T cell development October 28, Dan Stetson

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1 T cell development October 28, 2016 Dan Stetson 441 Lecture #13 Slide 1 of 29

2 Three lectures on T cells (Chapters 8, 9) Part 1 (Today): T cell development in the thymus Chapter 8, pages Part 2 (Monday): T cell activation Part 3 (Wednesday): T cell effector function 441 Lecture #13 Slide 2 of 29

3 Making useful and safe T cells How does a hematopoietic stem cell turn into a CD4 + MHC class IIrestricted helper T cell or a CD8 + MHC class I-restricted cytotoxic T cell that can respond to a pathogen but not to your own tissues? Too few pathogen-specific T cells: HIV/AIDS Persistence of autoreactive T cells: Autoimmune disease Type I diabetes: T cells attack insulin-producing cells in pancreas (Normal CD4 T cell count is cells/mm 3 ) 441 Lecture #13 Slide 3 of 29

4 T cell development proceeds through several steps Periphery (SP, LN) ~3 weeks Bone marrow-derived stem cell: TCR genes in germline configuration No coreceptor expression (CD4-, CD8-) Not yet committed to the T cell lineage Not immunocompetent Mature T cell: Rearranged TCR genes Either CD4+ or CD8+ Immunocompetent Self MHC-restricted Self tolerant 441 Lecture #13 Slide 4 of 29

5 What happens in the thymus? 1. Developing T cells commit to either the TCRαβ or TCRγδ lineage (involves rearranging completely different antigen receptor genes). 2. Allelic exclusion during gene rearrangement (one TCR/cell). 3. Keep thymocytes that can interact with your own MHC molecules: Positive selection. 4. Eliminate thymocytes that see self too well: Negative selection. Self to a TCR means self-peptides presented by their own MHC class I or class II molecules. 5. CD4 versus CD8 lineage commitment. 441 Lecture #13 Slide 5 of 29

6 What is so special about the thymus? T cell development is a sequential process that occurs while they move through different compartments of the thymus Thymocytes move outside-in as they develop Epithelial cells, macrophages, dendritic cells all very important Epithelial cell network supports thymocyte development: 441 Lecture #13 Slide 6 of 29 Stetson 10/28/2016

7 Both bone marrow-derived and epithelial cells are required to make T cells SCID mouse: DNA-PKcs mutant can t rearrange BCR,TCR genes Nude mouse: Transcription factor mutant can t develop thymic epithelial cells Normal peripheral T cells, all of nu/nu origin 441 Lecture #13 Slide 7 of 29

8 20% become γδ T cells (don t worry about these) (~5% of total Thymocytes) Thymocyte development overview 60% become αβ T cells...and another 20% NKT cells We are only going to focus on αβ T cells today 441 Lecture #13 Slide 8 of 29

9 Thymocyte maturation occurs in stages that can be defined by expression of CD4 and CD8 441 Lecture #13 Slide 9 of 29

10 Flow cytometric analysis of CD4/CD8 on thymocytes 441 Lecture #13 Slide 10 of 29

11 DN, DP, SP thymocytes localize to discrete regions 441 Lecture #13 Slide 11 of 29

12 T cell receptor β and α chain gene rearrangements Similar to Ig loci, but more Ds and Js are shown here Remaining Js between the V-J and the C are removed by RNA splicing D-J occurs first, then V DJ second in the TCRβ locus 441 Lecture #13 Slide 12 of 29

13 TCRβ and TCRα genes rearrange at distinct times RAG1/2 gene expression is activated in early DN thymocytes TCRβ rearranges FIRST, in DN T cells 441 Lecture #13 Slide 13 of 29

14 β selection checkpoint: tests for successful β chain VDJ gene rearrangement Newly generated TCRβ chain pairs with a surrogate α chain (ptα) encoded by a nonrearranged gene. This heterodimer is called the pre- TCR. Pre-TCR is expressed on cell surface in a complex with CD3 (TCR signaling components). Membrane expression of this complex delivers a signal via Lck kinase (without ligand engagement), resulting in: A. RAG genes are turned off B. Cell proliferation starts C. Expression of CD4 and CD8 441 Lecture #13 Slide 14 of 29

15 The consequences of β selection β selection results in an expanded population of DP thymocytes carrying functionally rearranged TCRβ genes ptα TCRβ It also ensures that the TCRβ genes are allelically excluded, such that each cell carries only one functionally rearranged TCRβ allele Each thymocyte has two tries to rearrange a TCRβ gene (two alleles). DN DP 441 Lecture #13 Slide 15 of 29

16 TCRα gene rearrangement begins after successful β selection Signals through the pre-tcr stop β rearrangement, drive proliferation and induce CD4/CD8 expression and TCRα transcription. ptα/tcrβ CD4 CD8 Rag genes are reexpressed and Vα-Jα rearrangement begins. Surface expression of TCRαβ occurs; positive and negative selection are initiated. surface TCRαβ lo CD4 CD8 441 Lecture #13 Slide 16 of 29

17 Second checkpoint in thymocyte maturation: Positive Selection TCRβ/pTα TCRβ/TCRα DN Checkpt #1: β selection DP Checkpt #2: positive selection Positive Selection tests for a functional TCR complex that is: 1) expressed on the cell surface 2) can interact with self MHC + self peptide 441 Lecture #13 Slide 17 of 29

18 The consequences of positive selection positive selection tests Am I a useful TCR? DP DP dull Positive selection drives: A. Extinction of RAG expression (no more V(D)J rearrangements) B. Upregulation of TCR expression C. Downregulation of CD4 and CD8 expression (DP dull ) 441 Lecture #13 Slide 18 of 29

19 What happens to cells that fail to make a useful TCR? Cells that do not receive a positively selecting signal eventually die by neglect. Finding the right match between TCR specificity and expressed MHC molecules is difficult! Continued Rag expression and delayed death by neglect give developing thymocytes more than one chance to get it right. Dead thymocytes (tens of millions per day) are cleared by macrophages: Apoptotic cells stained brown Macrophages stained blue 441 Lecture #13 Slide 19 of 29

20 Plan B for cells that fail positive selection Subsequent TCRα gene rearrangements can occur on both chromosomes (maximizes chances for positive selection) This makes allelic exclusion at TCRα genes less strict than at TCRβ Some T cells can have two TCRα chains paired with the same TCRβ chain 441 Lecture #13 Slide 20 of 29

21 Which cells in the thymus present MHC-peptide for positive selection? Choices: the main MHC I/IIexpressing cells in the thymus are: dendritic cells + macrophages (bone marrow derived) thymic epithelial cells Radiation bone marrow chimera experiments have shown that thymocytes are selected to recognize self- MHC/peptide by radioresistant thymic epithelial cells. These epithelial cells are in the cortex. 441 Lecture #13 Slide 21 of 29

22 The first two checkpoints of thymocyte maturation 1x %? % <APC-A>: CD4 CD % : β selection x10 5 <APC-Cy7-A>: CD8 CD8 2: positive selection? 2.2% What drives lineage commitment in DP T cells to become CD4+ or CD8+? 441 Lecture #13 Slide 22 of 29

23 Lineage commitment and positive selection work together positive selection Lineage commitment: Did I lose signaling strength? Upregulate CD4 NO CD4 + SP YES CD4 + CD8 + CD4 low CD8 low CD4 + CD8 low CD8 + SP Lineage commitment matches coreceptor expression (CD4 or CD8) with the MHC restriction specificity of the TCR (Class I or Class II) This choice is still not understood, but the TCR itself plays the principal role How do we know this? 441 Lecture #13 Slide 23 of 29

24 T cell receptor transgenic mice Imagine cloning out the rearranged TCRα and TCRβ genes from two mature T cells from the lymph node, one CD4+ and one CD8+: CD4 + CD8 + In mice transgenic for rearranged these TCRα and β chain genes, the transgenes are expressed early in T cell development. This early expression suppresses the rearrangement of endogenous loci. These mice generate a monoclonal T cell population (all T cells are identical) if the MHC on the thymic epithelial cells matches the MHC specificity of the TCR. 441 Lecture #13 Slide 24 of 29

25 The TCR/MHC interaction is the primary mediator of coreceptor choice Transgene-encoded TCR from CD8+ T cell Transgene-encoded TCR from CD4+ T cell CD8 + CD Lecture #13 Slide 25 of 29

26 The final checkpoint for maturing thymocytes: Negative selection Interaction with MHC-II + self peptide CD4 + Interaction with MHC-I + self peptide CD8 + SP STRONG interaction with MHC + self-peptide Apoptotic death: Clonal deletion CD4 + CD8 low Negative selection during SP stage asks Am I a safe TCR?? Removes thymocytes with a dangerously high affinity for self MHC + self peptide Mediates central tolerance so that mature T cells are not self-reactive 441 Lecture #13 Slide 26 of 29

27 TCR affinities must be different for positive and negative selection TCR Affinity for Ligand Positive Selection low Neglect Negative Selection Ligand Density high 441 Lecture #13 Slide 27 of 29

28 Which cells in thymus mediate negative selection? This experiment: graft tests whether T cells mature that are strongly reactive to MHC b MHC b graft onto MHC a mouse: rejection The only cells that express MHC b are the bone marrow-derived APCs in the thymus Any T cells that are strongly reactive to MHC b are negatively selected in this mouse and the graft is tolerated Therefore, self-reactive T cells are eliminated by MHC + self peptides expressed mainly by bone marrow-derived cells (dendritic cells mostly) 441 Lecture #13 Slide 28 of 29

29 When a T cell progenitor enters the thymus, what are the odds of making it out alive? 100 stem cells enter/day 5 x 10 7 cells generated/day (proliferation) 1 x 10 6 T cells exit/day ~98% of thymocytes that begin differentiation in the thymus also die in thymus most are eliminated at the DP stage due to a failure of positive selection. That is because the end result - a self MHC restricted, self tolerant T cell - is very difficult to achieve! 441 Lecture #13 Slide 29 of 29