GOD B cells and Antibodies April 2024.pptx

The Adaptive Immune response BMS2045 19th April 2024 9am LTD Part 3- Generation of Diversity, B cells and Prof Deborah DunnAntibodies Learnin g profile. Describe how a diverse repertoire of lymphocytes can be created. Name and describe the different effector functions of antibodies Know the differen...

The Adaptive Immune response BMS2045 19th April 2024 9am LTD Part 3- Generation of Diversity, B cells and Prof Deborah DunnAntibodies Learnin g profile. Describe how a diverse repertoire of lymphocytes can be created. Name and describe the different effector functions of antibodies Know the difference between Tdependent and T-independent B cell responses. Describe how B cells become activated, proliferate and mature including the role of T cell help and the germinal centre Know the difference between isotype class-switching, somatic hypermutation and affinity maturation Basic principle of Adaptive Immune System Start with very diverse repertoire so that there is more chance of finding a match to the pathogen Expand (and in some cases improve) the matched cell Maintain some of these matched cells for future “memory” against the pathogen What do we mean when we say a “Diverse repertoire ” Ie What defines B cell and T cell pathogen recognition? Comparison of B and T cell receptors summary T cell receptor B cell receptor gd or ab Heavy and light chain Membrane bound Membrane bound OR soluble as ANTIBODY Recognises peptide antigen presented to it by MHC on other cells Recognises antigens on whole molecules Can be different isotypes depending on the Fc region B cells: Start off as cells with the receptor on the surface of the cell (BCR). The receptor recognises whole antigen (without the need for it to be fragmented and presented) ANTIBODY can bind to whole pathogens and facilitate a range of functions –depending on its Fc region Differentiate into plasma cells that secrete the receptor as ANTIBODY Antibody is important because it: Neutralises toxins and viruses by blocking their interaction with cells Opsonises pathogens to promote phagocytosis and killing activity by other cells Activates the complement cascade which helps kill pathogens Agglutinates particles (pathogen debris, viruses etc) Mediates Antibody dependent cellmediated cytotoxicity (ADCC) Generatio n of lymphocyt e diversity Overview of B cell Development Initial generation of diversity (gene rearrangement and heterodimer formation) happens as the cells are first formed, before they see exogenous antigen. In the case of B cells this happens in the Bone marrow* In the case of T cells this happens in the Thymus * Actually called B cell because it was originally discovered in the Bursa Fabricus in chickens BCR/Antibody Structure Variable region with two binding sites which determine the ANTIGEN SPECIFICITY Fv region Fc region – constant region which determines the CLASS of the antibody and thereby the FUNCTION BCR/Antibody Structure Heavy BCR/Antibody Structure Light chain BCR/Antibody Structure Two identical heavy chains and two identical light chains Lymphocytes – Generation of Diversity B cell receptor (bone marrow) T cell receptor (Thymus) Gene Rearrangement Heavy and Light chain (x2) Hypermutation a with b g with d No Class Switching No Heterodimer formation VDJ gene rearrangement – heavy, beta, delta genes VJ gene rearrangement – light (kappa or lambda), alpha, gamma genes How many different B cell receptors (Antibodies) do we need for diversity? Human genome is 3 billion base pairs Much DNA is intergenic The total estimated number of human genes is only 20,000-25,000 Estimates of diversity in microbes are difficult, but tend to be in the order of half a million different species Diversity of pathogens We can’t make enough pattern recognition receptors to deal with all pathogens, especially since many pathogens can evolve faster than us. The solution - Gene Rearrangement One whole gene is made by combining different segments of genes. (Pick ‘n’ Mix) The enzymes that do this are RAG1 and RAG2 (Recombination Activating Genes), V regions D regions J regions Variable regions of the antibody are made up of two or three gene segments – VJ or VDJ depending whether it is the light chain or the heavy chain V J C EITHER kappa light chain OR lambda light chain + Heavy chain V DJ C Rearrangement of Immunoglobulin Genes IGHV regions IGHD regions IGHJ regions V region 5’- Fw1 CDR1 Fw2 CDR2 Fw3 D CDR3 region J -3’ Why Gene rearrangement?? It’s all in the numbers……………… Mathematics of combinatorial diversity IGHV regions 9 genes. A, B, C, D. 1.2.3. x.y = 9 proteins OR When 1 protein is encoded by a combination of group 1, group 2, group 3. A, B, C, D. 1.2.3. x.y = 24 proteins (i.e. 4x3x2) IGHD regions IGHJ regions Mathematics of combinatorial diversity 9 genes. A, B, C, D. 1.2.3. x.y = 9 proteins OR When 1 protein is encoded by a combination of group 1, group 2, group 3. A, B, C, D. 1.2.3. x.y = 24 proteins i.e. 4x3x2 A1x B1x C1x D1x 4 Mathematics of combinatorial diversity 9 genes. A, B, C, D. 1.2.3. x.y = 9 proteins OR When 1 protein is encoded by a combination of group 1, group 2, group 3. A, B, C, D. 1.2.3. x.y = 24 proteins i.e. 4x3x2 A1x B1x C1x D1x A2x B2x C2x D2x 4 x3 A3x B3x C3x D3x Mathematics of combinatorial diversity 9 genes. A, B, C, D. 1.2.3. x.y = 9 proteins OR When 1 protein is encoded by a combination of group 1, group 2, group 3. A, B, C, D. 1.2.3. x.y = 24 proteins i.e. 4x3x2 A1x B1x C1x D1x A2x B2x C2x D2x 4 x3 A3x B3x C3x D3x A1y B1y C1y D1y A2y B2y C2y D2y x2 A3y B3y C3y D3y Mathematic s of Ig Gene Rearrangem ent Junctional Region Diversity The actual number of different Ig gene possibilities is more than 5 million because the region where the different segments join together creates more diversity. This is also true for the T cell receptor Nucleotides can be accidentally removed from here V region D region J region Random nucleotides can be deliberately inserted here (by an enzyme called TdT for short. Terminal deoxynucleotidyl Transferase) C How many different antibodies can we really make? Estimates in the region of 10 billion …and that’s not all! We can “Class switch” a particular antibody to change its function After a B cell has seen antigen - Class switching Keep the ANTIGEN SPECIFICITY in the Variable region Switch the CLASS (and therefore FUNCTION) of the antibody. Classes and subclasses of Antibody Ig A Ig G Ig E Ig D Ig M IgG 1 IgG 2 IgG 3 IgG 4 IgD Membrane-bound form of Immunoglobulin on Naïve B cells (B cells early in development before they see antigen) The default immunoglobulin that B cells start with in development. IgM The Fc region enables the molecules to form pentamers – which is useful in forming immune complexes, but which means that the molecules are too large to diffuse into tissues or cross the placenta. Very efficient at activating complement through the classical activation pathway. IgG is the main antibody secreted into the blood after class switching. IgG It is very good at opsonisation – coating pathogens so that phagocytic cells can recognise them. Pathogens coated in IgG also become targets for killing by Natural killer cells – antibodydependent cellular cytotoxicity There are 4 subclasses of IgG (IgG1,IgG2 etc) Is the mucosal antibody – produced by B cells at mucosal surfaces and secreted into breast milk IgA It is actively secreted across mucosal surfaces It can form a dimer, which helps protect it from enzymatic breakdown (such as occurs in the gut), but generally exists in monomeric form in the plasma There are two Subtypes (IgA1, IgA2) IgE is important in parasitic infection. It binds to the surface of Mast cells via it’s Fc receptor – even in the absence of antigen IgE Antigen cross-linking of IgE on Mast cells causes the mast cells to degranulate Usually low in concentration, but higher in cases of allergy – especially immediate hypersensitivity Who wants to be an immunology millionaire? Post-activation B cell development B cell receptor (bone marrow) T cell receptor (Thymus) Gene Rearrangement Heavy and Light chain (x2) Hypermutation a with b g with d No Class Switching No Heterodimer formation Happen AFTER B cell activation Affinity maturation and Class switching Are unique to the B cell receptor/antibody Both take place in the specialised microenvironment of the Germinal Centre and require an enzyme called Activation Induced Cytidine Deaminase (AID) Both require help from outside the B cells in the form of cytokines and contact with other cells – especially T helper (Th2) cells B cell activat ion There are three types of antigen that can activate B cells: T-independent type I (TI-I) T-independent type II (TI-II) T-dependent (TD) B cell activati on A number of different receptors are involved: B cell receptor – specificity Innate immune receptors Co-stimulatory molecules Cytokine receptors Stimulation of these receptors is by antigen and by helper cells – e.g. CD4+ Tfh cells T-independent type I (TI-1) Polyclonal activators without needing reference to specific receptor. e.g. via TLRs “Pattern recognition receptors” Tindependent type II (TI-2) Large molecules with a repeating determinant (eg H. influenzae b, S. pneumoniae, N. meningitidis) which bind to multiple receptors on the cell surface causing cross linking and activation. Traditionally said that no memory B cells are formed. Can accept cytokine help – possibly from DC’s? Or T cells? T-dependent (TD) Protein antigens. The B cells process the protein and present the peptide antigen on the surface with MHC class II molecules in order to recruit T cell help for the response. Memory B cells are formed. B-T Interactions: The BCR captures antigen to present to the T cell Up-regulation of Co-stimulatory molecules TCR “Signal 1” Signalling B cell MHC II T cell CD4 Antigen internalisation and processing The T cell provides helpful signals to the B cell (eg CD40-CD40L (CD154)) “Signal 2” TNF-R family of molecules, involved in: CD40 B cell proliferation, B cell differentiation isotype switching upregulation of surface molecules contributing to antigen presentation and co-stimulation development of the germinal centre – inhibition of cell death Ac s e t a tiv CD40 ligand T-dependent B cell Development – the Germinal Centre A germinal centre in the spleen T CCL19 CCL21 CBCXCL12 CXCL13 CC MGZ MT IgD, Rhesus macaque spleen. T: T cell zone, MT: mantle cell zone, MGZ: Marginal Zone B cell selection B cells that recognise the pathogen expand V D J C Each member of this B cell family has a copy of the gene that encodes the particular specificity for antigen Keep these Worse bindin g As B cells expand they mutate their Ig genes The best ones are kept No chang e Better bindin g Germinal Centre Affinity Maturation Selection in LIGHT ZONE FDC Hypermutation in DARK ZONE Memory B cells Plasma cells B cells in here Centroblasts Centrocytes T cell Bcl2-ve BAFF limited Ag:Ab complex Centroblast Co-stimulation signals & cytokines Centrocyte FDC TFH Follicular helper T cell, Somatic hypermutation DELIBERATE mutation of Ig genes Multi- step process Starts with enzyme AID, at hotspots AID is also involved in class switching V Variable region with two binding sites which determine the ANTIGEN SPECIFICITY D J C Fc region – constant region which determines the ISOTYPE of the antibody and thereby the FUNCTION Arrangement of Constant region genes at the IgH locus V region 5’- Fw1 CDR1 Fw2 CDR2 Fw3 D J -3’ C CDR3 region Gene name Cm Cd Cg3 Cg1 Ca1 Cg2 Cg4 M D G3 G1 A1 G2 G4 IgG3 IgG1 IgA1 IgM IgD IgG2 Isotype produced IgG4 Ce E IgE Ca2 A2 IgA2 Overview of T dependent B cell response Extrafollicular region IgG Naïve T cell Germinal Centre Follicles IgM Mutated memory B cell Unmutated shortlived plasma cells Survival B cell blast RIP Naïve B cell Germinal centre B cells Hypermutation Mutated long lived plasma cell IgG Plasmablasts Switched memory Antigen specific memory Antigen specific IgG Further Readin g https://www.immunology.org/public-inform ation/bitesized-immunology/immune-devel opment/b-cell-activation-and-the-germinal -centre https://www.immunology.org/public-inform ation/bitesized-immunology/immune-devel opment/generation-b-cell-antibody-diversi ty Kuby