The Foxp3⁺ regulatory T cell: a jack of all trades, master of regulation

T_reg cells influence other T cells

Both in vitro and in vivo analyses suggest that T_reg cells can suppress the proliferation and/or cytokine production of T_eff cells. Additionally, T_reg cells prevent CD8⁺ cells from differentiating into cytolytic effector cells in vivo without affecting their proliferation or interferon-γ (IFN-γ) production²³. Because the suppression of T_eff cell proliferation by T_reg cells was observed in vitro in an APC-free system, it was suggested that T_reg cells suppress through direct contact with T_eff cells²⁴. A mechanism to explain the contact dependence of in vitro suppression has been proposed in a publication showing that T_reg cells have large amounts of cytoplamic cAMP and could deliver this potent immunosuppressive compound to T_eff cells by contact through gap junctions²⁵. Other studies have shown that T_reg cells can kill T_eff cells directly in culture through the release of granzyme B and perforin^26-29. However, imaging studies have shown that T_eff cells and T_reg cells do not interact stably during suppression in vivo and in vitro^30,31.

Because natural T_reg cells constitutively express CD25, the high-affinity receptor for IL-2, it has long been suspected that T_reg cells suppress by ‘sopping up’ IL-2 produced by T_eff cells thereby preventing their proliferation and differentiation. This hypothesis has been revisited in studies examining the physiological changes in T_eff cells after their encounter with T_reg cells³². T_eff cells undergo apoptosis after being exposed to T_reg cells. This form of T_eff cell death is distinct from those mediated by granzyme B or perforin mentioned earlier and is dependent on the proapoptotic factor Bim, expressed by T_eff cells. Preventing Bim-mediated cell death by either Bim deficiency or forced expression of Bcl-2, a protein shown to counteract the proapoptotic activity of Bim, renders the T_eff cells resistant to T_reg cell–mediated suppression in vitro and in vivo. In this context, it is notable that the galectins also mediate cell death by a perforin- and granzyme-independent mechanism, which suggests a potential link between these two pathways^33,34. Unfortunately, it is difficult to explain bystander suppression or infectious tolerance in vivo on the basis of these T_eff cell–killing activities.

In contrast, some studies suggest that T_reg cells can alter the differentiation of other T cells. For example, CD4⁺ T_eff cells differentiate into IL-10- or TGF-β-producing adaptive T_reg cells in the presence of T_reg cells in vitro and in vivo, an observation more consistent with the idea of bystander suppression and infectious tolerance^35-37.

T_reg cells alter APCs

A second approach to defining T_reg cell function has been to evaluate their effect on APCs. It has been shown that T_reg cells directly interact with antigen-presenting dendritic cells (DCs) in vivo within hours of transfer. The interaction of T_reg cells with DCs profoundly affects the ability of T_eff cells to subsequently engage and become activated by the same DCs. T_reg cells either abrogate the antigen-presenting activity of the DC or promote the secretion of suppressive factors by the target DC population. It has been shown that T_reg cells can stimulate APCs to upregulate the activity of indoleamine 2,3-dioxygenase (IDO), a potent immunosuppressive enzyme associated with pregnancy and tumor immune evasion^38,39. In those studies, IDO induction was found to depend on high expression of the inhibitory receptor CTLA-4 on T_reg cells⁴⁰. However, there is no clear evidence of the involvement of IDO in T_reg cell function in vivo or in vitro. The effect of CTLA-4 on T_reg cell function is also complex. Blocking CTLA-4 has no effect on in vitro suppression by T_reg cells, and in most circumstances, T_reg cells from CTLA-4-deficient mice show suppressive activity similar to that of cells from their wild-type littermates^41,42. However, in vivo CTLA-4 blockade abrogates T_reg cell–mediated suppression in mouse models of inflammatory bowl disease (IBD), autoimmune gastritis and transplantation (Table 1). In the setting of IBD, CTLA-4-deficient T_reg cells are able to prevent disease. However, unlike the protection afforded by wild-type T_reg cells, disease protection mediated by CTLA-4-deficient T_reg cells is dependent on IL-10 (ref. 43). That in vivo finding is consistent with in vitro analyses showing that CTLA-4-deficient T_reg cells are distinct from wild-type T_reg cells in the dependency of their suppressive function on TGF-β⁴². Finally, mice deficient in the recombination-activating gene product, when reconstituted with a mixture of CTLA-4-deficient and Foxp3-deficient bone marrow cells, live longer than mice reconstituted with either CTLA-4-deficient or Foxp3-deficient bone marrow cells alone, which shows that CTLA-4⁺ and Foxp3⁺ cells complement each other's function⁴⁴. These findings suggest that T_reg cells rely not on one mechanism for their function but instead use many alternative mechanisms to control unwanted autoimmune and uncontrolled adaptive immune responses.

Another relevant observation is the potential of T_reg cells to activate TGF-β on the surfaces of DCs. Latent TGF-β binds to DCs through interaction with α_Vβ₈ integrins on the basis of an Arg-Gly-Asp motif. This binding is essential for the provision of a substrate for TGF-β activation and prevention of colitis⁴⁵. Evidence has been presented that furin, an enzyme purported to cleave latent TGF-β, is made by T cells after stimulation with IL-12 and thus might influence the creation of inflammatory and regulatory milieus⁴⁶. Additionally, TGF-β, together with a subpopulation of DCs, promotes the differentiation of T_eff cells into Foxp3⁺ T_reg cells⁴⁷. Thus, elaborate cross-talk between T_reg cells and DCs lead to DC silencing, local TGF-β activation and expansion of the T_reg cell repertoire, all of which help to actively establish and reinforce immune quiescence and self-tolerance.

T_reg cell–derived suppressor molecules

A third and perhaps the most straightforward approach to understanding T_reg cell biology has been to identify T_reg cell–specific molecules that are directly responsible for their potent tolerogenic effect. Like the other two approaches, this approach has led to complex and contradictory results. Many studies seem to definitively describe a mechanism of action that is essential for T_reg cell function, whether IL-10, TGF-β, CTLA-4, granzyme B, perforin, IFN-γ, IL-9, heme oxygenase-1 (HO-1), cAMP, CD39, galectins or IL-35 (Table 1). Yet with the possible exception of CTLA-4 and TGF-β (the latter of which is also essential for T_reg cell development), disruption of the genes encoding these molecules does not result in the catastrophic effects found in T_reg cell–deficient mice. In an effort to categorize the various pathways and perhaps identify common characteristics, several are summarized below (Fig. 1).

Many studies have shown that T_reg cells can make suppressive cytokines such as IL-10 and TGF-β. Early in vitro studies demonstrated that neutralizing antibodies to IL-10 or TGF-β do not block T_reg cell activity and that T_reg cells from mice lacking IL-10 or TGF-β show similar suppressive activity^24,41,48. It should be pointed out, however, that although often dismissed, there were some early suggestions that TGF-β is involved in T_reg cell suppressive function in vitro^49,50. In contrast to those in vitro studies, it has been found that neutralizing IL-10 or TGF-β abolishes T_reg cell suppression in vivo in mouse models of IBD, type 1 diabetes, leishmania skin infection and transplantation (Table 1). In a few in vivo models, expression of IL-10 or TGF-β specifically in T_reg cells is required for T_reg cell function^51-53. However, in some other situations, the source of these suppressive cytokines is not limited to T_reg cells^37,54,55.

HO-1, which catalyzes the formation of carbon monoxide through heme degradation, has been linked to T_reg cell function^56,57. Similarly, CD39 and CD73, which are ‘preferentially’ expressed on the surfaces of T_reg cells, catalyze the generation of adenosine from the extracellular nucleotide ATP or ADP⁵⁸. Adenosine can bind to its receptor A2A expressed on T_eff cells to suppress their responses. Additionally, it has been suggested that several members of the galectin family of carbohydrate-binding proteins are involved in T_reg cell function^59-61. It has also been proposed that another cytokine, IL-35, is responsible for close-range suppression by T_reg cells. IL-35, a heterodimeric protein composed of the IL-12 p35 chain and Ebi3, is regulated in T_reg cells by Foxp3 (ref. 62). T_reg cells lacking either p35 or Ebi3 are less suppressive in vitro and are unable to control IBD induced by T_eff cells. Thus, it would seem that many molecules can mediate T_reg cell functions both in vitro and in certain in vivo settings.

The ‘big picture’

Although the results presented above seem overwhelming and confusing at first glance, the following theme emerges from the vast array of data obtained in various disease settings: T_reg cells probably use many mechanisms to control unwanted immune responses in vivo. Depending on the nature of the immune response, the eliciting agent, the immunological makeup of the host and the site of suppression, certain mechanism(s) may seem to dominate.

This phenomenon is most vividly exemplified by the IBD model⁶³. Reliance on IL-10 for T_reg cell–mediated control of IBD varies depending on how the disease is elicited and the timing of T_reg cell treatment. Control of more aggressive forms of disease induced by memory T cells or pathogenic bacteria and reversion of established disease requires IL-10 in addition to CTLA-4 and TGF-β. In contrast, in the absence of one of the suppressive mechanisms (such as CTLA-4), T_reg cells rely more on alternative suppressive functions such as IL-10 and TGF-β in vitro and in vivo^42,43. Thus, it is possible that T_reg cell–mediated control of IBD, a disease at the highly inflammation-prone mucosal surface, may require a wider array of suppressive mechanisms operating in synergy and thus that the presence of IL-10, TGF-β, CTLA-4, and IL-35 as well as IL-2 deprivations are necessary for complete protection. This scenario is mirrored in the transplant setting. Immune responses to organ transplants are one of the most vigorous forms of immune responses because of the exceptionally high frequency of alloreactive T cells. Thus, T_reg cells may depend on many suppressive mechanisms working in concert to keep alloimmune responses under control. In contrast, one or two mechanisms may suffice to control slowly progressing organ-specific autoimmune diseases such as type 1 diabetes and autoimmune gastritis. In these disease settings, when one pathway is blocked, alternative mechanisms may fully compensate; therefore, T_reg cell function might not be dependent on any one particular mechanism.

The site of T_reg cell action in vivo is certainly not limited to lymphoid organs. T_reg cells have been detected at sites of inflammation, and in many situations, their ability to migrate to and remain in inflamed tissues is important for their function in vivo⁵⁰. Moreover, the molecular mechanisms of suppression used by T_reg cells seem to differ depending on their localization in nonlymphoid versus lymphoid tissues. T_reg cell–mediated prevention of IBD induced by the transfer of naive T cells is confined to lymphoid organs and is dependent on TGF-β and CTLA-4. In contrast, T_reg cell–mediated control of colitogenic bacteria–induced IBD requires the presence of T_reg cells in lymph nodes and in the inflamed colon, as well as IL-10 (ref. 64). In addition, control of an acute immune response to leishmania is located mainly in lymphoid organs and is independent of IL-10, whereas control of a chronic immune response in the infected skin requires local retention of T_reg cells and their production of IL-10 (ref. 64). Finally, tumor-infiltrating T_reg cells suppress tumor immunity by a mechanism dependent on TGF-β and IL-10, unlike T_reg cells isolated from the peripheral blood of the same person⁶⁵. These data from various disease models collectively demonstrate differential requirements for distinct suppressive functions of T_reg cells at different tissue sites. In general, T_reg cells rely on more suppressive mechanisms to control responses at sites of inflammation than in lymphoid organs.

On the basis of the experimental evidence collected so far, we propose a three-tiered ‘sequential’ model of T_reg cell function in vivo (Fig. 1). In the steady state, T_reg cells exert ‘homeostatic control’ over the immune system in lymphoid organs to prevent the potential outgrowth of auto-reactive T cells. By virtue of their constitutive expression of CD25, T_reg cells effectively ‘sop up’ IL-2 produced by T_eff cells, thereby terminating local incidental T_eff cell activation while boosting T_reg cell fitness and function. This phenomenon helps to explain some of the antigen-non-specific functions of T_reg cells noted in several in vivo systems. T_reg cells also function in an antigen-specific way. The T_reg cell antigen receptor repertoire is skewed toward recognition of self antigens⁶⁶. In addition, T_reg cells show greater sensitivity to antigens than do their T_eff cell counterparts^47,67. These properties allow T_reg cells to effectively patrol the lymphoid organs to prevent potential priming of autoreactive T cells. Most likely the type of T_reg cell target in this setting is self antigen–presenting DCs. This contention is supported by the observation that T_reg cells are stably conjugated with tissue antigen–bearing DCs during in vivo suppression in lymph nodes. In addition, complete T_reg cell ablation in adult mice leads to the expansion of populations of activated DCs and to subsequent fatal multiorgan autoimmune disease¹⁸. The molecular mechanisms used by T_reg cells during this stage probably involve TGF-β and CTLA-4, as suggested by the IBD studies mentioned above⁶³. In addition, the massive lymphoproliferation noted in CTLA-4-deficient and TGF-β-deficient mice also supports the idea of an essential function for T_reg cells in normal immune homeostasis^68,69. Other mechanisms involving IL-10, IDO, HO-1 and CD39-CD73 may also contribute to T_reg cell function at this stage, but they are probably not essential, as indicated by the lack of overt changes in immune homeostasis in their absence. It is notable that one attribute of the galectins is their ability to bind to multiple ligands and soluble molecules because of the ‘sticky’ nature of the lectin moieties. Thus, it is possible that the galectins, by forming functional scaffolds that bind several of the small molecules purported to be mediators of T_reg cell suppression, help to establish a local immunosuppressive milieu that influences many cell types, such as T_eff cells, DCs, macrophages and B cells, to actively maintain immunological quiescence.

When steady-state self-tolerance is breached, as after Toll-like receptor engagement during infection⁷⁰ and in the presence of heightened frequencies of self-reactive T_eff cells in autoimmune and transplantation settings, T_reg cells become further activated to engage the second tier of ‘damage control’. After being activated, T_reg cells shed CD62L and upregulate the chemokine receptors and adhesion molecules needed to leave lymphoid organs and gain entry into inflamed tissues. Once in inflamed tissue, T_reg cells reactivated by tissue DCs upregulate CD25 and therefore become more effective at competing with T_eff cells for IL-2. Activated T_reg cells express more IL-10, CTLA-4, IL-35, TGF-β, HO-1 and CD39-CD73 and so are more potent suppressors. In addition, chronically activated T_reg cells have high expression of cytotoxic granules such as granzyme B, which enable T_reg cells to actively kill APCs and T_eff cells in extreme conditions that lead to resolution of the immune response.

Infectious tolerance is established during the final stage to stabilize the tolerant state. Tissue destruction associated with the immune response leads to the presentation of newly exposed tissue antigens on the T_reg cell–silenced DCs. Together with TGF-β, also a byproduct of immune activation, these tolerogenic DCs induce the differentiation of T_eff cells into adaptive T_reg cells, thus expanding the T_reg cell antigen receptor repertoire. In the intestine, it has been shown that CD103⁺ DCs have the unique ability to induce adaptive T_reg cells because of their ability to produce retinoic acid, which is needed to induce naive T cells to differentiate into Foxp3-expressing T_reg cells⁴⁷. TGF-β induces CD103 expression⁴⁵; thus, TGF-β may contribute to the induction and/or maintenance of these specialized DCs. This model is consistent with the ‘hygiene hypothesis’, proposed to explain the reciprocal rates of infection and atopic and autoimmune disease. Improved hygienic standards and a decrease in both infections and the resulting active immune responses may also deprive people of the opportunity to expand T_reg cell antigen receptor repertoires through infectious tolerance and thereby lead to higher rates of allergy and autoimmune disease.

Overall, T_reg cells probably use many mechanisms in vivo to prevent and terminate immune responses and to establish a regulatory milieu that will promote a long-lasting, durable state of tolerance. Short-range mechanisms such as cytokine deprivation and the secretion of molecules such as IL-10, adenosine, IL-35, galectins and carbon monoxide ‘shut down’ local T_eff cells during adaptive immune responses. These pathways are designed to act quickly and locally and in many cases are sufficient to dampen immunity. However, in the long term, T_reg cells create a regulatory environment by producing TGF-β and/or by substantially changing DC functions to promote new T_reg cell production. These processes promote bystander suppression and are critical to the creation of infectious tolerance that can spread beyond the local tissue and exert long-lasting influence over the immune system. It is notable that the some of the mechanisms that T_reg cells use to suppress immunity also enhance T_reg cell function. For example, by ‘sopping up’ IL-2, T_reg cells simultaneously starve T_eff cells and promote their own population expansion and survival. Similarly, the direct or indirect production of TGF-β by T_reg cells ‘feeds back’ to stabilize and enhance Foxp3 expression in T_reg cells, thereby boosting their suppressive function.

Concluding remarks

In conclusion, in vitro and in vivo T_reg cell function cannot be attributed to a single dominant pathway or molecule. Instead, T_reg cells use many suppressive mechanisms that can target various cell types depending on the circumstances and their surroundings. The versatility and adaptability of T_reg cells makes them true ‘masters of immune regulation’. The chief function of T_reg cells in a normal person is to maintain immune homeostasis in the lymphoid organs. After the onset of an immune response, T_reg cells use additional suppressive strategies to resolve inflammation and limit tissue damage. The dynamic interaction between T_reg cells and DCs is vital for T_reg cell function. This model provides a conceptual framework for the ongoing quest to discover more suppressive mechanisms used by T_reg cells, which are intricately involved in the pathogenesis of autoimmune disease, infectious disease, cancer and the rejection of transplanted organs. Better understanding of their biology will create opportunities for the development of new therapeutic intervention in these disease settings.