2 heavy, 2 light disulfide bonds light chains (к or λ) 1 variable (VL) domain 1 constant (CL) domain heavy chains (ɣ, μ, δ, α, or ε) 1 variable domain (VH) 3 (or 4) constant domains (CH1, CH2, CH3, CH4) 1  11/28/2023 Typical Antibody Structure Immunoglobulin Domains antigen-binding sites = VL + VH compact, stable, folded “sandwich motifs” AA sequence == antibody diversity (specificity) 100 - 110 AA 2 β sheets connected by loops hinge = flexible antigen-binding ability disulfide bonds, hydrophobic interactions proteolytic cleavage 2 Fab fragments (“antigen-binding”) 1 Fc fragment (“crystallizable”) cell-receptor (FcR) binding C’-binding Figure 4.7 The three-dimensional structure of immunoglobulin C and V domains Variable Domains hypervariable regions (HVs) AA loops most distal to C domains 3 per VL and VH form antigen-binding site at end of each Fab “arm” determine antibody specificity framework regions (FRs) β strands & other loops 4 per VL and VH structurally stable HVs = CDRs (complementarity-determining regions) Epitope (Antigenic Determinant) Antigen-Binding Site Shapes part of antigen to which an antibody binds pockets = bind small, compact epitopes grooves = bind short, linear epitopes multivalent antigens extended surfaces = bind curved (globular) epitopes multiple different epitopes multiple copies of same epitope knobs = bind pocket/grooved epitopes linear contiguous AA discontinuous noncontiguous AA 2  11/28/2023 Antibody Affinity Antibody Affinity antigen-binding strength increases during adaptive immune response due to somatic hypermutation and affinity maturation combination of noncovalent attractions van der Waals forces hydrophobic interactions electrostatic forces hydrogen bonds Figure 4.24 The surface and secreted forms of an immunoglobulin are derived from the same heavy-chain gene by alternative RNA processing From BCR to Secreted Antibodies BCR membrane-bound antibodies (IgM and IgD) secreted antibodies produced by alternate pattern of heavy-chain mRNA processing SC = secretion-coding exon used MC = membrane-coding exons SC = secretion-coding exon IgM Membrane-Bound IgM vs. Secreted IgM first antibody class produced & secreted membrane-bound IgM secreted IgM by plasma cells in lymph nodes, BM, spleen, & MALT monomer pentamer + J chain circulates in blood & lymph low affinity but high avidity avidity = overall strength of binding to multiple epitopes of an antigen 3  11/28/2023 Figure 4.30 Each human immunoglobulin isotype has specialized functions correlated with distinctive properties Secreted IgM 10 low affinity antigen-binding sites weakly neutralizing antibody 5 Fc regions excellent complement activator large size cannot easily enter tissues IgG Antibody Classes & Subclasses produced & secreted after IgM by plasma cells in lymph nodes, BM, spleen most abundant blood-borne ab two high-affinity ag-binding sites easily enter infected tissue IgG IgG Subclasses (IgG1, IgG2, IgG3, IgG4) smaller & more flexible than IgM vary in C region of heavy chain, especially in hinge region balance flexibility with susceptibility to proteolytic cleavage neutralization opsonization C’ activation crossing placenta entering extravascular sites 4  11/28/2023 Figure 4.34 The four subclasses of IgG have different and complementary functions IgG1 most abundant & versatile IgG subclass intermediate in flexibility intermediate in proteolytic susceptibility intermediate C’ activator produced in response to protein antigens IgG2 IgG3 second most abundant IgG subclass longest hinge region = most flexible less flexible most susceptible to proteolytic cleavage less susceptible to proteolytic cleavage less able to activate C’ best at activating C’ Fc region more accessible to C1q produced in response to repetitive carbohydrate antigens deficiency == recurrent infections == chronic lung disease especially important for defense against encapsulated bacteria IgG4 Monomeric IgA cannot activate C’ (Fc region binds C1q poorly) produced & secreted after IgM swaps “halves” with other IgG4 molecules by plasma cells in lymph nodes, BM, spleen hybrid antibodies with two different antigen-binding sites 2nd most abundant blood-borne ab only able to neutralize antigens anti-inflammatory / anti-allergen two high-affinity ag-binding sites blocks binding of allergen to IgG easily enter infected tissue 5  11/28/2023 Dimeric IgA Pentameric IgM, IgG, Monomeric IgA produced by plasma cells in MALT defend against microbes in bloodstream & in infected tissues secreted into gut, milk, tears, saliva secreted by plasma cells in lymph nodes, BM, spleen keeps commensal populations in check actively transported across epithelium of mucous membranes excellent neutralizing antibody Pentameric IgM and Dimeric IgA IgG and Dimeric IgA == Passive Immunity defend against microbes at mucosal surfaces Antepartum secreted by plasma cells in MALT IgG crosses placenta into fetal bloodstream IgG IgA protects against pathogens that enter fetus through damaged skin Postpartum dimeric IgA in breast milk binds to pathogens in gut protects against pathogens that enter fetus through mucosal surfaces Figure 9.22 The first year of life is a period when infants have a limited supply of IgG and are particularly vulnerable to infections Symptoms of genetic deficiencies appear here. Neutralizing Antibodies prevent attachment of antigens to body cells microbes, microbial toxins, animal venoms must have high-affinity binding sites (high avidity IgM antibodies = weakly neutralizing) IgG and monomeric IgA irreversibly bind to toxins in blood & tissues dimeric IgA prevent attachment of microbes at mucosal surfaces 6  11/28/2023 Neutralizing Antibodies: Examples anti-hemagglutinin antibodies prevent binding of influenza virus to respiratory epithelium new strain of S. pyogenes with different F protein == strep throat anti-adhesin antibodies limit numbers of bacteria colonizing mucous membranes anti-F protein antibodies limit binding of Streptococcus pyogenes to fibronectin on surface of respiratory epithelium Figure 9.27 Classes and subclasses of antibodies differ in their capacity to activate and fix complement C’-Activating Antibodies classical pathway activated by antigen-antibody complexes pentameric IgM only one IgM needed (5 Fc regions per IgM) binding of antigen exposes C1q binding site on Fc region of antibody IgG3 and IgG1 C1q binding site exposed even without antigen binding at least two IgG molecules needed (1 Fc region per IgG) Pentameric IgM IgG3 > IgG1 planar form single C1q binding site per IgG molecule cannot bind C1q exposed with or without antigen-binding staple form C1q must cross-link at least 2 IgG molecules to binding to antigen exposes become activated C1q binding site on Fc region 2 IgG on surface of microbe OR C1q attaches to IgM at 2 IgG on soluble multivalent antigen multiple points 7  11/28/2023 Classical Pathway Results Forms of C4 haplotype = genes inherited from one parent C3b-opsonized pathogen phagocytosed via CR1 C4A C4B Variations in C4 Haplotypes membrane attack complex lyses pathogen membrane binds to amino groups binds to hydroxyl C3a and C5a recruit inflammatory cells (neutrophils, monocytes) of pathogen groups of pathogen macromolecules macromolecules missing from 13% of missing from 18% of human chromosomes human chromosomes inc. risk of developing inc. susceptibility to SLE infection Immune Complexes (ICs) Immune Complexes (ICs) (Antigen-Antibody Complexes) erythrocytes (RBCs) form when IgG binds to soluble multivalent express CR1; outnumber WBCs 500:1 antigens remove most C3b-tagged immune complexes from blood toxins, pathogen degradation products, etc. macrophages in spleen, liver activate complement → C3b deposited on antigen remove immune complexes from RBCs and antibody release RBCs back into bloodstream C3b-coated immune complexes removed by phagocytes via Fc receptors and C’ receptors Circulating Immune Complexes aggregate if not removed from circulation deposit in basement membrane of small blood vessels especially in kidney complement-mediated damage to blood vessels & surrounding tissues 8  11/28/2023 Circulating Immune Complexes in Kidney IgE podocytes express CR1 binds to high-affinity receptor (FcɛRI) on … podocyte mast cells in epithelial tissue activated eosinophils on mucosal surfaces mesangial cells blood basophils eliminate ICs stimulate tissue repair induces degranulation upon antigen binding involved in … anti-parasite defense mechanisms allergies & asthma Anti-Parasite Defense Mechanisms Allergies & Asthma eosinophils bind to IgE-coated parasites mast cells bind IgE specific for non-harmful antigens (allergens) (can also bind to IgG-coated parasites) pollen, dust, shellfish, peanuts, etc. release toxins directly onto surface of parasite massive mast cell degranulation → inappropriate immune response basophils & mast cells bind IgE antibodies runny nose, itchy/watery eyes, sneezing, hives degranulate when parasite antigens bind to IgE release chemicals that stimulate smooth muscle anaphylaxis (life-threatening reaction) contraction sudden drop in blood pressure coughing, sneezing, vomiting, diarrhea → eject parasite sudden constriction of airways from body IgD Antibody Receptors = Fc Receptors secreted by plasma cells in upper bind to Fc (“stem”) region of antibodies respiratory tract especially tonsils transporting Fc receptors involved in antibody transcytosis binds to receptor on basophils induces degranulation upon antigen binding activating Fc receptors enhance phagocytosis & pathogen destruction increase efficiency of antigen-presentation interacts with commensal & pathogenic respiratory bacteria inhibiting Fc receptors decrease inflammatory response of effector cells 9  11/28/2023 Figure 9.17 The receptor FcRn transports IgG from the bloodstream into the extracellular spaces of tissues FcRn expressed on surface of endothelial cells structure similar to MHC-I molecules transports IgG from blood into tissues diverts pinocytosed IgG away from lysosomes releases IgG at basolateral surface of cell helps IgG cross placenta from mother to fetus Figure 9.18 Transcytosis of dimeric IgA antibody across epithelia is mediated by the poly-Ig receptor Polymeric Immunoglobulin Receptor (pIgR) transports IgA & IgM from basolateral surface to apical surface of mucosal epithelial cells by binding to J-chain secretory piece fragment of pIgR retains antibody at mucosal surface binds to mucins (glycoproteins in mucus) FcɛRI Fcɣ Receptors high-affinity receptor for IgE bind IgG molecules expressed on … FcɣRI mast cells (in CT) Each cell binds IgE below mucous membranes & epidermis around blood vessels molecules of different antigen specificities. FcɣRII basophils (in blood) FcɣRIIA activated eosinophils (at mucosal surfaces) FcɣRIIB (B1 and B2) = inhibiting cross-linking of FcɛRI-bound IgE by FcɣRIII parasite/antigen/allergen → degranulation FcɣRIIIA FcɣRIIIB 10  11/28/2023 Figure 9.35 The family of human Fc receptors that bind to IgG ITIM = immunoreceptor tyrosine-based inhibitory motif FcɣRI FcɣRI activating receptor specific for IgG only Fc receptor that can bind IgG with or without antigen α chain 3 Ig-like domains → bind CH2 & lower hinge regions of IgG binds IgG subclasses with different ɣ chain dimer affinity (IgG3 > IgG1 > IgG4 > IgG2) ITAM domains → cell signaling monocytes, macrophages, DCs major function: facilitate uptake & always express FcɣRI degradation of pathogens by phagocytes & APCs neutrophils, eosinophils FcɣRI expression induced at infection sites FcɣRII and FcɣRIII low-affinity IgG receptors = only 2 Ig-like domains only form stable interactions with pairs of IgG cross-linked by antigen 11  11/28/2023 FcɣRII: Three Different Genes Error in Text: Page 274 FcɣRIIA activating similar function as FcɣRI weakly activates ITIM = immunoreceptor most strongly FcɣRIIB2 tyrosine-based inhibitory motif inhibitory → dec. inflammatory response of macrophages, neutrophils, & eosinophils FcɣRIIB1 inhibitory → dec. inflammatory response mast cells & B cells IgG2 FcɣRIIA Variants responds best to polysaccharide antigens H131 (histidine at AA position 131) binds IgG2 effectively only weakly activates complement R131 (arginine at AA position 131) binds IgG2 ineffectively bound best by FcɣRIIA homozygotes → inc. risk of serious infections with Neisseria meningitidis Figure 3.40 C-reactive protein initiates the classical pathway of complement activation FcɣRI & FcɣRII also bind C-reactive protein (CRP) increases Streptococcus pneumoniae uptake & degradation by phagocytes 12  11/28/2023 FcɣRIII FcαRI activating receptor activating medium affinity receptor for expressed on macrophages, neutrophils, eosinophils, NKs monomeric IgA antibody-dependent cell-mediated cytotoxicity (ADCC) α chain NKs use FcɣRIII to bind to & kill cells coated with IgG1 or IgG3 2 Ig-like domains ɣ chain dimer ITAM domains → cell signaling expressed on mac, neut., act. eo, DC enhances phagocytosis of IgA-coated pathogens Figure 9.37 Comparison of structure and cellular distribution of the Fc receptors for IgG, IgE, and IgA Immunoassays use antibodies to detect & quantify a particular antigen latex bead agglutination precipitation / flocculation enzyme-linked immunosorbent assay (ELISA) lateral flow assays Western blot immunophenotyping / flow cytometry 13  11/28/2023 Polyclonal Antisera vs. Monoclonal Antibodies polyclonal mix of antibodies specific for different epitopes monoclonal one type of antibody specific for one epitope Figure 4.12 Production of a mouse monoclonal antibody Monoclonal Antibody Production 14  11/28/2023 Flow Cytometry Evolution of Monoclonal Antibodies distinguishes cells by cell mouse → chimeric → humanized → fully human surface (and cytoplasmic) molecules fluorescent-labelled antibodies “tag” different molecules (immunophenotyping) https://youtu.be/EQXPJ7eeesQ https://youtu.be/B2zreF2dnWk Figure 4.14 Monoclonal antibodies as treatments for disease https://en.wikipedia.org/wiki/List_of_therapeutic_monoclonal_antibodies Drug Brand Name Orthoclone OKT3 ProstaScint Blincyto Rituxan Simulect Siliq Herceptin Lemtrada or Campath Xolair Humira Yervoy Kevzara 15