Protein Tyrosine Phosphorylation Induced by Lysophosphatidic Acid in Rat
PLC activation Ca++ flux NF-AT / NFkB nuclear localization protein tyrosine phosphorylation
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PLC activation Ca++ flux NF-AT / NFkB nuclear localization protein tyrosine phosphorylation IL-2 production proliferation cytokine production TCR internalization IL-2 production proliferation T cell costimulation What is meant by “costimulation”? How does one define T cell activation? ervation: Signaling through the TCR is not ficient to drive IL-2 production and T cell prolife e other signal is needed. “Signal 2” “Costimulation” } Ability to become an effector ce
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}. IL-2 production proliferation. Ability to become an effector cell. T cell costimulation. What is meant by “costimulation”?. How does one define T cell activation?. PLC activation Ca++ flux NF-AT / NFkB nuclear localization protein tyrosine phosphorylation IL-2 production - PowerPoint PPT Presentation
Transcript of PLC activation Ca++ flux NF-AT / NFkB nuclear localization protein tyrosine phosphorylation
PLC activationCa++ fluxNF-AT / NFkB nuclear localizationprotein tyrosine phosphorylationIL-2 productionproliferationcytokine productionTCR internalization
IL-2 productionproliferation
T cell costimulation
What is meant by “costimulation”?
How does one define T cell activation?
Observation: Signaling through the TCR is notsufficient to drive IL-2 production and T cell proliferation.Some other signal is needed.
“Signal 2” “Costimulation”
} Ability to become an effector cell
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
Anergy
TCR signal only 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?
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.YXXM
IL-2
“Costimulation” results in stabilizing of IL-2 mRNA and transcription from IL-2 promoter. (Fraser et al. Science. 1991)
T cell Dendritic cell
CD28 : B7
What was costimulation? A more precise definition of costimulation.
1. 2. immediate 3. subsequent 4. regulatory
TCR CD28 : B7 CD40L : CD40ICOS : B7hothers
CTLA-4 : B7PD-1 : PD-L
(1b. adhesion)CD2 : LFA-3 and LFA-1 : ICAM (Bachmann et al. J. Exp. Med. 1999)
Homodimeric V-like Ig-domain receptors
Homodimeric V-/C- likeIg-domain containing proteins
Homo or heterotrimeric TNF like proteins
(Bernard et al. Transplantation. 2002)
Secretion ofinterleukin 12
Example of the stepwise function and temporal regulation of costimulatory receptor / ligand expression.
CD40
(Schwartz. Science.2001)
More about CD28
CD28 - constitutively expressed on the surface of T cells.In mice, all T cells express CD28. In humans, mostT cells (except for a sub-pop. of CD8+) express CD28.
B7-2
B7-1
CTLA-4 - higher specificity, higher affinity for B7-1. Not expressed at surface of naive T cell. Sequestered intracellularly, delivered to synapse quickly afterT cell activation.
} APC B cellsInduced by “innate immunesystem signals”. Stimulationby LPS, etc.
T cell
TCR
MHC
CD28
LFA-1
ICAM
APC
CD40
Signaling through TCR leads to activation of LFA-1
LPS
B7-2
B7-2 (and B7-1) expression is induced on APC in response to activators of the innate immune system activators
CD40L is upregulated on T cell in response to TCR/CD28 signaling
APC upregulates B7-2 in response to CD40 engagement
CD40 / CD28 pos. feedback loopAfter 2 days
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
CD28:B7-2(CD86), CTLA-4:B7-1(CD80) regulation in TH activation
(Bernard et al. Transplantation. 2002)
Nature of the CD28 costimulatory signal
How, when and where does CD28 have to functionin order to deliver a costimulatory-signal?
1.
2.
1. Proximal signal
2. Signal in trans
No unique CD28 signaling molecules have yet been identified.
QuickTime™ and aGIF decompressor
are needed to see this picture.
Review of the TCR intracellular signaling pathway
Nature of the CD28 costimulatory signal
How, when and where does CD28 have to functionin order to deliver a costimulatory-signal?
1.
2.
1. Proximal signal
2. Signal in trans
Both proximal signals and signaling in trans are able to deliverthe costimulatory signal.What does this mean about the nature of the costim. signal?
Mechanism of the CD28 costimulatory signal
1. Proximal functions
2. Augment the signal of the TCR
3. Can signal independently of TCR
Synapse (Adhesion)
Lipid raft (GEM) organization
PLC1 activation
SLP-76 phosphorylation
Itk phosphorylation
IL-2 production
Actin polymerization
NF-AT nucl. localizat.
QuickTime™ and aGIF decompressor
are needed to see this picture.
Proteins implicated in CD28 costimulation
TCR Proximal Effects mediated by CD28:B7 interaction
(Michel et al. Immunity. 2001)
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.
Conjugate formation
-C
D3
+
-CD
28
(Alonso and Milan. J. Cell Sci.2001)
-C
TL
A-4
Glycosphingolipid/cholesterolenriched microdomains (GEMs) are associated with proteins involved in TCR signal transduction
-CD3/-CD28treatment of T cellclones results in surface accumulation of GEMs
-CTLA-4 treatment of T cellclones prevents -CD3/-CD28stimulation from causing surfaceaccumulation of GEMs
TCR Proximal Effects
(Martin et al. J. Exp. Med. 2001)
(Viola et al. Science. 1999)
TCR Proximal Effects mediated by CD28:B7 interaction
(Martin et al. J. Exp. Med. 2001)
Ligation of CD28 on murine T cells results in upregulation of surface GEMsand increased T cell proliferation.
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
PLC and SLP-76 show CD28 costimulation dependent phosphorylation following TCR signaling.
(Michel et al. Immunity. 2001)
Phosphorylation of ZAP-70 and LAT is not dependant on CD28 costimulation
Signaling through CD28:B7 augments TCR generated signal
Thus, the lack of signaling from CD28 selectively affects TCR-directed phosphorylation of PLC1 and SLP-76 while sparing other more proximal events such as the phosphorylation of ZAP-70 and its substrate LAT.
CD
28
CD28 Signals independently of TCR
VAV and SLP-76 transfected into COScells and ligation of CD28
Molecules involved in TCR signaling and CD28 signal are inseparable.
CD28 is normally incapable of deliveringa signal when crosslinked on T cells without TCR signaling.
VAV and SLP-76 transfection into non-hematapoietic cells (COS) completes theT cell NF-AT signal transduction pathway.
CD28 crosslinking induces NF-AT nuclearlocalization
(Raab et al. Immunity. 2001)
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)
CD28 Signals independently of TCR
Mechanism of the CD28 costimulatory signal
1. Proximal functionsAdhesionGEM Raft Formation
2. Augment the signal of the TCRPLC pathwayPI3K activationSLP-76 adaptor proteinProvides active Itk kinase
3. Can signal independently of TCRInduce NF-AT nuclear localizationDrive IL-2 transcriptionPolymerize 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?
