BSS020N232Y Immunology B Cells 2019-20 (2020-21) PDF

Welcome back to BSS020N232Y Immunology The session will start at 9 a.m. The session will be recorded for later online viewing. You should see this slide and may hear a sound check few minutes before 9. Internet/sound/video...

Welcome back to BSS020N232Y Immunology The session will start at 9 a.m. The session will be recorded for later online viewing. You should see this slide and may hear a sound check few minutes before 9. Internet/sound/video issues? Try this first: Log in using fully updated Chrome browser Check internet connectivity under “My If my session crashes: Settings” You should be able to stay in the session. I will do my best to restore it within 15 min. Get closer to your WiFi If this does not work, feel free to leave. Close other applications on your computer I will then post a replacement lecture Help: Moodle or IT Helpdesk recording and email you when this is finished.  Recording starts now BSS020N232Y Immunology Adaptive immunity: B cells Robert Busch, PhD [email protected] Whitelands PH.1053 Usual contact hours: Mon-Wed 1-2, Fri 2-3 via Zoom On-site option on Fridays. Bookings via link HERE Reading Review lectures on immunology from Introduction to Human Disease (Year 1) and on T cells so far in this module Review lectures on cell signalling from Cell Biology (Year 1) Posted in the top section of the Immunology Moodle site Chapters on adaptive immunity, antibodies and B lymphocytes in any good immunology textbook E.g., Murphy et al., Janeway’s Immunobiology, 7th ed., Garland Science, 2008 or 5th edition of Immunobiology searchable online at http://www.ncbi.nlm.nih.gov/books/NBK10757/ or in Resource List Further reading on Moodle and linked in these slides 3 Review: Innate vs. adaptive immunity What is the key difference in the way that pathogens are recognised by the two mechanisms? “Stimulus, response! Stimulus, response! Don’t you ever think?” Use the chat. 4 (Gary Larson) Specific recognition of pathogens is mediated by lymphocyte antigen receptors Foreign antigen Soluble antibody Y Y APC (immunoglobulin) T-cell MHC Surface antigen peptide immuno- globulin Y Y receptor (B-cell antigen receptor) T cell B cell (matures in thymus) (matures in bone marrow) 5 This lecture Review structures of antibodies and specific interactions with antigen Genetic basis of antigen specificity Different classes of antibody and their different functions in immune defence Genetic basis of antibody classes B-cell activation and fate during immune responses B-cell development, tolerance and regulatory B cells Monoclonal antibodies in diagnosis and treatment of disease 6 Structural biology of Ab/Ag interactions 7 Architecture of an antibody 50 kDa Heavy lines = polypeptide chains Dashed lines = disulphide bonds intra-domain 25 kDa inter-chain Proteolytic fragmentation produces: ab = “antigen binding” c = “crystallisable” 8 Crystal structure of an anti-lysozyme antibody F(ab) fragment complexed with antigen https://www.rcsb.org/ Search “Fab antigen complex” Ab contacts Ag at a complementary surface formed by loops at one end of the F(ab) fragment. “Hypervariable loops” or “complementarity-determining regions” (CDR) 3 each from the two polypeptide chains (H and L) 9  Antibodies have a characteristic tertiary structure, comprising multiple immunoglobulin superfamily (IgSF) domains Loops bind Ag “CDR” 1 domain ≈ 100 AA ≈ 12.5 kDa Ab V region domain Strand connectivity and intra-domain disulphide bond 10 https://www.pnas.org/content/96/26/14672 Recognition of the model antigen, hen egg lysozyme, by different antibodies Antibodies bind surface patches on this protein antigen (stippled coloured surfaces) The bound patches are called “epitopes” Typically recognition of intact antigen in native conformation Structural changes in the epitope disrupt antibody binding Shape & charge complementarity, as in other protein interactions 11  The IgSF fold recurs in many different cell surface receptors, including many CD antigens Specific Ag binding via CDRs CD = “cluster of differentiation” – a nomenclature for surface antigens of haematopoietic cells Also found on other cell types, e.g. in the brain. 12 Summary: structural biology of antigen (Ag) recognition by antibodies B and T lymphocytes have cell surface receptors which bind Ag (respectively, BCR and TCR). BCR and TCR extracellular domain structures are based on the immunoglobulin superfamily fold, albeit in different arrangements. BCR is a Y-shaped, membrane-bound form of soluble antibody (immunoglobulin), composed of 2 copies each of a heavy (H) and light (L) chain. BCR recognises surface patches of intact foreign antigens (mostly protein or carbohydrate), usually in their native form. Contrast with TCR from previous lectures 13 Genetic basis of Ag specificity of Abs 14 Clonal selection of antigen-stimulated cells enables specificity and memory of adaptive immune responses 15 How can Ab diversity be genetically encoded? Antibodies can be produced against a virtually unlimited variety of chemical structures Abs made by individual B cells against different Ags have unique amino acid sequences Sequencing of Ig H chain genes in individual B lymphocytes allows repertoire estimates upwards of millions Further diversity added by Ig L chains Upper limit on B cell repertoire set by numbers of B cells: low billions https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-3083.2011.02609.x https://onlinelibrary.wiley.com/doi/epdf/10.1111/imr.12670 For other proteins, 1 gene codes for 1 protein (or a few variants) Yet human genomes contain only ≈ 23,000 protein-coding genes. 16  Two ways to create diversity Additive (Innate, e.g. TLR) Combinatorial (Adaptive, e.g. TCR, BCR) Coffee blends x coffee grinds x # shots x milk types x milk amount x froth amount x frivolous additives x sugar x serving temperature … 17  Inherited (germline) antigen receptor genes: some assembly required for the segments coding the variable (V) region In the germline configuration, Ig H and L chain genes cannot be expressed Expression requires prior genetic rearrangements These take place in individual lymphocytes 18 Somatic gene rearrangement produces different VL-JL and VH-DH-JH combinations in individual B cell = “V(D)J combinatorial diversity” 1. One of the DH and one of the JH gene elements are joined to one another 2. One of the VH segments is then joined to the DH element 3. One of the VL variants is joined to one of the JLs 19 Combinatorial diversity is one important source of BCR diversity 20 Only B and T cells rearrange the BCR/TCR genes RAG1/RAG2 genes expressed in early lymphocyte development Code for proteins that recognise specific short repeat DNA sequences on either side of each V, D, and J segment Recruit recombinase enzyme complex, involved in DNA repair by homologous recombination Intervening DNA is excised, forming ring structures (mechanistic detail, not for class test) 21 Imprecise joining at V(D)J junctions adds further diversity P = palindromic sequences N = non-templated sequences Deletion may also occur Length varies randomly 2/3 of these recombinations fail to preserve the reading frame of the gene across the V(D)J junction Only those that do give rise to functional BCR genes (mechanistic detail, not for class test) 22  The rearranged Ig genes are transcribed, introns spliced out, then translated The greatest diversity is concentrated in the V(D)J junctions, which code for the CDR3 loops of the Ig VH and VL domains 23 Summary: mechanisms that diversify the BCR repertoire in individual naïve B cells VJ recombination of the L chain … and its junctional diversity VDJ recombination of the H chain … and its junctional diversity The combination of the two chains The combined variability allows, in theory, the production of ≈ 1012- 1018 distinct BCR gene sequences (of which ≈ 3.5 x 106 arise from combinatorial diversity) – enough to account for BCR clonal diversity These mechanisms also act on TCR genes in developing T cells 24 Effector functions of antibody classes Or: how do antibodies help you to fight infection? Suggest some answers in the chat. 25 Fc portion of soluble antibodies dictates their defensive (“effector”) functions Different antibody “classes” are distinguished by the Fc portions of their constant regions Unrelated to antigenic specificity IgM, IgD, IgA, IgG, IgE (“GAMED”) Fc region Differ in: No. of CH domains Further assembly into multimers Participation in primary or secondary/memory responses Distribution in the body and effector function 27 Features of IgM First antibody to be secreted into blood plasma during primary response Production does not depend on T-cell help Secreted form is a pentameric, disulphide-bonded assembly of the “Y” H2L2 structure, stabilised by a J (joining) chain Low affinity (KD ≈ 10-6 M) but high valency (10 Ag binding sites), thus high avidity 970 kDa Best Ig class for activating classical complement pathway No binding to Fc receptors on phagocytes or mast cells – but opsonisation via complement receptors 28 Features of IgD Only membrane-bound, no secreted form IgM-expressing naïve B cells may or may not co- express IgD, as a differentiation marker Function? 29 Other Ig classes are produced mainly in secondary immune responses 30 Features of IgG Produced at end of primary responses and dominates secondary responses Depends on Th cell interaction with B cells for class switching and affinity maturation Circulates as H2L2 monomer in plasma Divalent, higher affinity (KD ≤ 10-9M) than IgM ≈ 150 kDa Four subclasses* exist (IgG1, IgG2, IgG3, IgG4) Activate the classical complement pathway Engage FcRg on phagocytes natural killer cells Can be transferred across the placenta, protecting the foetus Can neutralise viruses, toxins (* subclass variation not for revision) 31 Features of IgA Produced in secondary responses Depends on Th cell interaction with B cells for class switching and affinity maturation Secreted form is a monomer or a disulphide-bonded dimer of the “Y” H2L2 structure, stabilised by a J (joining) chain Both circulate in plasma but the dimer is secreted at mucosal surfaces (GI, respiratory, urogenital) bound to a “secretory component” polypeptide made by epithelial cells. Mucosal immunity Two subclasses* exist (IgA1, IgA2) Bind to FcRa on phagocytes; neutralising (* subclass variation not for revision) 32 Features of IgE Produced in secondary responses Depends on Th cell interaction with B cells for class switching and affinity maturation Secreted form is a monomer of the “Y” H2L2 structure Binds a dedicated FcRe found on basophils and mast cells with high affinity Crosslinking of FcRe-bound Ab by Ag triggers mast cell degranulation Important in allergy and anaphylaxis, and in defences against worm infection 33  Break! You will be in break out groups to socialise if you wish Recording will pause; you can turn on cam and mic 34 IgM and IgG antibodies binding to bacterial antigens boost complement activation Circulating antibodies and (“alternative” and “lectin” complement proteins enter via pathways do not require Ab) the inflammatory exudate Antibodies bind to bacterial surface antigens Complement components C1/2/4 assemble into a C3 convertase Boosts deposition (“fixation”) of C3b on bacteria, release of C3a to boost inflammation, and formation of the membrane- attack complex 35 Receptors for Ig Fc domains are expressed by immune cells Greek letters in the receptor name identify the Ig classes recognised Inhibitory Details not for revision 36 Uptake of IgG-coated bacteria by phagocytes via FcRg and complement receptors Low-affinity FcR: not triggered by monomeric free antibody Similarly, FcRa – monomeric IgA IgM can opsonize via complement/CR1 (not via FcR) 37 Killing of IgG antibody-coated target cells (e.g., virally infected) by natural killer cells “Antibody-dependent cellular cytotoxicity”, ADCC 38 Crosslinking of allergen-specific IgE pre-bound to FcRe on mast cells triggers histamine release – rapid allergic reactions 39 Summary: vital stats of the Immunoglobulin classes Not for revision: Variation between IgG and IgA subclasses Serum level and half-life Staphylococcal protein A binding 40 Genetic basis of antibody classes (isotypes) 41 How are antibodies with different constant regions (and thus different classes) produced? Again, Greek letters identify the appropriate Ig class. Coexpressed via alternative splicing Need another type of specialised DNA recombination, called “class switch recombination”, to be used in place of Cm Only one is used in any one cell 42 Alternative splicing allows coexpression of IgM and IgD in some naïve B cells Coexpressed via alternative splicing In all naïve B cells In some naïve B cells 43 Class switch recombination allows activated B cells to replace the C domains of their IgM antibodies by the C domains of IgG/E/A Class switch recombination is induced by cytokines from Th cells Different process from VDJ recombination, and thus independent of antigenic specificity Mechanism not for revision 44  B-cell activation and fate during immune responses 45 Overview of B-cell activation T cell help usually required Class switching Low-affinity IgM Somatic hypermutation No T-cell help Primary response Secondary response 46  BCR and coreceptor signalling activate B cells CD19, CD21: Triggered by antigen binding Commonly used markers of B lineage Antigen/BCR complex is internalised for antigen processing B-cells proliferate and differentiate into memory cells and antibody-secreting cells Other signals required unless BCR crosslinking is extensive By Altaileopard - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=12193205 Detailed signalling pathways not for revision 47 Major B-cell subsets 48 T cell help for B cells (T/B collaboration) (or other Ag) Antigen-activated follicular B cells and follicular helper T cells meet at the boundary of the T and B cell zones of lymph nodes (attracted by CXCL13, a chemokine) B cells uses BCR to capture specific antigen → peptides presented by MHC class II (see next lecture) together with B7 costimulation BAFF, IL-4 – survival and hypermutation IL-21 → plasma cell differentiation T cells provide surface ligands and Various → class switching to various Ig classes/isotypes cytokines (“help”) CD40L/CD40 – survival, proliferation 49 Somatic hypermutation of VL/VH gene regions allows affinity maturation of antibodies Yellow: silent Pink: neutral Red: decreases affinity for Ag → B cells die Blue: improves affinity for Ag → B cells survive, divide 50 The Germinal Centre reaction orchestrates T- cell help, class switching, affinity maturation http://pleiad.umdnj.edu/~dweiss/nor mal_node/germ_center_lo_image.html https://www.frontiersin.org/articles/10.3389/fimmu.2018.02469/full 51  Some activated B cells differentiate into antibody-secreting plasma cells Plasma cell B cell Bone marrow LN, spleen, blood ER- and chaperone-rich Transmission EM (detailed marker profile not for revision) 52 Differential RNA splicing allows plasma cells and plasmablasts to secrete soluble Ab B cells Plasma cells and plasmablasts 53 B-cell development and tolerance 54 B cells, like T cells, develop from a common lymphoid progenitor and acquire self tolerance Early stages of development occur in bone marrow. Note parallels with T-cell development in thymus “Positive selection” for expression of functional BCR (2 steps) H chain is rearranged first “surrogate light chains” allow transport of H chain to the cell surface, where it can provide survival signals L chain is then rearranged, replacing surrogate chains “Negative selection” eliminates strongly self-reactive B cells (“central tolerance”) Surviving B cells circulate to spleen and lymph nodes 55  B-1 cells may arise from a distinct progenitor and develop earlier in life Express CD5 (otherwise a T-cell antigen) Migrate to peritoneal cavity and intestine Make “natural antibodies”, IgM (and IgA) Less antibody diversity 56  Emerging research: regulatory B cells Roles in preventing immunopathology and autoimmunity Secrete anti-inflammatory cytokines Promote differentiation of regulatory T cell subsets https://www.cell.com/immunity/fulltext/S1074-7613(15)00134-X 57 Clinical applications of B-cell immunology (brief review) Vaccination Monoclonal antibody technology Diagnostic and research uses Treatment See also Intro to Human Disease notes 58 Immunization Vaccination: active immunization with non-pathogenic preparation of antigen Live attenuated, killed or subunit vaccines Highly successful prevention strategy for bacterial and viral infection Well-established safety/efficacy record of childhood vaccination series Adult vaccination targeting specific populations at risk E.g., HBV, HPV, influenza Protection often by antibodies Neutralisation of viruses or bacterial toxins by IgG and IgA Passive immunization by i.v. injection of anti-pathogen antibodies early in infection, e.g., rabies, tetanus, snake venoms, some viruses 59 Monoclonal antibodies Or use recombinant DNA techology 60 Antibodies (often monoclonals) in diagnostics: the enzyme-linked immunosorbent test (ELISA) Variants: 1. the “sandwich” ELISA (two antibodies specific for two different epitopes on the same antigen) E.g., testing for Ab 2 cytokines, A g serum proteins YAb 1 2. “indirect” ELISA (Ag/Ab complex is detected by incubation with a secondary antibody-enzyme conjugate) E.g., testing for human Ab 2 antibodies to viruses, such as A Ab 1 HIV g Investigations using monoclonal antibodies: looking for a particular antigen in tissues (“immunohistochemistry”) Beta cells (insulin) Alpha cells (glucagon) Brown staining indicates the presence of the appropriate hormones by binding of a specific monoclonal antibody! 62 Tests for neutralising antibody (nAb) Often predicts whether antibodies are effective in combating a viral infection Incubate viral samples with antibodies from patients or vaccines If neutralising Abs are present, infectivity is reduced (test with, e.g., plaque assay) Influenza nAbs can be tested by inhibition of haemagglutination, caused by a viral coat protein binding to surface carbohydrates of RBCs http://microbiologynotes.com/haemagglutination-assay-viral-quantitation/ 63 Monoclonal antibodies for therapy (“biologics”) Applications in malignancies and autoimmune diseases Monoclonal antibodies targeted against human antigens Normally made in another species – differences in antibody structure can stimulate the immune system of the treated patient Which antigens do the mAbs target? Haematologic malignancy (leukaemia, lymphoma) – deplete immune cells of the relevant lineage (including normal and malignant cells). Side effects: immune deficiency Various cancers – use checkpoint inhibitors – disable molecules that boost T- cell tolerance, improving chances of immune attack on the tumour. Side effects: autoimmune Autoimmune disease: deplete pathogenic cell populations OR block the action of key cytokines. Side effects: immune deficiency 64 Strategies for engineering antibodies to decrease inter-species differences 65 Monoclonal antibodies for therapy: anti-TNF in rheumatoid arthritis Tumour necrosis factor (TNF-a) is a key immune signalling molecule in rheumatoid inflammation and joint damage Anti-TNF monoclonal antibodies can be used to halt progression of damage in many patients 66 Anti-TNF therapies Infliximab is a chimeric antibody, in which the Fab' (variable) domain is of mouse origin and the Fc (constant) domain is of human origin. Adalimumab and golimumab are fully human IgG1 antibodies. Etanercept is a fusion protein between a human IgG1 Fc tail and TNF receptor 2. Certolizumab pegol consists of a humanized Fab' fragment bound to polyethylene glycol.  Summary: Antibodies and B cells Abs: Y-shaped structure with hypervariable loops making complementary interactions with specific carbohydrate or protein Ags Extraordinary variation in Ag specificity enabled by VDJ recombination and junctional diversity, creating clonal diversity among naïve B cells Affinity for Ag is refined by somatic hypermutation (SHM) after activation B-cells develop in BM, undergo + and – selection (tolerance), populate 2ry lymphoid organs; on activation & proliferation, some become memory cells, others go to BM as plasma cells B-1 cells emerge from foetal liver, populate the peritoneum, and make T-independent IgM responses RNA splicing and class-switch recombination allow antibodies of different subclasses to be produced in secondary responses IgM/(D) → IgG/A/E in FoB (but not MZB) cells → different effector functions Neutralisation, opsonisation, ADCC, mucosal immunity, anaphylaxis Class switching requires T-cell help and is accompanied by SHM in the GC reaction Antibodies are powerful tools for biomedical research, diagnosis and therapy 68 Thank you for your attention! 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