CLINICAL AND TRANSLATIONAL RESEARCH Expression of Regulatory T–Cell-Related Molecule Genes and Clinical Outcome in Kidney Transplant Recipients Cristiam M. Alvarez, 1,2 Gerhard Opelz, 2 Luis F. Garcia, 1 and Caner Su ¨sal 2,3 Background. Naturally occurring regulatory T cells have been associated with long-term allograft survival. We inves- tigated whether gene transcripts of Treg-related molecules are upregulated or downregulated in kidney transplant recipients with different clinical outcomes and may serve as markers of operative tolerance. Methods. Expression levels of transcription factor (forkhead box P3 [FOXP3], t-bet, and GATA3), regulatory molecule (cytotoxic T-lymphocyte antigen-4, glucocorticoid-induced tumor necrosis factor receptor-related protein, tribbles protein-1, and transforming growth factor-), and chemokine receptor (CCR7 and CXCR4) genes were measured in kidney graft recipients with long-term (9 years) stable renal function (LTS) or chronic rejection (ChrRx). Patients on dialysis and healthy individuals served as controls. Results. The level of FOXP3 transcripts was lower in ChrRx patients than in LTS patients (P0.01). The highest transforming growth factor- transcripts were observed in ChrRx and the highest CCR7 and CXCR4 transcripts were observed in LTS patients. In LTS patients, FOXP3 gene expression was associated with CXCR4 gene expression (P=0.015). FOXP3 and CCR7 transcript levels were higher in LTS patients without calcineurin inhibitor therapy than in LTS patients with calcineurin inhibitors. Conclusion. Our results suggest that high expression of FOXP3 and chemokine receptor genes in LTS patients are possible indicators of a regulatory process that contributes to long-term allograft acceptance. Markers that were increased in LTS patients were found to be decreased in ChrRx patients, suggesting that rejection may partly be the result of a lack of this regulatory process. FOXP3 and CCR7 and CXCR4 transcripts might be used as markers to distinguish patients who developed long-term allograft acceptance from patients who are prone to ChrRx. Keywords: Kidney transplantation, Long-term graft survival, Regulatory T cells, Gene expression, Transplantation tolerance. (Transplantation 2009;87: 857–863) A lthough induction of tolerance is one of the major goals of research in organ transplantation, the mechanisms underlying long-term allograft acceptance in humans under immunosuppression are not fully understood (1, 2). Patients with long-term allograft survival (LTS) in the absence of or on low doses of immunosuppression represent an important source of research for exploring the mechanisms involved in induction and maintenance of long-term allograft survival. Immune regulation by regulatory T cells (Tregs) has been implicated in the homeostasis of the immune response, control of alloimmune responses, and induction and mainte- nance of transplant tolerance in vivo (1). Tregs comprise a subset of T cells with the CD4 + CD25 high FOXP3 + phenotype (3). The transcription factor forkhead box P3 (FOXP3) has been recognized as the master regulator of Tregs in mice and in humans (4). Zheng et al. (5) demonstrated that FOXP3 acts not only as a transcriptional activator but also as a repressor that amplifies and stabilizes gene expression in Tregs and thus maintains the homeostasis in these cells. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) are additional molecules expressed on Tregs. Although involved in the regulatory function of Tregs, they are not exclusive to Tregs and are also expressed on other activated T cells (6). Another option to classify Tregs is their capacity to produce immunomodulatory cytokines such as interleukin (IL)-10 and transforming growth factor- (TGF-). IL-10 and TGF- were shown to support the mediation and mainte- nance of suppressive activity by Tregs (7, 8). Recently, che- mokine receptors have also been implicated in the function and the regulatory capacity of Tregs. In animals (9 –12) and humans (13, 14), it has been demonstrated that chemokines 1 Grupo de Inmunología Celular e Inmunogene ´tica, Facultad de Medicina, Instituto de Investigaciones Me ´dicas, Universidad de Antioquia, Medellín Colombia. 2 Department of Transplantation Immunology, Institute of Immunology, University of Heidelberg, Heidelberg, Germany. 3 Address correspondence to: Prof. Dr. Caner Su ¨sal, Department of Trans- plantation Immunology, Institute of Immunology, University of Heidel- berg, D-69120 Heidelberg, Germany. E-mail: caner.suesal@med.uni-heidelberg.de Received 18 July 2008. Revision requested 11 August 2008. Accepted 21 October 2008. Copyright © 2009 by Lippincott Williams & Wilkins ISSN 0041-1337/09/8706-857 DOI: 10.1097/TP.0b013e318199fa57 Transplantation • Volume 87, Number 6, March 27, 2009 857