Lymphocyte Signal Transduction

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Lymphocyte Signal Transduction

• Arnold B. Rabson
• CHINJ

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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

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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

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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
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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
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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
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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
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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
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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

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Recruitment and activation of the B Cell Receptor
(microclusters and complex assembly)
Harwood Batista, Immunity, 2008
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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.)

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B and T Cell Activationsignaling overview
Scharenberg et al. Nat Rev Immunol. 2007
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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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T Cell Receptor Activationan evolving story
Smith-Garvin et al, AnnRevImmunol., 2008
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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
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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
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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

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TCR Signaling CD4 enhancement, Lck activation
and recruitment and activation of Zap-70.
From Janeway, Fig.6.11 Immunobiology, Garland
Press, 2004
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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
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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-cellAPC signaling
• Lck phosphorylation-15 min
• But it takes 4hrs of contact for IL-2 and 10
  hours for maximal IL-2 synthesis.

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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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FromKoretzcky and Singer, STKE
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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 16296, 2004 Ann
Rev Imm. 2005
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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
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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
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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

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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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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
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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,109S67 2002
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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 41675, .2004
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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. 16376, 2004
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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
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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
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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 moleculesLAT, 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

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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
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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
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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

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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

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TCR Signaling-Summary
Schwartzberg Curr Opin Immunol 16296, 2004
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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 JAKSTAT pathway
• Activation of Ras-MAPk pathway
• Activation of PI3Kinase pathway
• Others..

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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)

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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

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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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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-kB 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-kB, AP-1 and JAK/STAT activation
• NF-kB activation/mutation in some tumors
• Zap-70 activation in B-CLL-worse prognosis

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Defective Signaling Immunodeficiencies

• Mutations in signaling components can lead to
  hereditary immunodeficiencies
• B cells BTK deficiency (B cell Tec kinase)
  Brutons X-linked agammaglobulinemia
• Loss of B cell maturation
• T cell activation defects
• Wiskott-AldrichWASP 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

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Some Additional References

• Nel, ., T-cell activation through the antigen
  receptor. part 1 J. Allergy and Clin. Immunol.
  109758-770 part 2 109901-905, 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. 27591-619,
  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 25701, 2006.
• Lineberry and Fathman. T Cell anergy where its
  LAT. Immunity 24501, 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. 7690,
  2007.
• Cronin SJF, Penninger JM From T cell activation
  signals to signaling control of anti-cancer
  immunity. Immunological Rev 220151, 2007
• Liu, YC, Penninger J, Karin M. Immunity by
  ubiquitylation a reversible process of
  modification. Nat.Rev. Immunol. 5941, 2005
• Choudhuri K and van der Merwe A. Molecular
  mechanisms involved in T cell receptor
  triggering. Sem Immunol. 19255, 2007.

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