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2. Nave T cell Clonal T cell progeny with effector functions 3. Key Points: 1.T cell activation occurs during a complex cell-cell interaction 2.Stimulation of a G OT cell results in cell cycle progression as well asenormous metabolic and synthetic changes as well as cell enlargement 3.Activation is initiated by the TCR, but also requires multiple otherreceptors involved in intercellular interactions 4.Key event is the production of lymphokines and the cellularresponse to them 5.End result is the clonal expansion and acquisition of effector functions 4. Complexity of the T Cell : Antigen Presenting Cell Interaction 5. T Cell Target Cell MTOC Polymerized Actin T Cells Polarize During Antigen- Specific Recognition 2.MTOC and microtubules polarize 3.Actin cytoskeleton polymerizes and polarizes 4.Requires TCR signals: Rac/Cdc42 activation and Ca 2+ elevation (calcineurin- and CaMK- independent events)5.Polarization of cytoskeletal elements contribute to polarized secretion of cytolytic granules or lymphokines 1.T cell polarizes, APC does not 6. Immunologic Synapse 7. SMAC (supramolecular activation complex)
c-SMAC c-SMAC p-SMAC TCR/CD3, CD2, CD28, PKC lck LFA-1, Talin, CD43, CD45, CD148
8. Formation of an Immunological Synapse Involves Coordinated Spatial Organization Red= ICAM-1 Green=MHC/pep Grakoui et al. (1999) Science285 :221. 9. Signaling Occurs in TCR Microclusters at the Periphery of the SynapseYokosuka et al., Nature Immunol. 6, 1253, 2005 10. Function(s) of the Immunological Synapse? 1.Signal Initiation :probably not P-tyr and Calcium responses precede mature synapse formation 2.Signal enhancement :perhaps But no biochemical correlate and mature synapsedoes not appear to be required(CD2-AP KO signals better than wt but does not form synapse) Peripheral microclusters appear to be most active signaling complexes 3.Directed secretion :fits well Directed secretion by CTL and Th cells makes teological sense and has been well documented 4.Receptor downmodulation :likely Recent modeling and CD2-AP KO data support this New data on TCR microclusters also supportive 11. 12. 13. 14. The TCR is an Oligomer and Utilizes CD3 andChain ITAMs to Initiate Signal Transduction TCRand 15.
16. Mechanisms for Signaling Ligand Occupancy States Across Membranes Cochran et al, TIBS, 28:304, 2001 GPCRs RTKs Antigen Receptors? TCR/CD4 Cytokine Receptors? Bacterial Asp Receptor 17.
18. Irving and Weiss, Cell, 1991 CD3 mAbs Against the TCR/CD3 Complex or ChimericReceptors can Induce Similar Signals and Responses 19. A Hetero-Oligomerization Model The TCR and CD4 coreceptor both interact with peptide/MHC complex, bringing the Lck kinase into close proximity with TCR. 20. Some recent data support a conformational change 21. Conformational Change in the TCR Constant Region A-B Loop in the LC13 TCR CD3 thought to bind here Ligated - red Unligated - cyan Kjer-Nielsen, et al., Immunity, 2003 22. A CD3 Proline Motif May Become Accessible During a Putative TCR Conformational Change:Models (Alarcon, et al,Cell , 2002;Gil, et al,J. Exp. Med ., 2005; Levin and Weiss,J. Exp. Med ., 2005) SH3 SH3 SH3 SH2 N c k actin cytoskeleton Pak kinase ???? 23. The TCR is an Oligomer and Utilizes ITAMs toInitiate Signal Transduction TCRand 24. 25. 26. Lipid rafts, GEMs, DIGs
27. 28. 29. CD45
30. PRIMED Dynamic Lck Regulation by Activating and InhibitoryTyrosine Phosphorylation CD45 Csk Receptor Stimulation CD45 or PTPN22 Y Y INACTIVE P Y ACTIVE P Y 31. ZAP-70 in TCR Signaling
32.
Deindl, et al., Cell, 2007 33. 34. Hypothetical Conformational Change in ZAP-70Upon ITAM Binding Contributes to Its Activation 35. New Model: Intramolecular Regulatory Switch in ZAP-70: Initiation of Activation by Lck-MediatedPhosphorylation of a Negative Regulatory Switch Brdicka, et al., Mol. Cell Biol. June 2005 36. 37. A Hetero-Oligomerization Model: 38. 39. 2 Adapters Are Phosphorylated by ZAP-70: Required for PLC and Ras Activation in T Cells LAT SLP-76 LAT function:Lipid raft recruitment ofeffectors SLP-76 function:Organization of signaling complex - Y D S T - Y P R G - Y P P V - Y E N E - Y H N P - Y L V V - Y V N V - Y V N V - Y E N L SH2 . . . Y E S P . . . . . Y E S P . . . . . Y E P P . . . . . Pro-rich 40. 41. Assembly of LAT and SLP-76 into a Signalosome in Lipid Rafts Is required for Multiple Downstream TCR Signaling Pathways Events requiring LAT & SLP-76 PLC 1 activation Ras activation Rac activation HPK1 activation 42. I t k / T e c / R l k K i n a s e S H 3 S H 2 PH T H Tec Kinases in T cells
Y Y 43. 44. Sequential Events in TCR Signal Transduction 45. PLC Activation Leads to Second Messenger Generation and Mobilization of Calcium as well as PKC and RasGRP Activation 46. Evidence for the Importance of the Phosphatidylinositol Pathway in T cells 1.Activating and synergistic effects of calcium ionophores and phorbol esters 2.Inhibitoryeffects of calcium chelators and PKC inhibitors 3.Positive effects of activating alleles of calcineurin and PKC or Ras) 4.Ability of heterologous G-protein coupled receptors that activate PLC beta but not Src or Syk PTKs to induce IL-2 gene activation 5.T cell activation defects in PKC theta mice 47. The Phosphatidylinositol Pathway: Calcium Mobilization after TCR Stimulation + EGTA Initial release from Intracellular stores (results from IP3 interacting with receptors) Depletion of intracellular stores results in a transmembrane flux of calcium, Icrac Downstream Consequences: Activation of Calcineurin(Ca/CaM-dependent serine/threonine phosphatase), thetarget of Cyclosporin A and FK506 Activation of Ca/CaM kinase Time (min) 1 Lewis Annu. Rev. Immuno. 2001 48. Marchant, Current Biology, 2005 STIM is the ER Calcium Sensor and Orai is a Component of SOC Orai Prakriya, et al.,, Nature, 2006 Depletion of intracellular stores results in altered function and distribution of ER resident protein STIM. STIM migrates in puncta towards plasma membrane where it regulates the recently identifiedIcracchannel, Orai, in the plasma membrane to allow a transmembrane flux of calcium 49. The Phosphatidylinositol Pathway: PKC activation after TCR Stimulation PKC - a large family of serine/threonine kinases Multiple isozymes in T cells DAG-regulated, phorbol ester responsive Some are calcium responsive PKC theta localizes to cSMAC, and KO mice have selective defect in NF-kB activation in T cells Function: TCR downregulation NFkB activation Activation of RasGRP 50. TCR Stimulation Activates Ras R a s ( G D P ) i n a c t i v e R a s ( G T P ) a c t i v e P O 4 R a s G A P N e u r o f i b r o m i n G r b 2 / S O S RasGRP G T P G D P R a s G E F s R a s G A P s R a s E f f e c t o r s : R a f - 1 K i n a s e P I - 3 K i n a s e G A P R h o G T P a s e s 51. The TCR can Activate Ras via at Least 2 Mechanisms Roose and Weiss, Nat. Immunol., 2000 52. 53. 54.