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

• Inhibition of T cell activation signaling: immunosuppression

• Diseases of immune signaling– Malignancies (T- and B-)– Immunodeficiencies

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

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

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

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


• 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


• 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.6.13Immunobiology, Garland Press, 2004

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

Harwood & Batista, Immunity, 2008

Recruitment and activation of the B Cell Receptor (microclusters and complex assembly)

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.)

B and T Cell Activationsignaling overview

Scharenberg et al. Nat Rev Immunol. 2007

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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Smith-Garvin et al, AnnRevImmunol., 2008

T Cell Receptor Activationan evolving story

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

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

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

TCR Signaling: CD4 enhancement, Lck activation and recruitment and

activation of Zap-70.

From Janeway, Fig.6.11Immunobiology, Garland Press, 2004

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

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.

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)


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From:Koretzcky and Singer, STKE

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

SchwartzbergCurr Opin Immunol16:296, 2004Ann Rev Imm. 2005

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

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

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

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– SOS assoc with GRB2 and LAT facilitates local activaion of Ras

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T Cell Receptor Activationof Calcium Signaling

•IP3 generation leads to Ca++ release from ER and extracellular Ca++ influx•Recently described Ca++ release mechanisms

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

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

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, chemokines, etc

Weil and Israel, Curr. Opin. Immunol.16:376, 2004


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 PKC

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


MAPK Activation in T cells

From Janeway, Immunobiology

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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

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


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


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

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

TCR Signaling-Summary

SchwartzbergCurr Opin Immunol16:296, 2004

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…..

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)

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

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

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 – Zap-70 activation in B-CLL-worse prognosis

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

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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Lymphocyte Signal Transduction