Consequences of TCR engagement

1. Consequences of TCR engagement Sara L. Colpitts October 7, 2014

2. How to become a T cell? Consequences of TCR engagement Sara L. Colpitts October 7, 2014

3. Basic terminology: • T cell – lymphocyte population identified by the expression of CD3 and a TCR • Thymocyte – developing T cells located in the thymus • Thymopoiesis – process by which thymocytes develop into mature T cells

4. Step 1: Arrival in the thymus • Migration of progenitor from the bone marrow to the thymus via the blood

5. What is the thymus? • Multi-lobed primary lymphoid organ • Located just above the heart

6. What is the thymus? • Multi-lobed primary lymphoid organ • Located just above the heart • Divided into an outer cortex and an inner medulla

7. Cellular architecture of the thymus

8. Cellular architecture of the thymus Thymic stroma

9. Cellular architecture of the thymus

11. Experimental manipulation of the thymus • Thymectomy – surgical removal of the thymus • nude mutation – failure to develop cortical epithelium of the thymus

12. Step 2: Initiation of T cell maturation • Changes in cell phenotype (i.e. upregulation or downregulation of surface proteins) reflect the state of functional maturation of the cell

13. Earliest thymocytes • Lack expression of surface molecules characteristic of T cells • T cell receptor genes are not yet rearranged = double-negative (DN) thymocytes • ~5% of all thymocytes

14. Earliest thymocytes • Phenotypic diversity within the DN stage DN1 DN2 CD44 (or Kit) DN4 DN3 CD25

15. Earliest thymocytes • Phenotypic diversity within the DN stage DN1 DN2 CD44 (or Kit) DN4 DN3 CD25

16. Step 3: TCR rearrangement • Rearrangement of the β chain followed by the α chain generates αβ T cells

18. β chain rearrangement • DN2 into DN3  Dβ to Jβ • DN3  Vβ to DJβ • Option #1: No successful rearrangement (after multiple attempts) leads to cell death at the DN3 stage • Option #2: β chain expression leads to…

19. pTα and Pre-TCR formation • pTα = pre-T-cell α; a surrogate α chain that binds with the β chain to form the pre-T-cell receptor (i.e. pre-TCR) • Equivalent to λ5 in B cells • Allows for the formation of a signaling complex with CD3 to initiate downstream signaling • No ligand is required for signaling

21. Effects of Pre-TCR formation • Downregulation of CD25 (transition to DN4) • Proliferation • Arrest of further β-chain gene rearrangement (i.e. allelic exclusion) • Upregulation of both CD4 and CD8 = double-positive (DP) thymocytes

22. α chain rearrangement • RAG genes are re-expressed following the period of intense cell proliferation (cells return to a smaller, resting size) • DP  Vα to Jα • δ chain gene is excised prohibiting any further rearrangement • Multiple successive attempts at rearrangement are possible

25. Identifying DN, DP, and SP thymocytes by flow cytometry CD8 CD4

26. Identifying DN, DP, and SP thymocytes by flow cytometry DP CD8 CD4 DN

29. Step 4: Selection • Positive selection = immature T cells must recognize self-peptide:self-MHC complexes to receive survival signals • Negative selection = immature T cells that recognize self-peptide:self-MHC too strongly are deleted (i.e. central tolerance)

30. MHC expression on thymic stroma drives positive selection a b b b a b a a • Irradiation kills all hematopoietic cells but not stromal/epithelial cells

31. MHC restriction • T cells can only recognize foreign peptide antigens in the context of the SAME MHC molecule that they were positively selected on in the thymus

32. Transgenic receptors have already undergone VDJ rearrangement and have a known MHC specificity

33. Step 5: Generation of single-positive (SP) thymocytes • If the TCR weakly interacts with self-peptide:self-MHC class I molecules, CD8 will remain expressed on the surface of the cell and CD4 expression will be turned off • If the TCR weakly interacts with self-peptide:self-MHC class II molecules, CD4 will remain expressed on the surface of the cell and CD8 expression will be turned off

36. Reciprocal transcription factors aide in CD4 versus CD8 commitment ThPOK Runx3 CD4+ SP CD8+ SP

37. Reciprocal transcription factors aide in CD4 versus CD8 commitment • CD4+CD8low ThPOK Runx3 CD4+ SP CD8+ SP

38. Reciprocal transcription factors aide in CD4 versus CD8 commitment • CD4+CD8low TCR signaling (Lck) ThPOK Runx3 CD4+ SP CD8+ SP

39. Reciprocal transcription factors aide in CD4 versus CD8 commitment • CD4+CD8low TCR signaling (Lck) ThPOK Runx3 CD4+ SP CD8+ SP

40. Reciprocal transcription factors aide in CD4 versus CD8 commitment • CD4+CD8low TCR signaling (Lck) ThPOK Runx3 CD4+ SP CD8+ SP

41. Negative selection • Involves interactions with ubiquitous self antigens and tissue-restricted self antigens • Can take place in both the thymic cortex and thymic medulla • Mediated largely by bone marrow-derived antigen presenting cells (dendritic cells/macrophages) but also thymic epithelium

42. Affinity hypothesis • Low-affinity interactions (i.e. weaker/shorter signaling) rescue the cell from death by neglect • High-affinity interactions (i.e. stronger/longer signaling) induce apoptosis

43. Step 6: Exiting the thymus

44. Getting from the thymus to secondary lymphoid tissues requires… • Expression of the S1P receptor draws thymocytes into the blood, which expresses a high concentration of S1P • Expression of CD62L promotes homing to the lymph nodes • Expression of CCR7 promotes localization in the T-cell zones of the spleen and lymph nodes

46. Questions?Email: colpitts@uchc.edu