2. Chapter 5 Understand how the thymus is the site of development for T cells How do cells commit to a lineage during T cell development How do we eliminate self-reactive T cells without eliminating the ability to recognize self-MHC

3. The Development of T Lymphocytes Two types of TCR 1. αβ T cells CD4 CD8 2. γδ T cells

4. T cells develop in the thymus T cells (T-lymphocytes) = thymus-dependent lymphocytes The thymus contains The thymus is a primary lymphoid organ because it is only involved in development, not fighting infection. a) Thymocytes (immature T cells) b) Thymic stroma (epithelial cells)

5. The Thymus 2 areas of the thymus: Cortex – outer, close-packed consists of ectodermal cells; can contain thymocytes and macrophages Medulla – inner, less dense consists of endodermal cells; contains thymocytes, dendritic cells, and macrophages Thymic anlage : The combination of the ectodermal and endodermal cells, colonized by progenitor cells from the bone marrow.

6. Thymus facts Fully developed at birth and increases in size until puberty Most active in the young Degrades after puberty (involution), being replaced with fat tissue Even after involution (~30 yrs. old) or a thymectomy immunity by T cells is not impaired significantly Mature T cell repertoire is long-lived and self- renewing

7. T cell markers T Cell receptor – αβ or γδ chains bind peptide antigens CD3 – complexes of CD3γε, CD3δε, and 2 ζ chains for signaling to the interior of the cell CD4 or CD8 – receptors that bind MHC molecules Progenitor cells entering the thymus have NO TCR, CD3, or CD4/CD8 surface receptors = immature thymocytes called double-negative thymocytes After β chain rearrangement, T cells express both CD4 and CD8 surface receptors = immature thymocytes called double-positive thymocytes Mature T cells express either CD4 or CD8 surface receptors = thymocytes called single-positive thymocytes

8. Order of TCR gene rearrangements controls the distribution of T cell lineages: 95% αβ T cells 5% γδ T cells TCR gene rearrangement

9. T cell lineages Pre T cell  (pT  ) - a place-holder

10. αβ TCR rearrangement β chain –rearranges first –has variable (V), diversity (D), and joining (J) gene segments –Can attempt gene rearrangements on both chromosomes or by a second rearrangement on the same chromosome Tandem DJ and V segments 80% of T cells make successful rearrangements –Successful rearrangement leads to expression of CD4 and CD8 α chain –rearranges second –has several variable (V) and joining (J) gene segments –Can undergo several gene rearrangements –Both α chain loci can rearrange leading to the potential for 2 different α chains and 2 different TCRs on a single cell

11. Gene rearrangement... In pictures

12. β chain rearrangement

13. α chain rearrangement

14. γδ T cells Express rearranged TCR’s, generate immunologic memory and induce dendritic cell maturation Effector functions similar to  T cells making them part of the Adaptive immune system HOWEVER, they have limitied TCR gene usage, TCRs act as pattern recognition receptors, and respond fast Might be a conserved, primitive form of immunity that bridges INNATE with ADAPTIVE

15. Proposed scheme to use  T cells for immunotherapy

16. T cell generation in mice The first γδ T cells express receptors based on the first V segment nearest the D segments (epidermis- yellow). Later the majority of γδ T cells express receptors with the other V segment (reproductive tract - red). After birth the αβ T cells dominate.

17. Positive and Negative Selection of T Cells Where: cortex, cortico-medullary junction (thymus) Who: double positive,  :  T cells What is being selected: the TCR Purpose Positive Selection: Select TCRs that recognize self MHCs - In periphery, T cells recognize foreign peptide that is presented by self MHCs -T cells must recognize (bind to) self MHCs to be activated by foreign peptide Negative Selection: Eliminate TCRs that recognize self -T cells recognizing self MHC containing self peptides are potentially autoreactive

18. Positive Selection (PS) TCR physically binds to MHC on APC Select T cells that can recognize self MHCs Primary thymic repertoire has bias toward general MHCs -select those TCR that recognize inherited self MHCs (6 MHC I and minimally 6 MHC II) Only 2 % of a given thymic repertoire can recognize self MHCs; PS: process of stimulation those T cell to mature POINT: Thymic T cells undergoing positive selection are pre- programmed to die unless they receive a signal to live and mature (98% DIE) How are thymocytes (T cells in the thymus) positively selected? thymic cortical epithelial cells express MHC I and MHC II on their surface MHC molecules not stable in absence of peptide; all contain self peptides  :  chains of TCR test all MHC complexes for ones they recognize (bind to) -if bound with 3-4 days: signal to live; if not, DIE by default -120,000 self peptides presented by 12 different MHC molecules; most involved in positive selection

19. Bone Marrow Transplant: Share HLA Allotypes Bone marrow transplant: destroy recipient bone marrow cells including hematopoietic stem cells Reconstitute all blood cells including lymphocytes with donor cells T cells selected on self (recipient) MHC (thymus) APCs developing in bone marrow are donor Recipient T cells can not recognize donor APCs, unless donor and recipient have some common (share) HLA (MHC) molecules Minimum: share one common MCH I molecule and one common MHC II molecule

20. Transition from Double Positive to Single Positive Choice of whether to become a CD4 + T cell or CD8 + T cell is determined during positive selection Single positive T cells: express either CD4 or CD8, but NOT both Developing T cell interacts with MHC I on thymic stromal epithelial cells: CD8 + Developing T cell interacts with MHC II on thymic epithelial cells: CD4 + Bare lymphocyte syndromes -patients lacking MHC I have only CD4 + cells -patients lacking MHC II have only CD8 + cells

21.  Chain Gene Rearrangement During Positive Selection  chains of the TCR can continue to rearrange to enhance chances of passing positive selection (reminiscent of light chain rearrangement: BCR) Permits development of single T cell with two different  chains Probability of both chains passing positive selection is extremely remote Result: only one functional TCR per T cell

22. Negative Selection Select cells for elimination that express TCRs that strongly recognize self MHC presenting self peptides Where: cortico-medullary junction Who is selecting: macrophages and dendritic cells How: strong binding of the TCR sends signal to DIE (apoptosis) Two individuals of completely different haplotypes have non-overlapping T cell repertoires: the T cell repertoire is highly personalized.

23. Mature, Naïve T Cells Meet Ag in Secondary Lymphoid Tissue Only 1-2% of immature T cells survive selection in the thymus Surviving cells leave, enter the periphery and circulate through secondary lymphoid tissue Naïve T cells are long lived and can circulate for years in the absence of Ag (antigen) Meet Ag in the T cell area of the secondary lymphoid tissue Ag stimulates naïve T cells Effector T cells CD8 + Cytotoxic T cellsCD4 + Helper T cells TH1TH2 Thymus: Leave Leave Stay Twice as many CD4 + T cells in the circulation than CD8 + T cells

24. Alloreactive T Cells 5-10% of T cell repertoire reacts strongly to allogenic cells (non-self MHC) Allograft rejection (kidney) T cell repertoire biased to recognize MHC molecules in general Elimination of those T cells that recognize self MHCs (negative selection) leaves an increased proportion of T cells recognizing non-self MHC (allogenic)

25. 12 Different MHC Isotypes is Optimal Double MHC isotype number; double those passing positive selection Double number of isotype number; geometrically increase those deleted by negative selection Too many MHC isotypes will significantly limit the T cell repertoire 12 isotypes appear optimal Note: Skip Fig. 5.15

26. Development of T Cell Tumors T cell tumors represent different stages of T cell development (like B cell tumors)

27. T Cell Development in the Thymus

28. Stages of  :  T Cell Development