Injection of donor-derived splenic dendritic cells plus a nondepleting anti-CD4 monoclonal antibody to prolong primary skin graft survival indefinitely and abrogate the production of donor-specific antibodies in the Fischer-to-Lewis rat combination.

We have previously shown that injection of donor-derived Fischer rat OX62+ dendritic cells plus an anti-CD4 monoclonal antibody generates donor-specific CD4+CD25+FoxP3+ regulatory T cells in Lewis rats spleens. The regulatory T cells indefinitely prolonged the survival of skin graft from Fischer rat and abrogated the antidonor antibody response. We have now shown that an injection of 2 × 10(6) donor-derived OX62+ dendritic cells plus 2 mg nondepleting anti-CD4 monoclonal antibody (W3/25) at 28 days before grafting induced indefinite skin graft survival in this combination; whereas an injection on day -1 prolonged it only to 50 days. This effect is donor specific. In both cases, we suppressed the antidonor antibody response. It is likely that the efficacy of this protocol is, at least in part, dependent on induction of donor-specific regulatory T cells, as suggested by previous data. The 28 days necessary to obtain tolerance of allogenic skin grafts may be due to the time required for the host to induce proliferation of donor-specific regulatory T cells.

Injection of donor-derived OX62+ splenic dendritic cells with anti-CD4 monoclonal antibody generates CD4+CD25+FOXP3+ regulatory T cells that prolong allograft skin survival indefinitely and abrogate production of donor-specific antibodies in a rat model.

OBJECTIVE: To examine in a rat model the ability of donor dendritic cells and anti-CD4 monoclonal antibody (mAb) to generate donor-specific CD4+CD25+ regulatory T cells (Tregs) and to evaluate the capacity of these Tregs to prolong skin allograft survival and abrogate the production of donor-specific antibodies after skin grafting. MATERIALS AND METHODS: OX62+ (nonplasmacytoid) splenic dendritic cells were isolated from Fischer rats using magnetic beads and injected (2 x 10(6)) into Lewis rat recipients with or without treatment with a nondepleting anti-CD4 (W3/25) mAb. After 4 weeks, splenic CD4+CD25+FOXP3+ T cells were harvested using magnetic beads from conditioned animals and injected (1 x 10(6)) into naïve Lewis recipients (day 1) before they received a skin graft from a Fischer (n = 4) or a third-party (Norway; n = 4) donor rat. Donor-specific antibodies were detected in recipient blood using flow cytometric cross-matches with donor lymphocytes from day 0 to day 30 after grafting. RESULTS: After injection of conditioned CD4+CD25+FOXP3+ T cells, Lewis recipients accepted skin grafts from Fischer donors indefinitely (>100 days) but rejected third-party skin grafts. Donor-specific antibodies were detected at low levels in only 1 recipient receiving conditioned Tregs before grafting. Naive Tregs did not prolong skin graft survival. CONCLUSION: These preliminary data suggest that splenic dendritic cells in combination with an anti-CD4 mAb induce donor-specific Tregs that indefinitely prolong allogeneic skin graft survival and inhibit donor-specific antibody production. Experiments are under way to determine whether this protocol can inhibit chronic lesions after heart transplantation in this model.