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• 1. Microbiology 204 TCR Signal Transduction Art Weiss October 9, 2009

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)

• Kupfer - deconvolution IF microscopy of T cell/APC interactions - organized, bulls-eye type structure

c-SMAC c-SMAC p-SMAC TCR/CD3, CD2, CD28, PKC lck LFA-1, Talin, CD43, CD45, CD148

• Non-activating, altered, peptides do not support the formation ofthese structures

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.

• How do TCRs transmit their ligand occupancy state across

• the plasma membrane?

• 2.Do TCRs undergo conformational changes when they bind ligand?

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.

• Support for the Oligomerization Model

• Bivalent (but usually not univalent Fab) mAbs can induce

• • T cell responses

• Many different mAbs can induce T cell signals and responses

• The more multivalent, the better (role of immobilization to surface

• or to Fc receptor)

• Chimeric receptors(ITAM containing chimeras)

• Hetero-oligomerization with coreceptors (ie. CD4) increases

• • • sensitivity to peptide/MHC molecule

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

• • • Cholesterol-rich microdomains in the plasma membrane

• • • (25 to 70 nm in diameter)

• • • Enriched in GPI-linked receptors and glycosphingolipids

• • • Enriched in many signaling molecules, including:

• • • PIP2 and PIP3, several GTPases including Ras,

• • • Src kinases, some heterotrimeric G-proteins

• • • Operationally defined based on detergent solubility

• • • Some of Lck (50% or less) partitions into lipid rafts

• • • (reversibility and dynamic changes?)

• • • May be a focal point of signal initiation or sequestration

• • • -A topic of considerable controversy!

27. 28. 29. CD45

• Receptor-like protein tyrosine phosphatase expressed on all nucleated hematopoietic cells

• Distinct isoforms expressed in a cell-type and developmental-stage specific manner

• Required for T cell and B cellantigen receptor signaling and for normal development - dephosphorylates negative regulatory tyrosine in Src kinases.

• Mutations associated with SCID and

• autoimmunity in humans and mice

• Contains tandemly duplicated phosphatase domains, but only domain 1 is functionally active.

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

• • Expressed only in T cells and NK cells

• • Unique N-terminal SH2 domain (incomplete; unlike Syk)

• • Cooperative binding to doubly phosphorylated ITAMs

• • Catalytic activity induced by phosphorylation of activation loop

• • Positive and negative sites of phosphorylation

• • Negative sites Positive sites (? Allosteric)

• • Y292-c-Cbl (Ub system) Y315-Vav

• • Y492-? Allosteric Y319-Lck, PLC gamma 1, Crk

• • Potential Substrates:LAT, Slp-76

• • ZAP-70 mutations:Cell line-loss of TCR signaling

• • Mouse KO - developmental arrest (DP -> SP)

• • Human SCID - No CD8 T cells

• • CD4 T cells with TCR signaling defect

• • SKG mutant - C-terminal SH2 mutant - Balb/c - RA-like disease

32.

• Key Features of the Full-Length ZAP-70 Structure

• Y315F and Y319F are sequestered

• by hydrophobic interactions involving

• SH2 linker region and C-terminal

• kinase domain - representing an auto-

• inhibitory mechanism

• The kinase domain is in an inactive

• conformation due to interactions stabilizing

• the hinge region

• Alignment of SH2 domains is

• not consistent with ITAM

• binding

• A conformational change likely

• occurs after ITAM binding

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

• Itk

• Expressed predominantly in T cells

• PH domain binds to PIP3 which recruits Itk to membrane

• Kinase domain is phosphorylated and activated by Lck

• TH and SH3 domains interact in basal state to inhibit kinase

• Autophosphorylation of Y180 adjacent to SH3 domain allows Itk to unfold,

• rendering SH2 and SH3 domains accessible

• Likely to phosphorylate and activate PLC 1

• KO in mice results in mild defect in PLC activation and Calcium flux

• • (redundant roles of other family members, Rlk and Tec, 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.