T Cell Development I: The Generation of TCR + Thymocytes

1. T Cell Development I: The Generation of TCR+ Thymocytes

2. Questions for the next 2 lectures: How do you generate a diverse T cell population with functional TCR rearrangements? How do you generate a T cell population that is self-MHC restricted? How do you ensure that those diverse T cell receptors are not-self reactive? How do you coordinate lineage specification with MHC specificity and coreceptor expression? •  vs.  T cell • CD4 vs. CD8

3. The pathway of T cell development T cells...precursors are born in the bone marrow and “educated” in the thymus. • The thymus “involutes” with age • DiGeorge Syndrome, Nude mice

4. Nude Mice A. Human (mammal) skin after 60 days. B. Cat (mammal) skin at 51 days. C. Chicken (bird) at 32 days; D. Chameleon (reptile) at 41 days. E. Fence lizard (reptile)at 28 days. F. Tree frog (amphibian) at 40 days.

5. The Thymus is required for T cell development scid mutation: DNA PK no gene rearrangement nude mutation: transcription factor required for terminal epithelial differentiation (whn) NO THYMUS NO B or T CELLS

6. T cell development occurs in the thymus

7. Figure 5-3 part 1 of 2

8. The epithelial cells & developing thymocytes

10. T cell development can be characterized using flow cytometry

11. Double-positives CD4 single-positive 88 8 CD4 Double-negatives CD8 single-positive 1 3 CD8 Real data

12. DN’s can be further subdivided into DN1 through DN4 CD25 (a chain of IL2R) “Gate” on CD4-CD8-, analyze for CD44 and CD25

13. CD25 The real data (gated on DN cells)

14. Double negative cells appear in the fetal thymus before double positive cells K. Hogquist

15. T cell populations during development and in the lymphoid tissue THYMUS SPLEEN CD4 CD8

16. Thymocytes at different developmental stages are found in distinct parts of the thymus Maturation

17. Today Thursday

18. Most cells fail to complete thymocyte maturation

19. Most developing thymocytes die in the thymus apoptotic cells (DNA fragmentation) macrophages Macrophages...garbage collectors, contain the dying cells

20. T cell development involves the sequential generation, assembly and testing of the newly rearranged TCR

21. TCR rearrangement is very similar to Ig rearrangement

22. First step: TCRbeta rearrangement Figure 5-6 part 1 of 3

23. Figure 5-6 part 2 of 3

24. How do you test for successful TCRbeta chain rearrangements if you have not rearranged TCRalpha? Pre-Talpha Pre-T cell receptor

25. TCR  chain is required for DN to DP transition:Experimental Evidence TCRa-/- TCRb-/- TCRa/b-/- TCRb/d-/- WT 108 108 <107 <107 106 Total thymocyte numbers Expressing TCRgd CD4 CD8

26. Does the pre-TCR have a ligand? Remove extracellular domains… …and examine T cell development (work of Nigel Killeen, UCSF)

27. Pre-TCR extracellular domains are not required! pTaT;bTreceptor restores CD4+8+ development and thymocyte expansion (Irving (1998) Science 280:905)

28. CD25 b-selection leads to proliferation DN2 Gated on CD4/CD8 DNs DN3 Proliferating cells are bigger Forward scatter (cell size)

29. TCR b locus has two D-J clusters Allows a 2nd rearrangement if 1st is nonproductive

30. We are here

31. Progression through development correlates with rearrangement

32. At the DP stage, TCRalpha rearrangement begins

33. TCR a locus has 50 Ja segments

34. TCRa locus-- replacement rearrangement

35. DP thymocytes express surface TCR CD3 lo cell-- pre-TCR CD3 hi cells-- TCR

36. TCR  chain is required for DP to SP transition:Experimental Evidence TCRa-/- TCRb-/- TCRa/b-/- TCRb/d-/- WT 108 108 <107 <107 106 Total thymocyte numbers Expressing TCRgd CD4 CD8

37. Differential expression of Src family kinases

38. Remember what Lck does

39. Role of Lck in T cell development RAG KO TCRTransgene RAG KO x Lck KO TCRTransgene RAG KO CD4 CD8 Total thymocyte number 0.4 x 106 288 x 106 10 x 106 30-fold reduction in DPs!

40.  vs  Thursday

41. A model for  versus  lineage commitment

42. TCR rearrangement removes the delta locus

43. TCR lineage comprises the majority of T cells 90% 10%

44. gdT cells are favored during early fetal development

45. Waves of  T cells with specific TCR usage develop early gestation

46. Waves of  T cells home to different tissues

47. gd cells bearing specific receptors end up in skin (Vg5), gut (Vg2), uterus (Vg6), etc. Limited junctional diversity: no N nucleotides (no TdT) The mechanisms controlling this limited rearrangement are poorly understood. Early waves of  T cells

48. Antigen recognition by  T cells is different than  T cells  T cells are not MHC restricted! Antigen is recognized directly, more like an antibody In some cases ligands for the  TCR are self proteins upregulated under stress conditions In humans, circulating  cells recognize a phospholipid antigen from Mycobacterium tuberculosis

49. Dendritic epidermal T cells (DETC) Vg5 gd T cells home to the skin and wedge among keratinocytes Involved in wound healing as well as tumor protection

50. DETCs promote wound healing