T Cell Receptor & T Cell Development

1. T Cell Receptor & T Cell Development

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. T lymphocyte: a key regulator of the immune system

4. T Lymphocytes • Arisefrom stem cells resident in the bone marrow and migrate to the thymus which serves as an indoctrination center where thymocytes must learn to distinguish self from nonself • Thymocytes that cannot make this induction are eliminated, those that can may further differentiate, mature, and graduate as T lymphocytes and enter the circulation

5. Origin, generation and differentiation of T cells

6. T Cell Development • T cell progenitors migrate from bone marrow and seed thymus. • T cell progenitors undergo differentiation to CD4, CD8 and NKT cells in thymus. • Mature CD4 and CD8 T cells circulate between blood and lymphoid tissues until they meet antigens presented on dendritic cells in lymphoid tissues. • T cells further undergo maturation to become functional memory or effector T cells in LT

7. Figure 5-2 Thymic involution: Human thymus is fully developed before birth and increases in size until puberty. It then progressively shrinks during adult life. Most thymectomized adults have no problem in T cell immunity because they have enough memory T cells in the periphery, and these T cells are long-lived.

8. T Cell Functions • Collectively, T cells display a number of diverse functions: - They often function to initiate, regulate, and fine-tune humoral immune response - They are effector cells responsible for various types of cell mediated immune responses like; DTH, contact sensitivity, transplantation immunity, and cytotoxicity

9. T Cell Surface Molecules • TCR: A very diverse heterodimerthat lacks a cytoplasmic tail that would allow direct cytoplasmic signaling once TCR binds an epitope • CD3 Complex: It is composed of a group of six invariant accessory molecules; one CD3 , one CD3 , two CD3ε, and an intracytoplasmic homodimer of ξ or CD247 chains • Cytoplasmic signaling occurs through CD3 that noncovalently associate with TCR

10. Figure 3-6 TCR Complex

11. The CD3 complex is essential for both cell surface expression of the TCR and for signal transduction once the TCR recognizes an antigen • Unlike antibodies that can readily bind free antigen, a TCR cannot bind soluble antigens, but only enzymatically cleaved fragments of larger peptides presented as peptide MHC (pMHC) complexes

12. CD4 or CD8: Most mature T cells express CD4 or CD8 molecules, they function as important co-receptors in association with the TCR • By binding to invariant portions of the MHC I (CD8) or MHC II (CD4), they serve to increase the interaction of the MHC-bound antigenic fragment and the TCR

13. Figure 3-10 The structures of CD4 and CD8

14. CD8 binds MHC class ICD4 binds MHC class II Most mature T cells are either CD4+ or CD8+. CD8 T cells kill cells infected with intracellular pathogens or tumor cells while CD4 T cells regulate (activate or suppress) other immune cells’ function.

15. TCR Vs Immunoglobulin • Both: • Bind antigen • Have Variable region and Constant region • Have a binding site that is a heterodimer (composed of 2 different chains) • TCRs act only as receptors • Igs act as receptors and effector molecules (soluble antigen-binding molecules)

16. Similarity between TCR and Ig

17. TCR Structure • The TCR is a member of the immunoglobulin supergene family and is composed of two polypeptide chains; a light α or  chain and a heavy β or chain • Each polypeptide chain of the heterodimer pair contains a variable and a constant region domain • The Vα and V regions are encoded by V and J gene clusters • The Vβ and Vregions are encoded by V, D, and J gene clusters • The D gene cluster provides an additional source of variation

18. Figure 3-7

19. a and b TCR gene loci (germline configuration)

20. The gene clusters undergo DNA rearrangement, similar to that already described for immunoglobulin genes, to synthesize αβ dimers or dimers • As with immunoglobulins, the constant domain of the α and β or  and  chains are encode by constant region genes (Cα and Cβ or C  or C ) • T cell receptors do not undergo any subsequent changes equivalent to isotype switch, and somatic hypermutation, important to generating diversity of immunoglobulins.

21. As might be imagined, in the random process of generating diversity, a variety of TCR specificities would be generated for peptides that one may never encounter during his lifetime • Three distinct categoriesof TCR specificities can be identified: - Those that recognize peptides that will never be encountered - Those that recognize peptides produced by potential pathogens or peptides of foreign origin - Those that recognize peptides that are produced by cells of self

22. Vn V2 V1 J C Germline TcR  Rearranged TcR 1° transcript TcR a gene rearrangement by SOMATIC RECOMBINATION Spliced TcR mRNA Rearrangement very similar to the IgL chains

23. Vn V2 V1 J C Vn+1 Productively rearranged TcR 1° transcript TcR a gene rearrangement RESCUE PATHWAY There is only a 1:3 chance of the join between the V and J region being in frame • chain tries for a second time to make a productive • join using new V and J elements

24. L & V x52 D1 J C1 D2 J C2 Germline TcR  TcR b gene rearrangement SOMATIC RECOMBINATION D-J Joining V-DJ joining Rearranged TcR 1° transcript C-VDJ joining Spliced TcR mRNA

25. D1 J C1 D2 J C2 V Germline TcR  D-J Joining V-DJ joining 2nd chance at V-DJ joining Need to remove non productive rearrangement TcR b gene rearrangement RESCUE PATHWAY There is a 1:3 chance of productive D-J rearrangement and a 1:3 chance of productive V D-J rearrangement (i.e only a 1:9 chance of a productive b chain rearrangement) Use (DJC)b2 elements

26. TCR gene rearrangements occur in the thymus The same RSS and the same enzymes are used to rearrange both the TCR genes and the Ig genes. P and N nucleotides are added at the junctions between rearranged segments n=70-80 n=52

27. TCR b Chain D region can be read in all frames V D J GTACTGCAGATT One addition and J is out of frame; the ATT start for J is lost (as are all appropriate downstream codons) V D J GTACCTGCAGATT Two addition and J is out of frame; the ATT start for J is lost (as are all appropriate downstream codons) V D J GTACCTGCAGGATT Three addition and J is in frame; the ATT start for J is is present (as are all appropriate downstream codons) V D J GTACCTGCAGGCATT J starts with ATTNo additions: J is in frame

28. -chain locus is first to be rearranged

29. Two chances for productive (=correct reading frame) rearrangement: b chain

30. -chain rearrangement

31. Multiple rounds of -chain rearrangement can rescue nonproductive TCR

32. TCR gene rearrangement generates the TCR repertoire Pre-TCR complex stops further gene rearrangement at b locus, and induces thymocyte proliferation Finally TCR+ DP cells are made

33. Germline configuration of g and d loci TCRd D, J and C exons are encoded in the intron between the the Vs and the Js of the TCRa locus. The V segments for TCRd (4 known) are mixed in with the V segments of the TCRa

35. Figure 3-8 part 2 of 2 Most gd T cells do not express CD4 or CD8. They are thought to be: First line of defense? Bridge between innate and adaptive responses?

36. Signals through the TCR and the pre-TCR compete to determine thymocyte lineage

37. Generation of diversity in the TcR COMBINATORIAL DIVERSITY Multiple germline segments In the human TcR Variable (V) segments: ~70, 52 Diversity (D) segments: 0, 2 Joining (J) segments: 61, 13 The need to pair  and  chains to form a binding site doubles the potential for diversity JUNCTIONAL DIVERSITY Addition of non-template encoded (N) and palindromic (P) nucleotides at imprecise joints made between V-D-J elements SOMATIC MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR

38. The Generation of Diversity (GOD)

39. T Cell Development • T cell precursors migrate from the bone marrow to enter the thymus as thymocytes, they express neither αβTCR nor CD4 or CD8 and are called double negative (DN) cells • DN cells proliferate in the subcapsular region of the thymus and differentiate to express low levels of newly generated αβTCR, both CD4 and CD8, and are called double positive (DP) cells

40. DP cells move inward to the deeper portion of the thymus, where they are fated to die within 3-4 days, unless their TCRs recognize an MHC class I or class II molecules on thymic dendritic cells. This process is called positive selection • Although the mechanism of positive selection is yet unclear, partial recognition of class II by CD4 or class I by CD8 molecules must occur • T cells that recognize self MHC molecules survive

41. A DP thymocyte with a TCR that engage MHC class I may become a CD8+ T cell and a DP thymocyte that recognizes MHC class II may become a CD4+ T cell • Class I and class II molecules are not displayed on cell surface unless they are loaded with a peptide • Only molecules of self origin are available on thymic APCs, and these are presented to the DP thymocyte in the deep or medullary area of the thymus

42. CD4+CD8+ DP cells: To be CD4 or CD8?

43. Thymocytes that show strong interaction with MHC molecules or pMHC complexes undergo apoptosis, a process known as negative selection • Thymocytes that survive both positive and negative selection migrate from the thymus to populate lymphoid tissues and organs as T cells

44. Figure 5-3 part 2 of 2

45. Each thymocyte maturation stage occurs at a distinct location of the thymus Young adult:~5x107 thymocytes produced/day ~1.5x106 mature cells leave/day

46. Figure 5-3 part 1 of 2 DN (CD4-CD8-) and DP (CD4+CD8+) Immature thymocytes are here Differentiation More mature SP (CD4+CD8-or CD8+CD4-) thymocytes are here

47. Positive Selection • Positive selection selects T cells that recognize peptides on self MHC • This is to assure that mature T cells can respond to antigen-presented on self MHC. • Self MHC I and II harboring self peptides on thymic epithelial cells recognize and activate TCRs on some DP thymocytes. • DP thymocytes should receive this signal within 3-4 days to survive, otherwise they undergo apoptosis.

48. Negative Selection • Negative selection eliminates T cells with TCRs that bind too strongly to self antigen/MHC complex (autoreactive cells are removed by this process) • Dendritic cells and macrophages in cortico-medullary junction mediate it. • Negative selection cannot eliminate T cells whose receptors are specific for self peptides that are present outside of the thymus • These cells enter circulation, but soon to be rendered anergic or unresponsive by other mechanims.

50. Does receptor occupancy explain positive and negative selection ? High occupancy Negative selection Low occupancy Survival