Regulatory CD25 + T Cells in Human Kidney Transplant Recipients ALAN D. SALAMA, NADER NAJAFIAN, MICHAEL R. CLARKSON, WILLIAM E. HARMON, and MOHAMED H. SAYEGH Laboratory of Immunogenetics and Transplantation, Renal Division, Brigham and Women’s Hospital; Nephrology Division, Children’s Hospital; Harvard Medical School, Boston, Massachusetts. Abstract. Recent evidence suggests that a population of pro- fessional regulatory cells, which limit immune responsiveness, exist in rodents and healthy human subjects. However, their role in disease states remains unclear. A proportion of renal transplant recipients do not demonstrate in vitro reactivity toward their mismatched donor-derived HLA-DR antigens; it was therefore hypothesized that this may be due to such reg- ulatory cells. A cohort of 23 renal transplant recipients was studied at a single institution. In patients with no history of acute rejection, 6 (40%) of 15 demonstrated regulation toward the mismatched HLA-DR allopeptides by CD25 + cells. By contrast, only one (12.5%) in eight of those with a history of acute rejection demonstrated regulation. Interestingly, if the patient assays were stratified according to initial in vitro im- mune responsiveness toward the mismatched allopeptides, 8 (47.1%) of 17 of patient assays with low allopeptide respon- siveness (alloreactive T cell frequencies less than 60/million) demonstrated regulation of indirect pathway alloresponses by CD25 + cells, whereas 0 of 8 with higher responses (frequen- cies greater than 60/million) demonstrated no such regulation (P  0.05 by  2 test). The regulatory cells are present in the circulation as early as 3 mo after transplantation and persist for a number of years, despite conventional immunosuppression. Furthermore, induction treatment with anti-IL-2R mAb did not prevent the development of these regulatory CD25 + cells. Data from two patients suggest that these cells may also play a role in preventing epitope shifting, implicated in the ongoing im- mune activation contributing to chronic rejection, and that loss of regulation in a given patient may precede an episode of rejection. The mechanisms by which tolerance is established toward self-antigens in healthy individuals or toward foreign alloanti- gens in transplant recipients remain incompletely understood. Experimental systems have demonstrated that there are a num- ber of non–mutually exclusive mechanisms acting on T lym- phocytes that can mediate such a state of immunologic unre- sponsiveness. These mechanisms include deletion of the pathogenic T cells (during immune development and also in the mature immune system), T cell anergy, and immune reg- ulation of effector T cells by soluble factors, such as cytokines, or by professional regulatory cells (1–3). Understanding the contribution that each of these mechanisms makes in human disease states is of great importance for the development of strategies to achieve clinical tolerance after its breakdown in autoimmune diseases or after transplantation. Recently, confirming early descriptions in rodent transplant models (4), several investigators have described the existence of a unique population of regulatory T cells, expressing both CD4 and CD25 (the IL-2 receptor  chain), which are hyporesponsive and have potent immune regulatory effects in vitro and in vivo (5–11). These cells can efficiently abrogate autoimmune disease and prevent transplant rejection in a number of different experimental animal models (4,7,12–17); more recently, their counterparts have been described in healthy human subjects (10,11,18 –21). Only a proportion of CD4 + CD25 + cells mediate immune regulation; to date, no unique cell markers have been discovered that reproduc- ibly identify these cells, although altered patterns of gene expres- sion may provide a clue (22). However, it appears that in humans at least, they constitute a small subset of CD4 + T cells, which express the highest levels of CD25 (CD4CD25 high cells) as well as other markers of previous T cell activation (23). The function of these cells in human disease or in clinical transplantation has not yet been elucidated. Transplant recipients have benefited from dramatic improve- ments in short-term graft survival; however, in the long-term, they eventually lose their grafts from chronic rejection, cur- rently the major cause of graft failure (24 –27). Chronic rejec- tion is a complex process that is believed to be largely immu- nologically mediated and related to ongoing graft injury initiated by alloreactive T cells, stimulated by processed al- loantigens shed from the graft (termed the indirect pathway of allorecognition) (25). Extensive animal studies indicate that indirect allorecognition is sufficient to affect acute skin allo- graft rejection (28 –30) and, in vascularized grafts, priming to the indirect pathway promotes development of chronic rejec- Received September 6, 2002. Accepted January 3, 2003. J. Harold Helderman served as Guest Editor and supervised the review and final disposition of this manuscript. Correspondence to Dr. Mohamed H. Sayegh, Laboratory of Immunogenetics and Transplantation, Brigham and Women’s Hospital, 75 Francis Street, Bos- ton 02115. Phone: 617-732-5259; Fax: 617 732 5254; E-mail: msayegh@rics. bwh.harvard.edu 1046-6673/1406-1643 Journal of the American Society of Nephrology Copyright © 2003 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000057540.98231.C1 J Am Soc Nephrol 14: 1643–1651, 2003