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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

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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

proliferation

cytokine production

TCR internalization

Observation: Signaling through the TCR is not

sufficient to drive IL-2 production and T cell proliferation.

Some other signal is needed.

“Signal 2”

“Costimulation”


Exogenously supplied IL-2

Anergic state can be “outgrown” by culture in the presence of IL-2 over time.

In the absenceof 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

TCR + costimulation

Anergy

(Jenkins and Schwartz. J Exp Med. 1987)


Originally referred to as a Tp44, and implicated for its role in T cell:APC adhesion. (Linsley et al. PNAS. 1990)

CD28

CD28 : B7

CD28 when binds its ligand, B7, transmits an intracellular signal via its cytoplasmic tail.

YXXM

“Costimulation” results in stabilizing of IL-2 mRNA and transcription from IL-2 promoter. (Fraser et al. Science. 1991)

IL-2

What drives/allows IL-2 production during T cell:APC interaction?

T cell

Dendritic cell


(1b. role in T cell:APC adhesion. adhesion)

CD2 : LFA-3 and LFA-1 : ICAM

(Bachmann et al. J. Exp. Med. 1999)

What was costimulation? A more precise definition of costimulation.

1.

2. immediate

3. subsequent

4. regulatory

TCR

CD28 : B7

CD40L : CD40

ICOS : B7h

others

CTLA-4 : B7

PD-1 : PD-L


Homodimeric V-/C- like role in T cell:APC adhesion.

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 costimulatory receptor / ligand expression.

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.

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B7-2

B7-1

Induced by “innate immune

system signals”. Stimulation

by LPS, etc.

APC B cells

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 T costimulatory receptor / ligand expression.H activation

CD40L is upregulated on T cell in

response to TCR/CD28 signaling

APC upregulates B7-2 in

response to CD40 engagement

CD40

After 2 days

CD40 / CD28 pos. feedback loop

ICAM

LPS

MHC

APC

TCR

T cell

LFA-1

B7-2

CD28

APC (activated DCs) start to

near the end of their life and

begin to express B7-1

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

T cell starts to massively

express CTLA-4

(Bernard et al. Transplantation. 2002)


Nature of the CD28 costimulatory signal costimulatory receptor / ligand expression.

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 costimulatory receptor / ligand expression.


Nature of the CD28 costimulatory signal costimulatory receptor / ligand expression.

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?


Synapse (Adhesion) costimulatory receptor / ligand expression.

Lipid raft (GEM) organization

PLCg1 activation

SLP-76 phosphorylation

Itk phosphorylation

IL-2 production

Actin polymerization

NF-AT nucl. localizat.

Mechanism of the CD28 costimulatory signal

1. Proximal functions

2. Augment the signal of the TCR

3. Can signal independently of TCR


Proteins implicated in CD28 costimulation costimulatory receptor / ligand expression.


TCR Proximal Effects mediated by CD28:B7 interaction costimulatory receptor / ligand expression.

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 costimulatory receptor / ligand expression.

Glycosphingolipid/cholesterol

enriched microdomains (GEMs)

are associated with proteins

involved in TCR signal

transduction

a-CD3/a-CD28

treatment of T cell

clones results in

surface accumulation

of GEMs

a-CD3 + a-CD 28

a-CTLA-4

(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 costimulatory receptor / ligand expression.

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 costimulatory receptor / ligand expression.

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 costimulatory receptor / ligand expression.

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 costimulatory receptor / ligand expression.

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


CD28 Signals independently of TCR costimulatory receptor / ligand expression.

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 non-

hematapoietic 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 costimulatory receptor / ligand expression.

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 costimulatory receptor / ligand expression.

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?


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