PowerPoint Presentation - How T cells recognize antigen: The T Cell Receptor (TCR)

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Signal Transduction
Transmission of external signals across the
membrane and conversion of these signals into
intracellular biochemical events
PeptideMHC
TCR
?
Changes in gene expression
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TCR signaling Big Picture
RECEPTOR
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Alterations in Gene expression
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?
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General principle of signal transduction Ligand
induced dimerization is the initiating step in
signal transduction
(or clustering)
Example Kit signaling
A kinase is an enzyme that attaches phosphate
groups to specific amino acids of proteins (Tyr,
Ser, Thr)
General principle Phosphorylation is a dynamic,
reversible way of modulating protein function
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How does the TCR transduce signals with such a
little tail?
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TCR associates with the CD3 Complex
Analogous to the Ig? and Ig? proteins that
associate with BCR on B cells
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ITAM
ITAMs are the essential basis of TCR signal
transduction ITAM Immune Tyrosine Activation
Motif ITAM structure is Y- X - X - I/L - X 6-8 -
Y - X - X -I/L
When a tyrosine within an ITAM is phosphorylated,
it creates a binding site for other signaling
molecules
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SH2 domains bind to specific phosphotyrosines
within ITAMs
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Protein Tyrosine Kinases in initial TCR signaling
Lck and Fyn - Src family kinases
Y
Y
SH3
SH2
Kinase
Regulatory Tyrosines
Zap-70 - Syk family kinase
Y
Kinase
SH2
SH2
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How do the ITAMs on CD3 get phosphorylated?
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Lck binds to the tails of CD4 and CD8
Lck
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Initiation of TCR signaling
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Initial events in TCR signaling

1. TCR CD4 or CD8 bind peptideMHC (clustering)
2. Lck and Fyn phosphorylate ITAMs on CD3 complex
3. ZAP-70 binds to P-ITAMs on CD3zeta chains
4. Lck and Fyn phosphorylate and activate ZAP-70
5. ZAP-70 initiates downstream signaling events

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Initial TCR signaling
RECEPTOR
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Regulation of early TCR signaling
Lck or Fyn
Phosphorylation is a dynamic, reversible way of
modulating protein function

• Activating and inhibitory Tyr-phosphorylation
  sites
• CD45 (B220) is a phosphatase which activates PTKs
  by dephosphorylating inhibitory P-Tyr.
• Csk is a kinase which inhibits PTKs by
  phosphorylating inhibitory Tyr

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The phosphatase CD45 regulates PTK activity
CD45 removes inhibitory phosphates from Lck and
Fyn
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Regulation of early TCR signaling
The plasma membrane is not homogeneous Lipid
rafts are enriched for saturated lipids and
cholesterol making them more rigid This
composition regulates which types of proteins can
localize to these rafts -most transmembrane
proteins (e.g. TCR) are excluded -Lck and Fyn
are enriched in lipid rafts
TCR
Fyn
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Lipid rafts help regulate T cell activation
If rafts are disrupted (by depleting
cholesterol), T cell activation is blocked
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Downstream TCR signaling
RECEPTOR
?
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ZAP-70 phosphorylates SLP-76 and LAT which leads
to activation of 3 downstream signaling pathways
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2
3
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(No Transcript)
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Zap-70 phosphorylates PLCg, activating it to
cleave PIP2
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IP3 binds to Ca2 channels and opens them
2nd messengers amplify initial signals
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TCR signaling Big Picture
RECEPTOR
Protein kinase C
Intracellular Ca2
MAP kinases
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2
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Alterations in Gene expression
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NFAT
?
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Immunosuppressive drugs block NF-AT activation
FK506 and cyclosporin block NF-AT activation by
inhibiting calcineurin
Essential drugs to prevent transplant rejection
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Cyclosporin
Tolypocladium inflatum, the white mold from Norway
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TCR signaling Big Picture
RECEPTOR
Protein kinase C
Intracellular Ca2
MAP kinases
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2
3
Alterations in Gene expression
?
NFAT
?
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IP3 binds to Ca2 channels and opens them
2nd messengers amplify initial signals
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Activating NF-kB
activates kinase
Ca
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TCR signaling Big Picture
RECEPTOR
Protein kinase C
Intracellular Ca2
MAP kinases
1
2
3
Alterations in Gene expression
?
NF-?B
NFAT
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ZAP-70 phosphorylates SLP-76 and LAT which leads
to activation of 3 downstream signaling pathways
1
2
3
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Ras is a small G-protein
GTPase activating protein
Guanine nucleotide exchange factor
GEF
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Ras activates the MAP kinase pathway
TCR activation
Raf (MAP kinase kinase kinase)
MEK (MAP kinase kinase)
MAP kinase
AP-1
(Transcription factor)
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RECEPTOR
Protein tyrosine kinase
Phospholipase C
Ras (G-proteins)
MAP kinases
Protein kinase C
Intracellular Ca2
The IL-2 gene promoter
NF-kB
AP-1
NF-AT
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Example of T cell activation
TCR binds peptideMHC
Induces expression of genes
T cells divides many times creating more T cells
with the same TCR
T cells differentiate and acquire new
characteristics
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IL-2 is one of hundreds of genes induced by TCR
signaling
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Mysteries in T cell antigen recognition

• Affinity of TCR for peptideMHC is quite low
• How is T cell-APC interaction maintained?
• How are low frequency peptideMHC complexes ever
  recognized?

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Initial T cell/APC contact is mediated by
adhesion molecules
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Clustering leads to formation of an
immunological synapse
B-Cell (Peptide-pulsed)
T-Cell
TCR, MHC adhesion molecules cluster at
interface between T cell and APC
8 minutes in 20 seconds of video
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Many additional receptor interactions contribute
to T cell activation
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Summary of TCR signaling
TCR signaling is initiated by TCR binding
peptideMHC and clustering of TCR with CD4/CD8
and other accessory molecules. Clustering
leads to activation of Src family protein
tyrosine kinases (Lck and Fyn) that phosphorylate
ITAMs of the CD3 complex In naïve T cells,
the TCR is excluded from lipid rafts. TCR
activation allows the TCR to enter lipid rafts
and associate with PTKs. T cell signaling
activates 3 general downstream pathways PLC? -
Ca2 - NFAT PLC? - PKC - NF?B Ras - MAPKs -
AP-1 Coreceptors and adhesion molecules
contribute to T cellAPC interactions. These
molecules are organized into an immunological
synapse which facilitates prolonged T cell
signaling.