CD4+ Tregs and immune control (original) (raw)
Without question the most remarkable feature of CD25+CD4+ Tregs is their ability to dampen immune responses. They appear capable of suppressing a wide variety of immune cells, encompassing those of both the innate (55–57) and the adaptive immune systems (58–60). This suppressive ability can be modeled in vitro by mixing of titrated numbers of highly purified CD25+CD4+ Tregs and responder cells, typically CD25–CD4+ T cells plus a T cell stimulus. Under such conditions, the CD25+ population suppresses both the proliferation and, more fundamentally, the IL-2 production of the CD25– cells in a dose-dependent manner (58, 59, 61). CD25+CD4+ Tregs themselves require TCR stimulation, and, it now seems, IL-2, to actually trigger their suppressive effects, but once this condition has been satisfied their ensuing suppression can act non–antigen-specifically (58, 59, 61). Therefore, suppression is an active process and can be directed against bystander cells. Curiously, CD25+CD4+ Tregs themselves are anergic in vitro, i.e., they do not proliferate or produce IL-2 in response to conventional T cell stimuli such as plate-or bead–bound anti-CD3, concanavlin A (ConA), or splenic APCs. This anergy can, however, be broken by a sufficiently potent stimulus, e.g., the addition of high-dose exogenous IL-2 or anti-CD28, or the use of mature DCs as APCs (27, 58, 59, 61–63). Some of these strong stimuli, particularly mature DCs, also perturb CD25+CD4+ Treg suppression both in vitro and in vivo (63, 64). At least in vitro, anergy seems to be the default state of naturally occurring Tregs, since they revert back to it once potent stimulation is withdrawn (58). In vivo, however, CD25+CD4+ Treg anergy is not readily observed; instead they seem to have a highly active rate of turnover (33, 65). It seems likely, then, that CD25+CD4+ Treg anergy is an in vitro phenomenon, merely reflecting an exacting set of activation requirements generally absent from cell culture.
Given that the ability to control immune responses is the cardinal feature of CD25+CD4+ Tregs, it is surprising that their mechanism(s) of suppression remains elusive. Essentially, Treg suppression can be divided into those mechanisms mediated by relatively far-reaching soluble factors and those requiring intimate cell contact. In vivo experiments based chiefly on the IBD model mentioned previously have demonstrated the importance of the immunomodulatory cytokines IL-10 and TGF-β (66). By blocking IL-10 signaling in vivo with an anti–IL-10 receptor mAb, it was possible to abrogate the normal colitis-preventative action of CD45RBlow cells (66). Similarly, CD45RBlow T cells from IL-10–/– mice lacked their otherwise intrinsic ability to protect from colitis and, moreover, were even colitogenic themselves when transferred alone (66). The importance of IL-10 is further underscored by the observation that IL-10–/– mice spontaneously develop colitis (67, 68). Examination of the in vivo role of TGF-β has generally painted a similar picture to that of IL-10, with Treg function being blocked by the presence of neutralizing anti–TGF-β mAbs (69). Some data also suggest that TGF-β may not necessarily act as a soluble factor but can also be found on the surface of activated CD25+CD4+ Tregs and may therefore act in a membrane-proximal manner (70). Interestingly, virtually all TGF-β+ CD25+CD4+ Tregs also express thrombospondin, a factor capable of converting normally latent TGF-β into its active form (71). There should be a note of caution regarding these in vitro studies on TGF-β, since a comprehensive analysis by a second group failed to demonstrate any role for it in vitro (72).
The confusion over a definitive CD25+CD4+ Treg suppression mechanism is compounded when viewed in the context of the in vitro data, since here the overwhelming evidence highlights direct cell-cell interaction, and not cytokines, as being critical (58, 59, 73). Several lines of evidence lead to this conclusion: with the exception of the study on membrane-bound TGF-β alluded to above, both anti–IL-10 and anti–TGF-β fail to perturb CD25+CD4+ Treg suppression (58, 59, 70, 72). Similarly, supernatants from suppressed cultures or activated CD25+CD4+ Tregs show no inherent suppressive activity, nor can suppression be observed across a semipermeable membrane (58, 59). Collectively, these in vitro observations therefore appear to obviate a role not just for IL-10 and TGF-β but for soluble factors in general.
The actual membrane events occurring during suppression that depends on CD25+CD4+ Treg contact have yet to be clarified. The most simplistic models propose competition for APCs and specific MHC/peptide antigenic complexes. Additionally, the constitutive expression of the high-affinity IL-2 receptor could make naturally occurring Tregs into an effective IL-2 sink, depriving potential autoreactive T cells of this essential growth factor (74). However, given the relative physiological scarcity of naturally occurring Tregs, it is perhaps unlikely that a simple competitive-adsorptive model alone could account for their suppressive action in vivo. Other models of CD25+CD4+ Treg suppression propose a more proactive and antagonistic form of suppression that relies on the expression of specific “inhibitory” molecules. The identity and indeed even the very existence of such an inhibitory molecule are uncertain, but 1 potential molecule could be Treg–expressed CTLA-4. Aside from its well-established high affinity for the costimulatory molecules B7.1 and B7.2 (CD80 and CD86, respectively), CTLA-4 has also recently been shown to trigger the induction of the enzyme indoleamine 2,3-dioxygenase (IDO) when interacting with its ligands on DCs (75–78). IDO catalyzes the conversion of tryptophan to kynurenine and other metabolites, which have potent immunosuppressive effects in the local environment of the DC. In this way, CD25+CD4+ Tregs may exert their suppression by proxy through their action on APCs. Another APC-centric mode of suppression could be via the perturbation of antigen-presenting capacity. In support of this concept, one report has demonstrated that purified CD25+CD4+ Tregs are able to downregulate the expression of both CD80 and CD86 on DCs, converting them into inefficient APCs (57). At any rate, CD25+CD4+ Tregs need not act exclusively via the APC, since they are quite capable of suppressing in the context of “APC-free” systems such as plate- or bead–bound antibodies or MHC/peptide tetramers (58, 79). At least in vitro, direct suppression of the target cell is therefore still also possible.
A provocative investigation into the membrane events involved in CD25+CD4+ Treg suppression was recently reported (80). This study suggested that engagement of CD80, and to a lesser extent CD86, on the responder T cell and not the APC was responsible for the transmission of a negative signal, and therefore these were the molecular targets through which Tregs exert their function (80). In support of this, the authors demonstrated that B7–/– responder cells were resistant to suppression in vitro and induced a fatal wasting disease refractory to cotransferred CD25+CD4+ Tregs (80). Again the obvious candidate Treg molecule for this inhibitory interaction would be CTLA-4, although this fails to explain the paradoxically intact suppression mediated by CTLA-4–/– CD25+CD4+ Tregs (27). The presence of B7 on conventional T cells has been known for several years, and it would be interesting, then, if this hitherto puzzling expression pattern were shown to play a role in Treg–mediated suppression (81, 82). While the identification of a membrane-bound CD25+CD4+ Treg–specific inhibitory molecule remains inconclusive, some very recent work has suggested that the CD4-related molecule LAG-3 may be important (CD223), though this awaits independent confirmation (83, 84). Proving a negative hypothesis is always a difficult task, but it may yet be shown that there are no truly unique Treg–associated molecules responsible for inhibition. Rather, the specialized functions of Tregs could simply be the product of known molecules acting semi-redundantly, which together generate a suppressive phenotype. An integrated summary of CD25+CD4+ Treg suppressive mechanisms is shown in Figure 2.
Possible CD25+CD4+ Treg suppression mechanisms in vivo. CD25+CD4+ Tregs may suppress their effector T cell targets (TR) by a number of proposed mechanisms. In vivo CD25+CD4+ Tregs may act in a cell contact–dependent manner by competing directly for stimulatory ligands on the APC, by sinking essential growth factors such as IL-2, or by directly transmitting an as-yet uncharacterized negative signal. Alternatively, they may use longer-range suppressive mechanisms by means of the cytokines IL-10 and TGF-β. Finally, CD25+CD4+ Tregs may act through the APC either by triggering IDO activity, resulting in the generation of immunosuppressive metabolites, or by perturbing the APC’s presenting capacity. Such mechanisms are not necessarily mutually exclusive, and more than 1 might operate in tandem.
Given the relative physiological scarcity of CD25+CD4+ Tregs, it seems likely that in vivo they would use mechanisms to amplify their suppressive action, and ones that are not normally fully appreciated under in vitro analysis. This could occur either by the modification of APCs as outlined above or by the “infectious” spreading of tolerance to conventional T cells. In accordance with this, some recent work has demonstrated that human CD25+CD4+ Tregs can confer a suppressive phenotype to conventional CD4+ T cells in a contact-dependent manner (52). These newly generated regulatory-like cells then suppress by means of IL-10 or TGF-β. This would constitute a mechanism of not only spreading a suppressive phenotype but also making it more efficient on a per-cell basis by engaging the action of soluble mediators. Most satisfyingly, this scenario could also finally reconcile some of the disparities observed between the in vitro and the in vivo mechanisms of CD25+CD4+ Treg suppression.