1. Lecture 9- T cell development Flow cytometry- how it works Thymic architecture and cells Thymic development I- generation of a TCR  chain Thymic development II-  vs  T cell development Positive selection of CD4 + CD8 +  T cells. Negative selection of high affinity autoreactive cells. Alloreactivity, recognition of the MHC molecules of others.

2. Fluorescence

3. Fluorescence 2 1-10 x 10 -9 seconds

4. Fluorescence 3

5. Figure 2-13

6. Fluorescein Couple to antibody Green fluorescent protein Examples of commonly used fluorescent probes

7. T cell development quality control- phase I  -chain selection As with B cell development, T cells develop in an ordered sequence. First, there is TCR  chain gene recombination, starting with D  -to-J . Next, V  -to-DJ rearrangement occurs. As rearrangement is random and error-prone,  -chain rearrangement is tested for functional protein using the surrogate chain mechanism (preT  ).

8. T cell development occurs in the thymus Neonatal removal of the thymus, or congenital lack of the thymus (DiGeorge Syndrome, nude mice) leads to T cell deficiency

9. Scanning electron micrograph of thymus Developing T cell Thymic stroma

10. Thymocyte maturation (approximate) Positive selection Negative selection V(D)J recombination

11. The first step in  T cell development, TCR  rearrangement and testing for protein.

12. Figure 5-6 part 1 of 3

13. Figure 5-6 part 2 of 3

15.  -chain+

16. Cells with a good first chain then proliferate a bit, then stop and redirect rearrangements at the second locus, with V  -to-J  recombination. At this time, preT  is no longer expressed. In contrast to B cells, T cells that make an intact receptor are screened for "positive selection" based on low affinity for self-MHC/self-peptides. Cells that have no affinity for self-MHC/self-peptide complexes are eventually lost by apoptosis. T cell development quality control- phase II

17. Thymocyte maturation (approximate) Positive selection Negative selection V(D)J recombination

19. The ability of CD8 T cells to recognize MHC class I bound peptides and CD4 T cells to see MHC class II bound peptides is not random, but is selected for during thymic education.

20. Before selection, cells expressing TCR  coexpress both CD4 and CD8 and are called "double positive cells". These cells, which are the major cell population in the thymus, mature when their TCRs see MHC (carrying self-peptides) with a low affinity. The coreceptor (CD8 or CD4) engaged regulates the fate decision to CD8 or CD4 "single positive" differentiation. Hence, CD8 cells have TCRs that were selected on class I, and CD4 cells have TCRs that were selected on MHC class II.

21. T cell populations during development and in the lymphoid tissue CD4 CD8 THYMUS SPLEEN Fluorescence color 1 Fluorescence color 2

22. Figure 5-5 T cells failing positive selection die in the cortex and are immediately engulfed by macrophages. Red stain shows dead cells, blue stain, macrophages.

23. Thymocyte maturation (approximate) Positive selection Negative selection V(D)J recombination

24. T cell development quality control- phase III Negative selection Cells that bind with too high an affinity for self-MHC/self- peptide complexes are lost, probably by apoptosis.

25. U. of Cambridge

26. Figure 5-3 part 2 of 2

27. Figure 5-13

28. TCR  CD8 + 4 + double positive thymocytes that fail to be positively selected can undergo many rounds of TCR  recombination in an attempt to generate a functional receptor.

30. Figure 5-19

31. T cell developmental stages are identified by cell surface markers and gene rearrangement status. A major checkpoint involves the testing of functional TCR  for ability to pair with the surrogate TCR  chain, preT .

32. Kuby et al. W. H. Freeman & Co. and Sumanas, Inc. Immunology, January, 1997

33. Molecular basis of direct allogeneic MHC recognition

34. Concepts in  T cell development TCR  cells develop in the thymus from bone marrow progenitors. TCR  is rearranged first and tested in association with pT . Cells that express TCR  protein then express CD4+CD8 and rearrange TCR  genes. Cells carrying appropriate  TCRs undergo positive selection, leading to expression of just CD4 or CD8. Autoreactive T cells undergo negative selection in the medulla. The result of selection is a skewing of the T cell repertoire that promotes MHC restricted recognition. Thymic selection also leads to skewing toward alloreactivity and high frequency reactivity to allografts.