Immunological Synapse Formation between T and APC

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Immunological Synapse Formation between T and APC
Science 2002 2951539
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Immunological Synapse
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Signal Transduction Determines Cell Response
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OUTLINE

• Role of cytokines in immune system and
  hematopoiesis
• Basic concepts of signal transduction
• JAK-STAT pathway in cytokine signaling
• Regulation of cytokine response
• Negative regulators for cytokine signal
  transduction, SOCS and PIAS families

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General Properties of Cytokines

• Polypeptides
• Produced in response to microbes and antigens
• Mediate and regulate immune and inflammatory
  responses
• Pleiotropic and redundant
• Regulate the synthesis and actions of other
  cytokines
• Actions are local or systemic (autocrine,
  paracrine, or endocrine)

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Cytokines/Chemokines Involved in Innate Immunity
Modified from Cell. Mol. Immunol. Abbas
Lichtman 5th Ed. 2003
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Cytokines/Chemokines Involved in Innate Immunity
Cytokine Producers TargetsEffects
TNFa IL-1 IL-12 IFNa/b IFNg Chemokine
MØ, DC, T cells Neutrophils activation
Endothelial cells activation
(inflammation) Hypothalamus
Fever Many cells apoptosis
MØ, endothelial Ditto
MØ, DC T cells NK IFNg
synthesis and CTL activity T
cells Th1 differentiation IFNa MØ
All cells antiviral state, increase MHC I
IFNb fibroblast NK cells activation NK,
NKT MØ activation (increased microbicidal) T
cells, CD8 B cells isotype switching to
IgG2A Many cells Increase MHC
I MHC II MØ, endothelial Leukocytes
chemotaxis, activation, Fibroblast, T cells
migration to tissues
Modified from Cell. Mol. Immunol. Abbas
Lichtman 5th Ed. 2003
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How Adaptive Immunity Works
Modified from Cell. Mol. Immunol. Abbas
Lichtman 5th Ed.
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Cytokines Involved in Adaptive Immunity
Cytokine Producers TargetsEffects
IL-2 IL-4 IL-5 Lymphotoxin IFNg
T cells T cells proliferation, cytokine
production B cells
proliferation, antibody production
NK proliferation and acitvation Th2 B
cells isotype switching to IgE T cells Th2
differentiation, proliferation
Th2 Eosinophils activation,
increase prod B cells
proliferation, IgA production T cells
Recruitment and activation of neutrophils
Th1, CD8 MØ activation (increased
microbicidal) NK, NKT B cells
isotype switching to IgG2A Many
cells Increase MHC I MHC II
Modified from Cell. Mol. Immunol. Abbas
Lichtman 5th Ed. 2003
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Roles of Cytokines in Hematopoiesis
Cell. Mol. Immunol. 2003 Abbas Lichtman 5th ed.
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What Happens When Ligands Bind to Receptors

• Conformational change of the receptors
• Opens ion channel
• Facilitates binding of intracellular signaling
  proteins such as chemokine receptor
• Dimerization (Oligomerization) of the receptors
• Bring signaling molecules into juxtaposition
• -induces post-translational modification
  such as phosphorylation in the receptors or
  signal meidators
• -activates downstream mediators

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Phosphorylation of Proteins as a Controling
Mechanism for Signal Transduction

• Advantages
• Rapid does not require new protein synthesis or
  protein degradation
• Reversible easily reversed by action of protein
  phosphatases
• Easy to relay signals phosphorylation on Tyr,
  Thr, or Ser creating binding sites for other
  proteins

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Tyrosine Phosphorylation Initiates Signaling(In
general)
PTK Protein Tyrosine Kinase PTP Protein
Tyrosine Phosphatase
Note Src-family kinase
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Regulation of Activity of Src Family Kinase
Src family B cells Lyn, Fyn, Blk T cells
Lck, Fyn,
Immunobiology 6th ed. 2005, Janeway et al.
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Major Events in Signal Transduction Mediated by
Receptor Tyrosine Kinase (RTK) or non-RTK

• Ligand-induced receptor dimerization
• Activation of kinases
• Activation of signal mediators
• Activation of transcription factors
• Translocation of transcription factors into
  nucleus and transactivation

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Cytokine Receptor-mediated Signaling Pathways
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Interferon-a Receptor Signaling Pathway
IFN?R
TYK2
JAK1
Cytosol
P
STAT2
STAT1
STAT3
Nucleus
STAT2
STAT1
STAT3
STAT3
STAT1
GAS
p48
GAS
GAS
STAT1
STAT1
STAT3
ISRE
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(No Transcript)
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Biochemical Approach for Elucidating IFN
Signaling Pathway
HeLa
Microsequencing and cDNA cloning
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Genetic Approach for Elucidating IFN Signaling
Pathway
Mol. Cell. Biol. 5th ed. 2004 Lodish et al.
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Complementation Groups for IFNa (U1-U6) or IFNg
(g1 and g2) responses
Response to Ligands
Complementing Protein
Complementation group
IFNa/b
IFNg
-

U1
TYK2
U2
IRF9(P48)


U3
STAT1
-
-
U4
JAK1
-
-
U5
IFNAR2
-

U6
STAT2
-

g1
IFNGR2
-

g2

JAK2
-
indicated that some genes do not respond,
whereas others do respond well
Q Why there is no STAT3 mutation in the
complementation group ?
Modified from Science 1994 2641415
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Animation for JAK-STAT signaling
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Structural and Functional Domains of JAK Family
JAK family JAK1, JAK2, JAK3 and TYK2
Nat. Rev. Mol. Cell Biol. 2002 3651
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Structural and Functional Domains of STATs
STAT family STAT1, STAT2, STAT3, STAT4,
STAT5A, STAT5B, and STAT6
Nat. Rev. Mol. Cell Biol. 2002 3651
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Activation of JAKs and STATs by Cytokines
Ligand Jak kinases STATs
IFN family Type I IFN- IFNa or IFNb Tyk2,
Jak1 STAT1, STAT2 Type II IFN-IFNg Jak1,
Jak2 STAT1
gC family IL-2 Jak1, Jak3 STAT5 IL-4 Jak1,
Jak3 STAT5 IL-7 Jak1, Jak3 STAT5
gp130 family IL-6 Jak2 STAT3 IL-11 Jak2 STAT
3
Modified from Gene 2002 2851
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Cytokine Receptor Families by Their Structures
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Molecular Structure of Class I Receptors
One chain for Ligand Binding One chain for
Signal transducing
Cytokine Growth Factor Rev 2001 1219
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Critical Roles of gC Family in Lymphocyte
Development and Function
Nat. Rev. Immunol.2001 1200
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SCID Resulting from Defects in IL-7Ra, JAK3 or gc
Chain
N. Eng. J. Med. 2000 3431313
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Phenotypes of JAK-Knockout Mice
Nat. Rev. Mol. Cell Biol. 2002 3651
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Prevention of Organ Allograft Rejection by a
Specific Janus Kinase 3 Inhibitor
Science 2003 302875
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Phenotypes of STAT Knockout Mice
Nat. Rev. Mol. Cell Biol. 2002 3651
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Impaired Response to IFNa/b and Lethal Viral
Disease in Human STAT1 Deficiency
Nature Genet. 2003 33388 (U. Paris)
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Negative Regulation of the JAK-STAT pathway

• Receptor-mediated endocytosis and degradation
• Dephosphorylation by tyrosine phosphatases
• Naturally occurring dominant negative STATs such
  as STAT1b and STAT3b that dont have
  transactivating domain
• Suppressor of cytokine signaling (SOCS) family
• Protein inhibitor of activated Stats (PIAS)

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Cell 2002 109S121-S131
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Negative Regulations of Cytokine Signaling
Nat. Rev. Immunity 2003 3900
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Structural and Functional Domains of SOCS Family
K Kinase inhibitory region
Nat. Rev. Immunol. 2002 2410
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Inhibitory Mechanisms of SOCS Molecules
Trend. Immunol. 2003 24659
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SOCS Family Members Target Signaling Proteins for
Degradation by Proteasome
Trends Biochem. Sci. 2002 27235
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(No Transcript)
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Liver Degeneration and Lymphoid Deficiencies in
Mice Lacking SOCS1
SOCS1-/-
SOCS1/
Proc. Natl. Acad. Sci. 1998 95 14395
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SOCS1 Is a Critical Inhibitor of IFNg Signaling
and Prevents Fatal Neonatal Actions of this
Cytokine
Cell 1999 98597
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Phenotypes of SOCS Knockout Mice
Nat. Rev. Mol. Cell Biol. 2002 3651
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Hypersensitivity of SOCS-1 KO Mice to LPS In Vivo
Immunity 2002 Vol. 17583
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SOCS1 Negatively Regulates TLR Signaling by
Mal/TIRAP Degradation
Forward
Nat. Immunol. 2006 7148
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TLR Signaling Pathways
back
Cell Death Diff. 2006 13816
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SOCS1 Is a Suppressor of Liver Fibrosis
and Hepatitis-induced Carcinogenesis
JEM 2004 1991701
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Structural and Functional Domains of PIAS Family
SIM
Nat. Rev. Immunol. 2005 5593
SAP Scaffold Attachment factor A and B, Acinus,
and PIAS RLD RING finger-like Zinc binding
domain AD Acidic domain SIM SUMO-
interacting motif S/T Serine and Threonine-rich
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PIAS-protein Regions Involved in Proteinprotein
Interactions
Nat. Rev. Immunol. 2005 5593
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Proposed Mechanisms for Inhibiting the JAKSTAT
Pathway by PIAS Proteins
Nat. Rev. Immunity 2003 3900
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Negative Regulation of PIAS on Gene
Transactivation
Nat. Rev. Immunol. 2005 5593
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Positive Regulation of PIAS on Gene
Transactivation
Nat. Rev. Immunol. 2005 5593
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Some but not All IFNa-inducible Genes Are
Enhanced in Pias1-/- MEF
Nat. Immunol. 2004 5891
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Enhanced Antiviral Response and Sensitivity to
LPS-induced Toxic Shock in Pias1-/- Mice
Nat. Immunol. 2004 5891
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Enhanced Expression of NF- kB-Regulated Genes in
Response to TNF-a and LPS in Pias1 null Cells
Mol. Cell. Biol. 2005, 251113
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PIAS1 Affects the Binding of p65 to the
Endogenous Promoters Revealed by ChIP Assays
Mol. Cell. Biol. 2005, 251113
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Specificity of PIAS1-mediated Transcriptional
Repression in STAT1 and NF-kB Signaling
Nat. Rev. Immunol. 2005 5593
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PIAS1/PIAS3 Activates Smad Transcriptional
Activity
Proc. Natl. Acad. Sci. USA 2004 10199
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• Suggested Readings
• JAK-STAT pathways and transcriptional activation
  in response to IFNs and other extracellular
  signaling proteins Science 1994 2641415
• 2. STAT transcriptional control and biologic
  impact.
• Nat. Rev. Mol. Cell Biol. 2002 3651
• 3. Regulation of gene-activation pathways by
  PIAS proteins in the immune system
• Nat. Rev. Immunol. 2005 5593
• Textbook
• Immunobiology Janeway 6th ed. 2005, Chapter 6
• Cellular and Molecular Immunology 5th Ed. 2003,
  by Abbas and Lichtman, Chapter 11

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Chromatin Immunoprecipitation (ChIP) for
Detecting Protein-DNA Interactions in Vivo
Add pre-blocked protein A/G beads
Break cells and sonicate chromatin
Cross-link protein to DNA
Add primary Ab
Immunoprecipitate and enrich chromatin
Degrade protein and reverse crosslinks
Detect specific DNA with PCR
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ipit