Evidence that FoxP3+ regulatory T cells may play a role in promoting long-term acceptance of composite tissue allotransplants.

Abstract

FoxP3/CD4/CD25 regulatory T cells (Treg) play an important role in maintaining peripheral tolerance and are potent suppressors of T-cell activation. In this study, we evaluated the role of Treg in peripheral tolerance to composite tissue allografts (CTA). Mixed allogeneic chimeric rats were prepared by preconditioning recipients with anti-αβ-T-cell receptor monoclonal antibody followed by total body irradiation. Animals received T-cell-depleted August Copenhagen Irish bone marrow cells followed by antilymphocyte serum and FK-506. A modified osteomyocutaneous hindlimb flap composed of bone and all limb tissue components was placed in animals with chimerism greater than or equal to 1% on day 28. Recipients with CTA surviving more than or equal to 6 months were evaluated for Treg. Skin samples from tolerant long-term allogeneic transplanted, syngeneic transplanted, rejected, and naïve animals were immunostained with fluorochrome-conjugated anti-FoxP3 and anti-CD4 monoclonal antibody and visualized under a laser confocal microscope. Significant CD4/FoxP3 Treg infiltrates were observed in tolerant donor-allograft skin samples. No graft infiltrating FoxP3 cells were observed in rejector, naïve, or skin from syngeneic CTA. In parallel experiments, mixed leukocyte reaction assays were performed to investigate the suppressor function of Treg cells. Splenocytes from tolerant, rejected, and naïve rats were sorted by flow cytometry for CD4/CD25 T cells. Treg demonstrated similar suppressive levels between the three groups. These data suggest that Treg may play an important role in maintenance of tolerance and promoting graft acceptance in long-term CTA acceptors and may explain the favorable outcomes observed in clinical CTA recipients.