1. CARRIE MICELI (cmiceli@ucla.edu) TCR RECOGNITION AND THYMOCYTE SELECTION I APRIL 27, 2005 Reading (for this and next lecture): 1) parts of Chapter 3; p115-131 and CH7 267-293 2) How T cells 'see' antigen Michelle Krogsgaard, Mark M Davis, Nature Immunology6, 239 - 245 (01 Mar 2005) Review 3) Proteases Processing an Thymic Selection, Cresswell, Science 280 394, (2000) 4) Signaling Life and Death in the Thymus, Werlin..Palmer Science (2003) 299:1859 5) Expression of Self Antigens in the Thymus: A little goes a long way. Anderson and Kuchroo. J Exp Med 198:11:1627 (2003) (second half on T regs not covered on midterm, revisited later)

2. V regions C regions The TCR resembles an antibody Fab fragment  and  chains are involved in antigen recognition CD3 and  chains are involved in signal transduction sites for Lck phosphorylation and SH2 docking connecting peptide Multiple carbohydrate addition sites on each chain

4. # of mature T cells in the periphery Elephants: 10 16 Humans: 10 11 Mice: 10 8 We make 10 18 TCR options, but can only use 10 11 at any given time. We select for those that are useful to ensure: 1) cooperative T cells (positive selection) 2) no autoreactivity (negative selection)

5. Fig. 3.27 The T-cell receptor binds to the MHC:peptide complex. Panel a: the T-cell receptor binds to the top of the MHC:peptide complex,straddling, in the case of the class I molecule shown here, both the  1 and  2 domain helices. The CDRs of the T-cell receptor are indicated in color; the CDR1 and CDR2 loops of the  chain in light and dark blue, respectively; and the CDR1 and CDR2 loops of the  chain in light and dark purple, respectively. The  chain CDR3 loop is in yellow while the  chain CDR3 loop is in green. The  chain HV4 loop is orange. Panel b: the outline of the T-cell receptor antigen-binding site (thick black line) is superimposed upon the top surface of the MHC:peptide complex (the peptide is shaded dull yellow). The T-cell receptor lies diagonally across the MHC:peptide complex, with the  and  CDR3 loops of the T-cell receptor (3a, 3b, yellow and green, respectively) contacting the center of the peptide. The  chain CDR1 and CDR2 loops (1a,2a, light and dark purple, respectively) contact the MHC helices at the amino terminus of the bound peptide, whereas the  chain CDR1 and CDR2 loops (1b, 2b, light and dark blue, respectively) make contact with the helices at the carboxy terminus of the bound peptide. Courtesy of I.A. Wilson. From Science 1996 274:209-219. © 1996

6. Fig. 5.18 Superantigens bind directly to TCRs and to MHC molecules. Nature 1996, 384:188. Particular superantigens bind to all TCRs containing a particular V . i.e.MMTV superantigen binds to all V  5 containing TCRs. The two enterotoxin molecules (actually SEC3 and SEB) are shown in turquoise and blue, binding to the  chain of the class II molecule (yellow) and to the  chain of the T-cell receptor (colored gray for the V  domain and pink for the C  domain).

7. Agonist, partial agonist, and antagonist peptide/MHC TCR ligands.  Elucidation of which peptides are presented to T cells by MHC.  By combining knowledge of the crystal structure of class I/ peptide complexes and using MHC binding assays and reactivity of antigen specific T cell clones investigators have been able to map MHC:peptide contact sites and TCR:peptide contact sites.  Replace each amino acid within an "agonist peptide" & determined whether the altered peptide still binds MHC and/or stimulated T cells (with specificity for agonist peptide/MHC).

8. – Some "altered peptide ligands” (APL) still bind MHC but are partially impaired/altered in their ability to bind/stimulate TCR on T cell clones ="partial agonist". –Other altered peptide ligands bind MHC and TCR, but don't stimulate the clones. We know they still able to bind the TCR to some degree because they are able to antagonize the ability of the "agonist peptide" to stimulate the T cell clone. --i.e. they some how interfere. Such peptides are referred to as "antagonist peptides". Partial agonists can also function as antagonists

9. Biacore studies have determined that agonist peptide/MHC ligands have longer dwell times that partial agonist peptide/MHC and that antagonists have even shorter dwell times.  While not entirely perfect the best correlation is with off rate. On rates of antagonists can be comparable (or in some instances better) than agonists, but off rate much faster. Do agonists induce a conformational change not induced by partial agonist/antagonist peptides ? Dimerization/multimerization? How does dwell time translate into abortive or productive signaling?..or alternate signaling?

10. Qualitative and Quantitative Differences in T Cell Receptor Binding ofAgonist and Antagonist Ligands. S. Munir Alam…...Nicholas R. J. Gascoigne 1, and Paul J. Travers Immunity 10, 227,1999 The kinetics of interaction between TCR and MHC-peptide show a general relationship between affinity and the biological response, but the reported kinetic differences between antigenic and antagonistic peptides are very small. Here, we show a remarkable difference in the kinetics of TCR interactions with strong agonist ligands at 37°C compared to 25°C. This difference is not seen with antagonist/positive selecting ligands. The interaction at 37°C shows biphasic binding kinetics best described by a model of TCR dimerization. The altered kinetics greatly increase the stability of complexes with agonist ligands, accounting for the large differences in biological response compared to other ligands. Thus,there may be an allosteric, as well as a kinetic, component to the discrimination between agonists and antagonists.

11. CD4 can dimerize CD8 can exist as an  homodimer or a  /  heterodimer.  cant get to the surface without  CD4 and CD8 are TCR CO- Receptors

12. Class II MHC restricted T’s express CD4; Class I MHC restricted Ts express CD8. CD4 binds class II MHC, CD8 binds class I. Crystal Struc -ture (not full length) and dimensions compatible w/ coordinate binding

13. CD4 and CD8 function as co-receptors by binding to the same MHC (ligand) as the TCR Distinction between co-stimulators and co-receptors (for T cells) CD8 binds class I MHC at a site distinct from TCR-while CD8/MHC I interactions do not require TCR binding, coordinate binding possible CD4 binds class II at a site distinct from TCR binding site- while CD4/class II don’t need TCR for binding, coordinate binding is possible Co-receptors function best when they can bind to the same MHC as the TCR –CD8/MHC I decreases TCR/peptide MHC off rate –CD4 and CD8 cytoplasmic tails bind to Lck, coreceptor binding activates and recruits Lck to the TCR signaling complex where it functions to phosphorylate ITAMs in CD3/  and ITAM associated ZAP70 (CD4 binds better than CD8)

16. Pseudo-dimer model for CD4 coreceptor function The full length crystal structure of CD4 indicates binding at almost a 90 degree angle. Therefore its more likely that CD4 binds to the immediately adjacent class II molecule. If that adjacent MHC II has endogenous peptide that can bind with some minimal affinity (remember positive selection?) then the increased avidity offered by the CD4 and Lck contribution to agonist bound receptor enables partial agonists to participate and enhance agonist ligand induced signaling. CD4 helps most at low concentrations. This model explains how a single agonist/peptide MHC can trigger TCR activation Direct observation of ligand recognition by T cells Irvine…Davis Nature 419,, p845, October 2002 Blocking CD4 interferes with Ca+ signal at low antigen- MHC concentrations

17.  T cells can detect a single peptide-MHC on an APC. However, at least for CD4+ T cells, monomeric ligands can’t stimulate. What gives? Constructed soluble peptide-MHC heterodimers in which one peptide is an agonist and the other an endogenous peptide. Some combinations of these heterodimers can stimulate specific T cells Activation depends on an intact CD4-binding site on agonist/MHC ligand, but not on endogenous peptide/MHC ligand. Validates the suggestion that the basic unit of CD4 T cell activation is a heterodimer of agonist peptide/MHC:endogenous peptide/MHC, stabilized by CD4. Need to modify the original pseudo-dimer model (CD4 on endog/MHC not important). Krogsgaard……Davis, M. M, Nature March 10, 2005

18. a, b, 5C.C7 T cells were loaded with fura-2 and a dose-response curve was determined for the percentage of cells with elevated Ca2+ signalling (340/380 nm; >150 cells; percentage of cells plusminus s.d., n = 5) when the T cells were mixed with the indicated pMHC dimers (a) or with lipid bilayers containing B7-1, ICAM-1 and combinations of the indicated proteins (b). c, d, A modified pseudodimer model, in which the binding of a TCR (TCR1) to agonist pMHC (red circle) leads to the recruitment of a second TCR (TCR2), mediated by a CD4 molecule associated with it. TCR2 binds to an endogenous pMHC (yellow circle), resulting in a five-member complex stable enough to initiate activation through the tyrosine kinase Lck, phosphorylating one or both CD3 clusters (not shown) K5= agonist peptide; ER60 and endogenous (self) peptide; m is mutant CD4 binding site CD4 Binding site on Agonist /MHC matters

19. Modified pseudo-dimer model “In this model, the binding of a TCR (TCR1) to an agonist ligand creates a 'hotspot' for activation stable enough to recruit TCR–CD4 complexes (TCR 2). TCR2 then binds to a co-agonist endogenous pMHC and the resulting pseudodimer (TCR1/agonist peptide/TCR2/endogenous peptide/CD4) triggers an activation cascade emanating from the key protein kinase Lck carried by the recruited CD4. We have previously shown that Lck is rate-limiting with respect to early T cell activation. We predict that the agonist ligand induces a significant conformational change to TCR1 that allows the subsequent recruitment of TCR–CD4 to occur. This may involve a twisting or piston-like motion of the TCR27-29, which may in turn facilitate CD4 binding, CD3 phosphorylation, or both. In conclusion, we show here that T cells can be stimulated by heterodimers containing one strong and one very weak ligand. These data indicate a direct role for both endogenous peptide–MHC complexes and CD4 in triggering T cell activation. It also provides a mechanistic basis for the suggestion that the purpose of thymic selection is not only to select a TCR repertoire that can recognize foreign peptide–MHC complexes, but one that can also use self-peptide–MHC complexes to achieve maximal sensitivity”

20. Nature Immunology march 24 2004 Purboo…Davis observe dynamics and spatial overlap of TCR, CD8, transducers and MHC clustering at the T cell:APC interface…and CD8 requirements T cell can detect single TCR engaged but need 3 for CTL activity and 10 for complete Ca flux and synapse formation CD8 needed for initial conjugate formation and at all densities –Controversial as to whether similar psudo dimer type model exists for CD8/class I restricted TCRs. Some evidence against invovlment of endogenous peptides class I restricted T cells (reis e sousa EJI) –Some evidence that monomer can stimulate (but might be artifact with monomers created)

22. T cell Development in the Thymus Changes in cell-surface molecules allow thymocyte populations at different stages of maturation to be distinguished. The most important cell-surface molecules for identifying thymocyte subpopulations have been CD4, CD8, and T-cell receptor complex molecules (CD3, and a and b chains). The earliest cell population in the thymus does not express any of these. As these cells do not express CD4 or CD8, they are called 'double-negative' thymocytes. (The g:d T cells found in the thymus also lack CD4 or CD8 but these are a minor population.) Maturation of a:b T cells occurs through stages where both CD4 and CD8 are expressed by the same cell, along with the pre-T-cell receptor (pTa:b) and later low levels of the T-cell receptor (a:b) itself. These cells are known as 'double-positive' thymocytes. Most thymocytes (~97%) die within the thymus after becoming small double-positive cells. Those whose receptors bind self MHC molecules lose expression of either CD4 or CD8 and increase the level of expression of the T- cell receptor. The outcome of this process is the 'single-positive' thymocytes, which, after maturation, are exported from the thymus as mature single-positive T cells. CD3-/low CD3/TCR low CD3/TCR hi

23. Normal/typical mouse thymus contains 5% double negatives 1% DN  T; 1% DN  T; 3% immature progenitors 78%CD4 CD8 double positives –1%each CD4loCD8hi; CD8loCD4hi “transitional cells 10% CD4 single positives 5% CD8 single positive

26. Thymocytes of different developmental stages are found in distinct parts of the thymus

27. How do we experimentally determine the progressive stages of cell differentiation? Method 1: Inject the sorted or fractionated double negative thymocytes and follow their development in vivo. The more mature a thymocyte is, the more rapidly it will give rise to single positive T cells. Method 2: Determine the extent of TCR  and  gene rearrangement in sorted populations by southern blots. Method 3: Study gene knockouts ie CD3 , , or RAG

29. Phases during which thymocytes undergo cell division = curved arrows. The extent of T cell development in thymus of mice (red ) or humans (blue) deficient for a few selected genes is shown. Stringent developmental blocks=continuous bars; leaky mutations =dashed bars. For mouse T cell development time line: 1, Ikaros; 2, c-kit + c and GATA-3; 3, winged-helix nude; 4, IL-7, IL-7R, c, JAK-3, and c-kit; 5, c +. pT; 6, RAG-1, RAG-2, SCID (DNA-PK catalytic subunit), Ku80, TCR enhancer, CD3-5 + CD3-/, TCRa + TCRb, Lck + Fyn, and ZAP-70 + Syk; 7, pT; 8, CD3-/, Vav, Lck, CD45, and TCF-1 + LEF-1; 9 MHC class II, CD4, and H-2M; 10b (only CD8+ T cells), MHC class I, 2m, TAP-1, and CD8; and 11, LKLF. For human T cell 9, TCF-1 + LEF-1; 10 (affecting CD4+ and CD8+ Ts), TCR enhancer, CD3, and ZAP-70; 10a (only CD4+ T cells), development time line: 1, reticular dysgenesis; 3, Di George syndrome; 4, SCID-X1 (c) and SCID JAK3; 6, RAG-1, RAG-2, and other SCID; 10a, MHC class II; and 10b, ZAP-70 and TAP-2.

31. Fig. 7.10 Developing T cells that undergo apoptosis are ingested by macrophages in the thymic cortex. A) a section through the thymic cortex and part of the medulla in which cells have been stained for apoptosis using TUNEL. Thymic cortex is to the right. Apoptotic cells are scattered throughout the cortex, rare in the medulla. b)higher magnif. stained red for apoptotic cells and blue for macrophages. Apoptotic cells are seen within macrophages. Sprent & Suhr. Nature 1994, 372:100. In normal B6 mouse can count only 0.5%-1% of total thymocytes undergoing apoptosis if assume Tunel positive cells only visible for an hour can account for 12-24% of cells dying. Conclusion: apoptosis is fast and so are those macrophages. Dynamic process

32. Why all the death? Quality control, receptor checking First test of quality is at preT  stage to see if  is functional (and can bind ligand? No ligand to date). Those that don’t probably die, definitely don’t expand. TCR  chain is required for thymus maturation.  deficient mice have 50X smaller thymus and lack DP thymocytes TCR  gets to the surface with a surrogate  chain (pT  ) which transduces a (lck/fyn dependent) signal for the cell to rapidly proliferate and continue development by rearranging alpha. This gives functionally rearranged  an opportunity to pair with several . Mice deficient for preT  or lck and fyn similar to  

33. Thymocytes from preT  -/- are blocked at the DN to DP transition. same as:  chain -/- Lck Fyn The pre-TCR requires pT  +  for surface expression and signaling a Lck and Fyn dependent proliferative burst.

34. After  TCR emerges on the surface it needs to be checked out for two reasons During “positive selection” cells expressing TCRs with a “minimal” ability to interact with peptide/MHC are selected on thymic epithelial cells to undergo a number of activation and differentiation events including rescue from apoptosis and maturation into CD4 or CD8 single positive cells. –Can a TCR bind self MHC at all –Is it worthy of a spot in the periphery (i.e. likely to be useful?) If no, die of neglect (via apoptosis), if yes receive development signals –Ensure “correct” coreceptor choice Class II restricted TCR develop to CD4+ (and helper function) Class I restricted TCR develop to CD8+ (and cytotoxic function) During “negative selection”, thymocytes with TCRs highly reactive with self MHC/peptide ligands presented on dendritic (and in some instances thymic epithelial cells) are purged from the repertoire. –Is a TCR highly auto-reactive and likely to attack self in the periphery? –If yes die as a result of active signal for apoptosis –Thymocytes make transition from most undifferentiated T cells to mature T immunocompetent cells. Tolerence must be induced here Only 1% get out alive

35. Strategies for studying selection/tolerance in the thymus The problem is (was) diversity/ T cells each have a different specificity To determine criterion for selection, one must be able to correlate specificity with outcome during development TCR transgenics to the rescue!! –Make a mouse with fully rearranged TCR  and  transgenes that produce  TCR with a given specificity –rearranged  chain expression inhibits endogenous  chain rearrangement via “allelic” exclusion mechanisms, so all T cells are TCR tg  + –  chain rearrangement not turned off simply by  /  surface expression. Rather, turned off with “proper” ligand binding (positive selection turns off RAG genes). Thus, “allelic exclusion” of  is leaky. –Can get rid of cells expressing endogenous  rearrangement By staining with a “clonotypic” antibody against Tg TCR or w/ anti-  chain By crossing to a Rag1/2-/- mice, rearrangement of endogenous genes impossible Superantigen expression in the thymus leads to deletion cells expressing particular Vb genes.

36. Positive and Negative Selection in the H-Y (Rag -/- ) Transgenic model Small thymus -negative selection CD8 low Tg T cells Normal thymus -positive selection CD8 hi Tg T cells T cell APC CD8 CD3 Tg TCR HY peptide MHC

37. C57BL/6 control male HY TCR transgenic Female HY TCR transgenic male Expression of TCR Tg in females with H-2D b results in development of CD8+T (increased CD8/CD4) Expression of TCR tg and autoantigen in males results in deletion of DP thymocytes and blocks development of Tg+ CD8 SP Harold von Boehmer HY TCR transgenic mice

38. In Rag-/-HY TCR transgenics all thymocytes express the TCR tg. More dramatic skewing to CD8 in females and deletion in males Female HY TCR tg CD4 0.6%; 5.8%; 1.2x10 6 101x10 6 35x10 6 21x10 6 2.8%; 91%; 75%; 26x10 6 11%; 2x10 6 5%; 1.5x10 6 9%; 2.7x10 6 CD4 CD8 Male HY TCR tg

39. CD8 4% 1% 79%, 20x10 6 15%, 2x10 6 2.6%, 0.7x10 6 81%, 15x10 6 9%, 0.9x10 6 CD4 Males Females Peripheral T cells from spleen of HY-TCR transgenic mice wt mice have 2:1 CD4:CD8 Normal levels of CD8 on CD8+ T cells HY/D b responsive (stimulated by male APCs) CD8 downregulated on those cells that do escape HY/D b nonresponsive

40. TCR  tg IE + sAg + apoptotic cells in the medula a,b,c,d) blue cortical epithelial cells, red apoptosis (TUNEL) A B C D E F d) blue, macrophages; red apoptosis I-E -, all others I-E + note:very little negative selection in response to other self antigens in I-E -

41. During positive selection CD4/CD8 coreceptor expression is coupled to T cell specificity Thymocytes expressing class II restricted TCRs develop into CD4 SP Thymocytes expressing class I restricted TCRs develop into CD8 SP CD4 development requires class II expression, only CD8s present in class II knockouts CD8 development requires class I expression, only CD4s present in class I knockouts

42. How is TCR specificity coupled to proper co- receptor expression and developmental program? Instructive Model: during positive selection TCR/CD4 engagement by MHC II sends an instructive signal for CD8 off/CD4 on; engagement by MHC I sends an instructive signal for CD4 off/CD8 on. What is the nature of this signal and how does it cue a developmental program which includes functional maturation (helper or CTL)? Stochastive/selective model:CD4 or CD8 is randomly turned off prior to positive selection. Those with “matching” TCR and coreceptor expression are selected during positive selection. Those that randomly made the wrong choice die of neglect.

43. How do we keep from negatively selecting everything that we positive select? What is the difference between a positively selecting TCR ligand and a negatively selecting ligand? What is the difference between a signal for positive selection and negative selection?

44. both TCR loci have rearranged and concurrently excised the TCR loci. The phases during which thymocytes are undergoing cell division are highlighted by curved arrows. The developmental stages shown correspond to those found in the mouse. 44, CD44; 25, CD25. (Bottom) The extent of T cell development in thymus of mice (red time line) or humans (blue time line) (85) deficient for a few selected genes is shown. Stringent developmental blocks are depicted as continuous bars, whereas leaky mutations are depicted as dashed bars. The bars interrupting the mouse T cell development time line correspond to mutations in the following: 1, Ikaros; 2, c-kit + c and GATA-3; 3, winged-helix nude; 4, IL-7, IL-7R, c, JAK-3, and c-kit; 5, c + pT; 6, RAG-1, RAG-2, SCID (DNA-PK catalytic subunit), Ku80, TCR enhancer, CD3-5 + CD3-/, TCR + TCR, Lck + Fyn, and ZAP-70 + Syk; 7, pT; 8, CD3-/, Vav, Lck, CD45, and TCF-1 + LEF-1; 9, TCF-1 + LEF-1; 10 (affecting both CD4+ and CD8+ T cells), TCR enhancer, CD3, and ZAP-70; 10a (affecting only CD4+ T cells), MHC class II, CD4, and H-2M; 10b (affecting only CD8+ T cells), MHC class I, 2m, TAP-1, and CD8; and 11, LKLF. The bars interrupting the human T cell development time line correspond to mutations in the following: 1, reticular dysgenesis; 3, Di George syndrome; 4, SCID-X1 (c) and SCID JAK3; 6, RAG-1, RAG-2, and other SCID; 10a, MHC class II; and 10b, ZAP-70 and TAP-2. Natural and Engineered Disorders of LymphocyteDevelopment Alain Fischer, Bernard Malissen Science 1998 280: 237-243 Figure 1. Genetic dissection of the T lymphocyte developmental pathway. (Top) In adult mice, bone marrow contains hematopoietic stem cells (HSC),which can give rise to all lymphoid populations through common lymphoid progenitors (CLP). Upon thymus colonization, these progenitors developinto cells that express low CD4 (CD4low). Next, these CD4low precursors lose CD4 expression to become TN (CD4CD8CD3) cells. On the basis of the expression of CD25 and CD44, mouse TN cells have been subdivided in three different subsets. Late TN cells can mature into CD4+CD8+ (DP) cells, some of which develop into CD4+CD8 or CD4CD8+ (SP) cells that exit from the thymus. During intrathymic differentiation, the genes encoding the TCR variable region are assembled by site-specific DNA recombination reactions. TCR gene rearrangements start around the transition to the CD44/low CD25+ TN stage, whereas the first TCR rearrangements are measurable close to the transition to the DP stage. The CD4low precursors can also produce NK cells and thymic DCs. In contrast, cells belonging to the next developmental stage (CD44+CD25+) cannot generate NK cells but still give rise to thymic DCs. Commitment to the T cell lineage occurs at the next TN stage (CD44/lowCD25+), coincident with the onset of TCR, TCR, and TCR gene rearrangements. Finally, the irreversible decision to become an rather than a T cell may not take place until

45. High Avidity Low Avidity