IL-10–producing T cells distinct from Th1 and Th2 cells have been described that undoubtedly play a role in regulating inflammatory pathologies, but factors determining their development are unknown. We report in this study the isolation of such a population of CD4+ T cells, that produce IL-10 and no IL-4, IL-5, or IFN-γ, which was able to prevent autoimmune disease when adoptively transferred in two mouse models of central nervous inflammation. This population was only induced when the immunosuppressive drugs VitD3 and Dex were used in combination, together with stimulation of the cells through their TCR, while using these drugs individually did not lead to the generation of such regulatory cells. We also showed that these IL-10–producing cells can develop independently of Th1 and Th2 polarizing cytokines which conversely were shown to inhibit the development of these regulatory T cells in vitro. Although not sufficient for the development of regulatory T cells, endogenous IL-10 delivered a positive signal to these IL-10–producing T cells during their generation with Vit/Dex. Furthermore, our data suggests that the development of such regulatory T cells occurs under conditions where the expression and/or activation of key transcription factors involved in Th1 and Th2 differentiation as well as NF-κB and AP-1 is minimal.
A likely in vivo counterpart of these regulatory T cells are the allospecific IL-10–producing T cells which have been suggested to account for tolerance in patients with SCID transplanted with HLA-mismatched hematopoietic stem cells (17
). Other studies have shown that chronic antigenic stimulation in vivo can lead to the generation of IL-10–producing T cells and suggest how these cells may play a physiological role in regulating undesirable immune responses (54
). Thus, this type of IL-10–producing regulatory T cell population may arise from chronic encounter of self-antigens on subsets of specialized APCs e.g., DCs, resident in particular organs or particular stages of maturity (18
), and in the absence or low activation of the innate immune response normally required for the generation of inflammatory responses required to eradicate pathogens (56
). In addition, on a functional basis, the mouse CD4+
T cells have been shown to act via IL-10 to inhibit inflammatory bowel disease (IBD) and this heterogeneous population is likely to contain such IL-10–producing cells but in addition also Th2 cells (5
). Although much evidence supports the existence of regulatory T cells in vivo, the signals required for their development are still largely unknown. It was recently reported by Jonuleit et al. that immature DCs can stimulate T cells toward a phenotype of IL-10 producers with low proliferative capacity (18
). Interestingly, both VitD3 and Dex have been shown to affect DC maturation (28
) which may account for the generation of regulatory T cells in our study. It is possible that endogenous levels of both VitD3 and glucocorticoids play a role in dampening immune responses to self-antigens during development thus avoiding the development of pathology inducing autoreactive T cells. Macrophages have been shown to synthesize VitD3 upon activation with LPS and IFN-γ (58
), suggesting that upon triggering of APCs by microbial factors VitD3 is synthesized by these cells and thus may regulate the production of inflammatory mediators. Furthermore, glucocorticoids released from the adrenal glands in response to stress can have profound effects on the immune system (59
). In addition, it has been demonstrated that genetic variation in the magnitude of such stress responses and the release of glucocorticoids can determine susceptibility to an experimental autoimmune disease (59
). Other immunosuppressive drugs may also favor regulatory cells development in this in vitro–driven situation (61
The Tr1 cells that were derived in vitro in the presence of exogenous IL-10 or in the presence of IL-4 with IL-10 (21
) present similarities but also have numerous differences with the single IL-10–producing cells derived in our study with VitD3 and Dex. First of all, Tr1 cells not only produce IL-10 but in addition a large amount of inflammatory cytokines such as IL-5 and IFN-γ (21
). To support this, Levings et al. recently defined human Tr1 cells as IL-10 and IFN-γ positive cells, and their use of IFN-α as a possible cofactor to enhance the development of these cells, suggested that IL-10 is not sufficient to generate human IL-10–producing T cells (23
). This is not the case with cells derived with VitD3/Dex described herein, which produce only IL-10 and no IFN-γ, IL-4, or IL-5 when cultured in the presence of anti–IL-4, anti–IL-12, and anti–IFN-γ ( A). Second, as shown in and as already suggested in other studies (22
), addition of exogenous IL-10 during antigenic stimulation led to heterogeneous populations of T cells producing IL-4 and thus resembling Th2 cells. Furthermore, culture of T cells in the presence of IL-10 but in the absence of IL-4 generates cells which produce no IL-10 ( A, and data not shown using IL-4 KO mice). This is totally in contrast with the IL-10–producing T cells driven in our study with Vit/Dex which produce IL-10 and no IL-4, IL-5, or IFN-γ, and furthermore whose development does not require, but can be inhibited by IL-4 (). Most importantly, the cells derived in our study with Vit/Dex in the absence of Th1 and Th2 polarizing cytokines proliferate, in contrast to the Tr1 cells which do not. The fact that Th1 and Th2 polarizing cytokines downregulated the number and purity of IL-10–producing regulatory T cells resulting from culture in Vit/Dex provides a novel concept regarding the control of regulatory T cells by Th1 and Th2 cells, and has significant implications for the physiological interplay between these cells during an immune response.
We have obtained IL-10–producing cells using a combination of two drugs, Vitamin D3 and Dexamethasone (Vit/Dex). Interestingly, despite their well documented role in inducing cytokine genes in both Th1 and Th2 subsets, none of the T-bet, erm, GATA-3, or c-maf genes appeared responsible for the specific activation of the IL-10 gene in the Vit/Dex-induced cells, as their expression was inhibited in T cells induced with Vit/Dex while IL-10 mRNA expression is dramatically induced in these cells. These results question whether one or few specific transcription factors are specific for the induction in vivo of such a population of regulatory cells. The use of the IL-10–producing cells derived in our study will of course be of advantage in order to answer this question. We also show herein that signaling pathways known to be affected by both these drugs including AP-1 and NF-κB activities are inhibited in these cells as compared with cells driven under neutral conditions. Taken together, this suggests that IL-10–producing regulatory T cells arise in vivo under conditions where the engagement of the signaling pathways previously described to be important for the regulation of cytokine genes produced by Th1 and Th2 cells is minimal. These observations that transcription factors and signaling pathways shown to be involved in the activation of Th1 and Th2-type cytokine genes and thus leading to inflammation, are reduced in the IL-10–producing regulatory T cells developed in our study support the concept that the effect of the immunosuppressive drugs on the development of regulatory T cells producing IL-10 is likely to mimic events that occur during development as autoreactive T cells encounter antigen in the periphery under tolerogenic conditions. Furthermore, our data suggest that in contrast to other cytokine genes, IL-10 gene regulation is not only refractory to the inhibitory action of VitD3 and Dex, but conversely is induced by the synergistic action of the signaling pathways induced by these drugs (). A reciprocal synergistic action of these drugs was also apparent with respect to the downregulation of IL-2 ( A, middle panel).
The use of regulatory T cells in adoptive immunotherapy has been hindered first by the contamination of such populations with Th1- or Th2-derived cytokines that may exacerbate inflammatory pathologies, and second by the inability to expand them in large numbers. The IL-10–producing regulatory T cells derived by the strategy presented here do not suffer these limitations. They could indeed be produced as a homogeneous population after stimulation of T cells with the antigen of interest, and for example adoptively transferred into patients suffering from GVHD, organ-specific autoimmune disorders, or other inflammatory diseases. Alternatively, such populations could be generated to respond either to a well-defined self-antigen or to an unrelated protein antigen provided that the T cells are appropriately triggered and thus could function as regulators via a bystander mechanism. Other studies describing T cells mediating bystander suppression of pathology-inducing T cells (EAE, diabetes, IBD) support the notion that regulatory T cells when located at the site of inflammation could effectively downregulate an immune response (21
). Generating regulatory T cells specific for a distinct antigen to that which the pathogenic T cells react to could be a strategy to inhibit inflammation and autoimmune manifestations where the antigen is unknown. However, this still needs to be addressed in clinical situations. Initially IL-10–producing T cells generated with VitD3/Dex are more likely to be used in situations where the antigen is well defined such as GVHD or organ transplantation.
There is a clear paradox of how regulatory T cells preferentially inhibit T cell responses to distinct antigens, while simultaneously allowing responses to pathogens to occur. Clearly there are various thresholds imposed to regulate the activation of T cells, including positive and negative signals delivered to T cells from the APCs that may differ according to the previous activations encountered by the T cell (64
). In addition, signals from the innate immune response required to overcome negative signals delivered to the T cell or the APC (66
), or the distribution of antigen and the recirculation patterns of T cells, may determine whether a T cell is activated or suppressed (65
). Finally, the ratio of regulatory T cells to effector T cells will undoubtedly also contribute to the outcome of whether an immune response or tolerance ensues (65
The relationship between IL-10–producing regulatory T cells and other described populations of regulatory T cells (9
) is still unknown. Our ability to generate homogeneous populations of regulatory cells in vitro with Vit/Dex will certainly be key in order to address this issue. Indeed, we have generated IL-10–producing T cells using CD4+
T cells from DO11.10 RAG−/−
mice, which are known not to contain CD4+
T cells can inhibit CD45RBhigh
T cell–mediated colitis via the action of both TGF-β and IL-10 (9
), suggesting a role for both cytokines in regulation of mucosal inflammation. A role for TGF-β has also been demonstrated for a number of T regulatory populations, including Th3 and Tr1 cells, in inhibition of autoimmune pathologies, gut inflammation, and/or proliferation of antigen-specific T cells (8
). In addition to its broad antiinflammatory properties, TGF-β has been shown to inhibit the development of both Th1 and Th2 responses (75
). As we now show that Th1 and Th2 cytokines can downregulate the development of IL-10–producing regulatory T cells, this may explain some of the effects of TGF-β to inhibit inflammatory pathologies, as well as its relationship with regulatory T cells (8
Our studies now demonstrate that IL-10–producing regulatory T cells require antigenic stimulation to function to regulate EAE. As protection was only observed when the antigen was delivered intracranially but not intraperitoneally, this strongly suggests that the regulatory cells require activation at the site of inflammation. Furthermore, the inhibition of EAE by these OVA-specific T cells was via a bystander fashion, as they are inhibiting autoreactive T cells responding to CNS-specific antigens and require IL-10 for their function.
In conclusion, our present findings describing the generation of homogeneous populations of IL-10–producing regulatory T cells has allowed us to determine some of the regulatory mechanisms underlying their development and function. Our study will facilitate their potential use in clinical intervention and will be key to further understand the molecular events required for the differentiation and function of these cells.