Regulation of immune reactions is critical in health and disease. Nowhere is this clearer than following allogeneic hematopoietic cell transplantation (HCT). Here the beneficial effects of graft-versus-tumor reactions are clearly demonstrated, as well as the detrimental morbidity and mortality of uncontrolled graft-versus-host disease (GVHD). Control of immune reactions is complex and involves many factors including blood flow, micro-environmental interactions, cytokines, chemokines and both pro-inflammatory and anti-inflammatory molecules. In addition it has become well recognized that specialized populations of cells, termed regulatory T cells, are critically involved in controlling immune reactions. Although the field of regulatory T cell biology has been explored for many years, the more recent identification of specific populations of cells capable of regulating immune responses with defined phenotype has opened our re-examination of the potential impact of these cellular populations on immune function. To date, a number of different cell populations have been characterized with defined ability to regulate immune reactions, which include two major cell populations, namely those of CD4+CD25+ regulatory T cells (Treg) [1Shevach E.M. CD4+ CD25+ suppressor T cells: more questions than answers.Nat Rev Immunol. 2002; 2: 389-400Crossref PubMed Scopus (1913) Google Scholar] and abTCR+ NK-T cells which express an invariant T cell receptor and are CD1 reactive [2Bendelac A. Lantz O. Quimby M.E. Yewdell J.W. Bennink J.R. Brutkiewicz R.R. CD1 recognition by mouse NK1+ T lymphocytes.Science. 1995; 268: 863-865Crossref PubMed Scopus (841) Google Scholar]. These cellular populations have generated enormous interest in the field of HCT since animal models have demonstrated the potential benefit of regulatory T cell biology. Numerous studies have demonstrated the potential benefit of regulatory T cells in the setting of allogeneic HCT [3Hoffmann R. Ermann J. Edinger M. Fathman C.G. Strober S. Donor type CD4+CD25+ regulatory T cells suppress lethal acute graft-versus-hosti disease after allogeneic bone marrow transplantation.Journal of Experimental Medicine. 2002; 196: 389-399Crossref PubMed Scopus (909) Google Scholar, 4Cohen J.L. Trenado A. Vasey D. Klatzmann D. Salomon B.L. CD4(+)CD25(+) immunoregulatory T Cells: new therapeutics for graft-versus-host disease.J Exp Med. 2002; 196: 401-406Crossref PubMed Scopus (601) Google Scholar, 5Taylor P.A. Lees C.J. Blazar B.R. The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality.Blood. 2002; 99: 3493-3499Crossref PubMed Scopus (898) Google Scholar]. These studies are best demonstrated by the adoptive transfer of defined numbers of Treg in the inoculum of cells administered in the setting of allogeneic HCT. Several groups have demonstrated that a 1:1 ratio of conventional CD4 and CD8 cells (Tcon) with Treg results in control of GVHD. This has been demonstrated across multiple mouse strains demonstrating that Treg are capable of controlling GVHD. Several studies have also demonstrated that in these experimental settings, that adjusting the ratio of Treg:Tcon results in maintenance of GVT responses [6Edinger M. Hoffmann P. Ermann J. Drago K. Fathman C.G. Strober S. Negrin R.S. CD4(+)CD25(+) regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation.Nat Med. 2003; 9: 1144-1150Crossref PubMed Scopus (1053) Google Scholar, 7Trenado A. Charlotte F. Fisson S. Yagello M. Klatzmann D. Salomon B.L. Cohen J.L. Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia.J Clin Invest. 2003; 112: 1688-1696Crossref PubMed Scopus (402) Google Scholar, 8Jones S.C. Murphy G.F. Korngold R. Post-hematopoietic cell transplantation control of graft-versus-host disease by donor CD425 T cells to allow an effective graft-versus-leukemia response.Biol Blood Marrow Transplant. 2003; 9: 243-256Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar]. The proposed mechanism of this observation is that the Treg control the alloreactive expansion of Tcon but not their activation. The massive expansion of Tcon is required for the disease process of GVHD whereas activation is more required for GVT. Therefore, by controlling the proliferation of alloreactive T cells, the clinical sequelae of GVHD can be controlled. Additional studies have demonstrated that Treg likely perform these functions in specific microenvironments since the specific lymphoid homing molecule CD62L is critically important in Treg function [9Ermann J. Hoffmann P. Edinger M. Dutt S. Blankenberg F.G. Higgins J.P. Negrin R.S. Fathman C.G. Strober S. Only the CD62L+ subpopulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD.Blood. 2005; 105 (Epub 2004 Nov 2216): 2220-2226Crossref PubMed Scopus (344) Google Scholar, 10Taylor P.A. Panoskaltsis-Mortari A. Swedin J.M. Lucas P.J. Gress R.E. Levine B.L. June C.H. Serody J.S. Blazar B.R. L-Selectin(hi) but not the L-selectin(lo) CD4+25+ T-regulatory cells are potent inhibitors of GVHD and BM graft rejection.Blood. 2004; 104: 3804-3812Crossref PubMed Scopus (295) Google Scholar]. Interestingly, both CD62L+ and CD62L− Treg are capable of controlling the mixed lymphocyte reaction in vitro. These studies demonstrate the importance of in vivo studies to assess Treg function which are quite accessible in murine models, however, extremely difficult in the clinic. The only in vitro functional assay which has been established for Treg is their ability to suppress the mixed lymphocyte reaction (MLR) which is a useful but somewhat limited assay of Treg function. T cell invariant NK-T cells upon activation through CD1 [11Kronenberg M. Toward an understanding of NKT cell biology: progress and paradoxes.Annu Rev Immunol. 2005; 23: 877-900Crossref PubMed Scopus (874) Google Scholar] produce high levels of IL-4 which has been demonstrated to be critically important in their biological function of controlling immune reactions [2Bendelac A. Lantz O. Quimby M.E. Yewdell J.W. Bennink J.R. Brutkiewicz R.R. CD1 recognition by mouse NK1+ T lymphocytes.Science. 1995; 268: 863-865Crossref PubMed Scopus (841) Google Scholar]. In this setting, the ratio of regulatory to invariant TCR NK-T cells has resulted in altering the environment such that these animals are markedly resistant to developing GVHD [12Lan F. Zeng D. Higuchi M. Higgins J.P. Strober S. Host conditioning with total lymphoid irradiation and antithymocyte globulin prevents graft-versus-host disease: the role of CD1-reactive natural killer T cells.Biol Blood Marrow Transplant. 2003; 9: 355-363Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar]. These strategies have been well established in murine models and reproduced by a number of different laboratories. The clinical translation of an approach to alter the ratio of Tcon to NK-T cells developed in murine models has resulted in a markedly low risk of acute GVHD [13Lowsky R. Takahashi T. Liu Y.P. Dejbakhsh-Jones S. Grumet F.C. Shizuru J.A. Laport G.G. Stockerl-Goldstein K.E. Johnston L.J. Hoppe R.T. Bloch D.A. Blume K.G. Negrin R.S. Strober S. Protective conditioning for acute graft-versus-host disease.N Engl J Med. 2005; 353: 1321-1331Crossref PubMed Scopus (282) Google Scholar]. For the remainder of this review we will focus on Treg phenotype and function. Regulatory T cells have been characterized by the cell surface phenotype of CD4+CD25+, however, to date there is no unique repertoire of cell surface molecules which unambiguously defines regulatory T cells. Of the CD4+CD25+ Treg, the CD25 high or bright population is clearly the population with most Treg function. Identification of the expression of the Foxp3 transcription factor which is tightly correlated with Treg function has been critically important in defining those populations of cells which are biologically active [1Shevach E.M. CD4+ CD25+ suppressor T cells: more questions than answers.Nat Rev Immunol. 2002; 2: 389-400Crossref PubMed Scopus (1913) Google Scholar, 14Fontenot J.D. Gavin M.A. Rudensky A.Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.Nat Immunol. 2003; 4: 330-336Crossref PubMed Scopus (6053) Google Scholar, 15Hori S. Nomura T. Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3.Science. 2003; 299 (Epub 2003 Jan 1059): 1057-1061Crossref PubMed Scopus (50) Google Scholar]. In numerous studies, the CD4+CD25+ high cells express Foxp3, whereas the CD4+CD25+ intermediate cells are a mixed population of CD4+CD25− cells and Foxp3 expressing cells (Figure 1). Therefore, expression of Foxp3 can also be used to identify Treg in biological samples. However, because this molecule is an intracellular transcription factor, it is not possible to use this marker for cell sorting functions. A number of studies have demonstrated that both murine and human cells express Foxp3 and there is on-going debate as to whether Foxp3 defines all the Treg+ populations or is merely a marker for a predominant Treg population [16Roncador G. Brown P.J. Maestre L. Hue S. Martinez-Torrecuadrada J.L. Ling K.L. Pratap S. Toms C. Fox B.C. Cerundolo V. Powrie F. Banham A.H. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level.Eur J Immunol. 2005; 35: 1681-1691Crossref PubMed Scopus (507) Google Scholar, 17Morgan M.E. van Bilsen J.H. Bakker A.M. Heemskerk B. Schilham M.W. Hartgers F.C. Elferink B.G. van der Zanden L. de Vries R.R. Huizinga T.W. Ottenhoff T.H. Toes R.E. Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans.Hum Immunol. 2005; 66: 13-20Crossref PubMed Scopus (360) Google Scholar]. In addition, recent studies have indicated that CD127− (IL-7 receptor) better defines the CD4+CD25+ Treg [18Liu W. Putnam A.L. Xu-Yu Z. Szot G.L. Lee M.R. Zhu S. Gottlieb P.A. Kapranov P. Gingeras T.R. de St Groth B.F. Clayberger C. Soper D.M. Ziegler S.F. Bluestone J.A. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells.J Exp Med. 2006; 203: 1701-1711Crossref PubMed Scopus (2068) Google Scholar, 19Seddiki N. Santner-Nanan B. Martinson J. Zaunders J. Sasson S. Landay A. Solomon M. Selby W. Alexander S.I. Nanan R. Kelleher A. Fazekas de St Groth B. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells.J Exp Med. 2006; 203: 1693-1700Crossref PubMed Scopus (1232) Google Scholar]. Therefore, cells with the phenotype of CD4+CD25+ IL-7 receptor negative defines the majority of cells in the population which express Foxp3 and have Treg function. These phenotypic markers are critically important to isolate cells to utilize in clinical studies. This population is rare accounting for ∼5-10% of the CD4+ cells and 1-4% of all T cells in the peripheral blood. The ability to isolate Treg is dependent upon a variety of different techniques. Since these cells are a relatively rare cell population, it is difficult to use cell sorting as the exclusive technique. A variety of groups have used magnetic bead separation for isolation of CD25+ cells. This results in significant enrichment of the CD4+CD25+ Treg cells, however, in these preparations, they remain only ∼50% Foxp3+. However, for other applications where a purified population of Treg is required, additional approaches may be necessary. One approach is to sort the CD25 high positive population, however, this has been associated with significant loss of Foxp3+ cells. As discussed above, the use of the CD127 or IL-7 receptor appears to be an improved approach whereby cells that are IL-7 receptor negative but CD4+CD25+ can be isolated by cell sorting techniques. In our hands, this represents the majority of Foxp3 expressing regulatory T cells and also results in isolating the majority of Foxp3+ cells within an apheresis sample. A number of groups have demonstrated that T cells, including Treg, can be expanded by ex vivo culture techniques [20Levings M. Sangregorio R. Roncarolo M. Human CD25+CD4+ T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function.Journal of Experimental Medicine. 2001; 193: 1295-1301Crossref PubMed Scopus (858) Google Scholar, 21Hoffmann P. Eder R. Kunz-Schughart L.A. Andreesen R. Edinger M. Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells.Blood. 2004; 104 (Epub 2004 Apr 2015): 895-903Crossref PubMed Scopus (425) Google Scholar]. Several different approaches have been utilized involving stimulation with anti-CD3 to activate T cell receptor and co-stimulation through CD28. TGFβ may also play an important role and IL-2 is absolutely required for Treg expansion [5Taylor P.A. Lees C.J. Blazar B.R. The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality.Blood. 2002; 99: 3493-3499Crossref PubMed Scopus (898) Google Scholar]. Other groups have used dendritic cell activation in these cultures to expand the Treg population. In some studies, expansion of up to 1,000-fold can be achieved over several weeks [21Hoffmann P. Eder R. Kunz-Schughart L.A. Andreesen R. Edinger M. Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells.Blood. 2004; 104 (Epub 2004 Apr 2015): 895-903Crossref PubMed Scopus (425) Google Scholar]. Of specific concern is that conventional T cells also rapidly expand under these culture conditions such that if the input cells are not highly purified for Treg that these cultures may be overgrown by conventional CD4 and CD8 cells using CD3xCD28 beads and IL-2. Several approaches can be performed to overcome this limitation. These include the use of highly purified input cells with CD127−CD4+CD25+ cells, by the addition of rapamycin which appears to control the expansion of conventional T cells preferentially over regulatory T cells [22Battaglia M. Stabilini A. Roncarolo M.G. Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells.Blood. 2005; 105: 4743-4748Crossref PubMed Scopus (945) Google Scholar] or through the isolation of CD45RA+ cells prior to expansion [23Hoffmann P. Eder R. Boeld T.J. Doser K. Piseshka B. Andreesen R. Edinger M. Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T cell lines upon in vitro expansion.Blood. 2006; Google Scholar]. A number of studies have been performed by measuring Treg in GVHD development by characterizing the content of Treg in the peripheral blood of patients who have undergone allogeneic HCT. These studies have resulted in mixed results in that early studies somewhat paradoxically reported a higher frequency of CD25 high positive T cells were associated with a higher incidence of acute and chronic GVHD [24Stanzani M. Martins S.L. Saliba R.M. St John L.S. Bryan S. Couriel D. McMannis J. Champlin R.E. Molldrem J.J. Komanduri K.V. CD25 expression on donor CD4+ or CD8+ T cells is associated with an increased risk for graft-versus-host disease after HLA-identical stem cell transplantation in humans.Blood. 2004; 103: 1140-1146Crossref PubMed Scopus (82) Google Scholar]. In addition, a second study showed that CD25bright T cell content in the peripheral blood of patients >100 days following transplantation was associated with a higher risk of graft-versus-host disease. However, it is important to note that upon activation conventional CD4+ cells also express CD25 so it is difficult, if not impossible, to differentiate between activated T cells and regulatory T cells. More recent retrospective analyses have incorporated Foxp3 expression along with cell phenotype. Several studies have shown a decrease in Foxp3 levels in the peripheral blood of patients with both acute and chronic GVHD [25Miura Y. Thoburn C.J. Bright E.C. Phelps M.L. Shin T. Matsui E.C. Matsui W.H. Arai S. Fuchs E.J. Vogelsang G.B. Jones R.J. Hess A.D. Association of Foxp3 regulatory gene expression with graft-versus-host disease.Blood. 2004; 104: 2187-2193Crossref PubMed Scopus (268) Google Scholar]. In one study the level of Foxp3 expression assessed by quantitative PCR correlated inversely with the incidence of chronic GVHD and increased as GVHD resolved [26Zorn E. Kim H.T. Lee S.J. Floyd B.H. Litsa D. Arumugarajah S. Bellucci R. Alyea E.P. Antin J.H. Soiffer R.J. Ritz J. Reduced frequency of FOXP3+ CD4+CD25+ regulatory T cells in patients with chronic graft-versus-host disease.Blood. 2005; 106: 2903-2911Crossref PubMed Scopus (384) Google Scholar]. In addition, a recent study demonstrated that the Treg content of the graft correlated with acute GVHD such that those patients with higher numbers of Treg had lower risk of acute GVHD as analyzed by phenotypic analysis and Foxp3 expression [27Rezvani K. Mielke S. Ahmadzadeh M. Kilical Y. Savani B.N. Zeilah J. Keyvanfar K. Montero A. Hensel N. Kurlander R. Barrett A.J. High donor FOXP3-positive regulatory T-cell (Treg) content is associated with a low risk of GVHD following HLA-matched allogeneic SCT.Blood. 2006; 108: 1291-1297Crossref PubMed Scopus (312) Google Scholar]. In summary, although there are somewhat mixed results in correlating the numbers of Foxp3 expressing cells, several studies have been supportive of the concept that higher numbers of Treg may correlate with lower GVHD risk although additional larger cohorts of patients are likely necessary for definitive conclusions. It should also be noted that in almost all of the murine studies Treg function is in tissues, either in nodal sites or in the peripheral tissues, yet in humans there is little access to these tissues for analysis. Therefore, the analysis of peripheral blood has some potential flaws and caveats which are difficult to control for in clearly articulating the role of Treg in human GVHD pathophysiology. Due to the profound effects of Treg on GVHD pathophysiology in murine models, their potential role in the clinic seems obvious. A major limitation to developing Treg is the relative paucity of these cell populations in the peripheral blood or bone marrow samples of donors or patients undergoing HCT. Clearly technical advances are required to isolate sufficient numbers of Treg which, as discussed above, can be isolated by magnetic bead selection and/or cell sorting techniques. In addition expansion of Treg may be necessary in some clinical situations to obtain sufficient numbers of cells. Nonetheless, a number of groups are exploring the clinical application of Treg in the clinic. One approach has been to use magnetic bead separation to enrich for Treg such that the ratio is roughly 1:1 with conventional CD4+CD25− cells. This approach is under exploration by Edinger, et al. in a safety trial with early demonstration that this is technically feasible and is well tolerated. Additional studies are proposed, for example in the haplo-identical setting, where adjusting the mixture of Treg with Tcon may result in control of GVHD and possibly improve GVL effects, as well as enhance immune reconstitution. One concern is that there is emerging evidence that different immunosuppressive medications may have profoundly different effects on Treg function [28Zeiser R. Nguyen V.H. Beilhack A. Buess M. Schulz S. Baker J. Contag C.H. Negrin R.S. Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent interleukin-2 production.Blood. 2006; 108: 390-399Crossref PubMed Scopus (410) Google Scholar]. Because of these issues it is important that different clinical approaches be explored by several different groups in an effort to translate the findings of Treg biology into the clinic. In summary, the explosion of information on the function of regulatory T cells and biological similarities between human and murine cells has made clinical application attractive. In allogeneic HCT, the goal will be to determine whether Treg are capable of controlling GVHD reactions while allowing for maintenance of GVT. The clinical translation is challenging due to the relative paucity of these cells. We look forward to future clinical trials exploring this exciting population of regulatory T cells in the transplant setting.