Transcript Sundberg

T cell costimulation
What is meant by “costimulation”?
How does one define T cell activation?
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IL-2 production
PLC
activation
Ability to become an effector cell
Ca++ flux
proliferation
NF-AT / NFkB nuclear localization
Observation:
Signaling
through the TCR is not
protein tyrosine
phosphorylation
sufficient
drive IL-2 production and T cell proliferation.
IL-2toproduction
Some proliferation
other signal is needed.
cytokine production
“Signal
2”
“Costimulation”
TCR
internalization
In the absence of costimulation, activation of the T cell through the
TCR results in a failure to proliferate and the induction of anergy.
IL-2
TCR signal only
Anergy
TCR + costimulation
Exogenously supplied IL-2
Anergic state can be “outgrown” by culture in the presence of IL-2
over time.
(Jenkins and Schwartz. J Exp Med. 1987)
What drives/allows IL-2 production during T cell:APC interaction?
T cell
Dendritic cell
CD28 : B7
CD28
Originally referred to as a Tp44, and implicated for its role
in T cell:APC adhesion. (Linsley et al. PNAS. 1990)
CD28 when binds its ligand, B7, transmits an intracellular
signal via its cytoplasmic tail.
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“Costimulation” results in stabilizing of IL-2 mRNA and
transcription from IL-2 promoter. (Fraser et al. Science. 1991)
IL-2
What was costimulation? A more precise definition of costimulation.
1.
TCR
2. immediate
3. subsequent
4. regulatory
CD28 : B7
CD40L : CD40
ICOS : B7h
others
CTLA-4 : B7
PD-1 : PD-L
(1b. adhesion)
CD2 : LFA-3 and LFA-1 : ICAM
(Bachmann et al. J. Exp. Med. 1999)
Homodimeric V-/C- like
Ig-domain containing proteins
Homo or heterotrimeric
TNF like proteins
Homodimeric V-like
Ig-domain receptors
(Bernard et al. Transplantation. 2002)
Example of the stepwise function and temporal regulation
of costimulatory receptor / ligand expression.
CD40
Secretion of
interleukin 12
(Schwartz. Science.2001)
More about CD28
CD28 - constitutively expressed on the surface of T cells.
In mice, all T cells express CD28. In humans, most
T cells (except for a sub-pop. of CD8+) express CD28.
B7-2
B7-1
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APC
B cells
Induced by “innate immune
system signals”. Stimulation
by LPS, etc.
CTLA-4 - higher specificity, higher affinity for B7-1. Not
expressed at surface of naive T cell. Sequestered
intracellularly, delivered to synapse quickly after
T cell activation.
CD28:B7-2(CD86), CTLA-4:B7-1(CD80) regulation in TH activation
CD40L is upregulated on T cell in
response to TCR/CD28 signaling
APC upregulates B7-2 in
response to CD40 engagement
CD40
ICAM
LPS
CD40 / CD28 pos. feedback loop
After 2 days
MHC
TCR
LFA-1
B7-2
T cell
CD28
Signaling through TCR leads
to activation of LFA-1
B7-2 (and B7-1) expression is
induced on APC in response
to activators of the innate
immune system activators
APC (activated DCs) start to
near the end of their life and
begin to express B7-1
T cell starts to massively
express CTLA-4
(Bernard et al. Transplantation. 2002)
Nature of the CD28 costimulatory signal
How, when and where does CD28 have to function
in order to deliver a costimulatory-signal?
1. Proximal signal
1.
2. Signal in trans
2.
No unique CD28 signaling molecules have yet been identified.
Review of the TCR intracellular signaling pathway
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Nature of the CD28 costimulatory signal
How, when and where does CD28 have to function
in order to deliver a costimulatory-signal?
1. Proximal signal
1.
2. Signal in trans
2.
Both proximal signals and signaling in trans are able to deliver
the costimulatory signal.
What does this mean about the nature of the costim. signal?
Mechanism of the CD28 costimulatory signal
1. Proximal functions
Synapse (Adhesion)
Lipid raft (GEM) organization
PLCg1 activation
2. Augment the signal of the TCR
SLP-76 phosphorylation
Itk phosphorylation
IL-2 production
3. Can signal independently of TCR
Actin polymerization
NF-AT nucl. localizat.
Proteins implicated in CD28 costimulation
QuickTime™ and a
GIF decompressor
are needed to see this picture.
TCR Proximal Effects mediated by CD28:B7 interaction
Conjugate formation
Jurkat T cells expressing no CD28, wild type CD28 or a CD28 cytoplasmic tail
deletion mutant were incubated with 531-B7 cells expressing MHC class II and
B7.1 and assayed for conjugate formation.
(Michel et al. Immunity. 2001)
TCR Proximal Effects
a-CTLA-4
a-CD3/a-CD28
treatment of T cell
clones results in
surface accumulation
of GEMs
a-CD3 + a-CD 28
Glycosphingolipid/cholesterol
enriched microdomains (GEMs)
are associated with proteins
involved in TCR signal
transduction
(Viola et al. Science. 1999)
a-CTLA-4 treatment of T cell
clones prevents a-CD3/a-CD28
stimulation from causing surface
accumulation of GEMs
(Martin et al. J. Exp. Med. 2001)
(Alonso and Milan. J. Cell Sci.2001)
TCR Proximal Effects mediated by CD28:B7 interaction
Ligation of CD28 on murine T cells results in upregulation of surface GEMs
and increased T cell proliferation.
(Martin et al. J. Exp. Med. 2001)
Signaling through CD28:B7 augments TCR generated signal
PLC and SLP-76 show CD28 costimulation dependent phosphorylation
following TCR signaling.
(Michel et al. Immunity. 2001)
Signaling through CD28:B7 augments TCR generated signal
Phosphorylation of ZAP-70 and LAT is not dependant on CD28 costimulation
PLC and SLP-76 show CD28 costimulation dependent phosphorylation
following TCR signaling.
(Michel et al. Immunity. 2001)
Signaling through CD28:B7 augments TCR generated signal
CD28
Thus, the lack of signaling from CD28 selectively affects TCR-directed
phosphorylation of PLCg1 and SLP-76 while sparing other more proximal
events such as the phosphorylation of ZAP-70 and its substrate LAT.
CD28 Signals independently of TCR
VAV and SLP-76 transfected into COS
cells and ligation of CD28
Molecules involved in TCR signaling and
CD28 signal are inseparable.
CD28 is normally incapable of delivering
a signal when crosslinked on T cells
without TCR signaling.
VAV and SLP-76 transfection into nonhematapoietic cells (COS) completes the
T cell NF-AT signal transduction pathway.
CD28 crosslinking induces NF-AT nuclear
localization
(Raab et al. Immunity. 2001)
CD28 Signals independently of TCR
Transcription of IL-2 in response to CD28 ligation
Jurkat T cells transfected w/
SLP-76
Vav-1
IL-2 reporter driving luciferase
(Raab et al. Immunity. 2001)
Mechanism of the CD28 costimulatory signal
1. Proximal functions
Adhesion
GEM Raft Formation
2. Augment the signal of the TCR
PLC pathway
PI3K activation
SLP-76 adaptor protein
Provides active Itk kinase
3. Can signal independently of TCR
Induce NF-AT nuclear localization
Drive IL-2 transcription
Polymerize actin at sites of CD28 ligation
Which of these functions is the second signal?
Is there “a second signal” per se, or is there only costimulation/coactivation?