CLINICS 2012;67(7):805-813 administration of FTY720 (Tx + FTY, n = 5) by gavage for 21 days, starting three days before the transplantation. We observed a significant (p-value = 0.0047) increase in the survival of allografts from F1 mice (BALB/c x C57BL/6) when the recipient C57BL/6 mice were treated with FTY720 (Figure 1A). The macroscopic examination shows that the first rejection (90% of skin necrosis) occurred on day 13 for the FTY720-treated mice, whereas the first rejection occurred as early as 9 days after transplantation in the non-treated mice (Figure 1B). FTY720 reduced the number of cells in the lymph nodes The total number of cells and percentage of regulatory T cells were analyzed in the lymph nodes from the Tx and Tx + FTY mice five days post-transplantation. Our results showed that the FTY720 treatment was able to significantly reduce both the number of cells and percentage of CD4+Foxp3+ T cells in the lymph nodes (Figure 2). FTY720 reduced the number of graft-infiltrating cells Five days after the skin transplantation, we analyzed the graft-infiltrating cells and observed that the FTY720 treat- ment reduced the number of graft-infiltrating cells and diminished the percentage of CD4+ T cells in the skin allograft. The FTY720 did not prevent the accumulation of Foxp3+ cells in the graft but did cause a decrease in the percentage of these cells (Figure 3). FTY720 treatment in skin allograft rejection Commodaro AG et al. Decrease in the cytokine levels in mice treated with FTY720 We evaluated the cytokine levels in the spleen cells submitted to in vitro stimulation with anti-CD3. The analysis of the 72-h culture supernatants by Bio-Plex showed decreased levels of IL-10, IL-6 and IL-17 secretion in the FTY720-treated mice compared with the non-treated mice (Figure 4). The IL-6 secretion was significantly decreased in the Tx+FTY group (p,0.05). qPCR analysis To confirm our results regarding cytokine levels, we evaluated the IL-10, IL-6, FOXP3, IL-23 and IL-27 gene expression in the spleen cells by real-time PCR. In agreement with our previous observation, there was a decrease in the IL-10 and IL-6 mRNA levels in the splenocytes of the FTY720-treated mice. In this group, we also observed a decrease in the IL-23 mRNA level but an increase in the IL-27 mRNA level. The FOXP3 expression was decreased, but this decrease did not reach statistical significance (Figure 5). In the skin, we found increased mRNA levels of IL-10, IL- 27, and IL-23 in mice treated with FTY720 (Figure 6), but this increase was not statistically significant. The FOXP3 expression was decreased in this group (p = 0.0948). DISCUSSION In the present study, we investigated the events asso- ciated with FTY720 treatment in mice submitted to skin Figure 4 - FTY720 decreases the cytokine production by splenocytes. Analysis of the IL-10, IL-6 and IL-17 secretion levels by the splenocytes. The spleen cells from the Tx non-treated and Tx + FTY720-treated mice were harvested at day 5 post-skin transplantation and stimulated in vitro with anti-CD3. The supernatants were collected 72 h later, and the IL-10, IL-6, and IL-17 levels were determined by multiplex ELISA. The results are expressed as the means ¡ SD (*p,0.05). 809