Transcript Signaling Through Immune System Receptors

Overview

Events controlled by signaling

Development, Activation, Homing, Death

Antigens, cytokines, chemokines, etc.

Antigen receptors:



Associated chains, ITAM’s, tyrosine kinases, adaptors,
downstream effectors (more ubiquitous)
Spatial organization: lipid rafts, immunol. synapse
(SMAC)
Other receptors: TNFr’s, chemokines, TLR’s
Signal Transduction
Pathways Relay
Information from
the Cell Surface to
the Nucleus
Crosslinking and receptor tyrosine kinases
Surface Ig Crosslinking --> B Cell Activation
Similarly, TCR crosslinking activates T cells
TCR/CD3/ Complex
and Surface Ig
with Ig and Ig
These are relatively stable complexes, and can isolated intact
by immuno-precipitation, when mild detergents are used
Assembly of the entire complex is necessary
for efficient expression at the cell surface
Irving and Weiss expt. demonstrating that 
crosslinking is sufficient for T cell activation
CD8
ecto
+ t.m.
transfect into
CD8 neg. T cell line
Cell (1991) 64: 891.
X-link with
anti-CD8 Ab
 intracellular
IL-2
Similar results seen by others with chimeras containing
intracellular domain of CD3 or Ig or Ig
How do TCR and surface Ig access intracellular
signaling pathways?


Unlike RTK’s, no intrinsic catalytic activity
Conserved motif with 2 tyrosines in , CD3 chains
and Ig- and Ig- proteins

First recognized by Michael Reth

Now called an ITAM

Immuno-receptor Tyrosine-based Activation Motif

Also found in receptors on NK and monocytic cells
TCR- and Surface Ig-Associated Proteins
Immuno-receptor tyrosine-based activation motif (ITAM)
D/E X X Y X X L X7 Y X X L
Src vs. Syk Family Tyrosine Kinases
SH2
T cells
Src
Yes
Fgr
Hck
Lyn
Blk
Fyn
Lck
SH2
catalytic
Syk - B cells, early T cell
development, NK cells,
platelets, monocyte lineage
B cells
ZAP-70 - Throughout T
cell development, NK cells
Early events in signaling through the TCR
- Sequential involvement of src and Syk family
src family
tyrosine kinases
Syk/ZAP-70 family
tyrosine kinase
Sequential tyrosine kinase activation in B cells
Multi-domain adaptors nucleate signaling complexes
SH2 (src-homology 2) :: phospho-tyrosine
SH3 :: proline-rich sequences
Different SH2 and SH3 domains have different specificities
Cytoplasmic adaptors of lymphocytes
Transmembrane adaptors are also intermediates in
non-receptor tyrosine kinase systems
Transmembrane lymphocyte adaptors
LAB/NTAL
B cells
Nucleation of signaling complex at LAT
Formation of this
complex results in
stable membrane
association of PLC-1,
activation of which is
critical for generation
of second messengers
Second messengers relay signals from
receptors at the plasma membrane
DAG and IP3 are second messengers
Small G protein activation is aided by
Guanine nucleotide Exchange Factors
Ras activation is also aided by a
guanine nucleotide releasing protein (GRP)
GEF
GRP
Small G proteins activate MAPK Cascades
MAPK = mitogen-activated protein kinase
Three major transcription factors downstream of the TCR
PIP2
1.Tyrosine phosphorylation
2. “Second messengers”
3. Downstream kinases
4. Transcription factors
1
3
2
IP3
second
messengers
transcription
factors
AP-1
activation
NF-kB
activation
Ways to study the contributions of signaling
proteins to T cell development and activation

Generate mouse knockout



Germ-line; tissue-specific; inducible
Generate mutant cell line

Chemical or radiation mutagenesis

RNAi - oligos or plasmid-based - variable
Each approach has advantages and drawbacks
Effect of SLP-76 deficiency on T cell development
LAT k.o. looks identical
Signaling defects in
SLP-76-deficient
Jurkat T cells
Ras/MAPK
IP3/Ca2+
Some Remaining Big-Picture Questions


Exactly how does crosslinking initiate the whole
signaling cascade?

Is crosslinking/dimerization actually sufficient?

Any role for conformational change?

Maybe a little of both…
Changes in local concentration of ITAMs, sub-cellular
localization (i.e. access to kinases and/or lack of
access to phosphatases ?
Lipid rafts and Ig/TCR signaling


Distribution of lipids in p.m. not uniform
High concentration of sphingolipids and cholesterol in
mobile ‘rafts’


Singer/Nicholson fluid mosaic model…
Proteins with certain lipid modifications partition
preferentially to lipid rafts

including some signaling molecules (lck, LAT)

Chemical disruption of rafts prevents activation

May be important for initiation of signaling
MIRR = multi-chain immune recognition receptor
SMAC or Immunologic Synapse
SMAC = supra-molecular activation cluster

Kupfer - deconvolution IF microscopy of T cell/APC interactions --> organized, bulls-eye type structure
p-SMAC
c-SMAC
c-SMAC
LFA-1/Talin, CD45
TCR/CD3, CD28, PKC q, lck
- Non-activating, altered, peptides do not support
the formation of these structures
- Precise functional role still controversial
SMAC or Immunologic Synapse
LFA-1
TCR
APC
T cell
LFA-1: an integrin; binds ICAM on APC
Negative regulation and down-regulation of
TCR and BCR signaling
 Internalization; trafficking to endosomes/lysosomes
 role for the immune synapse in this process?
 Ubiquitination --> proteasomal degradation of receptor


Cbl and other adaptors
Phosphatases - remove tyrosines, etc.

SHP-1 and others
Other types of receptors in lymphocyte biology

G-protein coupled receptors (GPCR)


e.g. chemokine receptors
Jak/Stat signaling

cytokine receptors

Toll-like receptors (DC activation, etc.)

TNF receptor family


TNFr, CD40, Fas (co-stimulation; death)
TGF- Receptor (Treg/Th17 development/function)
GPCR Signaling
PLC
ATP
PI4,5P2
DAG
Ca2+
(CREB, etc.)
Some Toll-Like receptors and their ligands
Major Signaling Pathways
Activated by TLR’s and IL-1R
MyD88-Independent Pathway
MAPKs
Cytokine Receptor Signaling
that
bind
TNFr family - death inducers
TNFr family - co-stimulators
ligands
co-stimulators
TNFr family - proximal signaling

Downstream pathways:

MAP Kinases + AP-1

IKK’s + NF- B


anti-apoptotic
Caspases (apoptosis)
TGF- Receptor Signlaing