CLINICS 2012;67(7):805-813 FTY720 treatment in skin allograft rejection Commodaro AG et al. Figure 6 - FTY720-treated mice present an increase in the relative gene expression (qPCR analysis) of IL-10, IL-23, and IL-27 in the skin grafts. The relative gene expression (qPCR analysis) of FOXP3, IL-10, IL-23, and IL-27 in the F1 skin grafts was evaluated in the Tx non- treated and Tx + FTY720-treated mice. The results are expressed as the means ¡ SD. skin graft, as well as Foxp3 protein expression in the lymph nodes and graft-infiltrating cells, was decreased in mice treated with FTY720. These findings show that FTY720 does not inhibit but rather decreases the expres- sion of Foxp3 systemically. The increase in skin allog- raft survival due to FTY720 without reaching indefinite tolerance could be justified by the following: the increased skin graft survival induced by FTY720 is mediated by other factors in addition to Foxp3 cells; and the decreased Foxp3 expression contributes to the rejection, even if over the long-term. In our model, the in vitro anti-CD3 stimulation of splenocytes from the mice submitted to skin transplanta- tion and treated with FTY720 exhibited decreased secretion of IL-10 and IL-6, which was confirmed by qPCR analysis. In addition, the splenocytes from FTY720-treated mice secreted significantly less IL-17 after in vitro anti-CD3 stimulation, and the evaluation of the mRNA in the spleen cells showed a decreased level of IL-23. Chen et al. showed an enhanced expression of Th17-related cytokines at the early stage of cornea rejection either in the draining lymph nodes (LNs) or in the allograft. They also found that responder cells in the draining LNs of the transplanted recipients secreted high levels of IL-6, TGF-b and IL-21 compared with the controls and suggested that these factors could drive na¨ıve T cells to differentiate into Th17 cells (27). Because Th17 cells have been implicated in renal allograft rejection (28) and IL-17 production has been shown to be dependent on IL-6 (29) and IL-23 levels (30), 811 the decreased expression of these cytokines in our model suggests that the prolonged skin allograft survival obtained by FTY720 treatment was at least in part due to Th17 phenotype impairment in the periphery rather than Foxp3 enhancement. In agreement, Mehling et al. showed a decrease in the number of Th17 cells from the blood of patients with multiple sclerosis treated with FTY720 and of patients in disease remission (31). However, whether FTY720 increases the Treg cell population is still uncertain. Wolf et al. showed in vitro and in vivo that FTY720 inhibits the proliferation of Treg cells without affecting their viability, phenotype or in vitro immunosuppression (32). Zhou et al. showed that the addition of FTY720 to the mixed lymphocyte reaction (MLR) in mouse (BALB/c and C57BL/6) splenocytes increased the proportion of CD4+CD25+ T regulatory cells. Additionally, FTY720 added to the MLR caused the suppression of lymphocyte pro- liferation, decreased the IL-2 levels in the supernatant, and induced lymphocyte apoptosis (33). Allograft acceptance/rejection is associated with multiple suppressor/effector mechanisms, and immunosuppressive drugs administered during transplantation have been shown to affect T cell populations. In agreement, Hester et al. observed, in the follow-up of kidney recipients induced with alemtuzumab and treated post-transplantation with several drugs such as sirolimus or sirolimus + mycophenolate mofetil, that rejection and the long-term maintenance of immunosuppression influence the number of circulating Treg and Th17 cells (34).