Peri‐graft porcine‐specific CD4+FoxP3+ regulatory T cells by CD40‐CD154 blockade prevented the rejection of porcine islet graft in diabetic mice

Background Anti‐CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4+ regulatory T cells (Tregs) in xeno‐immune tolerance. However, the characteristics of Tregs and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined. Method Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5 mg/mouse of anti‐CD154 mAb on 0, 1, 3, 5, and 7 days post‐transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients’ cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme‐linked immunosorbent spot (ELISPOT) assay, and mixed‐lymphocyte reaction. Results Short‐term anti‐CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n = 18). Immunohistochemical staining showed significantly higher ratio of CD4+Foxp3+ Tregs in the peri‐graft site, but not in the spleen and kidney‐draining lymph node of the recipient mice. Depletion of Tregs evoked graft rejection, and adoptive transfer of Tregs from anti‐CD154 mAb‐treated recipients provided protection to the graft from rejection. These Tregs showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen‐specific. Conclusions In this study, we showed that anti‐CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine‐specific CD4+Foxp3+Tregs in the peri‐graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation.