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PLC activation Ca++ flux NF-AT / NFkB nuclear localization protein tyrosine phosphorylation

}. 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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PLC activation Ca++ flux NF-AT / NFkB nuclear localization protein tyrosine phosphorylation

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  1. } 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”

  2. 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)

  3. 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

  4. (1b. 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

  5. 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)

  6. Example of the stepwise function and temporal regulation of costimulatory receptor / ligand expression. CD40 Secretion of interleukin 12 (Schwartz. Science.2001)

  7. 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 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.

  8. 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 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)

  9. 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.

  10. Review of the TCR intracellular signaling pathway

  11. 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?

  12. Synapse (Adhesion) 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

  13. Proteins implicated in CD28 costimulation

  14. 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)

  15. TCR Proximal Effects 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)

  16. 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)

  17. 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)

  18. 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)

  19. Signaling through CD28:B7 augments TCR generated signal 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

  20. 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)

  21. 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)

  22. 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?

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