A NOVEL PATHWAY OF IMMUNOSUPPRESIVE ACTIVITY OF RAPAMYCIN: SPECIFIC INHIBITION OF IL-12-MEDIATED STAT4 PHOSPHORYLATION IN DDRITIC CELLS

Abstract

O124* Aims: Rapamycin, a potent immunosuppressive reagent, blocks T cell signal transduction mediated by IL-2 and the costimulatory pathway. It also induces TCR desensitization due to prolonged engagement by alloantigens. However, its effect on APC has not been extensively studied. Methods: In this study, DC were exposed to rapamycin during propagation from B10 (H-2b) bone marrow (BM) in the presence of GMCSF and IL-4. DC were extensively washed at the end of culture. Surface molecule expression on DC was determined by flow cytometry. DC allostimulatory function were assessed by MLR and CTL in vitro, and influence of allograft rejection in vivo. Cytokine profiles were analyzed by ELISA and RNase protection assay. NE-κB activity and Jak 2, Stat4 activation were detected by gel shifting assay and western blotting. Results: DC exposure to rapamycin markedly inhibited their ability to stimulate allogeneic (C3H, H-2k) T cell proliferation. The antigen-specific CTL activity was suppressed by 90% compared with controls. This was associated with reduced production of both Thl and Th2 cytokines (INF-γ, IL-4, IL-5, IL-6 and IL-13). In contrast to administration of normal DC that accelerated the rejection of B10 cardiac allografts (MST 6 days vs 10 days in control, p<0.05), rapa-DC markedly prolonged allograft survival (MST 18 day, p<0.05 compared with controls). Exposure to rapamycin did not alter DC yield, and also did not inhibit DC NF-κB activity, therefore, did not affect expression of MHC, costimulatory molecules and JL-12. However, rapamycin inhibited IL-12 induced Jak2 and Stat4 phosphorylation in DC. IFN-γ production, which was mediated by Stat4 signal transduction pathway was also significantly decreased. Indeed, DC deficient in Stat4 elicited low MLR in allogeneic T cells. Interestingly, T cells exposed to rapamycin showed suppression of Jak2 activation induced by IL-12, but phosphorylated Stat4 levels remained high, similar to the controls, suggesting that inhibition of IL-12-mediated stat4 phosphorylation by rapamycin is likely to be DC specific. Conclusion: these data indicate that rapamycin does not affect DC maturation, but significantly impairs DC allostimulatory function, probably mediated by specific inhibition of DC Stat4 activation. Rapamycin suppresses both T and antigen presenting cells, but through different mechanisms.