This project seeks to document that Janus kinase 3 (Jak3), an enzyme exclusively associated with the common y chain (yc) receptor used by a limited number of T-cell growth factors (interleukin [IL] -2, -4, -7, -9-13, and -15) to trigger T, B, monocyte and natural killer cells, represents a more specific target for immunosuppression than the ubiquitously expressed proteins, calcineurin (CaN) or mammalian target of rapamycin (mTOR). We plan to demonstrate that Jak3 blockade specifically interrupts membrane-associated, as well as downstream, enzymatic and transcriptional 1-cell activation events mediated by Cytokine Signal 3, but not by Antigen Signal 1 or by Co-stimulatory Signal 2, using immunoprecipitation, Western blot, cold kinase, electrophoretic mobility shift/supershift, quantitative PCR, fluorescence activated scanning and ribonuclease protection assays. Furthermore, we postulate that Jak3 blockade not only impairs cell cycle progression but also promotes T-cell apoptosis, thereby exerting immunosuppressive effects to prolong the survival of heterotopic cardiac and life-sustaining renal transplants in rats. To confirm that the in vivo effects correlate with in vitro molecular profiles, the biochemical assays are performed on lymphocytes infiltrating irradiated spleen allografts transplanted into recipients treated with Jak3 antagonists. Further, we seek to show that the immunosuppressive effects of Jak3 inhibition are synergistic with agents that blockade either Signal l(CaN) or Signal 2 (CD28), but not Signal 3 (mTOR) using the median effect model to correlate outcomes with drug doses/concentrations. Due to the limited tissue expression profile of Jak3, we doubt that blockade of its activity causes or potentiates the nephrotoxic, myelosuppressive and/or lipotoxic effects of CaN or mTOR inhibitors. Although the proposed experiments utilize a selective dodecyl alicyclic ketone inhibitor of Jak3 but not Jak2, alternative approaches (aside from identification of other drugs) include the use of Jak3 antisense oligonucleotides and/or adoptive transfer of T-cells transfected with a dominant-negative, inactive Jak3 variant to reconstitute the immune system of irradiated hosts.
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