Isolation of regulatory T cells in the skin of a human hand-allograft, up to six years posttransplantation

A bilateral hand allotransplantation was performed in a patient six years ago. Whereas skin is known to be highly immunogenic, grafts have been well accepted up to now. Therefore, here we investigated the putative presence of regulatory T cells in the graft. Skin biopsies were performed at different time points and analyzed by immunochemistry. T cells were initially expanded with interleukin (IL)-2. In the latter biopsy, skin was directly analyzed by reverse-transcriptase polymerase chain reaction without any culture. When tested against donor mononuclear cells, donor-primed skin T cells demonstrated unresponsiveness and inhibited donor-directed blood T cell alloresponse. Moreover, their T-cell receptor-Vbeta repertoire was skewed, in contrast to that of peripheral blood T cells. Retrospectively, nuclear FoxP3 expression in skin was measured by immunohistochemistry and was found positive at that time, but appeared to increase with time. This result was supported by the measurement of FoxP3 messenger RNA (mRNA) expression in the latter fresh biopsy, which showed higher levels than that of blood, together with no expression of perforin mRNA, but increased expression of transforming growth factor-beta and IL-10. No FoxP3 mRNA expression was found in the contralateral leg, due to the absence of T cell infiltrate. This study shows the presence of the FoxP3 marker, in a well accepted human composite tissue allograft, up to six years posttransplantation. Because a suppressive cytokinic profile was also detected intragraft, in the absence of perforin mRNA expression, our data suggest that regulatory T cells could play a role in the long-term survival of this allograft.