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 prolongs organ allograft survival in experimental models, but the immunosuppressive mechanism(s) responsible remains unclear. A central, confounding issue is how to reconcile documentation of comparable levels of mRNA for IL-2 in suppressed versus control groups with obvious host hyporesponsiveness. We used a model of tolerance induction involving perioperative injection of donor spleen cells and injection of CsA at day 2 after transplant to analyze the serial expression of several proinflammatory cytokines relevant to development of alloresponsiveness within cardiac allografts and recipients' spleens. Four experimental groups of Lewis rats receiving vascularized heterotopic cardiac allografts from 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 Lewis rats receiving the combined spleen cell/CsA therapy (mean 64 days, with 40% of grafts surviving > 100 days, n = 15) compared with acute rejection at about 8 days (range 6-13, n = 20) in each of the 3 control groups (P < 0.0001). By comparison with acutely rejecting allografts in the control untreated group at day 7 post-Tx, allografts in rats receiving the combined perioperative spleen cell/CsA treatment showed (1) significantly reduced graft cellularity and interstitial edema; (2) significantly decreased features of immune activation, including infiltration by mononuclear cells expressing IL-2R or proliferating cell nuclear antigen; (3) decreased intragraft expression of the cytokines IL-2 and IFN-gamma; and (4) suppression of endothelial activation as evidenced by both failure of up-regulation of intracellular adhesion molecule-1 and maintenance of thrombomodulin expression by graft endothelium. Analysis of sections of recipients' spleens showed that spleen cell/CsA therapy led to significant reductions versus untreated controls, in expression of IL-2, IFN-gamma, and IL-2R. Similarly, mixed lymphocyte response cultures showed that responder cells from rats receiving combined therapy proliferated by 93-95% less than untreated animals. Our results suggest that the efficacy of this clinically relevant protocol is associated with suppression of IL-2 or IFN-gamma protein production, and that in the absence of such molecules, it appears that T cell-receptor occupancy by alloantigens readily induces a state of anergy in vivo.