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Lymphocyte Signal Transduction

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  • 1. Lymphocyte Signal Transduction Arnold B. Rabson CHINJ
  • 2. Lymphocyte Signal Transduction • Basic Common Principles: B and T cells • T Cell Activation Signaling – The Immunological Synapse – Membrane events – Signal transduction molecules – Transcriptional Regulation – Cytoskeletal Reorganization • Inhibition of T cell activation signaling: immunosuppression • Diseases of immune signaling – Malignancies (T- and B-) – Immunodeficiencies
  • 3. General Principles: Antigen Receptors • Multi-protein complexes: – Clonally variable antigen-binding chains • Ig • TCR – Constant chains involved in receptor assembly at cell surface and signal transduction
  • 4. Antigen Receptors: B cell • Monomeric Ig (spliced with TM domain, short cytoplasmic tail) • Invariant chains: – Ig – Ig – Provide membrane assembly – Provide cytoplasmic domains for signaling- ITAMs (immunoreceptor tyrosine-based activation motifs sites) of tyrosine phosphorylation and SH2 From Janeway, docking) Immunobiology Fig6.7, Garland Pub, 2001
  • 5. Antigen Receptors: T cell • TCR heterodimer with Ag binding domain • Invariant chains: – CD3 complex • CD3 • CD3 • CD3 – Zeta ( ) chain homodimer – Roles in assembly and signaling (ITAMs) • History: differential cloning and monoclonals From Janeway, against T cell clones Immunobiology Fig6.8, Garland Pub, 2001
  • 6. General Principles: Receptor Activation • Receptor Association – BCR and TCR move to “microclusters” during activation • BCR Cross-linking – Experiments: Effects of F(ab’)2 vs Fab fragments in inducing signal From Janeway, Immunobiology – Effects of further Fig 6.1, Garland Pub, 2001 cross-linking
  • 7. General Principles: Receptor Activation • Activation of receptor- associated tyrosine kinases – Activation of PTKs and cross- phosphorylation upon receptor engagement by ligand – Regulation by additional kinases (CSK-inactivation) and phosphatases (CD45- allows activation) which set threshold – Co-receptors increase sensitivity (B cells: CD19, Cd21,CD81; T cells: CD4, CD8) and output (CD28) – Phosphorylation of ITAMs From Janeway, Immunobiology (immunoreceptor tyrosine- Fig 6.9, Garland Pub, 2001 based activation motifs) by
  • 8. General Principles: Receptor Activation • Full ITAM phosphorylation (paired tyrosines in consensus seq) • Recruitment (via SH2s) and enzymatic activation of additional tyrosine kinases – Syk: B cells – Zap 70 (zeta associated protein) in T cells • Activated by Lck – Phosphorylated downstream adaptors and targets From Janeway, Fig.6.13 Immunobiology, Garland Press, 2004
  • 9. General Principles: Intracellular Signaling Amplification, Diversification, Feedback, Crosstalk • Recruitment of adaptor and effector signaling molecules to membrane following tyrosine kinase activation – Role of SH2 (binds to phosphotyrosine) and SH3 (binds to pro-rich) domains in recruitment – Association into lipid rafts – Tec kinase activation (leads to PLC activation) • Activation of Signaling Pathways – Phospholipase C- activation • Ca++ release • PKC activation – Small G protein activation • MAP kinase cascade
  • 10. Recruitment and activation of the B Cell Receptor (microclusters and complex assembly) Harwood & Batista, Immunity, 200
  • 11. General Principles: Transcriptional Regulation • Activation of critical transcription factors: – MAPK targets: • AP-1 • Ets regulation – NF- B – NFAT • Role of co-stimulation for transcriptional activation • Downstream target gene activation • Amplification signals (i.e. cytokine signaling: IL- 2, etc.)
  • 12. B and T Cell Activation signaling overview Scharenberg et al. Nat Rev Immunol. 2007
  • 13. Consequences of T-Cell Activation • Tremendous Proliferation – Antigen plus co-stimulatory signal (CD28) leads to entry into cell cycle and IL-2, IL-2R production. IL-2/IL-2R leads to progression through cell cycle. – Many rounds of proliferation (1 cell to thousands) • Differentiation into effector functions
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  • 16. T Cell Receptor Activation an evolving story Smith-Garvin et al, AnnRevImmunol., 200
  • 17. T Cell Activation: Early Steps • Prior to cell-cell contact, dephosphorylation predominates: ITAMs unphosphorylated • CD45 phosphatase complexes with CD4 • Maintains activation- competent state-removal of C- terminal Tyr-P of Lck • T-cell scans APC, upon encounter with ligand, synapse begins to form. • TCRs may pre-exist in microclusters • Davis: T cells can detect even a single peptide; 10 peptides for max response and stable synapse-without CD4-25-30 From Nel, J. Allerby, Clin Immunol, 2002
  • 18. Forming the Immunological Synapse • After Antigen recognition, LFA- 1/ICAM-1 interactions allow close cell-cell contact • TCR and acc. molecules transported to center of contact of T-Cell and APC (kinetic segregation theory) – Concentrates TCR, CD3, CD4, CD28 together – displaces CD45 phosphatase – Concentrates Lck, Fyn, PKC and adaptors, Favors kinase activation • Conformation change to cytoplasm tails of CD3 not understood – membrane dissociation of intracellular domain proline rich region? – Role of TCR aggregation? • Initiation of Signaling in TCR microclusters PRECEDES formation From Nel, J. Allerby, Clin Immunol, 2002
  • 19. The Immunological Synapse: Co-Receptors • For T cells: co-receptors bind to MHC of MHC- Ag peptide complex – CD4: MHC II – CD8: MHC I • Co-binding of TCR and co-receptor leads to lowered threshold for activation • Recruitment of Lck to TCR through association with CD4 or CD8 cytoplasmic tail • B cell co-receptor: CD19, CD21, CD81 complex – CD21 recognizes activated complement – CD19 constitutively associated
  • 20. TCR Signaling: CD4 enhancement, Lck activation and recruitment and activation of Zap-70. From Janeway, Fig.6.11 Immunobiology, Garland Press, 2004
  • 21. Co-Stimulatory Molecules: Role of CD28 (“second signal”) • Binds to B7-1 and B7-2 on APC: TCR threshold, signal • Intercellular tail associates with kinases, Lck, Tec, Itk and with adaptors,  phos. • Promotes association of TCR complex with lipid rafts (Vav role) • Enhances PKC activation and JNK kinase activation and downstream NF- B and JNK(MAPK) activation – CD28RE is composite AP- From Nel, J. Allerby, Clin Immunol, 2002
  • 22. The Immunological Synapse: Regulation • Co-stimulation: – CD28 binds to B7.1 or B7.2 • Negative Regulators – CTLA-4 binds to B7.1 or B7.2 • In T-cell, recruitment of membrane signaling molecules – Roles of lipid rafts – Microtubule organizing center – Actin reorganization • Kinetics of T-cell:APC signaling: – Lck phosphorylation-15 min – But it takes 4hrs of contact for IL-2 and 10 hours for maximal IL-2 synthesis.
  • 23. T Cell Activation: Early Steps • Formation of immunological synapse allows Lck to be activated by phosphorylation and to recruit other substrates through SH2 (Zap 70, SLP, LAT-76, Vav) • Phosphorylation of ITAMs (10/TCR complex) lead to ZAP- 70 binding (tandem SH2 domains) and phosphorylation by Lck • ITAM: consensus with 2 tyrosine substrates • Stoichiometry of ITAM phosphorylation and ZAP-70 recruitment depends on affinity of TCR-peptide interaction (I.e. amplification with greater affinity) From Nel, J. Allerby, Clin Immunol, 2002
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  • 25. From:Koretzcky and Singer, STKE
  • 26. Post-TCR Events: Recruitment of Amplifying Tyrosine Kinases • Activation of PI3 kinase by Lck post TCR and CD28 • TEC Kinases – Itk and Rlk/Txk in T cells, Btk in B cells – Plekstrin homology domain allows interactions with lipids (PI3K products) at membrane – Activated by Src kinases (Lck) upon TCR activation also downstream of CD28 SH2 and SH3 allows interactions with adaptor Schwartzberg proteins Curr Opin Immunol – Complex with LAT and SLP- 16:296, 2004 76 Activates PLC- Ann Rev Imm. 2005
  • 27. Post-TCR Events: Recruitment of Adaptor Proteins • SH2 (P-Tyr bind), SH3 (Pro-rich), PTB (phosphotyrosine binding) and Pleckstrin homology (PIP binding) domains • Organize Effector Proteins for activation of multiple pathways • Positive Regulators: – LAT- adaptor Linker for Activation of T cells: • required for TCR signaling • TM protein highly tyrosine phosphorylated by ZAP-70: • Recruits PLC , activates PLC with TEC • Recruits p85 PI3K • recruits Grbp2-SOS to activate Ras Gads From Nel, J. Allerby, Clin Immunol, 2002
  • 28. Post-TCR Events: Recruitment of Adaptor Proteins (cont.) • Positive Regulators cont. – SLP-76 SH2 domain leukocyte phosphoprotein, 76kD • required for TCR signaling • Recruitment and activates Itk (TEC family kinase-which activates PLC- ) • Recruits Gads: Grb2 related • Binds Vav-a GTP exchange factor • Binds Nck-involved in cytoskeleton reorganization • SLP-76/Vav/Nck activates Rac and PAK for cytoskeletal reorganization From Nel, J. Allerby, Clin Immunol, 2002
  • 29. Post-TCR Events: Recruitment of Adaptor Proteins (cont.) • Negative Regulators – Cbl • Substrate of TCR activated kinases • Ubiquitin ligase for kinases – Kinase degradation • Negative regulator of T cell signaling (turns off a signal) – PAG/Cbp • TM protein, localized to rafts • Neg. regulates Src family kinases by co-localizing with Csk, a PTK that inactivates Srcs by C-terminal phosphorylation
  • 30. Generation of Second Messengers: PLC- • Phospholipase C- : • SH2 domains for recruitment to Tyr kinases at membrane • Activated by LAT/SLP-76/Tec complex  tyrosine phosphorylation • Cleavage of PIP2 to IP3 and DAG (amplification) • IP3  increased intracellular Ca++ • DAG Protein kinase C and Ras activation – DAG recruits Ras GRP (guanyl nucleotide releasing protein) to membrane,, phosphorylated by PKC
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  • 32. T Cell Receptor Activation of Calcium Signaling •IP3 generation leads to Ca++ release from ER and extracellular Ca++ influx •Recently described Ca++ release mechanisms Smith-Garvin et al, AnnRevImmunol., 200
  • 33. Calcium Activation: Induction of NFAT • IP3 generation leads to Ca++ release from ER and extracellular Ca++ influx • Activation of calcineurin, a Ca++ serine phosphatase – Target of cyclosporin and FK506 • Dephosphorylation of NFAT • Nuclear translocation of NFAT • Complexes with AP-1 for many targets (composite binding sites)- integrates Ras and Ca++ signaling • Activation of IL-2 transcription • Cooperation with FoxP3, STATS- lineage specific T cell differentiation From Crabtree and Olsen, Cell,109:S67 2002
  • 34. PKC Activation • PKC activated by DAG at TCR site • Important role for CD28 in PKC activation • Roles of Lck phosphorylation (increases DAG binding) SLP-76 and Vav (also downstream of CD28) • Downstream induction of NF- B • Also activation of JNK cascade From Sedwick, Altman, Mol Immunol 41:675, .2004 – AP-1 induction
  • 35. T-Cell Receptor Activation of NF-kappaB • PKC activation by DAG, PI3K and Lck • PKC phosphorylates Carma1 • Complex of Carma1, Malt, Bcl- 10 downstream of PKC; activates IKK • CD28 activation leads to Vav activation of IKK • Downstream targets for proliferation, anti-apoptosis, cytokine signaling – IL-2, IL-2Ra, Bcl-XL, IAPs, Bfl-1,TNF, interleukins, Weil and Israel, Curr. Opin. Immunol. chemokines, etc 16:376, 2004
  • 36. T Cell Receptor Activation of Ras Signaling •TCR activation leads to rapid accumulation of active GTP-Ras •Recruitment of Grb- 2/SOS to LAT •DAG recruits Ras GRP (guanyl nucleotide releasing protein-a GEF) to membrane, phosphorylated by PKC Smith-Garvin et al, AnnRevImmunol., 200
  • 37. Generation of Second Messengers: Activation of Ras Pathway – TCR activation leads to rapid accumulation of active GTP-Ras – Activation of MAP kinase cascade • Roles of Raf, MEK1/MEK2, JNK and ERKs • CD28 activates JNK, Jun for IL-2 promoter (CD28 RE) • Roles of JNKs and p38 in specifying Th1, Th2 From Nel, J. Allerby, Clin Immunol, 2002
  • 38. MAPK Activation in T cells From Janeway, Immunobiology
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  • 41. Cytoskeletal Reorganization • Recruitment of TCR complexes (effect of CD28, LFA-1 as well as ligation of TCR) into lipid rafts (glycosphingolipid+ cholesterol enriched) for enhanced and sustained signaling • Dependent on actin/cytoskeletal reorganization – Concentrations of signaling molecules:LAT, Lck, etc., palmitoylated proteins – Formation of TCR/MHC complex arrays • Formation of supramolecular activation complex (SMAC) on inside of T cell • Signaling in microclusters precedes SMAC formation • SMAC provides balance of activating (amplifying) and inhibitory regulation
  • 42. Cytoskeletal Reorganization • Actin cytoskeleton re-organization with TCR capping to maintain sustained synapse signaling – Accumulation of F actin at immune synapse – Inhibition of actin polymerization abolishes TCR signaling – Mechanisms not clear: – CD28 induced Vav activation with dephosphorylation of ERM proteins and activation of Rho, CDC42 – Nck recruits WASp (Wiskott- Aldrich : T cell defects in patients) – Vav1 activates CDC42- dependent activation of WASp and Rac activation of WAVE2 leading to changes in actin polymerization From Nel, J. Allerby, Clin Immunol, 2002
  • 43. Cytoskeletal Reorganization • (cont.) TCR Stimulation leads to T cell polarization – Microtubule organizing center moves towards T cell-APC contact – Polarization essential for immunological synapse to form • TCR activation also activates integrins (inside-out signaling) – Mechanism poorly understood: • Activation of Rap leads to TCR- induced adhesion through ICAM- 1 From Nel, J. Allerby, Clin Immunol, 2002
  • 44. Regulation of Signaling • Immunologic synapse and SMAC set thresholds dependent on # and avidity of TCR-peptide interactions – High affinity interactions lead to strong activation – Low affinity leads to inhibition • CD28 as co-stimulator – recruits in PI-3 kinase-binds to p85 subunit, which recruits p110 catalytic: converts PIP2 to PIP3 • Docking site for PDK1 and for Akt activation • Akt activates NF-kB, increases proliferation – Vav-actin reorg, – assists in lipid raft polarization, – brings in Tecs-binds ITK, – aids in JNK and NF-kB induction
  • 45. Regulation of Signaling (cont.) • Cell Surface Receptors – CTLA-4 binds to B7.1 or B7.2 • Appears after T cell activation (24-48 hrs) • Inhibition of PTKs or recruitment of phosphatases (SHP- 1) • Competitive inhibition of CD28 • Intracellular Regulators – Regulation of Lck • Csk (C terminal src kinase) phosphorylates Lck and maintains inactive state. CD45 dephosphorylates to activate • Shp1 dephosphorylates active site of Lck, turning off signal (Shp1 deficient mice: autoimmunity) • Dok (downstream of kinase) adaptor proteins associate with negative regulators – Cbl: regulate protein stability through ubiquitin
  • 46. TCR Signaling-Summary Schwartzberg Curr Opin Immunol 16:296, 2004
  • 47. Amplification of T cell response: IL-2 Signaling • Activation of IL-2 and IL-2 R synthesis leads to potent amplification of T cell mitogenic response • Differentiation into armed effector cells • IL-2 signaling, cell proliferation and survival: – Activation of JAK:STAT pathway – Activation of Ras-MAPk pathway – Activation of PI3Kinase pathway – Others…..
  • 48. TCR Signaling and Anergy • TCR stimulation in the absence of CD28 signal induces anergy (no proliferation or IL-2 secretion) • Associated with increased Cbl leading to degradation of signaling components • Assoicated with reduced LAT recruitment and signal transduction (decreased PI3K, GADS-SLP76, Grb2 complex)
  • 49. Inhibiting T cell Activation: Immunosuppresive Therapy • Surface directed – Antibodies against key components • Anti-CD3-T cell depletion: Rx for organ transplant • Anti-CD4: T cell depletion: autoimmune disease (psoriasis) and organ transplants • CTLA-4 Ig: blocks CD28 engagement; early human trials • Altered peptide ligands for tolerization (autoimmunity): MS, allergies
  • 50. Inhibiting T cell Activation: Immunosuppresive Therapy • Signaling – Calcineurin and NFAT • Cyclosporin and tacrolimus (FK506): organ transplants, dermatitis, autoimmune disease, GVHD • Complex with cyclophilin or FBP-12, immunophilins that inhibit calcineurin – Rapamycin • Organ transplants • Binds FBP-12 but blocks mTOR, kinase involved in regulating cell growth and proliferation, downstream of IL-2 receptor – NF- B inhibition • Glucocorticoids (also block AP-1) • Experimental drugs as IKK or proteosome inhibitors
  • 51. Aberrant Signaling: • Oncogenesis Inappropriate B-or T cell activation can be contribute to oncogenesis (requires additional events) – HTLV Tax induces NF- B, ATFs, SRFs, induces IL-2, IL-2R – NF- B mutations in T and B cell lymphomas – Lck is T cell oncogene in mice and activated in some human B cell leukemias/lymphomas – EBV LMP-1 mimic to CD40 activation, induces sustained B cell activation and transformation through NF- B, AP-1 and JAK/STAT activation – NF- B activation/mutation in some tumors
  • 52. Defective Signaling: Immunodeficiencies • Mutations in signaling components can lead to hereditary immunodeficiencies – B cells: BTK deficiency (B cell Tec kinase): Bruton’s X-linked agammaglobulinemia • Loss of B cell maturation • T cell activation defects – Wiskott-Aldrich:WASP deficiency: T cells fail to respond to Ag crosslinking – Mutations in CD3 and CD3 – Mutations in Zap-70 – Failure to synthesize IL-2 • NFAT defects – X-linked SCID: IL-2R defect
  • 53. Some Additional References • *Nel, ., T-cell activation through the antigen receptor. part 1 J. Allergy and Clin. Immunol. 109:758-770; part 2 109:901-905, 2002 • *Singer and Koretzky. Control of T cell function by positive and negative regulators. Science 296: 1639, 2002. • * Smith-Garvin JE, Koretzky G, and Jordan MS. T Cell Activation. Annu Rev Immnol. 27:591-619, 2009. • Berg et al. Tec Family Kinases in T Lymphocyte Development and Function. Ann.Rev. Immunol. 23: 549, 2005. • Luehrmann and Ghosh. Antigen receptor signaling to nuclear factor kappaB. Immunity 25:701, 2006. • Lineberry and Fathman. T Cell anergy: where its LAT. Immunity 24:501, 2006. • Harwood ND and Batista FE. New insights into the early molecular events underlyiing B cell activation. Immunity 28: 610, 2008. • Feske S. Calcium signaling in lymphocyte activation and disease. Nat.Rev.Immunol. 7:690, 2007. • Cronin SJF, Penninger JM From T cell activation signals to signaling control of anti-cancer immunity. Immunological Rev 220:151, 2007 • Liu, YC, Penninger J, Karin M. Immunity by ubiquitylation: a reversible process of modification. Nat.Rev. Immunol. 5:941, 2005 • Choudhuri K and van der Merwe A. Molecular mechanisms involved in T cell receptor triggering. Sem Immunol. 19:255, 2007.
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