Inhibition of IL-12 Signaling Stat4/IFN- Pathway by Rapamycin is Associated with Impaired Dendritc Cell Function P.-H. Chiang, L. Wang, Y. Liang, X. Liang, S. Qian, J.J. Fung, C.A. Bonham, and L. Lu R APAMYCIN (Rapa) has a similar molecular structure to tacrolimus (FK506), binds to FK binding protein (FKBP) 12, and has both immunosuppressive and antipro- liferative properties. However, unlike tacrolimus, the Rapa- FKBP 12 complex has no effect on calcineurin phosphatase, suggesting that it has a unique mechanism of action distinct from that of the calcineurin inhibitors. 1 Almost all research concerning Rapa has focused on targeting T and B cells; its effect on antigen-presenting cells (APC) has not been extensively studied. Dendritic cells (DC), the most potent APC, have been shown to be critical in the initiation and regulation of immune responses. 2 In this study, we exam- ined the effect of Rapa on DC immunologic functions by exposure of DC to Rapa during their propagation from bone marrow (BM) precursors. We found that Rapa did not affect DC maturation, but significantly suppressed DC allostimulatory activity. This was not associated with inhi- bition of interleukin-12 (IL-12) production, but resulted in reduced expression of interferon- (IFN-), although IL-12 is a principal mediator of immune responses. Here, we report that Rapa decreases IFN- expression through inhi- bition of the Stat4 pathway. MATERIALS AND METHODS DC were propagated from B10 (H2 b ) BM in the presence of GM-CSF and IL-4. Expression of cell-surface antigens on DC was determined by flow cytometric analysis. DC allostimulatory activity was determined by one-way MLR and generation of CTL activity in vitro and in vivo by the influence of administration of DC (2  10 6 , IV, 7 days before transplant) on survival of B10 vascularized cardiac allografts in C3H (H2 k ) recipients. Cytokine expression was measured by enzyme-linked immunosorbent assay (ELISA) for proteins and RNase protection assay for mRNA. Cytoplasmic and nuclear proteins were resolved in 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis gels, electrotransferred to nitro- cellulose, and subjected to Western blot analysis. Anti-Stat4 anti- sera were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). Polyclonal Ab against phosphorylated Stat4 was obtained from Zymed (San Francisco, Calif). An NF-B oligonucleotide, which was used as probe, was supplied with the commercial electrophoretic motility shift assay (EMSA) kit (Promega, Madi- son, Wisc). The probe was end-labeled with [ 32 ]ATP. RESULTS Exposure of B10 DC to Rapa at 20 ng/mL induced significantly inhibited proliferative responses of C3H T cells. Antigen-specific cytotoxic activity of these T cells was examined in a 4-hour 51 Cr-release assay using EL4 (H2 b ) as targets. This is associated with reduced production of IFN- in the culture supernatants. T cells stimulated by DC that were exposed to Rapa displayed about 90% inhibition of antigen-specific CTL activity. In contrast to administration of normal DC that accelerated rejection of B10 cardiac allografts in C3H recipients (median survival time [MST] = 8, n=7, vs 13 days, n=4, in non-DC-treated controls, P  .05), injection of Rapa DC prolonged cardiac allograft survival (MST=16, n=7, P  .05 compared with normal DC-treated group). These data indicate that Rapa pro- foundly inhibits DC allostimulatory activity both in vitro and in vivo. Rapa did not significantly alter surface expres- sion of MHC; costimulatory molecules, including CD80, CD86 and CD40; and CD11c on DC, suggesting that it does not interfere with DC maturation. Addition of Rapa in DC culture did not inhibit IL-12 expression at either mRNA or protein levels in response to LPS stimulation. Rapa ap- peared also not to inhibit nuclear NF-B expression as determined by Western blot. However, ELISA data re- vealed that IFN- production elicited by exogenous IL-12 was markedly inhibited in DC treated with Rapa in a dose-dependent manner. This was associated with almost total blockade of Stat4 activation, as phosphorylation of Stat4 in DC, which was induced by exogenous IL-12, was completely inhibited in Rapa-treated DC. These data sug- gest that inhibition of IL-12-dependent IFN- expression in DC by treatment of Rapa is likely mediated by blockade of Stat 4 signaling pathway. DISCUSSION These observations demonstrate that Rapa significantly inhibits DC allostimulatory function both in vitro and in vivo. In contrast to normal DC that accelerate cardiac allograft survival, systemic administration of Rapa DC prolongs graft survival. The inhibitive allostimulatory activ- ity of Rapa-DC is unlikely attributed to prevention of their From the Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania. Address reprint requests to Dr Lina Lu, E1554, BST, University of Pittsburgh Medical Center, 200 Lothrop St. Pittsburgh, PA 15261. 0041-1345/02/$–see front matter © 2002 by Elsevier Science Inc. PII S0041-1345(02)02900-7 655 Avenue of the Americas, New York, NY 10010 1394 Transplantation Proceedings, 34, 1394 –1395 (2002)