Regulatory mechanism of peripheral tolerance: in vitro evidence for dominant suppression of host responses during the maintenance phase of tolerance to renal allografts in miniature swine

Background: Studies from our laboratory have demonstrated that a short course of cyclosporine leads to indefinite survival of renal allografts across an MHC class-1 barrier in miniature swine. We have recently reported that a peripheral regulatory mechanism appears to be involved in the maintenance of this tolerance since peripheral blood lymphocytes (PBL), exposed to donor antigen in vitro specifically suppressed the generation of anti-donor cytotoxic activity by recipient-matched naive PBL. We have now further investigated the mechanism of this phenomenon to determine the level at which such regulation occurs, and investigated the phenotypes of the cells involved in maintaining dominant suppression. Materials and methods: PBL from long-term tolerant animals ( > 6 months after renal transplantation) were pre-stimulated in vitro with donor-type PBL. These cells were then incubated with recipient-matched naive responders and donor-type PBL stimulators in MLR assays. The proliferative activity of the cells was measured by [H-3]-thymidine incorporation. Suppression was measured by inhibition of proliferation of naive cells in response to donor PBL when co-cultured with tolerant cells. Flow cytometry was used to study the phenotypes of cells that were present in cell cultures. Results: Primed PBL from tolerant animals markedly suppressed the proliferative response of recipient-matched naive cells to donor-matched stimulators in vitro. No suppression of proliferation was observed in response to third party stimulators, indicating that the suppression was donor-specific. Primed PBL from naive animals stimulated with donor antigen and co-cultured with unprimed recipient-matched naive cells also demonstrated reduced proliferative responses. However, this decrease in proliferation appeared to be due to a 'burn-out' phenomenon, as assessed by kinetic studies, rather than due to true suppression. Expression of CD25 increased on a sub-population of T cells from tolerant animals following priming with donor antigen. These cell then markedly inhibited further activation of CD25 positive cells in co-cultures with naive responder cells, suggesting a possible mechanism of suppression. Conclusion: These results suggest that the mechanism of tolerance to class-l-mismatched renal allografts, involves a population of regulatory cells that are capable of suppressing proliferative anti-donor responses. (C) 2003 Elsevier Science B.V. All rights reserved.