Role of STAT3 in CD4  CD25  FOXP3  Regulatory Lymphocyte Generation: Implications in Graft-versus-Host Disease and Antitumor Immunity 1 Jean-Rene ´ Pallandre,* Emilie Brillard,* Gilles Cre ´hange, † Amandine Radlovic,* Jean- Paul Remy-Martin,* Philippe Saas,* Pierre-Simon Rohrlich,* Xavier Pivot,* ‡ Xiang Ling, § Pierre Tiberghien,* and Christophe Borg 2 * ‡ Immunological tolerance is maintained by specialized subsets of T cells including CD4  CD25  FOXP3  regulatory cells (Treg). Previous studies established that Treg thymic differentiation or peripheral conversion depend on CD28 and Lck signaling. More- over, foxp3 gene transfer in murine CD4  CD25  T lymphocytes results in the acquisition of suppressive functions. However, molecular pathways leading to FOXP3 expression remain to be described. In this study, we investigated the molecular events driving FOXP3 expression. We demonstrated that CD28 activation in CD4  CD25  T lymphocytes leads to STAT3 Tyr 705 phos- phorylation in an Lck-dependent manner. STAT3 neutralization during naive peripheral CD4  CD25  T cell conversion into Treg through costimulation with TCR/CD28 and TGF-1, decreased FOXP3 expression, prevented the acquisition of suppressive functions and restored the ability of the converted lymphocytes to produce IL-2 and IFN-. Furthermore, we observed that STAT3 ablation using small interfering RNA strategies inhibited FOXP3 expression and suppressive functions among naturally differ- entiated CD4  CD25  T lymphocytes, suggesting a direct role of STAT3 in Treg phenotype and function maintenance. CD4  CD25  T lymphocytes transduced with specific STAT3 small interfering RNA were devoid of suppressive functions and failed to control the occurrence of acute graft-vs-host disease. Finally, STAT3 inhibition in CD4  lymphocytes enhanced the anti-tumor immunity conferred by a lymphocyte adoptive transfer. In summary, our findings determine that STAT3 is critical in the molecular pathway required for FOXP3 expression. STAT3 modulation should be taken into account when assessing how regulatory T cells contribute to inflammatory diseases and tumor immunosurveillance. The Journal of Immunology, 2007, 179: 7593–7604. S elf-tolerance is an important feature in the prevention of autoimmune diseases which relies on both central and pe- ripheral mechanisms. Central tolerance refers to the dele- tion of developing autoreactive T cells that bind with high-affinity to intrathymic self Ags. Despite this stringent selection, autoreac- tive T cells can evade the thymus. Peripheral regulatory mecha- nisms are then required for the prevention of autoimmunity (1–3). CD4 + CD25 + regulatory T cells (Treg) 3 have been recognized as a major population of suppressing T cells that maintain peripheral immune tolerance (4). Mature Treg can be identified by their con- stitutive expression of CD25, CTLA-4, and glucocorticoid-in- duced TNF receptor family related receptor (5–7). In addition, Treg differentiation requires the expression of the Forkhead box P3 (foxp3) transcription factor which remains the more specific Treg marker today (8 –10). Although naturally occurring Treg lymphocytes develop in the thymus, several reports have demonstrated that peripheral CD4 + CD25 - naive T cells can be converted into adaptive FOXP3 + Treg (11–14). Adaptive Treg can be induced in the pe- riphery throughout the course of immune responses. Several re- searchers have compared the TCR-V repertoires in CD4 + CD25 - CD45RO + memory T cells and CD45RO + Treg subsets to determine to what extend both populations express T cell receptors that belong to the same family. These studies high- lighted that these two populations are related (15–16). This raises the question of whether Treg might derive from memory CD4 + T cells. Then, the crucial question to be addressed is what molecular signaling leads to Foxp3 expression and confers regulatory func- tions to CD4 + CD25 - T cells. Walker et al. (17) have shown that TCR and CD28 stimulation of human CD4 + CD25 - T cells in- duces Foxp3 transcription and acquisition of regulatory activity in vitro. Moreover, mice deficient in either CD28 or B7 have a re- duced number of Treg in both the thymus and periphery (18 –19). Recently, Tai X et al. (20) have provided evidence in a rodent model that the Lck binding activity of CD28 is required for Foxp3 transcription and Treg cell differentiation. However, the precise *Institut National de la Sante ´ et de la Recherche Me ´dicale U645, EFS Bourgogne Franche Comte ´, University of Franche-Comte ´, Besanc ¸on, France; † CHU Jean Minjoz, Department of Radiotherapy, Besanc ¸on, France; ‡ Centre Hospitalo-Universitaire Jean Minjoz, Department of Medical Oncology, Besanc ¸on, France; and § Section of Mo- lecular Hematology and Therapy, Department of Bone Marrow Transplantation, Uni- versity of Texas, MD Anderson Cancer Center, Houston, TX 77030 Received for publication December 8, 2006. Accepted for publication September 30, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 J.-R.P. was supported by the Conseil Regional de Franche Comte ´, E.B. has received a fellowship from the Ligue Franc ¸aise Contre le Cancer, and A.R. was financed by a grant from the Conseil Scientifique of Etablissement Franc ¸ais du Sang (no. CS-2005- 14). This work has also been supported by Institut National de la Sante ´ et de la Recherche Me ´dicale, the Ligue Contre le Cancer, Comite ´ du Doubs. 2 Address correspondence and reprint requests to Dr. Christophe Borg, Institut Na- tional de la Sante ´ et de la Recherche Me ´dicale U645, Boulevard Fleming, 25000 Besanc ¸on, France. E-mail address: christophe.borg@efs.sante.fr 3 Abbreviations used in this paper: Treg, regulatory T cell; GvHD, graft-vs-host dis- ease; aGvHD, acute GvHD; Foxp3, forkhead box P3; siRNA, small interfering RNA; ALK, anaplastic lymphoma kinase; QRT-PCR, quantitative real-time PCR. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 The Journal of Immunology www.jimmunol.org