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Keywords:

  • Regulatory T (Treg) cells;
  • T cells;
  • Transcription factors

Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgments
  4. Conflict of interest
  5. References

Tr1 cells are non-Foxp3-expressing regulatory CD4+ T cells that execute suppressor functions by secreting the anti-inflammatory cytokine IL-10. Differentiation of this T-cell subset is facilitated by the heterodimeric cytokine IL-27, which can activate transcription factors such as c-Maf and Ahr to positively regulate the differentiation of Tr1 cells and their IL-10 production. In this issue of the European Journal of Immunology, an alternate transcriptional network regulated by IL-27 to induce IL-10 production in Tr1 cells is reported by Iwasaki et al. [Eur. J. Immunol. 2013. 43: 1063-1073]. This study shows that IL-27 initiates tandem activation of the transcription factors STAT3 and Egr-2 to induce il10 in Tr1 cells in a Blimp1-dependent fashion. These findings indicate a c-Maf/Ahr independent mechanism that activates IL-10 production by Tr1 cells and suggest that Il10 induction may depend on both the cytokine environment and the molecular context. Thus, Tr1 cells may be another example of the remarkable plasticity of CD4+ T cells and indeed may not constitute a separate lineage of CD4+ T cells but rather represent a developmental endpoint of several T helper cell differentiation pathways.

IL-27 belongs to the IL-12/IL-23 cytokine family and is predominantly secreted by antigen presenting cells upon Toll-like receptor stimulation [1, 2]. It is a heterodimeric cytokine composed of two subunits, IL-27p28 and EBV-induced protein-3 [3]. IL-27 signals through a heterodimeric receptor that consists of IL-27Rα (WSX-1) and gp130 [4, 5]. It activates the Jak/STAT and MAPK pathways and the expression of transcription factors that determine the outcome of IL-27 stimulation during CD4+ Th-cell differentiation [6]. IL-27 has been shown to activate STAT1, which can induce the transcription factor T-bet and IL-12Rβ2 expression to facilitate T helper (Th) 1 differentiation [7, 8]. Nevertheless, IL-27 was dispensable for Th1 differentiation as EBV-induced protein-3 or IL-27R deficient mice mounted a normal Th1 response [5]. Inflammation instigated by infection with pathogens was, however, exacerbated in the absence of IL-27Ra suggesting an anti-inflammatory role for IL-27 [5, 9]. This idea was further strengthened by the finding that IL-27 suppressed IL-2 production during the Th1 response [10] prompting several groups to investigate the anti-inflammatory role of IL-27 during immune response in more detail.

The first direct evidence supporting the anti-inflammatory role of IL-27 came from the observation that IL-27Ra deletion in mice leads to excessive Th17 responses in an experimental autoimmune encephalomyelitis (EAE) setting [11]. It was later discovered that IL-27 restrains Th17 inflammation by suppressing expression of Rorγt, the Th17 lineage-specific transcription factor, in a STAT1-dependent manner [12]. IL-27 also limits Th2-cell responses by suppressing the Th2 transcription factor GATA3 [13]. In 2007, several groups independently found that IL-27 plays a crucial role in the differentiation of IL-10-producing Tr1 cells [14-16]. Following this breakthrough, investigations to understand the molecular pathways activated by IL-27 intensified, and a battery of transcription factors with prominent and recessive roles in Tr1-cell differentiation were identified in the subsequent years. Using CD4+ T cells deficient for STAT3 and STAT1, Stumhofer et al. [15] demonstrated the crucial role of these two transcription factors in activating IL-10 production. Although STAT1 induces the transcription factor T-bet, it is dispensable for IL-27-induced IL-10 production [15]. Utilizing loss-of-function experiments Pot et al. [17] demonstrated the essential roles of three other molecules, c-Maf, IL-21, and ICOS, in IL-27-induced Tr1 differentiation. Interestingly, the transcription factor c-Maf has also been shown to activate IL-10 production in Th17 cells [18]. A recent study by Apetoh et al. [19] revealed high levels of aryl hydrocarbon receptor (Ahr) expression in Tr1 cells. IL-27 activates Ahr in Tr1 cells, and Ahr together with c-Maf transactivates Il10 and Il21 transcription [19] (Fig. 1). Furthermore, the interaction between GITR and its ligand GITRL has been found to increase IL-27 expression and induce Tr1-like cells [20].

image

Figure 1. Summary of the different transcription factors required for Il10 induction in T cells. The figure illustrates the current knowledge of the transcriptional control of il10 in different T-cell subsets. In Tr1 cells two independent transcriptional modules appear to activate il10 upon IL-27 stimulation. While c-Maf and Ahr are required for IL-10 production under certain conditions, and constitute the first module, Iwasaki et al. in this issue of the European Journal of Immunology show that STAT3 can activate a second pathway that leads to the Egr2-dependent induction of Blimp1 and Il10 induction. In Foxp3+ regulatory T (Treg) cells IRF4 mediates the transcriptional activation of Blimp1 IL-10 production depends on both IRF4 and Blimp1. Blimp1 has also been shown to play a critical role in activating il10 in CD8 T cells. In Th2 cells, however, Nfil3 and IRF4 are the major players involved in il10 production. The transcription factors in between brackets have not been tested in any particular cell type (also indicated by the question mark (?)).

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In addition to Tr1 cells, two other CD4+ T-cell subsets, namely Foxp3+ Treg cells and Th2 cells, are exquisitely equipped for IL-10 production [21]. While IL-10 production by Th2 cells is dependent of the expression of certain transcriptional regulators, including Nfil3 [22] and IRF4 [23] (Fig. 1), the molecular requirements for IL-10 produced by Treg cells have been less well studied. Work from our group has recently revealed that IL-10 production by Foxp3+ Treg cells is restricted to a distinct effector subset of these cells expressing high levels of the transcriptional regulator Blimp1 [24]. The differentiation of this Treg-cell subset was dependent on IRF4, and we demonstrated that IL-10 production required the activities of both IRF4 and Blimp1 [24] (Fig. 1).

In this issue of the European Journal of Immunology, Iwasaki et al. [25] report the dominant role of the transcription factor Egr2 in the induction of Blimp1 and IL-10 production in Tr1-like cells. The same group previously reported that Egr2 is essential for the differentiation of a Foxp3-negative CD4+ T-cell subset that coexpressed Lag3 and IL-10 [26], consistent with a Tr1 phenotype. In the current study Iwasaki et al. aimed to identify factors that activate Egr-2. IL-27 along with TCR stimulation was potent in inducing Egr-2, and Egr-2 expression co-localized with IL-10 in intracellular staining [25]. The authors demonstrate the requirement of IL-27 signaling by showing impaired Egr-2 induction in the absence of IL-27Ra. Using conditional deletion of Egr2 in T cells or overexpression of Egr-2 in CD4+ T cells they demonstrate modulation of Blimp1 (Prdm1) transcripts in an Egr-2-dependent manner [25]. Furthermore, Blimp1 induction was impaired in IL-27Ra-deficient CD4+ T cells. Together, these results demonstrate that Blimp1 expression in CD4+ T cells stimulated by TCR signals and IL-27 is dependent on Egr-2. Iwasaki et al. [25] also found that the Egr2-dependent Blimp1 expression was specific to IL-27 since the cytokines IL-12 or IL-23 which belong to the same cytokine family as IL-27 only modestly induced Blimp1. To determine if Egr-2 directly induces Blimp1, the authors performed promoter reporter assays and revealed direct enhancement of Blimp1 promoter activity by Egr-2. Importantly, they also demonstrate Egr-2 binding to the −1 000 region of the Blimp1 promoter using a ChIP assay [25]. Finally, using CD4+ T cells deficient for Blimp1 the authors show impairment of IL-10 production upon IL-27 stimulation but intact induction of Egr-2.

As STAT3 and STAT1 have been shown before to be important for IL-27-induced IL-10 production [15, 27], Iwasaki et al. [25] also investigated whether Egr-2 can be activated by either of these transcription factors. Interestingly, only STAT3-deficient cells showed impaired Egr-2 induction when stimulated with IL-27, while both STAT3 and STAT1 deficiency negatively impacted on IL-10 production. IL-6 and IFN-γ can activate STAT3 and STAT1, respectively, and the authors therefore examined whether these cytokines can also induce Egr-2. Only IL-6 induced Egr-2 levels comparable with those induced by IL-27 stimulation, however, IL-10 and Blimp1 levels were much lower upon IL-6 stimulation. Overall these results reveal a novel molecular pathway to IL-10 production operating in CD4+ T cells. This pathway is guided by the IL-27-induced activation of STAT3 and Egr2. Egr2 induces expression of Blimp1, which is critical for IL-10 expression in IL-27-stimulated CD4+ T cells.

Over the past few years a plethora of transcription factors have been reported to be involved in IL-10 production in diverse T-cell populations (reviewed in [21]). Although the cytokine requirements for Il10 induction in these cell types differ, there is overlap in the downstream transcription factors that activate Il10. c-Maf was originally considered a Th2-specific transcription factor, being identified as an inducer of IL-4, but it has now been shown to activate Il10 in Tr1 cells and Th17 cells [18] but not in Th2 cells [28]. IRF4, on the other hand, was demonstrated to be required for IL-10 production in cell types as disparate as Treg cells [24], Th2 cells [23] and possibly Th17 cells [29], suggesting that IRF4 belongs to a core group of transcription factors critical for Il10 induction. Blimp1, although expressed in several Th-cell subsets, has only been shown to activate IL-10 production in Treg cells [24], CD8+ T cells [30], and now in IL-27-stimulated CD4+ T cells [25]. Similar to c-Maf Blimp1 is not required for IL-10 production in Th2 cells [31]. In contrast, the bZIP transcription factor Nfil3 has been shown be critical for IL-10 production from Th2 cells but also from IL-27 stimulated Th1 cells [22], and Ahr was found to be required for IL-10 production by in vitro generated Tr1-like cells [19] (summarized in Fig. 1). Interestingly, Ahr is induced by TGF-β signaling, which recently has been demonstrated to repress Blimp1 expression [32], suggesting that different transcriptional modules may be active in IL-10-producing Tr1-like cells. Thus, it appears that Blimp1 regulates IL-10 production only in certain cytokine environments. It will be interesting to explore whether the Egr-2—Blimp1 axis is also operational in cell types other than Tr1 cells to mediate IL-10 production.

While STAT1 and STAT3 are both important for IL-10 production in Tr1 cells [15], the study of Iwasaki et al. [25] has only illuminated the downstream effects of STAT3. How STAT1 regulates IL-10 is still an open question. Similarly, how specificity is achieved in the presence of cytokines, such as IL-6, IL-21, IL-23, and IL-27 that all activate STAT3 phosphorylation but show different outcomes with respect to Blimp1 and Il10 induction, is a fascinating topic. It becomes increasingly apparent that the Il10 gene locus is highly promiscuous and has the flexibility of recruiting a multitude of factors that regulate its transcriptional activity [21]. As so often during cellular fate decisions, the cytokine milieu dictates the transcription factor choice and the outcome of the differentiation processes. Thus, the expression of specific cytokine receptors, availability of cytokines, differential phosphorylation of the STAT family proteins, and induction of transcriptional activators and/or antagonistic factors in a combinatorial fashion may determine the induction of Il10 in a context-specific manner. The expression of individual activators of Il10 such as Blimp1, c-Maf or Nfil3 alone may not be sufficient to induce IL-10. A “choice based on availability” theory may be applicable in this context. As TGF-β suppresses Blimp1 expression in Th17 cells [32], Blimp1 becomes unavailable and c-Maf and Ahr subsequently activate Il10. This can be viewed as a fail-safe mechanism to ensure stable IL-10 production during various inflammatory conditions or in different tissues.

Although IL-10-producing Tr1 cells were identified in the mid 1990s [33], their identity and ontogeny has remained elusive. Tr1 cells are typically identified as IFN-γ and IL-10 double-producing cells, which has led to the early hypothesis that they derive from Th1 cells. More recent evidence, however, has pointed to a link to Th17 cells, as it has been observed that many of the IL-10-producing cells in the central nervous system during the resolution of EAE or in the gut after polyclonal T-cell stimulation derive from IL-17+ precursors [34]. Such an ontogeny would also be supported by the similarities between the cytokine cues and transcription factors involved in the differentiation of Th17 cells and in vitro generated Tr1-like cells. While the cytokines and transcription factors that can induce IL-10 have been studied extensively in vitro, it is an open question as to what the requirements for Tr1-cell differentiation are in vivo. The results from Iwasaki et al. [25] have made the complicated transcriptional network controlling IL-10 expression more complex by introducing Egr-2 as a factor required for the induction of Blimp1-dependent IL-10 production. Importantly, their results and earlier data [15, 17, 19] suggest that multiple pathways are involved in the differentiation of Tr1-like cells. We therefore propose a model whereby Tr1 cells do not constitute a separate lineage of CD4+ T cells but rather represent a developmental endpoint of several T helper cell differentiation pathways (Fig. 1). Consequently, the transcriptional regulation of Tr1 differentiation and Il10 induction may depend on both the cytokine environment and the molecular context, and Tr1 cells may be another example of the remarkable plasticity of CD4+ T cells.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Acknowledgments
  4. Conflict of interest
  5. References

The authors thank the National Health and Medical Research Council (NHMRC) and the Australian Research Council (ARC) for financial support.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Acknowledgments
  4. Conflict of interest
  5. References

The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Acknowledgments
  4. Conflict of interest
  5. References
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Abbreviation
Ahr

aryl hydrocarbon receptor