CCR4 dependent migration of Foxp3+ Treg cells to skin grafts and draining lymph nodes is implicated in enhanced graft survival in CD200tg recipients

We have previously reported that transgenic overexpression of CD200 in either mouse skin graft donors or recipients significantly enhances skin allograft survival. By focused microarray analysis we showed this enhanced graft survival is associated with increased expression of Foxp3, GITR, CTLA-4 and CCR4 mRNA, all genes related to T_reg cell induction/function, and of Gata3, IL-4, IL-5, IL-13, and somewhat surprisingly, of T-bet, INF-γ and granzyme b. Gene-specific real-time PCR and immunohistochemistry analysis confirmed an increase in Foxp3⁺ T_reg cells in both the skin grafts and draining lymph nodes (DLNs) of CD200^tg recipient mice at both 7/14 days post engraftment, as well as providing evidence for increased expression of the ligands for CCR4, CCL17 and CCL22 in both locations. Following lentivirus-mediated shRNA treatment of Dox-treated CD200^tg mice to attenuate expression of CCR4 mRNA, the increased localization of T_reg cells in skin/DLN of CD200^tg recipients was abolished, and the enhanced graft survival similarly reversed. We conclude that enhanced CCR4 dependent migration of Foxp3⁺ T_reg to grafted tissue and DLNs is an essential step in the graft prolongation afforded by overexpression of CD200.