Regulatory Cells and Infectious Agents: Détentes Cordiale and Contraire

Catalyzed by the late Dick Gershon, the limelight shone on suppressor cells in the 1970s and early 1980s, only to be dimmed by molecular biology. Until recently, few braved to use the term, although the concept that immune responses were modulated by inhibitory systems remained viable. Suppressor cells, now usually called regulatory cells, bounced back with the observations of Sakaguchi et al. (1) that cells of the CD4⁺CD25⁺ phenotype suppressed T cell-mediated organ-specific autoimmune diseases. Confirmed and extended by other groups, CD4⁺CD25⁺ regulatory T cells (Treg)² are widely accepted to influence responses to self-Ags, some tumor Ags, and an extending number of exogenous Ags (2, 3, 4, 5, 6). Indeed, even molecular biology seems content with the new developments, because CD4⁺CD25⁺ T cells with regulatory activity express the signature transcription factor Foxp3 (7). In fact, if CD4⁺CD25⁻ T cells are transfected with Foxp3, they take on regulatory function (7). Currently, CD4⁺CD25⁺Foxp3⁺ T cells represent the most newsworthy Treg, but cells of several other phenotypes may also exert regulatory function and may be more important in some circumstances (see Table I). For example, IL-10-driven and -producing Tr1 cells appear to be the main regulators of responses to some pathogens (8). However, this review focuses only on CD4⁺CD25⁺Foxp3⁺ Treg and their role in infections. Cells of this phenotype are usually referred to as natural Treg and are considered to recognize self-Ags functioning to prevent autoimmunity (1, 2). However, cells of the same phenotype also regulate responses to exoantigens such as allergens (9), transplantation Ags (6), and microbial Ags (10). Currently, it is not clear whether the exoantigen-specific Treg are of the same lineage as self-reactive natural Treg or represent peripherally induced counterparts derived from CD4⁺CD25⁻Foxp3⁻ cells. This issue has been discussed in other articles (11, 12).

Numerous recent observations have demonstrated that functional immunity to several microbes is influenced by CD4⁺CD25⁺ Treg (see Table II). Some of the first observations were made with the parasitic pathogen Leishmania major (10, 13). In this study, Treg were demonstrated to act as crucial participants in a host-pathogen détente. As is usually the case with parasitic infections, immunity involves persistent nonclinically evident infection (so-called concomitant immunity or premunition) (14). This is certainly the case in C57BL/6 mice infected cutaneously with Leishmania. Such immune animals retain a low-level infection at the cutaneous infection site (10). Sacks and colleagues (10) made the fascinating observation that CD4⁺CD25⁺ Treg too persist at the site. Furthermore, if removed, the animals could now discard the infection but as a consequence lost their immunity to reinfection. The interpretation that Tregs were limiting the ability of Th1 CD4⁺CD25⁻ effector cells to clear infection was confirmed by adoptive transfers. In fact, the transfer of effector cells alone could clear infection, but transfers that included Treg along with the effectors resulted in subclinical parasite persistence and, as a bonus, immunity to exogenous reinfection. Thus, the fine balance in function between Treg and defensive T cells allowed persistence, enabling the parasite to spread potentially to other animals. It also kept the host lesion free and resistant to reinfection—clearly a détente cordiale.

The détente between Leishmania and resistant mouse strains remains the best example of a Treg-orchestrated benevolent arrangement between host and parasite. Other situations are less cordial, as is the case of Leishmania infection in susceptible mouse strains such as BALB/c (13, 15). In these so-called “nonhealer” mice, cutaneous infection with the parasite results in progressive lesions and a lack of concomitant immunity. The process appears to be initiated by an early IL-4-producing response by effector CD4⁺ T cells that recognize a specific Ag (RACK 1) expressed by Leishmania (13). In fact, the extent of the response, and its ultimate tissue-damaging consequences, is held in check by CD4⁺CD25⁺ Tregs (13), because depleting such cells before infection results in more intense lesions. Adoptive transfer experiments revealed a similar picture. SCID mice receiving Treg-depleted splenocytes developed more severe infection than those given intact cells. Clearly in this situation Treg play a useful role in Leishmania pathogenesis, but this is inadequate to arrive at a détente cordiale. It is evident that humans too can show major differences in expression of cutaneous Leishmania, although usually they adopt the phenotype expressed by resistant B6 mice (10). Currently, it is not known whether Treg play any role in the pathogenesis of Leishmania in humans.

Many virus infections, particularly those that persist, may suppress one or more functions of the immune response (16, 17). Such suppression may help explain how some viruses persist. The suppression can also result in susceptibility to intercurrent infection and sequelae such as neoplasia and perhaps autoimmunity (18). Multiple mechanisms have been advocated to explain viral-induced immunosuppression (17, 19), including most recently the activity of CD4⁺CD25⁺ Treg (20). First hints came from studies on chronic Friend virus leukemia infection of mice (21). Such animals have suppressed CD8⁺ T cell-mediated antitumor immunity, an effect transferable to naive animals with CD4⁺ T cells. Recently, the same investigator group realized that cells responsible for the suppression were mainly CD4⁺CD25⁺ Treg, and that these cells also acted to suppress the antiviral protective effect of CD8⁺ T cells in persistently infected animals (20). Released from such suppression, the CD8⁺ T cells could markedly reduce viral levels during persistence, an effect best shown by adoptive transfer. The situation in chronic Friend virus leukemia clearly favors the pathogen over the host. We may consider this a détente contraire.

Instances wherein the Treg response acts to the detriment of the host in virus infection are increasingly being recognized (see Table II). Conceivably, the Treg system could play some role in the immune response to all infectious agents, but most cases reported so far involve chronic or persistent infections (Table II). However, one example occurs in the acute cytolytic virus infection by HSV in mice (5). With HSV infection, Suvas et al. (5) showed by multiple measures of CD8⁺ and CD4⁺ anti-HSV T cell responses that their magnitude was elevated 2- to 3-fold if mice were depleted of Treg before infection. The presence of Treg served to hamper responses to HSV in both the acute and memory phases (5). By means of adoptive transfer experiments in either Rag^−/− or immunocompetent mice, the presence of Treg was shown to impair the antiviral protective effects of CD8⁺ T cells against viral challenge. Curiously, the anti-HSV CD8⁺ T cell response to some HSV vaccine preparations was also hampered by Treg (our unpublished observation). This was noted both in situations where Treg were depleted before primary immunization as well as when depleted before boosting. Hence, Treg may influence the recall responsiveness of memory T cells, an effect also observed with memory responses to the bacterium Listeria monocytogenes (22). Currently, it is not known whether Treg influence anti-HSV responses in humans, the natural host for the virus infection. Conceivably, the activities of Treg may be one of the contributing reasons why it has been so difficult to produce effective HSV vaccines, an issue that warrants further investigation.

Two of the most important persistent infections afflicting mankind that also lack effective vaccines are HIV and hepatitis C virus (HCV). Effective immunity can be achieved, at least temporarily, against both agents by an antiviral CD8⁺ T cell response (23, 24). Ultimately, this fails, especially in HIV infection (24). Whether induction, and subsequent domination by a Treg response, is one explanation for the failure to control HIV requires investigation. Evidence from studies on HCV indicates that the expansion of CD4⁺CD25⁺ Treg may account for the suppression of effector CD8⁺ T cell immunity observed ex vivo in those with persistent viremia (25). In HIV, the issue of Treg in immunity was recently analyzed indirectly by Nixon and coworkers (26). They indicated a possible role for CD4⁺CD25⁺ T cells by showing that depletion from peripheral blood cells enhanced in vitro HIV T cell-specific responsiveness. The results of another group indicated that HIV-infected dendritic cells may induce Treg in vitro (27). The role of Treg in HIV pathogenesis requires further study.

From an evolutionary perspective, one might wonder why the Treg mechanism was developed and retained if it only serves to facilitate the microbe. However, the Treg system may function on the whole in a benevolent manner by limiting collateral tissue damage caused by protective immune responses against pathogens. Thus, it has long been recognized that protective cellular immune responses may also damage tissues, i.e., delayed-type hypersensitivity reactions. With some pathogens, or in circumstances in which there is underlying immune dysfunction, tissue damage can be intense and chronic. One of the first recognized examples of this was intestinal hyperinflammation caused by enteric microbes in mice that lacked CD4⁺CD25⁺ Treg (28, 29). Another example is the pulmonary inflammatory disease that occurs in immunodeficient mice infected with Pneumocystis carinii (30) where Treg also function to inhibit the extent of inflammation. More recently, a clear role for Treg was shown in the blinding immunoinflammatory lesion caused by ocular infection with HSV (31). Studies in both mice and humans have indicated that these keratitis lesions represent CD4⁺ T cell-mediated immunopathological reactions within the cornea (31). Their severity was modulated by Tregs, because depletion resulted in more severe disease and lesions in animals infected with significantly lower doses of virus (31). Indeed, at low infection doses, intact mice showed a détente cordiale situation, because lesions failed to occur, virus could replicate, and animals became immune to reinfection. The Treg response appeared to act at the stage of immune induction, limiting the magnitude of anti-HSV-specific CD4⁺ T cell responses as well as the migration of CD4⁺ effector T cells to the ocular site. Treg also seemed to modulate inflammation within the keratitis lesions limiting their severity, perhaps by secreting IL-10. We can conclude from studies on herpetic keratitis that Treg clearly function to benefit the host and control the extent of tissue damage. It might be that Treg play similar protective roles in all instances of microbe-induced immunopathology.

The realization that cells with regulatory activity influence the response to microbes may open a new chapter for immune intervention. However, before this becomes practical, several fundamental questions require solution. For example, it is neither clear at present how microbes are recognized by Treg nor the origin of cells that become Treg for microbial Ags. Initial studies emphasized that natural Treg developed in the thymus and that these recognized self-Ags (32). Conceivably, this population also includes in their repertoire exoantigens unique or possibly cross-reactive with self. Alternatively, microbe Ag-regulating cells may be induced from CD4⁺CD25⁻Foxp3⁻ precursors (see Fig. 1).

From a therapeutic viewpoint, the origin of cells may not matter. What is important is whether or not it is possible to deplete or expand microbe-reactive Treg. This task is made complex by the observation that Treg do not express a unique surface phenotype permitting selective deletion or activation by specific ligands. For example, CD25⁺ and glucocorticoid-induced TNFR (GITR), markers characteristic of Treg (33), are also expressed on activated T cells (34). In détente contraire situations it would be useful to deplete Treg, so enhancing protective immunity. Ideally, this should be accomplished in an Ag-specific way, because global depletion could result in complications such as autoimmunity (35). One of the most promising in vivo approaches to blunt the function of Treg is to exploit the fact that, among resting cells, they are the principal cell type that expresses the molecule GITR. Engagement of GITR with its ligand, or with an agonist Ab against it, inactivates Treg function (33). However, ways to achieve this maneuver in an Ag-specific fashion need to be developed. Conceivably, if vaccines could be designed that blunt Treg as well as induce protective responses, they would be more effective.

Induction and/or activation of Treg also represents a therapeutic objective in those circumstances that result in tissue damage. Once again, approaches have been described that achieve activation of all Treg, but practical situations might usually require Ag-specific manipulation. With regard to polyclonal activation, one approach that could prove useful is the observation that nonmitogenic anti-CD3 mAb may activate Treg selectively (36). This approach was shown to control autoimmune diabetes in the natural NOD mouse model as well as the virus-induced transgenic model for diabetes (36, 37). An alternative approach to activate Treg may be to use TGFβ. Accordingly, treatment of CD4⁺CD25⁻ T cells with TGFβ in vitro was shown to generate Foxp3⁺CD25⁺ Treg that acted to modulate lesions in an experimental asthma model (38).

The long-term objective will be to find ways of inducing and activating Ag-specific Treg either in vivo or ex vivo, and then using such cells for immunotherapy. Some progress has been made on both fronts, although not to modulate responses to microbes. As regards ex vivo expansion, Horwitz et al. (39) has shown in both humans and murine systems that alloantigen-reactive naive CD4⁺ T cells can be activated by TGFβ and IL-2 to become potent CD4⁺CD25⁺Foxp3⁺ regulatory cells. Such cells mediate Ag-specific regulatory effects in vitro and, in murine systems, also function in vivo (40). The general approach is under consideration for use in the control of human autoimmune disease and perhaps the control of allografts.

Other approaches have also been described that result in the induction of Ag-specific Treg both in vitro and in vivo (41, 42). For example, the von Boehmer laboratory (42) made the exciting observation that exoantigen-specific Treg can be induced in vivo by prolonged exposure to very low doses of a peptide Ag derived from the hemagglutinin protein of influenza virus. Treated mice developed Foxp3⁺ Treg and failed to respond to subsequent immunization with the peptide. It will be interesting to see whether the Treg activation also succeeds in modulating the response to infection with influenza virus.

Generating pathogen-specific Treg requires development. To achieve this objective requires a further understanding of how microbes induce or activate Treg. Whether or not microbial Ag-specific Treg precursors form part of the T cell repertoire has not been formally demonstrated. Moreover, the induction/activation process may involve a combination of Ag-specific and nonspecific ligands. Thus, complex microbes such as bacteria and large viruses are known to express pathogen-associated molecular patterns that are recognized by TLR. In line with these ideas, recently Treg were shown to express a TLR profile that distinguished them from effector T cells (43). Moreover, TLR ligand stimulation activated Treg (43). It would seem likely that induction and activation of Treg by microbes may involve both Ag-specific and polyclonal activation. We need to understand such issues if we are to identify means of expanding or deleting microbe-reactive Treg in clinical situations.

Finally, because the Foxp3 transcription factor appears characteristic of Treg, once the spectrum of molecules it transcribes become identified, manipulation of one or more of these may become a practical approach. In conclusion, suppressor cells are back, and we know they are involved in microbial pathogenesis in both beneficial and malevolent circumstances. The challenge now is to learn how to effectively manipulate Treg activity in vivo to help the host effectively manage infection.

We thank Mark Sangster, Chris Pack, and Steve Stohlman, who helped to improve this manuscript.