Linked suppression across an MHC-mismatched barrier in a miniature swine kidney transplantation model

We have demonstrated previously that a 12-day course of FK506 permits the induction of tolerance to fully MHC-mismatched renal transplants in miniature swine. In the present study, we examined the mechanism of this tolerance by assessing the possibility that the survival of one-haplotype mismatched third-party kidneys might be prolonged via linked suppression. Ten SLA ^d/d miniature swine received fully MHC-mismatched renal allografts from SLA^c/c donors with 12 days of FK506. Six animals received second SLA^c/c kidneys without immunosuppression to confirm tolerance. Regulatory mechanisms were assessed by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis coculture assays and ELISA for regulatory cytokines. Linked suppression was investigated by transplanting SLA^a/c or SLA^a/d allografts into long-term tolerant recipients without immunosuppression. All recipients showed donor-specific unresponsiveness in standard cell-mediated lympholysis and MLR assays. Tolerant cells prestimulated with donor Ag and then cocultured with naive recipient MHC-matched cells inhibited antidonor responses, confirming the presence of regulatory cells. ELISA and MLR assays showed that TGF-β2 was involved in mediating the suppression in vitro. SLA^a/d renal allografts transplanted into tolerant recipients were rejected by postoperative day 8 (median, 7 days; range, 6-8). In contrast, SLA^a/c allografts showed markedly prolonged survival (median, 52 days; range, 28-78; p = 0.0246), suggesting linked suppression. Animals not challenged with a second donormatched graft did not manifest linked suppression consistent with in vitro data showing that re-exposure to tolerated Ags is important for generation of regulatory cells. To our knowledge, these data represent the first evidence of linked suppression across fully MHCmismatched barriers in a large animal model.