CLINICS 2012;67(7):805-813 BASIC RESEARCH DOI:10.6061/clinics/2012(07)17 The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection Alessandra Gonc¸alves Commodaro,I,II Juliana Figueredo Pedregosa,III Jean Pierre Peron,IV Wesley Branda˜ o,IV Luiz Vicente Rizzo,II Valquiria BuenoV I Vision Institute, Federal University of Sa˜ o Paulo (UNIFESP), S a˜o Paulo/SP, Brazil. II Hospital Israelita Albert Einstein, S a˜o Paulo/SP, Brazil. III Nephrology Division, Federal University of S a˜o Paulo (UNIFESP), S a˜o Paulo/SP, Brazil. IV Faculdade de Medicina da Universidade de S a˜o Paulo, Department of Immunology, Sa˜ o Paulo/SP, Brazil. V Immunology Division, Federal University of S a˜o Paulo (UNIFESP), S a˜o Paulo/SP, Brazil. OBJECTIVES: FTY720 modulates CD4+T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment. METHODS: F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis. RESULTS: The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4+ graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft. CONCLUSIONS: Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment. This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection. KEYWORDS: Transplantation; Rejection; Tolerance; FTY720; Foxp3; Th17. Commodaro AG, Pedregosa JF, Peron JP, Branda˜ o W, Rizzo LV, Bueno V. The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection. Clinics. 2012;67(7):805-813. Received for publication on November 23, 2011; First review completed on February 20, 2012; Accepted for publication on March 14, 2012 E-mail: valquiria@nefro.epm.br Tel.: 55 11 5549 6073 INTRODUCTION The rejection responses to allografts are initiated by a specific T-cell recognition of antigens expressed on the cells within the graft (1-3). T-cell activation after allograft transplantation requires both antigen presentation and co- stimulatory signals (4,5), and, once they occur, several other factors are involved, leading to effector or regulatory immune responses. Recently, the immune responses to allografts have been reported to be modulated by regulatory T cells, such as CD4+CD25+ T cells, which comprise 5-10% of Copyright 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non- commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. the peripheral CD4+ T cells in adult rodents and humans (6). These cells play an important role in the maintenance of self-tolerance, prevention of autoimmune diseases (7), and regulation of allogeneic immune responses (8,9). Regulatory natural T cells (nTregs) are generated in the thymus, defined by their high expression of the IL-2 receptor a chain (CD25) and thought to be controlled by the forkhead box P3 (Foxp3) transcription factor. The population of thymus-derived Treg cells is supplemented with a popula- tion of inducible Tregs (iTregs) generated from na¨ıve T cells activated in the presence of TGF-b. iTregs are believed to be functionally similar to thymus-derived nTregs (10). In contrast with Treg-generated transplantation tolerance, the Th17 cell population has been associated with allograft rejection (11). Th17s are described as T cells producing interleukin-17 that are dependent on signals from cytokines, such as IL-6 (12). Moreover, the Th17 response is propa- gated by the cytokines IL-23 and IL-21 (13,14). 805