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Development

Development. ab T lymphocyte Development. Where do T cells come from?. 1. Multipotent Lymphoid Progenitors Migrate from the Bone Marrow to the Thymus. 2. In the Thymus, the Lymphoid Progenitors Differentiate to pre-T Cells and are Educated to Differentiate Self from Non-self.

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Development

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  1. Development

  2. ab T lymphocyte Development

  3. Where do T cells come from? 1. Multipotent Lymphoid Progenitors Migrate from the Bone Marrow to the Thymus 2. In the Thymus, the Lymphoid Progenitors Differentiate to pre-T Cells and are Educated to Differentiate Self from Non-self 3. Positively Selected T Cells Emigrate from the Thymus to Mediate and Effect the Cognate Immune Response

  4. T Lymphocyte Maturation in the Thymus

  5. The CD4 by CD8 FACS Plot as an Indicator of Normal (and Abnormal) Thymocyte Development CD4 CD8

  6. Ikaros controls commitment to Lymphoid lineage (Ikarus DN) Bone Marrow Thymus

  7. Notch 1 is required for T cell lineage commitment Control Notch1-/- CD4 Ratke et al, Immunity, 1999

  8. How Notch Signaling Works Ligands expressed In the thymus

  9. Reciprocal Regulation Pax-5 induces the expression Of B lineage genes (CD19, BLNK) And represses the expression of Notch

  10. But really location controls the T cell versus B cell Lineage Choice So what controls homing of CLPs to the thymus? And what controls the expression of these homing receptors?

  11. T Lymphocyte Maturation in the Thymus

  12. As T Lymphocytes Develop, They Migrate From the Thymic M/C junction to the Cortex to the Medulla

  13. DN1 Cells in the Thymus Are Not Committed to become T Lymphocytes • TCR loci are in germline configuration • Cell can differentiate to become a B lymphocyte, Natural Killer cell, or Dendritic cell (Michie et al, JI, 2000; Ikawa et al, JEM, 1999;Sanchez et al, JEM, 1994)

  14. The DN2 Subset of Thymocytes are More Committed, but Not Quite Exclusive • TCR loci are in germline configuration • DN2 thymocytes may give rise to dendritic cell(Moore et al, 1995; Wu et al 1996; Ardavin et al, Nature 1993; Shortman et al, Imm Rev. 1998) • DN2 thymocytes no longer differentiate to the NK cell or B lymphocyte lineages

  15. DN3 Cells are Committed to the T Lineage • Downregulation of CD44 expression • Upregulation of RAG genes • V-D-J recombination of TCRb chain locus • Expression of pre-TCRa chain

  16. The pre-TCRa chain (DN3) • Invariant Type I TM protein • Contains only one Ig Domain • Physically associated with TCRb chain • Signaling appears to be ligand-independent • TCRb chain in this complex lacking extracellular domain is sufficient to allow progression to DP stage From Fundamental Immunology, 4th ed. (Paul)

  17. Targeted Gene Mutants Unable to Mature to the DP Stage

  18. After Assembly of a Functional pre-TCR • Shut down of TCRb rearrangement; TCRb allelic exclusion • Onset of proliferation/expansion • Differentiation to DN4, CD8ISP, and then DP

  19. Three Models of ab vs. gd Lineage Determination • Earliest (fetal) waves of T cells are mostly gd • Later production is skewed toward ab lineage • Conflicting Data Supporting Each Model • Mature ab T cells have rearranged g locus; gd’s have rearranged b locus • Even DN4 cells (which have selected a b chain) can become gd lymphocytes in transfer experiments From Fundamental Immunology, 4th ed. (Paul)

  20. BMPSmad IL-7 Jak3/ SCF c-kit Wnt Tcf/Lef CXCR4 Notch

  21. V(D)J Recombination in developing T cells • b chain occurs first (DN3 stage) – VDJ • a chain occurs second – VJ, no D region • Allelic exclusion • Combinatory diversity of the adaptive immune system • This is a big problem

  22. Fates Awaiting a DP Thymocyte Default (95%)

  23. Positive Selection: Host MHC determines CTL immune responsiveness (Bevan, Nature, 1977) Balb/c H-2d/b BM -> H-2b/d Balb/c H-2d/b BM -> H-2d Balb/c H-2d/b BM -> H-2b immunize with B10H-2d/b splenocytes restim in vitro look for MHC restricted cytotoxicity d(with peptides) b(with peptides) B10(H-2d) B10(H-2b) H-2d/b BM -> H-2b/d +++ +++ H-2d/b BM -> H-2d +++ + H-2d/b BM -> H-2b + +++

  24. Evidence for MHC Restriction of CD4+ and CD8+ T Lymphocytes is Apparent in MHC KO Mice CD8 CD4 (Grusby et al, PNAS, 1993)

  25. Negative SelectionHY TCR Tg is negatively selected by a male specific antigen HY TCR female HY TCR Male (Kisielow et al, Nature, 1988)

  26. Negative and Positively selecting peptides different in there Kd (Alam et al, Nature, 1996)

  27. Model of Thymocyte Education

  28. Signal Transduction in thymocyte development

  29. TCR Signal Transduction

  30. But what are the down stream effectors?

  31. Which downstream pathways contribute to thymocyte selection?

  32. Do Positive and Negative Selection Signals Activate the Same Signal Transduction Pathways?

  33. Disruption of the ERK Pathway Interferes With Positive but not Negative Selection (an effect on differentiation and proliferation? • Also: • DN Mek1 • Alberola-Ila et al, 1995 • Alberola-Ila et al, 1996 • DN p21ras • Swan et al, 1995 • Alberola-Ila et al, 1996 • DN Raf • O’Shea et al, 1996 • RasGRP • Dower et al, 2000 • Indirect (ERK Induction from CD3d chain) • Werlen et al, 2000 • Delgado et al, 2000 ERK1 ERK1 Pages et al 1999 Science 286:1376

  34. Calcineurin B is required for positive selection Neilson et al, 2004

  35. Cnb1deficient and Control Thymocytes are Equally Sensitive to TCR Crosslinking and Peptide Complexed with MHC Viability Neilson et al, 2004

  36. Which downstream pathways contribute to thymocyte selection? (positive selection) (positive selection)

  37. Vav GEF is required for positive selection

  38. Active Rac1 changes positive to negative selection Female Female/active Rac1 (Gomez et al, Immunity, 2001)

  39. Data = Explanation? • Synapse during negative selection (Richie et al, Immunity, 2002) • Synapse formation in peripheral T cells requires Vav/actin dynamics (Holsinger et al, Curr. Bio, 1998; Wulfing et al, PNAS, 2000) • Positive selection likely does not form a synapse (Peter Ebert unpublished) • Therefore, lacking Vav/Rac etc. changes negative to positive selection via an effect on synapse formation and subsequent signaling

  40. Which downstream pathways contribute to thymocyte selection? (positive selection) (Modulator Both) (positive selection)

  41. Negative Selection is Impaired in DP Thymocytes Deficient in the Pro-Apoptotic Bcl-2 Family Member Bim Bouillet et al 2002 Nature 618:108

  42. Three Models for the CD4 vs CD8 Choice From Fundamental Immunology, 4th ed. (Paul)

  43. Modulation of lck activity can alter CD4/8 lineage commitment (based on the fact that the cytoplasmic tail of CD4 binds to more lck than CD8) Dn-lck Act-lck

  44. Other studies indicate that the duration of signal controls CD4 versus CD8 with CD4 requiring longer (Yasutomo et al, Nature, 2000)

  45. Regulatory T cells may develop in the Thymus HA specific TCR Transgenic mouse Mouse expressing HA peptide TCR Tg CD25

  46. 2D model

  47. Hypothetical 3D model of thymocyte development

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