Regulatory T cells in HIV infection: pathogenic or protective participants in the immune response?

Introduction Suppressor or regulatory T cells (Tregs) have enjoyed a checkered history since the first report of in-vivo suppressor T-cell activity in 1971 [1]. Widespread acceptance of Tregs as a bona fide T-cell subset came only with the identification of a surface marker, CD25 (the alpha chain of the IL-2 receptor), that was constitutively expressed by CD4 T cells with suppressive ability in vivo[2,3]. The demonstration that purified CD4+CD25+ T cells could suppress the proliferation of CD4+CD25− T cells in vitro provided a crucial confirmation of the link between phenotype and function [4]. Importantly, the in-vitro suppression assay could also be applied in human disease [5]. The past decade has seen an explosion of research interest in Tregs, as the tools of modern cellular and molecular immunology have been applied to understanding immune regulation in mouse and humans [6–10]. In particular, the discovery that the transcription factor Foxp3 is highly expressed by Tregs in both species, and is required for their development and function [11–14], has opened the way for elegant studies of Treg biology in mouse models [15] and for more reliable identification of Tregs in the human [16]. This review will briefly describe the current state of understanding of normal Treg biology, most of which is still based on murine models, and will then detail how Tregs are affected by HIV infection, and how they may influence the course of HIV disease. Identification of regulatory T cells Murine studies As mentioned above, the expression of CD4 and CD25 was initially used to define Tregs in the mouse [3]. As activated CD4 T cells also express CD25, purified CD4+CD25+ Tregs were likely to be contaminated with an unknown proportion of activated conventional T cells. Such contamination did not present a major problem in animals raised in pathogen-free conditions, as the number of conventional CD4 T cells expressing CD25 was usually small compared with the number of Tregs. Murine studies have continued to use CD25 as the principal marker for the isolation of viable Tregs, whereas FoxP3 expression is more commonly regarded as the most definitive marker after cell fixation (Table 1).Table 1: Regulatory T cell phenotype in mouse and human.Human studies In contrast to the mouse, human circulating effector/memory CD4 T cells express CD25 at a level only slightly lower than that of Tregs [17], and this finding has presented a major obstacle to the accurate identification of human Tregs. To avoid massive contamination of purified human Tregs with memory cells, investigators initially chose to study the relatively pure minority Treg subpopulation expressing the highest level of CD25 [5]. Human Tregs have also been identified by the expression of a number of molecules that, although not exclusive to the Treg subset, are either more highly or more uniformly expressed when compared with conventional CD4 T cells (Table 1). These molecules include members of two families of co-stimulatory molecules: the CD28 superfamily, represented by cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and inducible co-stimulator protein, and the tumor necrosis factor receptor superfamily, represented by CD27, OX40, 4-1BB and glucocorticoid-induced tumor necrosis factor receptor (GITR) family-related protein. In addition, human Tregs have been subdivided according to their activation state. Most Tregs in neonates are CD45RA-positive, whereas the majority in adults express CD45RO. The presence of a discrete population of CD45RA-positive Tregs in adults has also recently been described by several groups [17–19]. These naive Tregs probably comprise that part of the peripheral Treg repertoire that has not received sufficient T-cell receptor stimulation to convert to the expression of CD45RO. In general, CD45RA-positive Tregs express CD25 and co-stimulatory molecules at lower levels than CD45RO-positive Tregs, indicative of a lower ‘activation’ status [17]. Two recent technical advances have improved the identification and isolation of the majority of human Tregs that do not express the highest levels of CD25. The generation of anti-FoxP3 monoclonal antibodies allowed Tregs to be identified after fixation and permeabilization [16], whereas the surface CD127loCD25+ phenotype was shown to be highly correlated with intracellular Foxp3 expression, providing a means of purifying viable human Tregs [20,21]. It should be noted, however, that FoxP3 expression is not always indicative of regulatory status within human CD4 T cells [17,40–44]. Mechanism of regulatory T cell function Murine studies Most of our knowledge of in-vivo Treg function comes from murine models. Tregs are involved in virtually every stage of the adaptive immune response. They thus not only prevent the activation of self-reactive cells, but downregulate responses to foreign antigen at the expansion and effector stages (Table 2). Blocking Treg function in vivo has been shown to stimulate rejection of otherwise tolerated tumors [45] and to allow the clearance of chronic infections under some experimental conditions [46]. Despite their potent in-vivo activity, the mechanism of murine Treg suppressive activity remains unclear, with evidence both for and against the involvement of transforming growth factor beta (TGF-β), IL-10, CTLA-4, target cell killing and the induction of indoleamine 2,3-dioxygenase (IDO) production by dendritic cells and macrophages [25,26,47–52]. IDO is an enzyme that degrades tryptophan to kynurenine. IDO activity not only deprives T cells of an essential amino acid, but drives the production of immunosuppressive tryptophan metabolites [53].Table 2: Potential effects of regulatory T cells on immune responses and on HIV infection in particular.Suppression of CD25− responder T-cell proliferation to an artificial polyclonal stimulus such as anti-CD3 monoclonal antibody has been widely adopted as the definitive assay for murine Treg function. In these cell co-culture assays, the secretion of TGF-β and IL-10 by Tregs can usually be detected. In-vitro suppression is not, however, dependent on soluble factors, but requires physical contact between Tregs and responder T cells, involving an as yet undefined molecular mechanism [54]. On the basis of these findings, it was surprising that murine Tregs mediating in-vivo regulatory function were recently shown not to contact responder T cells [55]. To add to concerns about the validity of the in-vitro suppression assay, a number of recent reports have detected normal in-vitro suppressive function mediated by Tregs derived from gene knockout mice with major deficits in self-tolerance and in-vivo regulatory function [56,57]. In-vitro interactions between murine Tregs and dendritic cells have been reported to reduce dendritic cell expression of co-stimulatory molecules, which could explain how they downregulate multiple aspects of immune activation and differentiation [58–60]. Whether this is achieved by means of soluble mediators or cell–cell interaction remains an open question. Treg–dendritic cell interaction mediated via CTLA-4 ligation of CD80/86 has been shown to induce IDO production by dendritic cells [52]. This mechanism accounts at most for only part of the spectrum of Treg functions, because IDO knockout mice do not manifest an autoimmune phenotype [61], in contrast to FoxP3 knockout mice with no Tregs [62]. Human studies Human studies of Treg function have of necessity been limited to in-vitro testing. Human Tregs can suppress polyclonal stimulation of conventional T cells (usually CD25− cells stimulated with anti-CD3 with or without anti-CD28 or allogeneic non-T cells as antigen-presenting cells). Like murine Tregs, human Tregs make soluble immunosuppressive mediators such as TGF-β and IL-10, but require cell–cell contact with target cells to function in vitro. Once again, the molecular mechanism underlying suppression is still highly controversial. In the early studies of Baecher-Allen et al.[5] reliable suppressive activity was limited to the CD25high population. With the removal of contaminating CD25intCD45RO+ conventional T cells, we have now shown that CD25int Tregs have equally potent suppressive activity [20], as have CD45RA-positive naive Tregs [17]. It seems counterintuitive that naive and effector memory Tregs should have the same functional activity, given the clear in-vitro differences between conventional naive and effector memory cells. Furthering our understanding of how Tregs function at a cellular and molecular level is clearly one of the major challenges currently facing the field. Selection and specificity of regulatory T cells Murine studies Elegant genetic studies using green fluorescent protein to mark Foxp3+ Tregs in vivo have shown that Tregs arise in the thymus after selection for intermediate affinity recognition of self-antigen, which drives the expression of CD25 and then Foxp3 [15]. Tregs can also be generated de novo from naive peripheral CD25−CD4+ T cells after prolonged low avidity stimulation [63] or in skin graft recipients treated with non-depleting anti-CD4 antibodies [64], but the ongoing contributions of these peripheral pathways to the adult repertoire of Tregs remain unknown. In vitro, TGF-β can drive the differentiation of naive murine CD25−CD4+ T cells to a Foxp3+ suppressive phenotype [65] and recent in-vitro and in-vivo evidence supports a role for a combination of TGF-β and all-trans retinoic acid, a metabolite of vitamin A, in de novo Treg differentiation, particularly in the gut [66–69]. The antiself specificity of peripheral Tregs has not been formally demonstrated in normal mice, because of the technical difficulty inherent in detecting these intermediate affinity T-cell receptor interactions. Foxp3+ Tregs with defined specificity for foreign antigen have, however, been isolated from peripheral lymphoid and non-lymphoid tissues. In particular, murine Tregs specific for Leishmania major[46,70], and allo-major histocompatibility complex antigens [71] have been well characterized. These cells could be derived from self-reactive thymic Tregs that crossreact against foreign antigen, or from peripheral Foxp3− cells. It was recently demonstrated that at least some of the Leishmania-specific FoxP3+Tregs are the descendants of peripheral CD25+CD4+ T cells [70], but whether they first expressed FoxP3 in the thymus or in the periphery remains unknown. Human studies FoxP3+ T cells are present in human thymus and cord blood [17], suggesting a similar differentiation and selection mechanism to that in the mouse. Whereas FoxP3 expression appears to lag behind the expression of CD25 in the mouse, approximately half the FoxP3+ cells in the human infant thymus are CD25−[17], once again underlining the differences in FoxP3 expression between the species. Self-reactive specificities within human Treg populations have not been demonstrated experimentally, but several studies have documented reactivity against well-defined foreign antigens, including hepatitis C virus (HCV) [72]. Once again, whether antiforeign reactivity represents a crossreactive specificity of a Treg with antiself specificity, or whether it reflects differentiation from a peripheral antiforeign conventional T cell is not known. In-vitro differentiation from naive human CD4 T cells in the presence of TGF-β [73], vitamin D [74] or prostaglandin E2 [75] induces FoxP3 expression and suppressive function, and these pathways may also be used to generate Tregs in vivo, particularly during ongoing responses in the gut. Our own unpublished data (Fazekas de St Groth, Smialkowski and Dervish) derived from paired biopsies of involved versus uninvolved bowel mucosa in ulcerative colitis patients have shown a positive correlation between the presence of an inflammatory infiltrate and both the percentage and absolute number of CD4+FoxP3+ T cells. These data are consistent with a model in which the in FoxP3+ T cells within the gut in conditions such as inflammatory bowel disease are to the inflammatory as a to the effects of (Table 2). involved in regulatory T cell biology in vivo using mice have provided the required to generate and normal populations of Tregs. these is essential we are to how Tregs are involved in immune responses to such as IL-2 Murine studies IL-2 is required for normal Treg function in to role in the of Treg IL-2 or have regulatory although T-cell are only The expression of CD25 by Foxp3+ cells is in mice, and the of IL-2 CD25 expression via the and Foxp3 expression cell IL-2 is essential for normal Treg function in vitro, as demonstrated by in which the of monoclonal antibodies suppression in of Treg and conventional Human studies involving the of IL-2 have reported in circulating Treg particularly within the that human Treg is also by IL-2 IL-2 also FoxP3 expression within CD4+CD25+ T cells in vitro via the growth factor beta Murine studies The role of TGF-β in Treg biology is in TGF-β or receptor and at a conventional and regulatory T cells make TGF-β and the normal of self-tolerance requires that both also express functional TGF-β Whether TGF-β is involved in mediating Treg function remains TGF-β of murine Foxp3− cells can induce both Foxp3 expression and regulatory function in Human studies TGF-β can also convert human Foxp3− cells to a FoxP3+ phenotype in with the TGF-β levels in the of Tregs in the gut mucosa have been documented in human inflammatory bowel disease and in HIV infection As mentioned above, our own data from patients that Tregs in proportion to the of in the bowel suggesting that they are to inflammatory in Tregs however, not a of bowel as Treg in the gut are in graft versus disease Whether human gut Tregs are in from Foxp3− cells or derived from circulating Foxp3+ cells remains to be although recent reports the effects of TGF-β and retinoic in the murine gut T-lymphocyte-associated protein 4 and indoleamine 2,3-dioxygenase Murine studies mice a that can be by the of in that Tregs normal regulatory function in This finding is because CTLA-4 a than a positive should than the function of cells that express It has been that CTLA-4 in Tregs and conventional T cells. such function of CTLA-4 is to induce dendritic cells to express thus T-cell proliferation as mentioned [52]. in-vitro evidence that IDO may also function to induce de novo FoxP3 expression in CD4 T cells, suggesting that CTLA-4 and IDO may be part of a positive between dendritic cells and Tregs Human studies antibody is in as a of immune responses such as and have been in a number of patients in consistent with the role of CTLA-4 as a of Treg function these are with for CTLA-4 expression during induction of Foxp3 in naive human CD4 T cells has recently been demonstrated in CTLA-4 with the peripheral of Treg generation in to role in the function of Tregs in the is also evidence for the link between CTLA-4 and IDO mentioned CTLA-4 has thus been shown to induce the production of IDO by human dendritic cells and IDO can convert human CD4+CD25− T cells to cells and regulatory T cells is an receptor expressed on the surface of effector T cells in both the mouse and human It has recently been shown that the expression of by T cells in chronic infections such as HIV is with cytotoxic activity The expression of in Foxp3+ Tregs is to the but surface expression can be by activation in The role of expression in Treg function is currently not known. Regulatory T cells as part of a normal in is evidence that FoxP3+ Tregs are as a proportion of CD4 T cells at inflammatory in both the mouse and The evidence for such is in As mentioned above, whether such cells are derived from the thymus or are in the periphery is unknown. IDO expression is also at these and may be part of the positive described regulatory T cells prevent clearance of Murine Leishmania Leishmania skin in mice a proportion of Tregs and a viable [46]. The of Leishmania-specific T cells of Tregs can in mice, suggesting that Tregs prevent clearance under normal conditions [46]. Importantly, however, Tregs prevent of the skin are they in in mouse Human hepatitis C virus It has been that Treg activity may be with the induction and of the based on evidence that Treg are in the peripheral blood of patients and that the specificity of Tregs for antigens can be demonstrated [72]. however, enzyme levels and with lower than Treg consistent with a than role of Tregs in In Tregs are equally in chronic and animals It thus appears that Tregs are for the state in Regulatory T cells in HIV It has been that Treg activity may be for a state in HIV infection is by an early of immune function multiple cell which could be by regulatory Regulatory T-cell in HIV infection The number of Tregs detected in from patients is by a number of factors, including the the stage of infection, and whether has been a number of reports of Treg in peripheral and mucosa of patients and The has been by the use of Treg in with their of (Table 1). the use of a CD4+CD25+ in levels of contamination by activated CD4 T cells, on the status of the the Treg by to in normal and by an unknown proportion in Foxp3 expression was only by chain for Foxp3 usually without Treg such as CTLA-4 and have also been used as of Treg number and activity, but once again are also expressed by a subset of activated CD4 T cells. The use of antibody for Treg identification and (Table should the with which we can Treg phenotype and function in HIV studies of regulatory T cell number in HIV and is to as more studies of Tregs in HIV are and compared with the data from the In lymphoid such as the a in Tregs, detected by for Foxp3 expression, has been reported (Table of CD25 by these Foxp3+ cells is lower than in blood or mucosa Like the from an number of Tregs and dendritic cells with a phenotype and the ability to induce Foxp3 expression in In the which is a of HIV and infection with early and prolonged CD4 T-cell the absolute number of Tregs during infection by HIV and to of CD4 T cells express Foxp3 in some that may explain in Tregs have recently been reported 1). et have provided evidence that T cells are relatively in HIV infection, compared with conventional T cells. of Tregs may be a of the ability of FoxP3 to transcription from the HIV by the activation of factor et recently that HIV could Foxp3 expression in T cells, their et have that one of the effects of on dendritic cells is to CTLA-4 expression and the suppressive of Tregs. is that the is a to at the to the in 1: HIV may regulatory T cells during infection at of and early such as the and an that regulatory T cell activity and green an that Treg may to or absolute in Tregs. Tregs have of HIV than cells and may at the of infection HIV Treg FoxP3 expression and of Tregs via a In addition, HIV Treg expression of cytotoxic T-lymphocyte-associated protein 4 and suppressive activity whereas immune activation and induce Treg proliferation as part of a involving transforming growth factor beta Tregs induce indoleamine 2,3-dioxygenase (IDO) expression in dendritic cells, which in induces de novo FoxP3 expression in of Tregs between peripheral and lymphoid such as and to in Tregs in blood and lymphoid the both the effects of HIV and and the in immune activation and of has been shown to reduce the number of Tregs in and mucosa but not in Whether this is a of or a of is not yet blood In peripheral a number of groups have reported in the percentage of Tregs within the CD4 T-cell in of whereas have seen no (Table In general, with the in Tregs as a proportion of CD4 T cells have CD4 T-cell and although the Treg in this to be highly Treg to after the of consistent with the data from the gut and studies have shown in FoxP3 in peripheral blood T cells of patients with or low CD4 T-cell in the proportion of T cells. in FoxP3 expression cell that regulatory function may be The may be to a in which has been shown to FoxP3 transcription FoxP3 levels with of regulatory T cells in HIV infection studies of Treg function involved the of in-vitro peripheral blood T-cell responses to HIV and to such as and with and without the of Tregs, and in some with the of purified CD4+CD25+ T cells (Table These studies that Treg activity was in HIV infection, but did not compared with the level in studies of peripheral blood regulatory activity a in patients with and low CD4 T-cell were from studies of of were HIV Tregs and a in T-cell responses after Treg than recently study however, provided definitive evidence that Tregs in potent suppressive activity in disease and in the of study the in FoxP3 expression and regulatory activity in the blood of studies of Treg function in HIV is still whether Treg activity an at of infection such as the gut (Table 2). functional studies will be required to the of Tregs at on the clearance of virus within the as a Regulatory T cells in models of HIV between in Treg activity and the of infection of a species, and and green both species, have to the of whether Tregs or prevent disease. infection in green is by CD4+CD25+ T-cell FoxP3 expression and TGF-β compared with in of infection in and however, a positive than a correlation between immune Treg activity, TGF-β levels and et have reported in the of T cells in the T cell of in infection of by in T cells and In more in Treg and immune activation were seen in of monoclonal antibody of with a in an in effector function and a in IDO and TGF-β levels of Treg activity, the that Tregs were a in a model in which an with HIV in humans was in mice with human peripheral blood and given an of with the IDO to an in T cells and the of suggesting a role for IDO by Tregs, in chronic infection Regulatory T cells and the of that target regulatory T cells Treg or activity in vivo is likely to specific T-cell whether or not Tregs are for and the to that target Tregs is most in the of with and an IL-2 in studies have the for Treg to autoimmune including of the and The gut is the of major the role that Tregs in normal immune in the gut. in appears to be both the most potent and the most likely to probably because it Treg activity and in activated T cells. The positive effects of monoclonal antibody of with for the of to HIV effector T-cell activity by may also be it can be demonstrated that the activity of Tregs is not by this Treg could be for use at stages of HIV infection, either or in combination with in in the of the of that in with as in some of the or with such as antibodies to the co-stimulator 4-1BB that the between regulatory T cells and T-cell IL-2 low and IL-2 Treg particularly in the and the activation status of CD45RO-positive Tregs As mentioned above, IL-2 is to the expression of which is with Treg activity in vitro. IL-2 however, also a potent of T-cell activity, particularly when conventional CD4 T-cell are The effects of IL-2 may at stages of HIV infection, on the between regulatory and effector As most conventional T cells express levels of receptor whereas Tregs do not, the of T-cell during without Treg or receptor expression by conventional T cells however, low in HIV infection as a of in to levels In addition, has been reported to HIV although this may be to T-cell In the first report of the effects of in patients both CD4 and T-cell whereas the percentage of Tregs of has also shown when with in CD4 and T-cell in the naive and memory The use of in combination with IL-2 or thus the of the of regulation in HIV disease. In the between Tregs and conventional T cells is a complex Tregs are involved in virtually every immune and to function as a of and the activation Whereas the removal of Tregs virtually every immune this at a that of which may manifest as autoimmune disease or an in the disease. to more about the and between Tregs, conventional T cells and HIV to whether Treg will in the of and whether it should be with such as the of of