Donor-derived soluble MHC antigens plus low-dose cyclosporine induce transplantation unresponsiveness independent of the thymus by down-regulating T cell-mediated alloresponses in a rat transplantation model.

BACKGROUND: In vitro, soluble MHC (sMHC) antigens modulate and induce apoptosis in alloreactive and antigen-specific T cells, demonstrating their potency to regulate T cell-mediated immune responses. However, their efficacy to regulate immunological responses in vivo remains unclear. Here, we report that repetitive intraperitoneal injection of recombinant Lewis rat-derived MHC class I antigens in Dark Agouti (DA) rats modulates alloreactivity. METHODS: RT1.A1 (Lewis derived) genes were cloned into mammalian expression vectors, and RT1.Aa (DA derived) genes were used to transfect a rat myeloma cell line. RT1.A1 molecules were injected intraperitoneally in DA recipients that subsequently underwent transplantation with Lewis-derived cardiac allografts. RESULTS: Soluble class I antigens were secreted by the transfected cells and were shown to be heterodimeric, peptide-loaded, and conformationally folded. Injection of donor-derived soluble MHC significantly reduced the ability of recipient animals to mount a cytotoxic T-cell response to donor-derived tissue. More interestingly, this treatment significantly prolonged donor-graft survival and allowed 60% of treated animals to develop graft tolerance (>120 days), when donor sMHC were combined with a single subtherapeutic dosage of cyclosporine. Thymectomy of recipient animals before transplantation did not interfere with induction of peripheral tolerance. CONCLUSIONS: Donor-derived sMHC are potential tolerogens for down-regulating the cytotoxic T-cell response of animals that undergo transplantation. Thus, these data provide for the first time a rationale for the application of directly injected sMHC in vivo to down-regulate immunological responses and aid the induction of graft tolerance.

Co-transplantation of donor-derived hepatocytes induces long-term tolerance to cardiac allografts in a rat model.

BACKGROUND: Liver allografts transplanted between MHC-disparate mice, rats, and swine are spontaneously accepted in most strain combinations without requirement for immunosuppression. The underlying mechanism has, however, remained elusive. Here, we demonstrate that co-transplantation of donor-derived hepatocytes protect Lewis (RT1.A1) cardiac allografts from acute and chronic rejection in DA (RT1.Aa) recipients indefinitely. METHODS: Livers of donor Lewis rats were harvested and the hepatocytes separated from hepatic leukocytes by collagenase digestion and gradient separation. DA recipient animals were transplanted Lewis cardiac allografts and simultaneously intraportally infused either Lewis-derived hepatocytes or hepatic leukocytes. Recipient animals were either not further treated or received a single dose of 15 mg/kg cyclosporine. RESULTS: Donor hepatocytes alone significantly protected syngeneic cardiac allografts from rejection, whereas hepatic leukocytes failed to influence graft survival. In combination with cyclosporine, recipient cardiac allografts were indefinitely protected from rejection. Graft-infiltrating cells in tolerant animals presented as clusters of CD4+ T cells and stained mostly positive for interleukin-4, whereas graft-infiltrating cells in rejected allografts were predominantly positive for interferon-gamma. Adoptive transfer of splenocytes derived from tolerant animals protected Lewis cardiac allografts from rejection in DA recipients without immunosuppression. In contrast, hepatic leukocytes protected only 50% of the allografts from rejection. CONCLUSION: We propose that donor hepatocytes induce permanent engraftment of syngeneic allografts by establishing a Th2 type alloresponse that is transferable to new graft recipients. The results of this study demonstrate that liver parenchymal cells significantly mediate spontaneously liver-induced tolerance.

Oral feeding of an immunodominant MHC donor-derived synthetic class I peptide prolongs graft survival of heterotopic cardiac allografts in a high-responder rat strain combination.

The efficacy of two synthetic major histocompatibility complex (MHC)-derived DA (RT1.Aa) 25-mer peptides (residues 56-80 and 96-120) to modulate alloreactivity was tested in Lewis (RT1.A1) responder animals. The DA peptide 56-80, but not peptide 96-120, induced delayed-type hypersensitivity (DTH). DTH was significantly reduced by oral feeding of peptide 56-80, P = 0.004. In addition, oral feeding of this peptide in combination with a short course of cyclosporin A (CsA) prolonged graft survival of 60% of heterotope transplanted DA cardiac allografts in Lewis recipient rats. Long-term survivors developed low levels of allo-antibodies against donor tissue as compared to rejecting animals and increased levels of interleukin-4 (IL-4) within the allograft. Similarly, IL-4-secreting splenocytes were identified by flow cytometry in these animals, indicating a Th2-type cytokine pattern. However, graft survival was particularly limited to cardiac allografts because donor-type skin grafts were acutely rejected in tolerant animals. It is interesting that residue alignment of peptide 56-80 to the motif of the RT1.A1 molecule showed a preferred class I motif within this sequence, suggesting indirect presentation of this peptide to recipient T cells. Thus, peptide 56-80 appears to represent a dominant epitope that can be exploited for establishing tolerance in this transplantation strain combination.