Introduction. Introduction. Lymphocyte development (maturation)

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1 Introduction Abbas Chapter 8: Lymphocyte Development and the Rearrangement and Expression of Antigen Receptor Genes Christina Ciaccio, MD Children s Mercy Hospital January 5, 2009 Lymphocyte development (maturation) The process by which lymphocyte progenitors in central lymphoid organs such as the thymus and bone marrow differentiate into mature lymphocytes that populate peripheral lymphoid tissues Introduction Immune repertoire Collection of antigen receptors and specificities expressed by B and T lymphocytes Overview of Lymphocyte Development Central lymphoid organs Commitment of progenitor cells Rearrangement of antigen receptor genes and expression of antigen receptor proteins Selection events that preserve cells that produced correct antigen receptor and eliminates autoreactive cells Proliferation of immature cells Differentiation of B and T cells into distinct populations

2 Commitment to B/T cells Maturation of B cells occurs in the bone marrow and fetal liver Fetal liver gives rise to B-1 cells Bone marrow gives rise to follicular (circulating) B cells Maturation of T cells occurs in the thymus γδ T cells originate in the fetal liver αβ T cells originate in the bone marrow Commitment to T/B cells Early proliferation is stimulated by IL-7 Produced in the stromal cells in the bone marrow and thymus Mutations in the IL-7 receptor leads to SCIDS (common γ chain) Receptor Gene Rearrangement In B cells, the first receptor gene to be rearranged is the Ig heavy chain In T cells, the β chain is rearranged first If rearrangement is successful, next chain can rearrange If rearrangement is not successful, cell dies by apoptosis Gene Rearrangment Positive selection Preservation of useful specificities Negative selection Process that eliminates (or alters) developing lymphocytes whose antigen receptors bind strongly to self In T cells, this occurs by clonal deletion In B cells, this is corrected by receptor editing

3 Gene Rearrangement Genes are generated by rearrangement in individual lymphocytes of Variable (V) region Diversity (D) region Joining (J) region V(D)J rearrangement V(D)J Recombination DNA recombination event Mediated by the coordinated activities of several enzymes Some of which are found only in developing lymphocyte Others are ubiquitous DNA double strand break repair enzyme V(D)J Recombination Enzymes recognize DNA sequences called recombination signal sequences (RSS) Located 3 of each V segment, 5 of each J segment, and each side of the D segment Highly conserved heptamer Usually CACAGTG Followed by nonconserved nucleotides Followed by AT rich stretch of 9 nucleotides

4 V(D)J Recombination Most gene rearrangements occur by deletion 50% of κ light chain rearrangements occur by inversion V(D)J Recombination Synapsis Portions of the antigen receptor chromosome are made accessible to the recombination machinery, and two 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 V(D)J Rearrangement Cleavage Double-strand breaks are enzymatically generated at RSS-coding sequence junctions using machinery that is lymphoid specific V(D)J Rearrangement Coding and Processing The broken coding ends (but not the signal/rss ends) are modified by the addition or removal of bases, and thus greater diversity is generated V(D)J Rearrangement Joining The broken coding ends as well as the signal ends are brought together and ligated by a double-strand break repair process found in all cells that is called non-homologous end joining

5 V(D)J Rearrangement RAG 1/RAG 2 Recognizes DNA sequence at the junction b/w a heptamar and a coding segment and cleaves it Also hold the hairpin ends and the blunt ends together prior to the modification of the coding ends V(D)J Rearrangement Ku70 and Ku80 DNA end binding proteins that bind ot the breaks and recruit DNA-PK, a DNA repair enzyme Artemis An endonuclease that opens the hairpins DNA Ligase IV Ligases broken ends Terminal deoxynucleotidyl transferase (TdT) Catalyzes the addition of junctional N nucleotides Generation of Diversity Combinatorial diversity Different combinations of V, D, and J segments that are possible as a result of somatic recombination of DNA Junctional diversity Largest contribution to the diversity of antigen receptors Removal or addition of nucleotides between segments at the time of joining Generation of Diversity Greatest variability is at the junction of the V and C regions, which is called CDR3

6 B Cell Development Pro-B cell Earliest bone marrow committed cell CD19+ First recombination of Ig genes in heavy chain Pre-B cell Has productive Igµ Has surrogate light chain B Cell Development Pre-B cell receptor µ Surrogate light chain Igα Igβ Allelic exclusion An individual B cell can express heavy chain proteins encoded by only one of the two inherited alleles B Cell Development Bruton s tyrosine kinase (Btk) Delivers signals from the pre-bcr that mediates survival, proliferation, and maturation beyond pre-b cell stage B Cell Development Immature B Cell Expresses IgM CD19+ Leave bone marrow Receptor editing B cells that recognize self antigens with high avidity RAG genes are reactivated Additional light chain VJ recombination Expression of a new, different light chain

7 B Cell Development Mature B cell aka follicular B cell IgM IgD Ability to recirculate from one lymphoid organ to another in B cell follicles Naïve B Cell Subsets B-1 Develop from fetal liver Express CD5 (Ly-1) Found in peritoneum and mucosal sites Limited repertoire Secrete IgM (natural Ab) that react with microbial polysaccharides and lipids May also secrete IgA Analogous to γδ T cells B Cell Subsets Marginal Zone B cells Marginal sinus in the spleen Similar to B-1 cells T cell independent responses T Cell Maturation Thymocytes Developing T cells in the thymus First found in subcapsular sinus and outer cortical region Migrate through cortex where most of the maturation occurs Migrate to medulla and exit the thymus

8 T Cell Maturation Pro-T cells Double negative thymocytes New arrival from bone marrow No TCR, CD3, ζ chains, CD4, CD8 DJ rearrangements Pre-T cells V to DJ rearrangement TCR β chain Pre-Tα T Cell Maturation Double Positive T Cell CD4+ CD8+ CCR7 (guides to medulla) TCRα gene rearrangement May occur on both chromosomes Up to 30% of mature T cells have 2 different α chains (but only one β chain) T Cell Maturation Double Positive Thymocytes αβtcr, CD3 and ζ chains are all required for surface expression Positive and Negative Selection Single Positive Thymocytes Immature Lose CD4 or CD8 designation Naïve mature T cells Leave thymus T Cell Maturation Positive selection Process in which thymocytes whose TCRs bind with low avidity to self peptide-self MHC complexes are stimulated to survive Negative selection Process in which thymocytes whose TCRs bind strongly to self peptide antigens in association with self MHC molecules are deleted

9 T Cell Maturation Central Tolerance Tolerance induces in immature lymphocytes by recognition of self antigens in the generative (or central) lymphoid organs T Cell Maturation Autoimmune Regulator (AIRE) Secreted by medullary epithelial cells Induces expression of a number of tissue-specific genes in the thymus makin a host of tissue-specific peptides available for presentation in the thymus to double-positive and single positive T cells during negative selection Animation / /animation/T_cell.swf Summary B and T cells arise from a common bone marrow derived precursor that becomes committed to the lymphocytes lineage B cell maturation proceeds in the bone marrow T cells progenitors migrate to the thymus Early maturation is characterized by cell proliferation which is induced by IL-7 Summary B and T cell maturation involves the somatic rearrangement of antigen receptor gene segments and the initial expression of Ig heavy chain proteins in B cell precursors and TCR β molecules in T cell precursors Summary The expression of pre-antigen receptors and antigen receptors is essential for survival and maturation of developing lymphocytes and for selection processes that lead to a diverse repertoire of useful antigen specificities

10 Summary The antigen receptors of B and T cells are encoded by genes formed by the somatic rearrangement of a limited number of gene segments that are spatially segregated in the germline antigen receptor loci There are separate loci encoding the Ig heavy chain, Ig κ light chain, Ig λ light chain, TCR α, β, and γchains