MECHANISM OF A CLINICALLY RELEVANT PROTOCOL TO INDUCE TOLERANCE OF CARDIAC ALLOGRAFTS - PERIOPERATIVE DONOR SPLEEN-CELLS PLUS CYCLOSPORINE SUPPRESS IL-2 AND INTERFERON-GAMMA PRODUCTION

Many groups have reported that preoperative injection of donor-derived whole spleen cells or major histocompatibility complex antigens prolo ngs organ allograft survival in experimental models, but the immunosup pressive mechanism(s) responsible remains unclear. A central, confound ing issue is how to reconcile documentation of comparable levels of mR NA for IL-2 in suppressed versus control groups with obvious host hypo responsiveness. We used a model of tolerance induction involving perio perative injection of donor spleen cells and injection of CsA at day 2 after transplant to analyze the serial expression of several proinfla mmatory cytokines relevant to development of allpresponsiveness within cardiac allografts and recipients' spleens. Four experimental groups of Lewis rats receiving vascularized heterotopic cardiac allografts fr om Brown Norway (BN) donors were evaluated: (1) untreated controls; (2 ) animals receiving intraoperative injection of donor BN spleen cells; (3) those receiving a single injection of CsA on day 2 post-Tx; and ( 4) animals given the combination of intraoperative BN spleen cells and CsA on day 2 post-Tx. Graft survival was significantly prolonged in L ewis rats receiving the combined spleen cell/CsA therapy (mean 64 days , with 40% of grafts surviving > 100 days, n=15) compared with acute r ejection at about 8 days (range 6-13, n=20) in each of the 3 control g roups (P<0.0001). By comparison with acutely rejecting allografts in t he control untreated group at day 7 post-Tx,allografts in rats receivi ng the combined perioperative spleen cell/CsA treatment showed (1) sig nificantly reduced graft cellularity and interstitial edema; (2) signi ficantly decreased features of immune activation, including infiltrati on by mononuclear cells expressing IL-BR or proliferating cell nuclear antigen; (3) decreased intragraft expression of the cytokines IL-2 an d IFN-gamma; and (4) suppression of endothelial activation as evidence d by both failure of up-regulation of intracellular adhesion molecule- 1 and maintenance of thrombomodulin expression by graft endothelium. A nalysis of sections of recipients' spleens showed that spleen cell/CsA therapy led to significant reductions versus untreated controls, in e xpression of IL-2, IFN-gamma, and IL-2R. Similarly, mixed lymphocyte r esponse cultures showed that responder cells from rats receiving combi ned therapy proliferated by 93-95% less than untreated animals. Our re sults suggest that the efficacy of this clinically relevant protocol i s associated with suppression of IL-2 or IFN-gamma protein production, and that in the absence of such molecules, it appears that T cell-rec eptor occupancy by alloantigens readily induces a state of anergy in v ivo.