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Lecture 9- T cell development •Flow cytometry- how it works •Thymic architecture and cells •Thymic development I- generation of a TCRb chain •Thymic development II- gd vs ab T cell development •Positive selection of CD4+CD8+ ab T cells. •Negative selection of high affinity autoreactive cells. •Alloreactivity, recognition of the MHC molecules of others. Fluorescence Fluorescence 2 1-10 x 10-9 seconds Fluorescence 3 Figure 2-13 Examples of commonly used fluorescent probes Green fluorescent protein Fluorescein Couple to antibody T cell development quality control- phase I b-chain selection • As with B cell development, T cells develop in an ordered sequence. • First, there is TCRb chain gene recombination, starting with Db-to-Jb. • Next, Vb-to-DJ rearrangement occurs. As rearrangement is random and error-prone, b-chain rearrangement is tested for functional protein using the surrogate chain mechanism (preTa). T cell development occurs in the thymus Neonatal removal of the thymus, or congenital lack of the thymus (DiGeorge Syndrome, nude mice) leads to T cell deficiency Scanning electron micrograph of thymus Developing T cell Thymic stroma Thymocyte maturation (approximate) V(D)J recombination Positive selection Negative selection The first step in ab T cell development, TCRb rearrangement and testing for protein. Figure 5-6 part 1 of 3 Figure 5-6 part 2 of 3 b-chain+ T cell development quality control- phase II • Cells with a good first chain then proliferate a bit, then stop and redirect rearrangements at the second locus, with Va-to-Ja recombination. At this time, preTa is no longer expressed. • In contrast to B cells, T cells that make an intact receptor are screened for "positive selection" based on low affinity for self-MHC/self-peptides. • Cells that have no affinity for self-MHC/self-peptide complexes are eventually lost by apoptosis. Thymocyte maturation (approximate) V(D)J recombination Positive selection Negative selection The ability of CD8 T cells to recognize MHC class I bound peptides and CD4 T cells to see MHC class II bound peptides is not random, but is selected for during thymic education. • Before selection, cells expressing TCR a/b coexpress both CD4 and CD8 and are called "double positive cells". • These cells, which are the major cell population in the thymus, mature when their TCRs see MHC (carrying self-peptides) with a low affinity. • The coreceptor (CD8 or CD4) engaged regulates the fate decision to CD8 or CD4 "single positive" differentiation. • Hence, CD8 cells have TCRs that were selected on class I, and CD4 cells have TCRs that were selected on MHC class II. T cell populations during development and in the lymphoid tissue THYMUS Fluorescence color 2 CD4 SPLEEN Fluorescence color 1 CD8 T cells failing positive selection die in the cortex and are immediately engulfed by macrophages. Red stain shows dead cells, blue stain, macrophages. Figure 5-5 Thymocyte maturation (approximate) V(D)J recombination Positive selection Negative selection T cell development quality control- phase III Negative selection •Cells that bind with too high an affinity for self-MHC/selfpeptide complexes are lost, probably by apoptosis. U. of Cambridge Figure 5-3 part 2 of 2 Figure 5-13 TCRa/b CD8+4+ double positive thymocytes that fail to be positively selected can undergo many rounds of TCRa recombination in an attempt to generate a functional receptor. Figure 5-19 T cell developmental stages are identified by cell surface markers and gene rearrangement status. A major checkpoint involves the testing of functional TCRb for ability to pair with the surrogate TCRa chain, preTa. Kuby et al. W. H. Freeman & Co. and Sumanas, Inc. Immunology, January, 1997 Molecular basis of direct allogeneic MHC recognition Concepts in a/b T cell development • TCRa/b cells develop in the thymus from bone marrow progenitors. • TCRb is rearranged first and tested in association with pTa. • Cells that express TCRb protein then express CD4+CD8 and rearrange TCRa genes. • Cells carrying appropriate a/b TCRs undergo positive selection, leading to expression of just CD4 or CD8. • Autoreactive T cells undergo negative selection in the medulla. • The result of selection is a skewing of the T cell repertoire that promotes MHC restricted recognition. • Thymic selection also leads to skewing toward alloreactivity and high frequency reactivity to allografts.