Commitment Positive selection I Positive selection II Allelic exclusion Negative selection T B Harvard-MIT Division of Health Sciences and TechnologyHST.176: Cellular and Molecular ImmunologyCourse Director: Dr. Shiv Pillaipro -T NK B, T, NK, LD LD T, NK, LD B αβ γδA schematic view of an HLA class II molecule peptide α1 β1 β2α2HELPER T CELL Nucleus Endoplasmic reticulum T cell receptor CD4 MHC class II membrane immunoglobulin protein antigen CLIPPeptide -CLIP exchange mediated by HLA-DM Invariant chainA schematic view of an HLA class I molecule α1 α2 α3 β 2-microglobulin Transmembrane domain Cytoplasmic tail peptide Heavy chainNucleus Endoplasmic reticulumCytosolic antigen Proteasome MHC class I molecules Signals CD8 T cell receptor CYTOTOXIC T CELL TAPMHC RESTRICTIONαβ T CELL DEVELOPMENT 1. Development occurs in the thymus2. T cells with receptors biased towardsself MHC must be generated 3. Self-reactive T cells must be tolerized4. CD4 T cells which see MHC class II-peptide complexes and CD8 T cells which recognize MHC class I-peptide complexes must be generatedHSC "Lymphoid" restricted Multipotent progenitor B, T, NK, LD CD3-CD4lo CD8-TCRβ germlineOligopotent progenitor TCRγ germlineT, NK, LD ?SCF, IL-7, Flk-2/Flt-3 ligand TN CD44+ CD25-T lineage committed TCRβ/γ germline TN CD44+ CD25+ Rag gene expression Progenitors of both αβ TN CD44-CD25+ TCRβ/γ/δ rearranged and γδ lineagesThymic versus extra-thymic development 1. Most αβ T cell develop in the thymus 2. Feedback effect: recently generated mature αβ T cells inhibit the further development of precursors 3. The thymus provides a compartmentalization functionseparating precursors from mature T cells 4. Many γδ T cells and some αβ T cells develop in extra-thymic sites. Intestinal "cryptopatches" are an important site for the development of intra-epithelial γδ and αβ T cellsThymic Epithelium formed from endoderm of third pharyngeal pouch and ectoderm of third branchial cleft. whn is required for thymic epithelial cell differentiation and the ability of the thymus to attract lymphoid progenitors. Proper formation of cortex requires development of early T cells. A block at the pro-Tp (CD44 + CD25-) stage Thymic progenitors andleads to cortical disruption. Bone-marrow derived stromal components A block at the Early pre-T (CD44 -CD25+) stage leads to specific medullary defects A block at the CD4 +CD8+ DP stage also leads to medullary defects Cortex Medulla Migration of thymic progenitors starts soon after. Bone-marrow also provides macrophages and dendritic cells. Stromal development completed by end of gestation. Cell-cell and ECM-cell interactions, IL-7, and SCF contribute to T cell commitment and development. Stroma necessary for positive and negative selection Developing T cells are required for the architectural development of the cortex and the medullaT CELL RECEPTOR BINDING SITES Model 1: Initial repertoire like Ig Thymic education selects a small fraction of cells Model 2: Initial repertoire already has structural bias for MHCPRE-TCR SIGNALING CD4 +8+ CD4 + CD8 + NEGATIVE SELECTION POSITIVE SELECTION II POSITIVE SELECTION II Vα-J α pre-T IL-7 SIGNALING SCF/C-KIT SIGNALING pro-T TCR CD8 CD4 POSITIVE SELECTION I Non-productive Death by Neglect β chain rearrangement Double Negative Double positive Single positive REARRANGEMENT ELIMINATION RESPONDERSNo known ligand β pTα γ ε ε δ ζζ ITAM CD3δ not essential for pre-TCR signaling Src-family kinases (Lck and Fyn) Syk family kinases (Syk and ZAP70) 1) Survival and proliferation signals 2) Allelic exclusion at TCR β locus 3) Induction of TCR α rearrangement d) Shut-off of pTα expressionCortex Medulla ICAM-1 ?Glucocorticoid levels high ICAM-1 and B7-1 ?Glucocorticoid levels low Cathepsin-L Cathepsin-S POSITIVE SELECTION NEGATIVE SELECTIONHigh affinity Low affinity No affinity of TCR TCR- TCR-MHC for MHC MHC interactions interactions "negative "positive "death by neglect" selection" selection"Lineage Commitment - CD4+ versus CD8+ Stochastic versus instructive TCR Signal strength Duration of signaling Notch attenuates signal strength?DP CD5lo CD69lo DP CD5hi CD69hi Bcl-2lo TCRlo CD4 committed CD8 committed TCR/CD3 signaling TCR and CD8 signalsDefault 4+8lo Notch signaling DP CD5lo CD69lo Bcl-2hi TCRhi DP CD5lo CD69lo Bcl-2hi TCRhi DP cell DP cell 4+8lo 4lo8+ SP CD4 SP CD8 TCR sees MHC class II SP CD4 TCR sees MHC class I TCR sees MHC class I TCR sees MHC class II SP CD8 Selected CD8 cellsSelected CD4 cellsγδ T cells 1. Not educated in the thymus2. Often develop extrathymically3. Most IELs (intraepitheliallymphocytes) are γδ T cells 4. Either use CD8 or no coreceptor5. Innate immunity?STOCHASTIC INSTRUCTIVE pro-γδ pro-αβ pro-T pro-T γδ in-frame β in-frame βNP γδNP γδ lineage αβ lineage γδ in-frame γδ NP β in-frame γδ lineage αβ lineage β NP- -Vα-JαLatepre-T- -Pre-TCR SIGNALING (only in αβ committed cells) CD44+CD25 CD44+CD25+ CD44 CD25+ CD44 CD25 c-Kit + c-Kit + c-Kit -/lo c-Kit --T Small pre-T cycling Large pre-T IL-7/γc required for TNK p r o In αβ committed T cells TCR γ expression is downregulated; TCR δ segments are deleted during TCR α rearrangement survival of progenitors γδ T CELL DEVELOPMENT γδ REQUIRES IL-15 REQUIRES TCR TO BE TRIGGERED VIA MICA AND MICB NO NEGATIVE SELECTION STEP KNOWNVα/Vδ Dδ Jδ Eδ Cδ Vδ5 TEA Jα Cα Eα LCR BEAD-1