T-Regulatory Cells in Human Transplantation

The study of Bunnag et al. (1Bunnag S, Allanach K, Jhangri GS et al. FOXP3 expression in human kidney transplant biopsies is associated with rejection and time post-transplant but not with favourable outcomes. Am J Transplant 2008.Google Scholar) in this issue of American Journal of Transplantation is one of a small but growing number of studies investigating T-regulatory (T-reg) cells in clinical transplantation. The transcription factor forkhead box P3 (FOXP3) amplifies and stabilizes differentiation and function of T-reg cells (2Long E Wood KJ Understanding FOXP3: Progress towards achieving transplantation tolerance.Transplantation. 2007; 84: 459-461Crossref PubMed Scopus (41) Google Scholar), and sustained FOXP3 expression is required for lineage stability. Bunnag et al. (1Bunnag S, Allanach K, Jhangri GS et al. FOXP3 expression in human kidney transplant biopsies is associated with rejection and time post-transplant but not with favourable outcomes. Am J Transplant 2008.Google Scholar) have appropriately chosen this has a relatively specific marker for T-reg cells. Earlier studies of T-reg not including FOXP3 positivity as a criterion were likely too inclusive. However, though preferentially expressed in CD4+CD25+ T cells associated with suppressive function, most human T cells transiently express FOXP3 during activation (3Mantel PY Ouaked N Ruckert B et al.Molecular mechanisms underlying FOXP3 induction in human T cells.J Immunol. 2006; 176: 3593-3602Crossref PubMed Scopus (346) Google Scholar). Thus, detection of FOXP3 in inflamed allografts is not necessarily a specific marker for T-reg, and though correlated with inflammation/rejection, can be found in control kidneys, as noted in the study by Bunnag et al. (1Bunnag S, Allanach K, Jhangri GS et al. FOXP3 expression in human kidney transplant biopsies is associated with rejection and time post-transplant but not with favourable outcomes. Am J Transplant 2008.Google Scholar). Early clinical observations of T-reg in human transplantation include reports of positive correlation between graft survival and circulating T-reg cells/regulatory function (not using FOXP3 positivity as a criterion) in lung (4Meloni F Vitulo P Bianco AM et al.Regulatory CD4+CD25+ T cells in the peripheral blood of lung transplant recipients; correlation with transplant outcome.Tranplantation. 2004; 15: 762-766Crossref Scopus (120) Google Scholar), liver (5Demirkiran A Kok A Kwekkeboom J et al.Low circulating regulatory T-cell levels after acute rejection in liver transplantation.Liver Transpl. 2006; 12: 277-284Crossref PubMed Scopus (129) Google Scholar) and kidney (6Salama AD Najafian N Clarkson MR Harmon WE Sayegh MH Regulatory CD25+ T cells in human kidney transplant recipients.J Am Soc Nephrol. 2003; 14: 1643-1651Crossref PubMed Scopus (205) Google Scholar) allograft patients. CD4+ T-reg have also been reported in patients who developed spontaneous tolerance to liver or kidney allografts (7Van Buskirk AM Burlingham WJ Jankowska-Gan E et al.Human allograft acceptance is associated with immune regulation.J Clin Invest. 2000; 106: 145-155Crossref PubMed Scopus (229) Google Scholar) and in peripheral blood of human liver transplant recipients weaned from immunosuppression (8Yoshizawa A Ito A Li Y et al.The roles of CD25+CD4+ regulatory T cells in operational tolerance after living donor liver transplantation.Transpl Proc. 2005; 37: 37-39Crossref PubMed Scopus (75) Google Scholar). Of course, circulating cells may not reflect intragraft events, and examination of tissue is critical for advancing understanding of T-reg function in allografts. FOXP3 mRNA expression is positively correlated with rejection in human cardiac allograft biopsies, though not in the peripheral blood (9Dijke IE Caliskan K Korevaar SS et al.FOXP3 mRNA expression analysis on the peripheral blood and allograft of heart transplant patients.Transpl Immunol. 2008; 18: 250-254Crossref PubMed Scopus (34) Google Scholar). There are several recent studies of T-reg in renal transplantation (10Grimbert P Mansour H Desvaux D et al.The regulatory/cytotoxic graft-infiltrating T cells differentiate renal allograft borderline change from acute rejection.Transplantation. 2007; 83: 341-346Crossref PubMed Scopus (50) Google Scholar, 11Muthukumar T Dadhania D Ding R et al.Messenger RNA for FOXP3 in the urine of renal-allograft recipients.N Engl J Med. 2005; 353: 2342-2351Crossref PubMed Scopus (491) Google Scholar, 12Veronese F Rotman S Smith RN et al.Pathological and clinical correlates of FOXP3+ cells in renal allografts during acute rejection.Am J Transplant. 2007; 7: 914-922Crossref PubMed Scopus (152) Google Scholar, reviewed by Bunnag et al. (1Bunnag S, Allanach K, Jhangri GS et al. FOXP3 expression in human kidney transplant biopsies is associated with rejection and time post-transplant but not with favourable outcomes. Am J Transplant 2008.Google Scholar)). The study of Bunnag et al. (1Bunnag S, Allanach K, Jhangri GS et al. FOXP3 expression in human kidney transplant biopsies is associated with rejection and time post-transplant but not with favourable outcomes. Am J Transplant 2008.Google Scholar) adds to these early observations in clinical transplantation. FOXP3 mRNA in biopsy tissue was independently associated with rejection, younger donor age and longer time posttransplant, but not with measures of graft outcome, perhaps because of the context of rejection. (The authors cite the recent study of protocol biopsies without rejection in which higher numbers of FOXP3+CD4+CD25+ cells were associated with donor-specific hyporesponsiveness, less chronic change and better renal function (13Bestard O Cruzado JM Mestre M et al.Achieving donor-specific hyporesponsiveness is associated with FOXP3+ regulatory T cell recruitment in human renal allograft infiltrates.J Immunol. 2007; 179: 4901-4909Crossref PubMed Scopus (141) Google Scholar)). In this study, there was no significant correlation between FOXP3 mRNA and immunohistochemical detection of FOXP3+ cells. This could be due to sampling error, always an issue in biopsy tissue, since two different cores were used for these two detection methods. However, it could also reflect a discrepancy between cells expressing FOXP3 message and those with protein expression, and raises the issue of which are the ‘true’ regulatory cells. The literature to this point includes studies generally using one or the other of these detection methods. Studies like this one, incorporating both, may provide additional insights not provided by either modality alone. As noted by Allegre et al. (14Allegre M-L Florquin S Goldman M Cellular mechanisms underlying acute graft rejection: Time for reassessment.Curr Opin Immunol. 2007; 19: 563-568Crossref PubMed Scopus (58) Google Scholar), the redundancy and complexity of mechanisms of rejection underlies the difficulty in identifying biomarkers for prediction of graft outcome, and the induction of tolerance. Also, potential local control mechanisms within the graft include not only several population of lymphocytes (15Shevach EM From vanilla to 28 flavors: Multiple varieties of T regulatory cells.Immunity. 2006; 25: 195-201Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), but also mast cells (16Lu LF Lind EF Gondek DC et al.Mast cells are essential intermediaries in regulatory T-cell tolerance.Nature. 2006; 442: 997-1002Crossref PubMed Scopus (643) Google Scholar). Given this complexity, it is not surprising that no definite correlate of FOXP3 expression with short-term outcome could be defined in the study of Bunnag et al. (1Bunnag S, Allanach K, Jhangri GS et al. FOXP3 expression in human kidney transplant biopsies is associated with rejection and time post-transplant but not with favourable outcomes. Am J Transplant 2008.Google Scholar). On longer-term follow-up, nonalloimmune and/or noninflamamtory processes may well supervene to influence outcome, making associations even more obsure. In addition, a variety of individual factors, including sex, age and a range of gene polymorphisms potentially affect ability to maintains FOXP3 expression and regulatory phenotype in humans (2Long E Wood KJ Understanding FOXP3: Progress towards achieving transplantation tolerance.Transplantation. 2007; 84: 459-461Crossref PubMed Scopus (41) Google Scholar). That said, clinical studies seeking associations and correlations must continue, to advance our currently rudimentary understanding of suppressor mechanisms in the clinical setting. Positive association of FOXP3 mRNA with time posttransplant in the study by Bunnag et al. (1Bunnag S, Allanach K, Jhangri GS et al. FOXP3 expression in human kidney transplant biopsies is associated with rejection and time post-transplant but not with favourable outcomes. Am J Transplant 2008.Google Scholar) is very interesting. Future clinical studies of FOXP3 expression must take the variable of time posttransplant into account, perhaps as a dichotomous rather than continuous variable, as suggested by the authors. Bunnag et al. (1Bunnag S, Allanach K, Jhangri GS et al. FOXP3 expression in human kidney transplant biopsies is associated with rejection and time post-transplant but not with favourable outcomes. Am J Transplant 2008.Google Scholar) interpret this association as a reflection of time-dependent entry of regulatory T cells into sites of chronic inflammation; alternatively, FOXP+ cells could differentiate in situ over time from precursor cells. Of course, this is speculative, as the ‘chronicity’ of the inflammation cannot be established by single biopsy samples with a significant component of activated effector infiltrate. Other primary variables might explain more FOXP3 expression in later allograft biopsies, including less immunosuppression over time. Considerable evidence suggests that endogenous immune regulatory processes including tolerance involve immune cells potentially affected by immunosuppression. T-cell receptor-mediated expression of FOXP3 is blocked by cyclosporine A but not steroids or rapamycin (3Mantel PY Ouaked N Ruckert B et al.Molecular mechanisms underlying FOXP3 induction in human T cells.J Immunol. 2006; 176: 3593-3602Crossref PubMed Scopus (346) Google Scholar), and CNI but not rapamycin reduce the percentage of FOXP3+CD4+CD25+ cells in recipients of renal allografts (17Segundo DS Ruiz JC Izquierdo M et al.Calcineurin inhibitors, but not rapamycin, reduce percentage of CD4+CD25+FOXP3+ regulatory T cells in renal transplant recipients.Transplantation. 2006; 82: 550-557Crossref PubMed Scopus (210) Google Scholar). Anti-T-cell antibody therapy may also deplete T-reg in humans (18Pearl JP Parris J Hale DA et al.Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following antibody-mediated T cell depletion.Am J Transplant. 2005; 5: 465-474Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). On the other hand, rapamycin has been shown to promote expansion of human T-reg in vitro (19Battaglia M Stabilini A Migliavacca B Horejs-Hoeck J Kaupper T Roncarolo MG Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients.J Immunol. 2006; 177: 8338-8347Crossref PubMed Scopus (595) Google Scholar). Effects of immunosuppression on regulatory cells are of concern for both naturally occurring and potentially passively transferred regulatory cells administered therapeutically as well. Transduction strategies or allo-antigen stimulation in the presence of Treg-inducing conditions may ultimately provide a large enough pool of allo-specific regulatory cells to provide a therapeutic tool in clinical transplantation (20Kang SM Tang Q Bluestone JA CD4+CD25+ regulatory T cells in transplantation: Progress, challenges and prospects.Am J Transplant. 2007; 7: 1457-1463Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 21Akl A Luo S Wood KJ Induction of transplantation tolerance—the potential of regulatory T cells.Transpl Immunol. 2005; 14: 225-230Crossref PubMed Scopus (55) Google Scholar, 22Roncarolo M-G Battaglia M Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans.Nature Rev Immunol. 2007; 7: 585-598Crossref PubMed Scopus (458) Google Scholar). Ongoing clinical trials using adaptive transfer of T-reg cells in bone marrow transplantation should advance optimization of these strategies. However, translation of experimental studies into the clinical arena will likely not be straightforward (20Kang SM Tang Q Bluestone JA CD4+CD25+ regulatory T cells in transplantation: Progress, challenges and prospects.Am J Transplant. 2007; 7: 1457-1463Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar,22Roncarolo M-G Battaglia M Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans.Nature Rev Immunol. 2007; 7: 585-598Crossref PubMed Scopus (458) Google Scholar). The survey study by Bunnag et al. (1Bunnag S, Allanach K, Jhangri GS et al. FOXP3 expression in human kidney transplant biopsies is associated with rejection and time post-transplant but not with favourable outcomes. Am J Transplant 2008.Google Scholar) investigating FOXP3 expression in clinical transplantation is part of the necessary groundwork in what will be a long and fraught process toward the goal of harnessing these innate regulatory mechanisms.