Prolonged, Low-Dose Anti-Thymocyte Globulin, Combined with CTLA4-Ig, Promotes Engraftment in a Stringent Transplant Model Francesca D’Addio 1,2. , Olaf Boenisch 1. , Ciara N. Magee 1. , Melissa Y. Yeung 1 , Xueli Yuan 1 , Bechara Mfarrej 1 , Andrea Vergani 1,2 , Mohammed Javeed Ansari 1,3 , Paolo Fiorina 1,2 , Nader Najafian 1 * 1 Renal Division, Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Transplantation Medicine Division, San Raffaele Hospital, Milan, Italy, 3 Divisions of Nephrology and Organ Transplantation, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America Abstract Background: Despite significant nephrotoxicity, calcineurin inhibitors (CNIs) remain the cornerstone of immunosuppression in solid organ transplantation. We, along with others, have reported tolerogenic properties of anti-thymocyte globulin (ATG, ThymoglobulinH), evinced by its ability both to spare Tregs from depletion in vivo and, when administered at low, non- depleting doses, to expand Tregs ex vivo. Clinical trials investigating B7/CD28 blockade (LEA29Y, Belatacept) in kidney transplant recipients have proven that the replacement of toxic CNI use is feasible in selected populations. Methods: Rabbit polyclonal anti-murine thymocyte globulin (mATG) was administered as induction and/or prolonged, low- dose therapy, in combination with CTLA4-Ig, in a stringent, fully MHC-mismatched murine skin transplant model to assess graft survival and mechanisms of action. Results: Prolonged, low-dose mATG, combined with CTLA4-Ig, effectively promotes engraftment in a stringent transplant model. Our data demonstrate that mATG achieves graft acceptance primarily by promoting Tregs, while CTLA4-Ig enhances mATG function by limiting activation of the effector T cell pool in the early stages of treatment, and by inhibiting production of anti-rabbit antibodies in the maintenance phase, thereby promoting regulation of alloreactivity. Conclusion: These data provide the rationale for development of novel, CNI-free clinical protocols in human transplant recipients. Citation: D’Addio F, Boenisch O, Magee CN, Yeung MY, Yuan X, et al. (2013) Prolonged, Low-Dose Anti-Thymocyte Globulin, Combined with CTLA4-Ig, Promotes Engraftment in a Stringent Transplant Model. PLoS ONE 8(1): e53797. doi:10.1371/journal.pone.0053797 Editor: Clive M. Gray, University of Cape Town, South Africa Received October 9, 2012; Accepted December 3, 2012; Published January 10, 2013 Copyright: ß 2013 D’Addio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health 1KO8AI064335-01A2 and a grant from Genzyme. FD was supported by the ERA-EDTA Fellowship Grant and the SISQT-Biotest Fellowship Grant. MJA was supported by a NIH Grant K08 AI080836-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Paolo Fiorina is a co-author of this paper and he is currently an academic editor for PLOS ONE. However, the authors would like also to re- assure the Editorial Board that this did not alter their adherence to all the PLOS ONE policies on sharing data and materials. Genzyme as a commercial source and funder. Genzyme provided us with the major compound tested in this paper, the murine anti-thymocyte globulin (mATG). This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: nnajafian@rics.bwh.harvard.edu . These authors contributed equally to this work. Introduction The introduction of calcineurin inhibitors (CNIs) into clinical use positively influenced short-term graft survival by reducing acute rejection rates [1]. CNI-containing immunosuppressive regimens are, however, associated with significant morbidity, including infection and neoplasia, whilst CNI-induced nephrotox- icity remains a major barrier to long-term renal allograft survival, and the most common cause of end-stage renal disease in recipients of other solid organ transplants [2,3]. The achievement of a state of selective hyporesponsiveness towards donor alloan- tigens whilst maintaining recipient immunocompetence is, there- fore, a major goal in clinical transplantation. The alloimmune response is a complex interplay between pathogenic/inflammatory and regulatory/anti-inflammatory im- mune mechanisms; the supremacy of either process determines whether the ultimate fate of the allograft is rejection or tolerance, respectively [4–6]. Previous studies in renal transplant patients demonstrated that Tregs regulate the alloimmune response and contribute to alloantigen hyporesponsiveness [7,8]. Various compounds, including ThymoglobulinH, alemtuzumab and siroli- mus, are capable of inducing and expanding Tregs in vitro, both in animals and humans [9–13]. The administration of standard, depleting doses of murine homologue of polyclonal antithymocyte globulin, mATG, has recently been shown to promote the generation of Tregs capable of significant suppression both in vitro and in a graft-versus-host disease (GVHD) model [14], and to PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e53797