Lecture 7: Generation of Lymphocyte Antigen Receptors PDF

D Lecture 7 Generation of Lymphocyte Antigen Receptors 09/26/2024 Learning Objectives Primary Ig gene rearrangement TCR gene rearrangement and expression General logic: randomly generate a large repertoire, then select for the useful clone...

D Lecture 7 Generation of Lymphocyte Antigen Receptors 09/26/2024 Learning Objectives Primary Ig gene rearrangement TCR gene rearrangement and expression General logic: randomly generate a large repertoire, then select for the useful clone Pools of B-cell clones High-affinity B-cell clone Clonal B-cell Selection Antigen The CDR3 region originates from two or more individual gene segments that are joined during lymphocyte development Variable regions of the receptor chains are not directly encoded as a complete immunoglobulin domain by a single DNA segment. The variable regions are encoded by gene segments that each encode only a part of the Ig domain. During the development of each lymphocyte, these gene segments are rearranged by a process of somatic DNA recombination to form a complete and unique variable- region coding sequence. This process is known generally as gene rearrangement. A V gene segment encodes the CDR1 and CDR2 loops, and the CDR3 loop is formed by sequences from the end of the V gene segment and the beginning of the J gene segment, and by nucleotides added or lost when these gene segments are joined during lymphocyte development Germline organization of human Ig gene loci Three separate loci encode all of the Ig H chains, the Ig L chains, and the Ig L chains. At the 5’ end of each Ig locus, there is a cluster of V gene segments. At varying distances 3’ of the V gene segments are several J segments. Between V and J segments in Ig H chain, there are additional D segments. The C (constant) region genes are at 3’ end of J segments – single C and 4 C Fig. 5.5 V regions are constructed from gene segments VJ VDJ V = variable D = diversity J = joining Fig. 5.3 Combinatorial diversity Light chains Heavy chain Segment   H V 35 30 45 D 23 J 5 5 6 Possible V light chains = 35x5 = 175 Possible V light chains = 30x5 = 150 Possible V heavy chains = 45x23x6 = 6210 Possible unique V regions = 175x6210 = 1,086,750 Possible unique V regions = 150x6210 = 931,500 Total gene segments = 149 Fig. 5.4 Theoretical Combinatorial Diversity = 2,018,250 Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences Fig. 5.6 The 12/23 Rule V gene segments are joined by recombination [V(D)J Recombination] V(D)J recombinase recognizes 12/23 RSS and brings the exon together Recombination of V and J exons occurs by deletion of intervening DNA and ligation of the V and J segments Red arrows indicate the sites where germline sequences are cleaved before their ligation to other Ig gene segments Image Credit: Cellular and Molecular Immunology. 10 th Ed. Abbas, Lichtman, Pillai Enzymatic steps in V(D)J Recombination STEP 1: Synapsis – the process by which two distant selected coding segments and their adjacent RSSs are brought together by a chromosomal looping event and held in position for subsequent cleavage, processing, and joining. STEP 2: Cleavage – A lymphocyte-specific enzyme called the V(D)J recombinase creates double-stranded breaks at RSS-coding sequence junctions. The V(D)J recombinase is composed of two molecules each of two different proteins called RAG1 and RAG2 (recombination-activating gene). RAG genes are expressed only in developing B and T cells. RAG protein is inactivated in proliferating cells Fig. 5.8 Enzymatic steps in V(D)J Recombination STEP 3: Hairpin Opening and End Processing – After the formation of double-strand breaks, hairpins must be opened at coding junctions, and nucleotides may be added to or removed from coding ends to create even greater diversification. Artemis is an endonuclease that opens up the hairpins. A lymphoid-specific enzyme, called terminal deoxynucleotidyl transferase (TdT), adds nucleotides to broken DNA ends. STEP 4: Joining—The broken cording ends and signal ends are brought together and ligated by a double-strand break repair process found in all cells called nonhomologous end joining (NHEJ). Several ubiquitous proteins participate in NHEJ. KU70 and KU80 bind to the breaks and recruit the catalytic subunit of DNA-dependent protein kinase (DNA- PK), a DNA repair enzyme. DNA-PK also phosphorylates and activates Artemis. Ligation of the processed broken ends is mediated by DNA ligase IV and XRCC4. Fig. 5.8 Diversity of antigen receptor genes V D J segments segments segments Image Credit: Cellular and Molecular Immunology. 10 th Ed. Abbas, Lichtman, Pillai The introduction of P– and N- nucleotides diversifies the joints between gene segments – Junctional Diversity 1. Formation of DNA hairpins leads to ligation of heptamer sequences, forming a signal joint (not shown). 2. Artemis:DNA-PK cleaves DNA hairpin at random sites, producing single-strand DNA ends (indicated by arrows). 3. Single-stranded ends may have complementary nucleotides, forming short palindromic sequences (e.g., TCGA, ATAT). 4. Presence of terminal deoxynucleotidyl transferase (TdT) adds random nucleotides (N) to single-strand ends (indicated by shaded box). Fig. 5.11 The introduction of P– and N- nucleotides diversifies the joints between gene segments – Junctional Diversity 5. Two single-strand ends pair together. 6. Unpaired nucleotides are trimmed by exonucleases. 7. Final coding joint repaired via DNA synthesis and ligation, retaining P- and N-nucleotides (light blue shading). 8. Random insertion of P- and N-nucleotides creates unique markers, useful for tracking individual B-cell clones in studies like somatic hypermutation. Fig. 5.11 IgM and IgD are derived from the same pre-mRNA transcript a and are both expressed on the surface of mature B cells B cells expressing IgM and IgD have not undergone class switching, which requires irreversible changes to the DNA. Instead, these B cells produce a long primary mRNA transcript that is differentially spliced to yield either of two distinct mRNA molecule Fig. 5.12 Transmembrane and secreted forms of immunoglobulin are generated from different heavy-chain mRNA transcripts Fig. 5.13 TCR Diversity The germline organization of the human T-cell receptor  and  loci. Fig. 5.14 TRC - and -chain gene rearrangement and expression Fig. 5.15 Recombination signal sequences flank TCR gene segments – the 12/23 rule Fig. 5.16 Is this recombination possible? Is this recombination possible? Checkpoint questions for Lecture #7 1. What are the similarities and differences between somatic recombination in T cells and somatic recombination in B cells? 2. What key component of an immunoglobulin is removed by alternative splicing to produce soluble, secreted immunoglobulins? 3. Deficiencies in RAG-1 or RAG-2 cause a form of SCID in which patients lack B cells and T cells. Why is this the case? 4. How is the V domain of the heavy chain and light chain genes broken up? 5. Describe the process of making heavy chains. 6. Describe the process of making light chains. 7. What are the mechanisms by which B cells generate receptor diversity? Assigned Readings Assigned Readings Chapter 5 5-1, 5-2, 5-3, 5-4, 5-5, 5-7, 5-8 InQuizitive 20240926 Readings Due Oct 3rd, noon