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Lymphocyte Signal Transduction

Lymphocyte Signal Transduction. Arnold B. Rabson CHINJ. 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

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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 docking) From Janeway, 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 against T cell clones From Janeway, 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 • Effects of further cross-linking From Janeway, Immunobiology Fig 6.1, Garland Pub, 2001

  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 (immunoreceptor tyrosine-based activation motifs) by receptor-associated tyrosine kinases From Janeway, Immunobiology Fig 6.9, Garland Pub, 2001

  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 SignalingAmplification, 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, 2008

  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 Activationsignaling 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 Activationan evolving story Smith-Garvin et al, AnnRevImmunol., 2008

  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 needed 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 of Immunological Synapse 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-1/NF-B site 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 proteins • Complex with LAT and SLP-76 Activates PLC- • Roles in actin reorganization, migration, adhesion Schwartzberg Curr Opin Immunol 16:296, 2004 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 • Palmitoylated: rafts 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 q and Ras activation • DAG recruits Ras GRP (guanyl nucleotide releasing protein) to membrane,, phosphorylated by PKCq • SOS assoc with GRB2 and LAT facilitates local activaion of Ras

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  32. T Cell Receptor Activationof Calcium Signaling • IP3 generation leads to Ca++ release from ER and extracellular Ca++ influx • Recently described Ca++ release mechanisms Smith-Garvin et al, AnnRevImmunol., 2008

  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 • AP-1 induction From Sedwick, Altman, Mol Immunol 41:675, .2004

  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 IKKa • Downstream targets for proliferation, anti-apoptosis, cytokine signaling • IL-2, IL-2Ra, Bcl-XL, IAPs, Bfl-1,TNF, interleukins, chemokines, etc Weil and Israel, Curr. Opin. Immunol. 16:376, 2004

  36. T Cell Receptor Activationof 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 PKCq Smith-Garvin et al, AnnRevImmunol., 2008

  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 • Actin as a scaffold for signaling-PKC recruitment 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 ligase activity

  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

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