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Summary of Thymic Development

Summary of Thymic Development. Zuniga- Pflucker , NRI, 2004. What do we mean by the term “Lineage Commitment”?. What do we mean by the term “Lineage Commitment”? Commitment represents the loss in the ability of a cell to make alternative lineage choices under permissive conditions.

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Summary of Thymic Development

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  1. Summary of Thymic Development Zuniga-Pflucker, NRI, 2004

  2. What do we mean by the term “Lineage Commitment”?

  3. What do we mean by the term “Lineage Commitment”?Commitment represents the loss in the ability of a cell to make alternative lineage choices under permissive conditions.

  4. Name the Lineage Commitment Choices that are made during T cell Development

  5. Lineage Commitment Decisions During T Cell Development • To become a T cell • To become an TCRab cell vs a TCRgd T cell • To become a CD4 or CD8 T cell • To become one of the minor T cells subset NK T cell CDaa T cell nTreg

  6. First Lineage Commitment Decision in T Cell DevelopmentTo be, or not to be, a T cell

  7. Seeding the Thymus from Precursors in the Blood Bhandoola, et al., Immunity, 26:678-689, 2007 HSC Hematopoietic Stem Cell MPP Multipotent Progenitor ELP Early Lymphoid Progenitor CLP Common Lymphoid Progenitor CMP Common Myeloid Progenitor CTP Circulating T cell Progenitor TSP Thymus Settling Progenitors ETP Early Thymic Progenitor

  8. Is the ETP, recently arrived in the thymus, already committed to the T cell lineage?

  9. Alternative Fates of the Early Thymic Progenitor Yui and Rothernberg, NRI, 2014 A critical role for Notch in T cell lineage commitment

  10. Zuniga-Pflucker, NRI, 2004

  11. How would you demonstrate the importance of Notch in T cell lineage commitment of ETPs?

  12. OP9 stromal cells transfected with Deltex-1 can support T Cell Lineage Commitment Zuniga-Pflucker, NRI, 2004 Schmitt, et al, JEM, 2004

  13. ab T Cell Development Yui and Rothernberg, NRI, 2014

  14. Notch signaling is critical for the induction of multiple transcription factors Yui and Rothernberg, NRI, 2014

  15. Early Notch signaling induces TCF-1 (Tcf7) and Gata3 at The DN1 -> DN2 transition Yui and Rothernberg, NRI, 2014

  16. How would you test the importance of one of the transcription factors (for instance, TCF-1) that is upregulated during early Notch signaling for T cell lineage development?

  17. Impaired development of TCF-1-deficient BM HSC into CD25+ (i.e.,DN2) thymocytes in OP9 cultures Weber, et al., Nature, 2011 Defects also observed in vivo using competitive repopulation studies of WT vs TCF-1 KO BM

  18. Evolving Transcriptional Networks as Notch Influences Early T Cell Development Yui and Rothernberg, NRI, 2014

  19. DN Cells (CD4-/CD8-) DN1 DN2 DN3 DN4

  20. Sequential Rearrangement of TCR ab Genes DN DP Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

  21. The pre-TCR is Expressed in DN cells g Pre-Ta functions as a surrogate for the a chain during thymic development Expressed in DN cell and heterodimerizes with a functional b chain - assists in quality control for b chain rearrangement The pre-Ta/b chain dimer promotes increased CD3 expression and induces a ligand- independent signal, perhaps because of constitutive localization to lipid rafts or constitutive dimerization (unusual preTalpha structure), that is responsible for maturation and probably shut off RAG expression and further rearrangement, resulting in  chain allelic exclusion

  22. How would you test the ligand-independency of signaling by the pre-TCR?

  23. Reconstitution of rag1-/- mice with various forms of the pre-TCR Irving, et al., Science, 1998

  24. Ligand-Independent Dimerization of the pre-TCR Pang, et al, Nature 2010 Extended structure of Pre-Ta compared to TCR Ca Dimer of Heterodimers of pre-Ta and TCR b

  25. Two Lineages of Cells Expressing Distinct TCRs Develop in the Thymus: Stages of gd and ab T Cell Development Modified from Ciofani and Zuniga-Pflucker, Nature Rev. Immunol., 2010 (C-Kit)

  26. Two Lineages of T cells (cont.) Stochastic Instructive model Recent data suggest that receptor expression results in stronger signal that can provides instructional cue for cell to become  lineage ( Reviewed in Ciofani, et al., Nat. Rev. Immunol. 2010)

  27. Why might the pre-TCR and the TCRgd signal strengths be differentdifferently?

  28. Strength of signal: a fundamental mechanism for cell fate specification Immunological ReviewsVolume 209, Issue 1, pages 170-175, 31 JAN 2006 DOI: 10.1111/j.0105-2896.2006.00356.xhttp://onlinelibrary.wiley.com/doi/10.1111/j.0105-2896.2006.00356.x/full#f3

  29. CD44- CD25+ DN3 Reciprocal regulation of Syk and ZAP-70 expression during thymocyte development Palacios & Weiss, JEM, 2007 WT DN3 -selection Development TCR selection

  30. Strength of signal: a fundamental mechanism for cell fate specification Immunological ReviewsVolume 209, Issue 1, pages 170-175, 31 JAN 2006 DOI: 10.1111/j.0105-2896.2006.00356.xhttp://onlinelibrary.wiley.com/doi/10.1111/j.0105-2896.2006.00356.x/full#f1

  31. How might you test the signaling hypothesis?

  32. Starting with a TCRgd Transgene: Increasing signaling strength by elimination of a negative regulator, CD5, favors gd lineage commitment Hayes, et al., Immunity, 2005

  33. Instructing ab vs gd Lineage Commitment via Strength of Signal Ciofani and Zuniga-Pflucker, Nat. Rev. Immunol. 2010

  34. Some caveats to the strength of signaling Stochastic Instructive model SOX13, an SRY-related HMG-box transcription factor, is preferentially expressed in gd T cells. Its KO, decreases gd development. Its over-expression in DN thymocytesimpairs DN -> DP transition and ab T cell development. Bcl11b, a zinc finger transcription factor, is preferentially expressed in ab lineage T cells and is induced in DN2a-DN2b. It is low in the gd lineage. The KO of Bcl11b has little effect on gd T cell development but completely impairs ab lineage T cell development. There’s more to come.

  35. Checkpoints in Thymocyte Development Modified from Carpenter and Bosselut, Nature Immunology 2010 Notch Pre-TCR TCRab

  36. Linking CD4 (helper) to CD8 (killer) T cell lineage commitment to the recognition of class I versus class II MHC ensures that T cell effector functions are directed appropriately. MHC class I pathway samples intracellular antigens (e.g. viruses, intracellular bacteria). MHC class II pathway samples extracellular antigens Helper T cells regulate the activity of other cells of the immune system that have endocytosed pathogens. Cytotoxic CD8 T cells can kill invaded host cells before pathogens can replicate and spread.

  37. Recognition of MHC-1 or MHC-2 during positive selection in the thymus determines the CD4 versus CD8 T cell lineage choice.

  38. CD4 CD8 Recognition of MHC-1 or MHC-2 during positive selection in the thymus determines the CD4 versus CD8 T cell lineage choice. MHC-1 MHC-2 CD8 CD4 Early evidence of the link between TCR specificity for MHC-1 vs. MHC-2 and the CD4 vs. CD8 lineage choice: TCR transgenic mice and MHC gene ko mice.

  39. Generation of transgenic mice bearing rearranged TCR genes with defined specificity provided important tools for study of positive selection.

  40. T cell receptor transgenic mice: Antigen (e.g., ovalbumin) Rest and restimulate with Ag and cytokines, rest, repeat 9 days T cells + DCs + ovalbumin plate at 1 cell/well Test for antigen specificity grow and expand clone the TCR alpha and beta chain genes from the T cell clone

  41. T cell receptor alpha- & beta-chain genes specific for MHC class I or MHC class II OT-1 - CD8 T cells AND - CD4 T cells All T cells will express the same TCR receptor

  42. Increased development of CD4 SP thymocytes and T cells in mice expressing a rearranged class II MHC specific transgene CD4 CD8

  43. Generating H-Y TCR transgenic mice (anti-male peptide TCR) Male H-2b cells Rest and restimulate with Ag and cytokines, rest, repeat 9 days Female H-2b mouse Expand anti-male CD8 T cells plate at 1 cell/well Test for antigen specificity grow and expand clone the TCR alpha and beta chain genes from the T cell clone

  44. Increased development of CD8 SP thymocytes and T cells in mice expressing a rearranged class I MHC specific transgene 5% 37% 46% 10%

  45. TCR specificity for MHC I or II determines CD4 versus CD8 lineage commitment OT1, HY, F5, etc OT2, DO11.10, AND, etc

  46. . . . . . . . . .. . . . . . . . . . .. . … . . … . . MHC deficient mice provide evidence for positive selection. Lack of MHC class II expressionprevents development of CD4 cells WT CD4 CD8 MHC Class II o And lack of MHC class I expression (b2-microglobulin deficient mice) prevents development of CD8 T cells. MHC class I and II double deficient mice lack both CD4 and CD8 mature T cells, but have normal numbers of DP thymocytes. CD4 CD8

  47. CD4 CD8 MHC-1 MHC-2 CD8 CD4 What are the gene expression programs that determine the CD4 or CD8 T cell fate? How are distinct gene expression programs linked to TCR recognition of MHC class I or class II?

  48. CD4 CD8 MHC-1 MHC-2 CD8 CD4 What are the gene expression programs that determine the CD4 or CD8 T cell fate? How are distinct gene expression programs linked to TCR recognition of MHC class I or class II?

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