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? 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” } 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 onlyTCR + 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 cellDendritic cell CD28 : B7

What was costimulation? A more precise definition of costimulation immediate3. subsequent4. regulatory TCRCD28 : B7CD40L : 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-like Ig-domain receptors Homodimeric V-/C- like Ig-domain containing proteins Homo or heterotrimeric TNF like proteins (Bernard et al. Transplantation. 2002)

Secretion of interleukin 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, most T 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 after T cell activation. } APC B cells Induced by “innate immune system signals”. Stimulation by 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 loop After 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 T H activation (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? Proximal signal 2. Signal in trans No unique CD28 signaling molecules have yet been identified.

Review of the TCR intracellular signaling pathway

Nature of the CD28 costimulatory signal How, when and where does CD28 have to function in order to deliver a costimulatory-signal? Proximal signal 2. Signal in trans 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 2. Augment the signal of the TCR 3. Can signal independently of TCR Synapse (Adhesion) Lipid raft (GEM) organization PLC  1 activation SLP-76 phosphorylation Itk phosphorylation IL-2 production Actin polymerization NF-AT nucl. localizat.

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

 -CD3 +  -CD 28 (Alonso and Milan. J. Cell Sci.2001)  -CTLA-4 Glycosphingolipid/cholesterol enriched microdomains (GEMs) are associated with proteins involved in TCR signal transduction  -CD3/  -CD28 treatment of T cell clones results in surface accumulation of GEMs  -CTLA-4 treatment of T cell clones prevents  -CD3/  -CD28 stimulation from causing surface accumulation 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 GEMs and 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 PLC  1 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 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)

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 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?