lect 20 - antibodies_SM_090623 4.pdf

https://teaching.ncl.ac.uk/bms/wiki/images/3/33/Antibody.jpg Antibodies Shannon Murray, MS PhD BMS 5308 Lecture #20 [email protected] Special Acknowledgement • Thanks to Dr. Johnson for sharing her slides, some of which I have modified. Her acknowledgement was as follows: • “This lecture has...

https://teaching.ncl.ac.uk/bms/wiki/images/3/33/Antibody.jpg Antibodies Shannon Murray, MS PhD BMS 5308 Lecture #20 [email protected] Special Acknowledgement • Thanks to Dr. Johnson for sharing her slides, some of which I have modified. Her acknowledgement was as follows: • “This lecture has been given by Dr. Renee Prater to the OMS students for most of the 20 years that she taught at VCOM-VC. I acknowledge her collegiality and experience as I have used her slide set as inspiration for this lecture. “ Lecture Objectives • • • • • • • • • Identify the main types of humoral immune responses. Define antibodies and recognize their general structure and function. Identify the antibody isotypes and discriminate the structure and function of each. Define epitope, determinant, affinity, avidity, antigen, immunogen, hapten, T-dependent antigen and T-independent antigen. Recall the steps and mechanisms involved in the generation of antibody diversity during the development of B lymphocytes into naïve B cells. Explain the process and rationale behind receptor editing in the bone marrow. Analyze the significance of affinity maturation, somatic hypermutation, and isotype switching. Compare and contrast T-dependent and T-independent antigens. Characterize a T cell-independent B cell response and the antigens that elicit this response. Reading References • Basic Immunology, 6th ed., Abbas, Lichtman, and Pillai – Chapter 2 – "Antibodies", "Production of Diverse Ag Receptors", and "Maturation of Selection of B Lymphocytes" sections – Chapter 7 – "Phases and Types of Humoral Immune Responses", "Heavy Chain Isotype (Class) Switching", "Affinity Maturation", and "Antibody Responses to T-Independent Ags“ sections • Cellular and Molecular Immunology, 10th ed., Abbas, Lichtman, and Pillai – Chapter 5 – “Antibodies and Antigens“ – Chapter 7 – “The B Lymphocyte Ag Receptor Complex“ – Chapter 8 – “Lymphocyte Development and Ag Receptor Gene Rearrangement“ • Immunology for Medical Students, 3rd ed., Helbert – – – – Chapter 4 – "Antigen and Antibody Structure“ Chapter 5 – "Antibody-Antigen Interaction“ Chapter 6 – "Antibody Diversity“ Chapter 14 – "B Cell Development" Types of Humoral Immune Responses • Humoral immunity – acellular response utilizing macromolecules in the body’s fluids • Primary target – extracellular pathogens and dangers ‒ Little to no effectiveness against intracellular targets • Two major forms of humoral immunity ‒ Complement ‒ Antibodies https://i2.wp.com/www.stomponstep1.com/wp-content/uploads/2014/09/flow-chart.png?zoom=1.5&resize=474%2C350 Antibodies: General Structure https://teaching.ncl.ac.uk/bms/wiki/images/3/33/Antibody.jpg How are Antigen-Specific, High-Affinity Antibodies of Different Isotypes Generated? Terminology of Antigenicity https://s3-us-west-2.amazonaws.com/courses-images/wp-content/uploads/sites/ 1094/2016/11/03172844/OSC_Microbio_20_01_Epitope.jpg • Antigen (Ag) – a substance that specifically binds an antibody or T cell receptor • Immunogen – an Ag that elicits a T cell or B cell response • Epitope or determinant – smaller portion of Ag that is specifically bound by T cell or Ab ‒ A single Ag usually has multiple epitopes ‒ Some epitopes can be dominant and elicit greater T/ B cell responses (number of magnitude); called “immunodominant epitopes” Properties of Antigens • An immunogen is ALWAYS an Ag, but an Ag may not necessarily be an immunogen. • An Ag MAY or MAY NOT elicit an adaptive immune response ‒ i.e., can bind TCR or BCR without activating the cell • Typically, a protein or glycoprotein, but may also be a polysaccharide or glycolipid • General order of immunogenicity: protein >> carbohydrate >> lipid, haptens, amino acids, DNA • What makes a good antigen? ‒ Highly folded ‒ Unevenly charged ‒ Greater than 1kD in size, most over 100 kD https://www.microbiologybook.org/mayer/immunogenicity.gif Haptens • • • • • • Haptens = small part of Ag that is bound by Ab; generally, <1 kD in size Unable to initiate an immune response by itself MUST be attached (conjugated) to a larger molecule to be immunogenic Larger “helper” molecule often called the carrier and is often a protein Ab can also be made to carrier protein Common haptens – penicillin, warfarin, dander, industrial chemicals, sodium benzoate, titanium oxide, polyethylene glycol • Examples of carrier proteins – albumin (BSA, ovalbumin), keyhole limpet hemocyanin https://clinicalgate.com/wp-content/uploads/2015/03/ B9780323045940000201_f020-003-9780323045940.jpg https://www.creative-diagnostics.com/images/Anti-Hapten-Antibody-Production-1.jpg A Snapshot of Haptens and B Cells • Basis – lack of sufficient avidity of hapten to trigger B cell activation • “Natural reaction” – drug (e.g., penicillin) binding to host protein • “Designed reaction” – vaccine Ag (e.g., meningitis linked to carrier protein (e.g., albumin, DT)) • No BCR crosslinking • No B cell activation • No Ab against hapten • BCR are crosslinked • B cells activated • Hapten-specific Ab made https://onlinelibrary.wiley.com/cms/asset/3c4429eec6cb-4db0-99e8-bc949de640fd/voxs12469-fig-0001- Image made by Dr. Prater Valency versus Affinity versus Avidity • Valency = number of binding sites • Affinity = binding strength between a single antigenic determinant (epitope) and a single antibody binding site • Avidity = total binding strength of all antibody-epitope pairs in multivalent Ag-Ab interactions • So, Ab molecules with more binding sites will potentially have greater avidity • However, not all Ab binding sites have equal affinity – some bind better (high affinity) and some bind worse (low affinity) https://images.novusbio.com/design/affinity-avidity.jpg Antibodies and Cross-Reactivity https://www.cusabio.com/manage/upload/202109/antibody-cross-reaction.png • Ab produced against 1 Ag can bind a different, BUT STRUCTURALLY SIMILAR, Ag ‒ Can also occur with T cells, but at a lower frequency due to the way TCR versus BCR recognize their respective antigens • Called cross-reactivity or molecular mimicry • Key – enough contact in binding to generate sufficient affinity to activate B cell • Cross-reactivity can play a role in autoimmunity (lecture 24). https://creativemeddoses.com/wp-content/uploads/2019/11/dilemma-of-antibody-JPGscaled.jpg Antibodies: General Structure • Immunoglobulin (Ig) domain ‒ Globular domain containing disulfide bond between cysteine residues ‒ Common structural motif in immunology • Fab region – variable region that binds Ag, formed by BOTH heavy and light chain ‒ Antigen-binding site – highly variable region at Nterminal of heavy and light chain that makes direct contact with Ag • Constant or Fc region – more conserved, mediates Ag-independent functions (e.g., opsonization through Fc receptors) Figure 4-2 – Basic Immunology, 6th ed., 2020 Recall: Generation of B Cell Receptors • Generation of diversity • Need 100+ trillion Ag-ic specificities to fight off all infections, but total genome is only ~35,000 genes! • Cannot encode each antibody sequence in a unique gene • “Cut-and-paste” system of VDJ recombination of chromosome to generate diversity in immune response using a limited number of genes • VDJ recombinase includes RAG1 or RAG2 enzyme (+ DNA ligase and other enzymes) • Recognizes RSS to select V, D, J, C alleles • Allelic exclusion – starts on one chromosome and inhibits recombination on second chromosome; to maintain clonality Figure 4-12 – Basic Immunology, 6th ed., 2020 Recall: BCR Combinatorial and Junctional Diversity • Combinatorial – different combinations of V, D, and J genes • Junctional – gaps left by RAG recombination filled in by nucleotides ‒ P nucleotides – “paired” with template ‒ N nucleotides – random and non-templated Figure 8-10 – Cellular and Molecular Immunology, 10th ed., 2022 Figure 8-11 – Cell. Mol. Immunology, 10th ed., 2022 Figure 8-12 – Cell. Mol. Immunology, 10th ed., 2022 Antibody Binds Antigen https://www.austincc.edu/apreview/ NursingPics/ImmunoPics/Picture30.jpg Figure 4-4 – Basic Immunology, 6th ed., 2020 • Monomeric immunogloblin molecule ‒ 2 heavy chains + 2 light chains ‒ Valency = 2 ‒ Whole monomeric Ig – ~150 kD; heavy chain – ~50 kD; light chain – ~25 kD (either kappa or lambda) • Antigen binding site – see last slide too! ‒ CDR (or HVR) ‒ “Lock and key” fit – high Ag-ic specificity • Hinge region – structural flexibility; helps in binding Figure 5-7 – Basic Immunology, 6th ed., 2020 Antibodies: General Functions • Antibodies have great variety of functions ‒ Some indirect – e.g., activating complement which generates anaphylatoxins and opsonins that promote inflammation and phagocytosis, respectively ‒ Some direct – e.g., neutralization • More in lecture 21 ‒ Details on functions ‒ Impact of Ig isotype Figure 8-1, Panel A – Basic Immunology, 6th ed., 2020 B Cell Receptor Complex • Recall cytoplasmic tail of BCR is too short to allow docking and assembly of signaling molecules needed to transmit B cell activation signals • Therefore, BCR must be able to associate with accessory molecules and transmit Ag binding signal to them for B cell activation to occur • BCR accessory molecules – Igα and Igβ – Can transmit activation signal due to presence of ITAM domain in cytoplasmic tail – Interact with Fc portion of BCR Figure 4-1 – Basic Immunology, 6th ed., 2020 Figure 4-10 – Basic Immunology, 6th ed., 2020 Recall: B Cell Development • Points of differentiation from T cell development – Heavy chain paired with “placeholder” pseudolight chain at first checkpoint (pre-BCR) – B cell is not mature when it leaves bone marrow – Gets multiple chances to make a functional BCR due to process of receptor editing – Alters immunoglobulin molecule WITHOUT CHANGING SPECIFICITY to optimize function – Affinity maturation and isotype switching • Receptor editing – When B cell fails a checkpoint test, a new light chain is made Figure 4-14 – Basic Immunology, 6th ed., 2020 Phases of a B Cell Response • Most details about this response in next hour (lecture 21) • Impacts on antibodies in this process ‒ Clonal expansion (next hour) ‒ Affinity maturation ‒ Isotype switching • WHY??? To optimize Ab functionality during the response → to get the most effective and efficient clearance of pathogen Figure 7-1 – Basic Immunology, 6th ed., 2020 Affinity Maturation • Once B cell is activated and antibody is being secreted, optimizing functionality of Ig molecule continues through two processes – Affinity maturation Figure 7-14 – Basic Immunology, 6th ed., 2020 – Isotype switching • Affinity maturation or somatic hypermutation – Spontaneous mutations occur as B cell proliferates – Mutations that increase affinity outcompete and survive – Mutations that decrease affinity lose the competition and die by neglect (lack of signal) – DOES NOT change antigenic specificity, but only binding affinity for that same Ag • Mutations occur with across time of response and with each exposure to Ag Figure 12-17 – Cellular and Molecular Immunology, 10th ed., 2022 Isotype Switching – Why? • Specialization for function • Isotypes ‒ IgM – C’ activation ‒ IgG1, IgG2, IgG3, IgG4 – opsonization, C’ activation (different isotypes – different pathways) ‒ IgA1 and IgA2 – mucosal responses (ltd differences between isotypes) ‒ IgE – mast cell responses Figure 7-12 – Basic Immunology, 6th ed., 2020 Comparative Structure of Antibody Isotypes • Monomers = single Ig molecule secreted – IgG, IgE, IgA, and IgD monomers found in blood • Multimers = multiple Ig molecules joined and secreted – Bound together by J (joining) chain – IgM pentamers in circulation – IgA monomers in circulation; only dimers secreted to mucosa Figure 4-3 – Basic Immunology, 6th ed., 2020 https://www.tebu-bio.com/blog/wp-content/uploads/2018/11/Fig-2-The-5-major-classes-ofimmunoglobulin.png Isotype Switching – Comparative Functions https://www.researchgate.net/figure/Structure-and-function-of-human-Ig-isotypes_tbl1_6795381 Isotype Switching – How? • B cells activated → secrete IgM • B cells encounter CD4 Th cells → receive costimulation and cytokines • Activation-induced cytidine deaminase (AID) produced • DNA recombination mediated by AID that cuts VDJ from Cµ gene to a new constant gene • Intervening DNA (i.e., constant regions) looped out and excised (lost) • New isotype secreted • An irreversible process Figure 7-13 – Basic Immunology, 6th ed., 2020 Figure 12-14 – Cellular and Molecular Immunology, 10th ed., 2022 The Results of Isotype Switching The DNA sequence is altered, so the isotype of BOTH the membrane-anchored form (i.e., the BCR) and the secreted form (i.e., the antibody) are permanently changed. https://www.cell.com/cell/pdf/S0092-8674(19)30278-8.pdf Choosing the Right Isotype • Similar to CD4 Th cells, B cells have a 3-signal activation hypothesis (next lecture) • Cytokine environment during activation is 3rd signal • Cytokine-induced signaling then turns on signals to activate or inhibit particular isotype switching Major Inducing Cytokines https://o.quizlet.com/.ETaflC2QME5O53KicKDIw.png Comparative Valency and Affinity of Antibody Isotypes Recall slide 10 above! Let’s expand on that know that we know about the immunoglobulin isotypes • Valency – how many epitopes the antibody molecule can bind. So… ‒ Valency of IgG, IgE, IgD, and IgA monomer = 2 (1 × 2) ‒ Valency of IgA dimer = 4 (2 × 2) ‒ Valency of IgM pentamer = 10 (5 × 2) • Affinity – binding strength antibody to a particular antigen ‒ More mature antibodies have higher affinity. ‒ IgG, IgE, IgA > IgM, IgD • Avidity – combination of affinity and valency (how many ABS present in that Ab and how strongly Ag binds to the ABS) RECALL: ABS = Antigen Binding Site Figure 5-14 – Cellular and Molecular Immunology, 10th ed., 2022 B Cell Activation Details in next lecture (#21), but here are the basics! • B cell encounters Ag • Type of Ag determines whether T cell help is needed for B cell activation ‒ T-dependent antigen ‒ T-independent antigen • If T cell help needed, have: ‒ CD40-CD40 ligand costimulation ‒ Cytokine signal(s) Figure 7-1 – Basic Immunology, 6th ed., 2020 T Cell Dependence in B Cell Responses • Nature of antigen determines if T cell help is needed for B cell activation – T-dependent Ag – protein or glycoprotein usually – T-independent Ag – polysaccharide, glycolipid, or rarely protein • Each initiates different activation and differentiation programs in B cells Figure 7-2 – Basic Immunology, 6th ed., 2020 Figure 7-18 – Basic Immunology, 6th ed., 2020 T-Dependent Antigens and B Cells • Proteins and some glycoproteins • Process ‒ Ag binds B cell, is processed, and presented on MHC-II ‒ Ag binds mac or DC, is processed, and presented on MHC-II ‒ Ag-loaded B cell binds activated CD4 Th cell that provides o Costimulation o Cytokine ‒ B cell fully activated → o Secretes IgM → o Switches to IgG, IgE, or IgA → o Becomes memory B cell Figure 3-17, panel B – Basic Immunology, 6th ed., 2020 T-Independent B Cell Activation • Polysaccharides (e.g., LPS), lipids, glycolipids ‒ KEY – have repetitive structure • Process ‒ Repeated Ag binds multiple receptors on surface of B cell ‒ Crosslinking of receptors activates B cell directly ‒ Begins to secrete IgM • Production is generally short-lived, but… • Compound (i.e., Ag) generally stable, so continues to stimulate response • DOES NOT (see notes) https://images.slideplayer.com/ 27/8936467/slides/slide_2.jpg ‒ Isotype switch ‒ Differentiate to memory cells https://microbeonline.com/t-dependent-antigen-and-t-independentantigen/ Subsets of T-Independent Antigens • Based on which receptors on B cell are crosslinked • T-independent 1 (TI-1) Ag – Cross-linking of BCR PLUS another receptor(s) – Most frequently, TLR are the 2nd receptor crosslinked • T-independent 2 (TI-2) Ag – Crosslinking of BCR ONLY – Often occurs in marginal zone of 2° lymph tissue (e.g., spleen, LN) – Activated DC (less commonly mac, NK) secrete cytokine that can induce isotype switching and plasma cell differentiation https://images.slideplayer.com/24/7552515/slides/slide_5.jpg Summary I • What are the humoral immune responses? ‒ Innate – complement cascades ‒ Adaptive – antibody responses • Differentiate antigen, epitope, immunogen, hapten, affinity, and avidity. • What are antibody isotypes and the structural elements in monomeric and multimeric forms of each isotype? Know valency of each Ab form. ‒ Antibody isotype – altered heavy chain for specialized function, driven by cytokine signals during B cell activation, variable region (antibody binding site) does not change ‒ Monomer – 2 heavy chains and 2 light chains with variable regions that bind Ag and more conserved constant region for assembly of signaling complex and specialized functions, hinge region that provides flexibility for binding ‒ Multimers – IgM – pentamers; IgA – dimers; monomers of multimer bound together by J (joining) chain • Recall processes that contribute to the generation of diversity. ‒ Combinatorial diversity – mixing and matching of V, D, and J gene segments to make BCRs of different specificities ‒ Junctional diversity – addition of random nucleotides to fill in gaps of VDJ recombination events left during ligation Summary II • What are the major steps and significance of B cell development? ‒ VDJ recombination to mu constant region, checkpoints with receptor editing to be sure BCR is functional and does not react to self; migration to LN or spleen; activation upon contact with Ag ‒ Continued contact with Ag drives somatic hypermutation (point mutations) that leads to affinity maturation and then to isotype switching ‒ Purpose – greatest Ag-ic diversity in B cell responses with highest affinity and specialized functionality (isotype switching) so response is appropriately matched to pathogen of infection • Compare and contrast T-independent and T-dependent antigens. ‒ T-independent – generally polysaccharide, lipid, or glycolipid; binds and crosslinks multiple BCR or innate receptors. If sufficient receptors are cross-linked, BCR activation occurs producing a short-lived IgM responses. Generally does NOT isotypes switch, affinity mature, or make plasma or memory cells. ‒ T-dependent – protein or glycoprotein; binds BCR and some Ag endocytosed and processed for loading to MHC-II; presents to CD4 Th cell and gets costim and cytokine help from T cell which allows affinity maturation, isotype switching, differentiation of plasma cells, and generation of memory cells for long-lived Ab response References • Basic Immunology, Abbas, Lichtman, and Pillai; 6th ed., 2020 • Immunology for Medical Students, Helbert, 3rd ed., 2017 • Cellular and Molecular Immunology, Abbas, Lichtman, and Pillai, 10th ed., 2022 • Journal articles and publications as noted at images on each slide • ComBank question bank at www.truelearn.com B Cells in Action! • https://www.youtube.com/watch? v=548wQ5C6ufQ&list=PLS_8RsEvYc3XEoHlXgrhg3uEY6WFc9DtL&index=14 • https://www.youtube.com/watch?v=iIJulGXWm6Q – T-independent B cells • https://www.youtube.com/watch?v=_qF2jEe7ZWc – T-dependent B cells • https://www.youtube.com/watch? v=L32Na8fGjzA&list=PLS_8RsEvYc3XEoHlXgrhg3uEY6WFc9DtL&index=12 – B cell activation, clonal expansion, plasma cells, Ab secretion • https://www.youtube.com/watch?v=gyTHXjVUPWw – isotype switching (NOTE: this is a little more detail than you need to know, e.g., the signaling process) • https://www.youtube.com/watch?v=qGsyBwDVnTU&list=PLBF42DFD0B2C1E726 – affinity maturation SUPPLEMENTAL SLIDES Isotype Switching and Ig Functions https://www.researchgate.net/figure/3-Human-Antibody-