Regulatory T Cell Therapy Combined with Donor-Specific T Cell Deletion Promotes Long-Term Survival in Mouse Model of Allogeneic Islet Transplantation: Preconditioning Requirements and Mechanisms of Action

Abstract

Regulatory T cell (Treg) prevention of transplant rejection requires drastic change of the balance of donor antigen-reactive Tregs to conventional T (Tconv) cells at the time of transplantation. To accomplish this, we preconditioned recipient mice using donor-specific transfusion (DST) followed by cyclophosphamide (Cy) treatment to reduce donor-reactive T cells before Treg infusion. This DST+Cy approach deleted 70-80% of donor-reactive T cells, but failed to prolong islet allograft survival. Infusion of 5 × 106 Tregs with direct donor reactivity led to long-term survival of BALB/c islets in 70% of the DST+Cy conditioned C57BL/6 recipients. Equal numbers of polyclonal Tregs significantly prolonged graft survival, but 90% of the recipients rejected their grafts by 100 days. However, increasing the numbers of polyclonal Tregs to 25 × 106 was able to confer long-term graft protection in 90% of the recipients. Mice receiving Treg therapy showed marked reduction of CD8+ T cells, but not CD4+ T cells, and concomitant increase of Tregs in the grafts during the first 14 days after transplantation. Mice with long-term protected grafts harbored donor-reactive T cells in the periphery and but had minimal T cells without detectable therapeutic Tregs within the grafts. Majority of the leukocytes present in the protected grafts were F4/80+CD11c-macrophages. However, the original islet grafts remained to be protected while the recipients were challenged with and rejected skin grafts from the same donor strain. Therapeutic Tregs altered the balance as well as the size of lymphocytic infiltrates early after transplantation likely through altering local inflammatory milieu, and conferred long-term graft protection locally in the grafts via infectious tolerance. Together, these results demonstrate a clinically viable approach to harness the tolerogenic property of Tregs for inducing long-term allograft survival and suggest Treg-mediated protection and biomarkers for Treg-induced tolerance reside locally in the grafts.