The Generation of Lymphocyte Antigen Receptors PDF

The generation of Lymphocyte Antigen Receptors Literature: Chapter 5, Janeway’s Immunobiology ETH Zurich Lecture on “Pharmaceutical Immunology I” Prof. Dr. Cornelia Halin Winter 535-0830-00L HS 2024  Revision from Cha...

The generation of Lymphocyte Antigen Receptors Literature: Chapter 5, Janeway’s Immunobiology ETH Zurich Lecture on “Pharmaceutical Immunology I” Prof. Dr. Cornelia Halin Winter 535-0830-00L HS 2024  Revision from Chapter 1 What you already know…. The adaptive immune response relies on clonal selection and expansion: Upon recognizing foreign antigen, an antigen-specific T cell or B cell clone is selected: It proliferates and differentiates into an effector cell: (Plasma B cells, T helper cell, cytotoxic T cell) Human antibody repertoire: ≈ 1013 clones possible Human T cell receptor repertoire: ≈ 1018 clones possible Þ The diversity of the human antibody and TCR repertoire is not encoded by individual genes! This Chapter explains how the diversity in the T cell and B cell receptor repertoire is generated 1 Content 1) Primary immunoglobulin gene rearrangement 2) T-cell receptor gene rearrangement 3) Structural variation in immunoglobulin constant regions 2 1) Primary immunoglobulin gene rearrangement What we learnt in Chapter 4: The hypervariable regions lie in discrete loops of the folded structure Hypervariable regions are also called complementarity- determining regions (CDRs) => named CDRs because the surface they form is complementary to that of the antigen they bind 3 CDRs in the heavy chain and 3 CDRs in the light chain variable domain may contact the antigen 3 => total of 6 CDRs  1) Primary immunoglobulin gene rearrangement In a mature B cell the variable domain of an antibody is encoded by a single V-region exon Variable domains have the typical immunoglobulin fold composed of nine β sheets The antibody-binding site is formed by three loops of amino acids known as CDR1, CDR2, and CDR3 (or hypervariable regions HV1, HV2, and HV3) The CDRs (red) are separated from each other by framework regions (yellow), which are mostly the same in different antibodies. 4  1) Primary immunoglobulin gene rearrangement The CDR3 (HV3) originates from two or three individual gene segments The different variable regions (VH and VL) are not encoded in their final version in the germline. During development, B cells and T cells undergo rearrangement of the gene locus encoding their B cell and T cell receptor. The variable region exons are rearranged from 2 or 3 different gene segments (so- called V, D and J segments). V-segment: encodes the CDR1 & CDR2 J-segment: encodes the CDR3 and last framework region D: segment: only present in VH The joint region (border) of these segments fall into the CDR3. The CDR3 of the VH or VL chain therefore is highly variable between different B cells. 5  1) Primary immunoglobulin gene rearrangement The V-region genes are constructed from V, (D) and J gene segments Light chain V-region genes are constructed from V and J segments Heavy chain V-region genes are constructed from V, D and J segment The light chain C region is encoded in a separate exon The heavy chain C- region is encoded in multiple separate exons Heavy and light chain C regions are joined to the V regions by splicing of the mRNA after transcription 6  1) Primary immunoglobulin gene rearrangement Recombination of the heavy chain V-region The recombination process that generates a complete heavy chain V region occurs in two separate stages: First, a DH gene segment (“D” stands for diversity!) is joined to a JH gene segment Then a VH gene segment rearranges to DJH to make a complete VH region exon The border region of the V(D)J segments lie in the CDR3 “The border region of the V(D)J segments lie within the CDR3 loop. This has important consequences….namely? 7  1) Primary immunoglobulin gene rearrangement Multiple V, (D) and J segments are present at the immunoglobulin heavy and light chain loci The random selection of V, (D) and J segments produces the high variability between V regions of immunoglobulins. Some gene segments have accumulated mutations that prevent them from encoding a functional protein: these are termed ‘pseudogenes.’ Rearrangements that incorporate a pseudogene and will be nonfunctional. The genes are organised into three clusters or genetic loci: - heavy chain locus - K (kapa) or l (lambda) light chain loci 8  1) Primary immunoglobulin gene rearrangement The germline organization of the immunoglobulin heavy- and light-chain loci in the human genome There are three sets of immunoglobulin chains — the heavy chain, and two equivalent types of light chains, the κ and λ chains The immunoglobulin gene segments that encode these chains are organized into three clusters or genetic loci: the κ, λ and heavy-chain loci each locus can assemble a complete V-region sequence 9  1) Primary immunoglobulin gene rearrangement Heavy chain locus contains several constant regions Heavy chain locus contains a series of C regions arrayed one after the other each corresponds to a different Cµ : constant heavy chain sequence of IgM Cd : constant heavy chain sequence of IgD immunoglobulin isotype: IgM, IgD, IgG, IgE, Cg1-2a: constant heavy chain sequence of IgG3, IgA IgG1,IgG2b, IgG2a Ce : constant heavy chain sequence of IgE Ca : constant heavy chain sequence of IgA The isotype (C region chosen) will determine the effector functions of the antibody molecules The first isotypes produced by B cells that leave the bone marrow are IgM and IgD. (e.g. to IgE) Expression of other isotypes (e.g. IgG) is regulated by DNA rearrangements that occur once the mature B cell gets activated later in life, in a secondary lymphoid organ. This process is referred to as “class switching” (Chapter 10). 10  1) Primary immunoglobulin gene rearrangement Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences DNA rearrangement is guided by conserved noncoding DNA sequences, i.e. conserved recombination signal sequences (RSSs) that flank the gene segments encoding V, D and J regions where the recombination needs to take place. => similar mechanism for heavy- and light-chain locus Typically, the coding joint junction is imprecise; i.e. nucleotides can be added or lost between joined segments during the rearrangement process. This adds to the variability in 11 the V-region sequence, called junctional diversity  1) Primary immunoglobulin gene rearrangement Enzymatic steps in RAG-dependent V(D)J rearrangement The complex of enzymes that act in concert to carry out somatic V(D)J recombination is termed the V(D)J recombinase The lymphoid-specific components of the recombinase are called RAG-1 and RAG-2, and they are encoded by two recombination-activating genes, RAG1 and RAG2 RAG1 and RAG2 are essential for V(D)J recombination Only expressed during lymphocyte development (i.e. while assembly of antigen receptors occurring) 12 CDR3: diverse due to junctional diversity! RAG-1/2 V J Magnification from previous slide (Fig. 5.8) CDR1 and CDR2 are encoded within the V gene segment CDR3 in the VL comprises the border of V and J gene segments CDR3 in the VH, formed by V,D and J gene segments In both VH and VL, the diversity of CDR3 is significantly increased by the addition and deletion of nucleotides during the formation of the junctions between gene segments. => imprecise opening of the hairpins! CDR3: Creation of additional diversity by random addition and subtraction of nucleotides at the gene segment junctions How it works (no need to remember): RAG proteins generate DNA hairpins at the coding ends of the V, D, or J segments, after which another enzyme (artemis) catalyzes a single-stranded cleavage at a random point within the coding sequence but near where the hairpin was first formed P-nucleotides are so called because they make up palindromic sequences added to the ends of the gene segments Random N-nucleotides are added by the enzyme terminal deoxynucleotidyl transferase (TdT) “random addition and subtraction of nucleotides” Surely, this will create diversity, but….. what could be further consequences?  1) Primary immunoglobulin gene rearrangement Diversity of the immunoglobulin repertoire is generated by 4 processes 1. There are multiple versions of each V (D) J segment present, allowing for different combinations. This combinatorial diversity is responsible for a substantial part of the diversity of V regions 2. Junctional diversity is introduced at the joints between the different gene segments as a result of the addition and subtraction of nucleotides by the recombination process. 3. Many different combinations of VH and VL (which together form the antigen-binding site) are possible => Approx. 1011 different receptors possible! 4. Finally, once B cells get activated in secondary lymphoid organs, somatic hypermutation introduces point mutations into the rearranged V-region genes, leading to a further enhancement of diversity and binding affinity. (Chapter 10) 15 Take-Home Messages The antigen receptors of lymphocytes are remarkably diverse the same basic mechanism account for this diversity in developing T and B cells immunoglobulin and T-cell receptor chains are assembled by somatic recombination from sets of separate gene segments that together encode the variable region; V, (D), J segments Diversity is generated by 3 mechanisms: V(D) J joining imprecise joining at the junctions of the segments (e.g. addition of an amino acid) association of two different chains to form a complete T cell receptor or antibody Important enzymes mediating recombination: RAG-1/RAG-2 16 (and Artemis, TdT, DNA ligase)  2) T-cell receptor gene rearrangement 2) T-cell receptor (TCR) gene rearrangement TCR α and β chains each consist of a variable (V) amino-terminal region and a constant (C) region The TCR’s variable domains (Va and Vb) can be superimposed with an antibody’s VH and VL The TCR gene segments are arranged similarly as immunoglobulin gene segments and by the same enzymes a-chain locus: V (70-80) and J (61) segments b-chain locus: V (52), D (2) and J (7) segments 17  2) T-cell receptor gene rearrangement TCR α and β chain gene rearrangement and expression The organization of the TCR gene segments is broadly homologous to that of the immunoglobulin gene segments The gene segments are organized into a TCRα and TCRb locus The T-cell receptor gene segments rearrange during T-cell development to form complete V-domain exons 18  2) T-cell receptor gene rearrangement The TCR gene segments are arranged similarly as immunoglobulin gene segments and by the same enzymes Similarities: As in the immunoglobulin gene rearrangement the main enzymes involved are RAG1/2, Artemis, TdT etc. The CDR1 and CDR2 regions are encoded in the V gene segment, whereas the CDR3 is created by V(D)J joining Differences: Less diverse C region The C regions of the TCRα and TCRβ loci are much simpler than those of the immunoglobulin heavy-chain locus: The T-cell receptor C-region genes encode only transmembrane polypeptides. There is only 1 Cα gene, and although there are 2 Cβ genes, they are very closely homologous and functionally equivalent => This is in contrast to the different isotypes of antibodies. 19  2) T-cell receptor gene rearrangement Recombination signal sequences flank T-cell and B cell receptor gene segments T-cell receptor gene rearrangement takes place in the thymus The mechanics of gene rearrangement are similar for B and T cells The T-cell receptor gene segments are flanked by 12-bp and 23-bp spacer recombination signal sequences (RSSs) that are homologous to those flanking immunoglobulin gene segments What might be the phenotype of RAG-1 or RAG-2 deficiency? 20  2) T-cell receptor gene rearrangement The most variable parts of the T-cell receptor (TCR) interact with the peptide of a peptide:MHC complex Remember: The TCR does not only bind the antigenic peptide but also the MHC molecule => The TCR binds the peptide: MHC complex The less variable CDR1 and CDR2 loops of a T-cell receptor mainly contact the relatively less variable MHC component of the ligand The highly variable CDR3 regions mainly contact the unique peptide component 21  2) T-cell receptor gene rearrangement Summary: TCRs T cell receptors (TCRs) of a:b T cells are structurally similar to immunoglobulins and are encoded by homologous genes. TCRs are assembled by somatic recombination from gene segments (V(D)J), in analogy to immunoglobulins. The greatest part of T cells diversity lies in the junction of the V(D)J segments, which form the CDR3. The CDR3 is the part that makes most contact with the peptide of the peptide:MHC complex. 22 3. Structural variation in the immunoglobulin constant regions We have focused on the mechanisms of assembly of the V regions for immunoglobulins and T-cell receptors (TCRs). => Now we turn to the C regions. Immunoglobulin C regions: can be made as both a trans- membrane receptor and a secreted antibody The heavy-chain locus encodes different C regions (CH). They determine the antibody isotype, depending on the C region used by the heavy chain The light-chain C regions (CL) provide only structural attachment for V regions, and there seem to be no functional differences between ”l and k light chains” TCR C regions: support the V regions and anchor the receptor into the membrane do not vary after assembly of a complete receptor gene 23  3) Structural variation in immunoglobulin constant regions Differences between the immunoglobulin isotypes: Number of constant heavy chain domains (4 in IgM and IgE) Presence of hinge region (replaced by an additional C-domain in IgM and IgE) Glycosylation sites: IgGs are only glycosylated at one site on heavy chain (Asn 297) => important for ADCC (therapeutic activity so some anti-cancer Abs) Presence and location of disulfide bonds help to protect the body from infection in different ways => different effector functions 24  The isotype determine the effector functions of antibodies Neutralisation: IgA, IgG Opsonization: IgG Complement activation: IgM, (IgG) discussed in Chapter 10 Activation of mast cells: IgE Antibody-dependent cellular cytotoxicity (ADCC): IgG Transport across placenta (IgG) or into secreted fluids (gut, saliva, tears: IgA) What determines the ability of the individual isotype to exert a certain effector function? 25  3) Structural variation in immunoglobulin constant regions Summary of the physical and functional properties of the human immunoglobulin isotypes 26  3) Structural variation in immunoglobulin constant regions IgM IgM is the first class of secreted immunoglobulin produced after activation of a B cell. The IgM has a high molecular weight, as it is secreted as a pentamer. It is present in bloodstream, but not in tissues (too big to efficiently extravasate). The IgMs initially secreted in an immune response have a low affinity (not yet subjected to affinity maturation) – but this is compensated IgM pentamer by high avidity IgM is an excellent activator of complement  3) Structural variation in immunoglobulin constant regions IgD Immediately 3' to the µ gene lies the δ gene, which encodes the C region of the IgD heavy chain. Alternative splicing product of initial RNA IgD is co-expressed with IgM on the surface of almost all mature B cells, but is secreted in only small amounts by plasma cells The unique function of IgD is still unclear and a matter of active research. 28  3) Structural variation in immunoglobulin constant regions IgG most abundant immunoglobulins found as four subclasses (IgG1, IgG2, IgG3, and IgG4) that are named by decreasing order of their abundance in serum. different subclasses vary in their effector function efficiency: complement activation, opsonization, or ADCC (antibody-dependent cellular cytotoxicity (Chapter 10)) all therapeutic antibodies are IgGs exceptionally long half-life of 2-4 weeks transferred across the placenta (from mother to foetus) 29  3) Structural variation in immunoglobulin constant regions IgE and IgA IgA can be found in the bloodstream, but it also acts in the defense of mucosal surfaces is secreted into the gut and respiratory tract, and also into mother’s milk. can be secreted either as a monomer or as a dimer. IgE contain an extra CH domain instead of the hinge-region induce mast cell degranulation or activation of eosinophils and basophiles involved in defense against multicellular parasites, but also in allergic diseases like allergic asthma or rhinitis. 30  3) Structural variation in immunoglobulin constant regions IgM and IgA can form multimers by interacting with the J chain Usually synthesized as multimers in association with an additional polypeptide chain, the J chain Dimeric IgA: The monomers have disulfide bonds to the J chain as well as to each other Pentameric IgM: Monomers are crosslinked with disulfide bonds to each other and to the J chain 31  2) T-cell receptor gene rearrangement Summary: immunoglobulin constant region The classes of immunoglobulins are defined by their heavy-chain C regions, so called isotypes: IgM, IgD, IgA, IgE, IgG Heavy chain C regions are present in a cluster downstream (3‘) of the V(D)J segments A productively rearranged V-region initially pairs with µ and δ CH genes, which are co-expressed in naive B cells by alternative splicing from an mRNA transcript containing both exons. Antibodies can be expressed as surface bound („B Cell Receptors“) or soluble molecules. This is achieved by differential splicing of mRNA to either include a hydrophobic transmembrane region or a secretable tailpiece The antibody that a B cell secretes upon activation recognizes the antigen that initially activated the B cell – but the constant parts may vary (isotype switching). 32