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Foxp3 is a key transcription factor involved in the generation and function of regulatory T (Treg) cells. Transforming growth factor β (TGF-β) induces Foxp3, which generates inducible Foxp3+ Treg cells from naïve T cells, and interleukin 6 (IL-6) inhibits the generation of inducible Treg cells and induces T helper cells that produce IL-17 (TH-17 cells). However, a role for IL-4 in the generation of TGF-β-induced Treg cells and/or the generation of effector CD4+ T helper cells has not been studied. Here, we show that IL-4 blocked the generation of TGF-β-induced Foxp3+ Treg cells. Instead, IL-4 induced a population of T helper cells that predominantly produce IL-9 and IL-10. The IL-9+IL-10+ T cells did not exhibit any regulatory properties in spite of producing large quantities of IL-10. Adoptive transfer of IL-9+IL-10+producing T cells into RAG-1-deficient mice induced colitis and peripheral neuritis. Interestingly, the severity of tissue inflammation was aggravated when IL-9+IL-10+ T cells were co-transferred with CD45RBhi CD4+ effector T cells into RAG-1-deficient mice, which indicated that IL-9+IL-10+ T cells do not display any suppressive function and therefore constitute a unique population of IL-10-producing helper-effector T cells that promote tissue inflammation.
Upon encounter of specific antigen, naïve CD4+ T cells get activated, expand and differentiate into various effector T cell subsets: T helper (TH) 1, TH2 or TH-17, which are characterized by distinct patterns of cytokine secretion. These subsets of T cells have specific effector functions and recruit different cell types at the site of inflammation by the virtue of cytokines that they produce. T-bet, GATA-3 and ROR-γt are the transcription factors required for the generation of TH1, TH2 and TH-17 cells respectively 1–3. Whereas TH1 cells clear intracellular pathogens and mediate autoimmune tissue inflammation, TH2 cells are required for the clearance of extracellular pathogens, and an exaggerated TH2 response induces asthma, allergy and atopy. On the other hand, TH-17 cells appear to have a role in controlling both intracellular and extracellular pathogens and in orchestrating autoimmune tissue inflammation4,5. Since exaggerated response of TH1, TH2 and TH-17 cells can induce tissue inflammation, the maintenance of immune homeostasis and prevention of immunopathology requires tight regulatory mechanisms to control these effector T cells by subsets of regulatory T cells. Different types of T cells have been described that mediate these regulatory functions in vivo and prevent autoimmunity and tissue inflammation. The CD4+CD25+Foxp3+ and IL-10-producing Tr1 cells play a crucial role to regulate effector T cell functions6,7. Natural occurring Treg cells, which are generated in the thymus and express a transcription factor, forkhead box transcription factor (Foxp3) (A002750) http://www.signaling-gateway.org/molecule/query?afcsid=A002750, inhibit effector T cells and play a crucial role in the maintenance of peripheral tolerance8. In addition, emerging data suggest that Foxp3+ T cells can also be generated in the peripheral immune compartment 9–11. However, the in vivo factors that generate Foxp3+ induced Treg cells in the periphery are still unclear.
In vitro, TGF-β (A002271) (http://www.signaling-gateway.org/molecule/query?afcsid=A002271) together with TCR and co-stimulatory signals can induce Foxp3 and generate Treg cells from naïve T cells 12. TGF-β appears to be a crucial factor in this process, as mice deficient in TGF-β1 show a defect in the maintenance Foxp3+ Treg cells and develop a lethal lymphoproliferative disorder characterized by enhanced TH1 and TH2 effector responses. In addition, Foxp3+ T cells from mutant “FILIG” mice which display attenuated expression of Foxp3, produce increased amounts of TH2 cytokines13, demonstrating that Foxp3 negatively regulates the TH2 developmental program. How Foxp3 regulates TH2 responses, whether it is cell intrinsic where Foxp3 suppresses induction of TH2 specific transcription factors or whether Treg cells suppress TH2 cells indirectly has not been directly tested. Initial reports14,15 suggest that Foxp3 can bind to the TH2 lineage specific transcription factor GATA-3, but the consequence of this interaction is not well understood. One outcome of this interaction, however, clearly is that Foxp3 can directly inhibit the development of TH2 cells.
In addition to Foxp3+ Treg cells, T regulatory type 1 (Tr1) cells can suppress effector T cell function and tissue inflammation independently of Foxp3 expression. Tr1 cells predominantly produce IL-10 (A001243) (http://www.signaling-gateway.org/molecule/query?afcsid=A001243) and suppress immune responses in a contact-independent manner via the production of IL-10. Although IL-10 was initially shown to induce Tr1 cell differentiation, the Tr1 cells thus generated do not proliferate due to the suppressive nature of IL-10. This observation suggested that factors other than IL-10 might be important for the differentiation and expansion of IL-10-secreting Tr1 cells. Several recent papers, including one from our laboratory, demonstrated that IL-27 together with TGF-β induces the differentiation of Tr1 cells 16,17. IL-10-producing Tr1 cells thus generated have potent immunosuppressive properties in that they not only inhibit autoimmune disease and tissue inflammation, but also contribute to successful engraftment of HLA-mismatched bone marrow transplant6. On the other hand, IL-10-producing T cells are not always anti-inflammatory, and under some circumstances they have been shown to promote autoimmunity 18,19. It has not been elucidated yet whether these IL-10-producing cells co-produce other cytokines that negate their anti-inflammatory function of IL-10 and, what are the differences between these pro- and anti-inflammatory IL-10-producing cells.
In this study we show that IL-4, a TH2 differentiating cytokine, inhibits TGF-β-induced Foxp3 expression and thus suppresses the de novo generation of Foxp3+ Treg cells. Unexpectedly, the two cytokines together (TGF-β plus IL-4) induce the generation of predominantly Foxp3−IL-9+IL-10+ T cells that, unlike Tr1 cells, do not suppress T cell responses even though they produce IL-10. More surprisingly, the IL-9+IL-10+ T cells not only induce the development of colitis but also induce peripheral neuritis when co-transferred with CD45RBhi CD4+ effector T cells into RAG-1-deficient mice. IL-9 was initially cloned as a T cell growth factor whose receptor shares the common γ-chain with IL-2, IL-4, IL-7, IL-15 and IL-2120, and was shown to be involved in TH2 type immunity against helminthic infection21. Our data suggest that T cells differentiating in the presence of IL-4 and TGF-β, undergo a unique developmental program characterized by production of IL-10 and IL-9, which may not only subvert the immunosuppressive function of IL-10-producing cells, but also promote tissue inflammation, probably by regulating the expansion and/or function of other effector T cells.
TGF-β induces the de novo generation of Foxp3+ inducible Treg cells (iTreg cells) from naïve CD4+ T cells. We have previously shown that both IL-6 and IL-27 inhibit TGF-β mediated induction of Foxp3 12,16. To test the effect of IL-4 on the induction of Foxp3 by TGF-β, we used GFP− naïve CD4+ T cells from mice with a bicistronic enhanced green fluorescent protein (EGFP) reporter cloned into the endogenous Foxp3 locus (Foxp3-GFP mice) 12. As expected, TCR stimulation in the presence of TGF-β, induced Foxp3-GFP+ expression in up to 80% of T cells, and this was inhibited (reduced to 22%) in the presence of IL-4; thus, IL-4 actively inhibits the induction of Foxp3 in the presence of TGF-β (Fig. 1a). To understand the mechanism by which IL-4 mediates the inhibition of Foxp3 expression, we tested the cytokines produced from T cells activated in the presence of TGF-β plus IL-4. Naïve CD4+ T cells stimulated in the presence of IL-4 differentiated into TH2 cells and produced IL-10, IL-5, IL-13 and very low levels of IL-9. The combination of TGF-β with IL-4 inhibited IL-4, IL-13 and IL-5 production while sparing the IL-10 production from these T cells. Furthermore, activation in the presence of TGF-β plus IL-4 also induced the secretion of large amounts of IL-9 (Fig. 1b,c and Supplementary Fig. 1). Importantly, T cells expanded in the presence of TGF-β plus IL-4 produced as much IL-10 as TH2-differentiated T cells, but instead of IL-4 but they co-produced IL-9 with IL-10 (Fig. 1b and Supplementary Fig. 2a). To study whether APCs contribute to the generation of this unique subset of IL-9+IL-10+ cells or whether IL-9 was being produced by APCs, we also activated naïve CD4+ T cells with plate-bound anti-CD3 and anti-CD28 (without any APCs) in the presence of TGF-β together with IL-4. Even in the absence of APCs, IL-4 inhibited the induction of Foxp3 induced by TGF-β and induced T cells that co-produced IL-9 and IL-10 (Supplementary Fig. 2a).
To characterize the source of IL-10 production at the cellular level, we performed intracellular cytokine staining and compared T cells activated in the presence of TGF-β, IL-4 or both. Activation of naïve T cells in the presence of IL-4 did not induce the expression of Foxp3-GFP, but induced intracellular IL-4 and IL-10 production as expected, as a part of TH2 development. Neither IFN-γ nor IL-17 production was observed from responding T cells. However, addition of IL-4 to TGF-β inhibited Foxp3 expression and induced a predominant production of IL-10 from these cells with a dramatic decrease in other TH2 cytokines production including IL-4 (Fig. 1c). We could not perform staining for IL-9 at the single cell level due to lack of appropriate antibody reagents. Therefore, co-expression of IL-10 and IL-9 could not be established at the cellular level in the T cells differentiated by TGF-β plus IL-4. However, the T cells differentiated in the presence of TGF-β plus IL-4 produced both IL-9 and IL-10 as assessed by ELISA and quantitative real time PCR (Fig. 1b and Supplementary Fig. 1b–d).
To determine whether IL-9 production is dependent on IL-10 in the TGF-β plus IL-4-differentiated T cells, naïve T cells from IL-10-GFP reporter mice22 were differentiated in the presence of TGF-β plus IL-4. Then, IL-10-GFP+ and IL-10-GFP− T cells were sorted by flow cytometry based on GFP expression. We could detect IL-9 mRNA expression in IL-10-GFP+ and IL-10-GFP− expressing cells (Supplementary Fig. 2a), although maximal expression was only observed when IL-10-GFP+ and IL-10-GFP− cells were together. These TGF-β plus IL-4 differentiated T cells co-produce IL-9 and IL-10 and we could not detect any IL-10+IL-17+ or IL-10+IFN-γ+ double producing cells (Supplementary Fig. 2b). However, small numbers of IL-4+IL-10+ double producers (about 8%) are present in the cells activated in the presence of TGF-β plus IL-4, especially when T cells are activated in presence of low amounts of TGF-β. Titration of TGF-β had an important effect on IL-4 production, the percentage of IL-4+IL-10+ cells decreased with the increasing amounts of TGF-β (•••••• •••• •••••). The IL-4+IL-10+ double producers probably represent a transitional state since IL-4 is extinguished with increasing amounts of TGF-β (Supplementary Fig. 2b). These results show that like IL-6 and IL-27, IL-4 is also able to inhibit the induction of Foxp3 and induce a predominant production of IL-9 and IL-10 from the responding T cells.
Previous studies have shown that IL-10-producing Tr1 cells do not proliferate well and appear to acquire an anergic phenotype. However, IL-9+IL-10+ T cells induced in the presence of TGF-β plus IL-4 were not anergic and vigorously proliferated when activated by CD3 antibody (Fig. 1d). We could not detect, in the T cells differentiated with TGF-β plus IL-4, the expression of T-bet, GATA-3, Foxp3 and ROR-γt, lineage specific transcription factors necessary for the generation of TH1, TH2, Treg and TH-17 cells respectively (Fig. 1e). We previously hypothesized that TGF-β may act together with other cytokines to induce new T cell subsets 23 as exemplified by the observation that TGF-β in combination with IL-6/IL-21 or IL-27 induced TH-17 and Tr1 respectively. Similar to TGF-β and IL-6 that cross-regulate each other’s function and give rise to unique TH-17 subset, TGF-β in combination with IL-4 inhibited the expression of TH1, TH2, TH-17 and Treg cells specific transcription factors (Fig. 1e).
To understand the role of IL-4 in the active inhibition of TGF-β-induced Foxp3, naïve T cells from IL-4 transgenic mice were cultured in the presence of different concentrations of TGF-β. Surprisingly, TGF-β, ••••••••••••••••••••, did not induce the generation of Foxp3+ Treg cells, suggesting that IL-4 may play a dominant role in inhibiting the induction of Foxp3 expression (Fig. 2a). Upon binding to its receptor, IL-4 induces several signaling pathways including insulin receptor substrate (IRS)-2 and STAT6, which form a signaling complex in association with the cytoplasmic tail of the IL-4 receptor 24. However, STAT6 is a dominant pathway of TH2 differentiation because it induces GATA-3, the master transcription factor for TH2 differentiation. To study whether STAT6 is involved in inducing IL-4-mediated Foxp3 inhibition, we crossed STAT6-deficient mice with the Foxp3-GFP ‘knock-in’ mice to generate a STAT6KO.Foxp3-GFP strain.
Naïve CD4+ T cells from Foxp3-GFP and STAT6KO.Foxp3-GFP mice were purified and cultured in the presence of TGF-β, IL-4 or a combination of these two cytokines. Unlike wild-type Foxp3-GFP T cells in which IL-4 inhibited the induction of Foxp3, STAT6KO.Foxp3-GFP T cells expressed higher levels of Foxp3 (80%) even in the presence of TGF-β plus IL-4 (Fig. 2b). Furthermore, when we analyzed the cytokines produced by the STAT6KO cells stimulated in the presence of TGF-β plus IL-4, IL-10 and IL-9 was not induced but these cells continued to express Foxp3 in the presence of TGF-β plus IL-4 (Fig. 2c,d). Since GATA-3 is downstream of STAT6, we also analyzed the induction of IL-9 and IL-10 by TGF-β plus IL-4 in the conditional GATA-3-deficient mice and similar to the STAT6-deficient mice, GATA-3-deficient mice could not produce IL-9 or IL-10 upon differentiation in the presence of TGF-β plus IL-4 (Fig. 2e). The effect of STAT6 and GATA-3 in the induction of IL-9+IL-10+ T cells may not be a direct but an indirect effect, since in the absence of STAT6-GATA-3 signaling, TGF-β-induced Foxp3 can not be antagonized by IL-4 resulting in the predominant induction of Foxp3+, which may not allow differentiation of T cells to produce IL-9 and IL-10. Altogether, these data suggest that the inhibition of Foxp3 expression by IL-4, is mediated through IL-4–STAT6 pathway. Thus, the de novo generation of Foxp3+ iTreg cells is likely modulated by IL-4 through the activation of STAT6 GATA-3 pathway. We next explored whether IL-4 affected the function of already established naturally occurring Foxp3+Treg cells via STAT6. When we cultured sorted Foxp3− T effector cells with either Foxp3-GFP+ or STAT6KO.Foxp3-GFP+ Treg cells, Foxp3-GFP+ Treg cells lost a substantial part of their suppressive ability in the presence of IL-4 whereas STAT6KO.Foxp3-GFP+ Treg cells still remained partly suppressive despite the presence of IL-4 (40 % vs 70% suppression) (Fig. 2f). Thus, the IL-4–STAT6 pathway not only affects the generation of induced Treg cells but also regulates the suppressive capacity of natural-occurring Treg cells due to a mechanism that is intrinsic to Treg cells.
To assess whether GATA-3 could affect Foxp3 expression, we isolated CD4+ T cells from GATA-3 conditional deficient mice and activated them in the presence of TGF-β. On day 4 after activation, T cells were harvested and mRNA was extracted and analyzed for expression of GATA-3 and Foxp3 by quantitative PCR. In GATA-3-deficient CD4+ T cells, TGF-β induced Foxp3 mRNA expression was 3-fold higher than in wild-type CD4+ T cells. On the other hand, the addition of TGF-β decreased GATA-3 expression in wild type CD4+ T cells suggesting a possible inter regulation between these two transcription factors (Fig. 3a).
Because of the correlation between Foxp3 and GATA-3 expression, we sought to determine whether the two transcription factors could physically interact and associate with each other. We used two different approaches to test these possibilities. The first approach was to co-transfect Flag-tagged GATA-3 and renilla-luciferase-tagged Foxp3 cDNA into HEK-293 cells. After lysis of the cells, GATA-3 (or a control protein) was immunoprecipitated using anti-Flag and Foxp3 pull down was assessed in the immunoprecipitate by monitoring the renilla-luciferase expression by luminescence. Precipitation of GATA-3 strongly increased the luciferase activity compared to negative control (empty vector) or other transcription factor such as STAT6 (Fig. 3b) suggesting GATA-3 pulled down luciferase-tagged-Foxp3. We further confirmed this result by performing co-immunoprecipitation assays. For this purpose, Flag-GATA-3 was co-transfected with Myc-Foxp3. After lysis of the cells, immunoprecipitation was performed using anti-FLAG coated beads. To detect whether Foxp3 was co-precipitated with GATA-3, the samples were run on a SDS-PAGE gel and after transfer, the membrane was blotted using anti-myc. GATA-3 was co-immunoprecipitated with Foxp3 showing the direct association of the two proteins (data not shown). These assays clearly demonstrated that GATA-3 and Foxp3 could physically associate with each other.
Next, we sought to determine the functional significance of this interaction. We assessed whether Foxp3 could affect TH2 differentiation. Foxp3 was cloned into a retroviral vector (MSCV) and was transduced into activated CD4+ T cells differentiated under TH2 polarization conditions. T cells expressing Foxp3 were sorted by flow cytometry, RNA was extracted and analyzed for the expression of various cytokines. In parallel, a cohort of cells from each transduction condition was maintained in culture for 2–3 days, re-stimulated and the supernatant was collected for cytokine analysis by ELISA.
Whereas T cells transduced with a control retrovirus vector expressed high levels of IL-10, IL-13 and IL-4 with modest levels of IL-5 transcripts, consistent with T cells differentiated into TH2 effector cells; T cells transduced with Foxp3 dramatically reduced their expression of IL-4, IL-15 and IL-13 but retained substantial expression of IL-10. Similarly, at the protein level, control-transduced T cells produced IL-4 with low levels of IL-2 whereas Foxp3-transduced T cells showed a decreased production of IL-4 and an increase in the production of IL-10 (Fig. 3c). This is consistent with our initial observation that activation of T cells in the presence of IL-4 plus TGF-β results in the inhibition of IL-4, IL-5, IL-13 but spares IL-10 production from these cells. This would suggest that Foxp3 interferes with the expression of genes controlled by GATA-3, presumably by binding or interfering with its ability to transactivate specific TH2 genes. Specifically, these results suggest that the interaction of Foxp3 with GATA-3 inhibits its ability to transactivate promoters that are under the control of GATA-3 such as IL-4, IL-5 and IL-13 but not IL-10, which is most likely controlled by a different transcription factor.
To address this hypothesis, we analyzed the ability of Foxp3 to inhibit GATA-3-mediated transactivation of an Il5 promoter-luciferase construct. Jurkat cells were transfected with GATA-3, Foxp3 or both GATA-3 and Foxp3 in the presence of the Il5-luciferase construct and luciferase activity was measured after activation of the cells with PMA plus ionomycin. As expected, GATA-3 strongly transactivated Il5 promoter-luciferase activity, whereas this effect was reduced by 50 % when Foxp3 was co-expressed (Fig. 3d). Altogether these results show that Foxp3 can directly interact with GATA-3, and this association inhibits GATA-3-mediated transactivation of Il5, which is one of its target genes.
Since IL-10-producing Tr1 cells have been shown to suppress T cell proliferation in vitro and to inhibit tissue inflammation in vivo 25, we wanted to determine whether naïve T cells differentiated in the presence of TGF-β plus IL-4 that produce IL-9 and IL-10 are also suppressive. Although, Foxp3+ Treg cells are anergic and inhibited effector T cell proliferation in vitro, T cells generated in the presence of TGF-β plus IL-4 were not anergic nor suppressive, as they vigorously proliferated on their own and together with effector T cells further enhanced T cell proliferation (Fig. 4a). The addition of anti-IL-9 or anti-IL-10 neutralizing antibodies to the cultures of effector T cells with IL-10.GFP+ T cells generated by TGF-β plus IL-4, decreased proliferation but did not completely abolish it (Fig. 4a). These data suggest that unlike Foxp3+ Treg cells and IL-10-producing Tr1 cells, T cells induced by TGF-β plus IL-4 do not show any suppressive effects in vitro but readily proliferate and therefore, behave like effector T cells.
It is clear that Tr1 cells not only suppress proliferation in vitro but IL-10-producing cells can suppress induction of colitis in vivo as well 26,27. Therefore, to further characterize IL-9+IL-10+ T cells generated in the presence of both TGF-β and IL-4, we assessed their function in vivo in a T-cell transfer model of colitis. Naïve T cells purified from IL-10.GFP reporter mice were activated in the presence of TGF-β and IL-4. At day 4, the TGF-β + IL-4 treated IL-10+GFP+ T cells sorted by flow cytometry based on GFP expression. RAG-1-deficient mice were reconstituted with either effector T cells (Foxp3−/CD45RBhi T cells) (Group 1, n = 8), effector T cells together with (TGF-β + IL-4 stimulated IL-10.GFP+ T cells (‘T4-IL-10.GFP+ T cells’) (Group 2 n = 8), (T4-IL-10.GFP+ T cells alone (Group 3, n = 8) or effector T cells together with naturally-occurring Treg cells (Group 4, n = 6), and the development of colitis was followed by monitoring the body weight of the mice and scored as described previously28.
Histopathologic analysis was also performed to evaluate the induction and severity of the disease. All the mice reconstituted with effector T cells did not lose weight significantly but developed severe colitis as observed by histopathology with an average colitis score of 7.5 ± 3.5 (Fig. 4c). As a control to show that effector T cells can indeed be regulated, mice that received both effector T cells and natural-occurring Treg cells were protected from both clinical and histological disease (average colitis score 3.7 ± 1.5). Interestingly, recipient mice of T cells cultured in the presence of TGF-β plus IL-4 showed a weight loss and the development of a more moderate colitis (average colitis score 5.7 ± 2.6). Furthermore, when effector T cells were co-transferred with IL-9+IL-10+ T cells generated in the presence of TGF-β and IL-4 (T4-IL-10.GFP+ cells), the mice showed the greatest weight loss and histopathology revealed the development of a more severe colitis compared to mice transferred with effector T cells alone (average colitis score: 8.8 ± 2.3) (Fig. 4c). Surprisingly, however, the mice receiving either T4-IL-10.GFP+ T cells or both effector T cells (CD45RBhi) with the T4-IL-10.GFP+ T cells, additionally showed signs of clinical neurological disease characterized by impaired motor coordination with flaccid monoparesis or asymmetric paraparesis of the hind limbs. Muscular strength and tonus in forelimbs and tail did not appear to be affected. These clinical signs clearly differed from the typical manifestations of experimental autoimmune encephalitis (EAE), i.e., limp tail and ascending paralysis, and suggested rather a peripheral neuropathy. Consistent with this clinical appearance, histopathological analysis showed evidence of peripheral neuritis in the mice that received both effector T cells and T4-IL-10.GFP+ T cells or T4-IL-10.GFP+ T cells alone. However, consistent with the clinical picture, no inflammation in brain or spinal cord was observed (Fig. 4c). At week 8–9 after transfer and after the development of colitis, animals were sacrificed, cells were retrieved from lymph nodes and cytokine production was analyzed by intracellular cytokine staining. T cells from the RAG-1-deficient mice that received only the effector T cell (CD45RBhi) produced mostly IFN-γ and very little IL-17 (Supplementary Fig. 3a). However, T cells from the mice receiving both effector (CD45RBhi) and T4-IL-10.GFP+ T cells showed production of both IL-17 and IFN-γ, but the production of IL-17 predominated over IFN-γ in these cells. And while the expression of both IL-17 and IFN-γ was observed in IL-10.GFP+ and IL-10.GFP− cells, over a third of the IL-10.GFP+ cells co-produced both IL-17 and IFN-γ (Supplementary Fig. 3b). These data suggest that IL-9–IL-10-producing T cells generated in the presence of TGF-β and IL-4 do not act as regulatory T cells but rather they potentiate functions of effector T cells, resulting in additional tissue inflammation not induced by the transfer of effector T cells alone.
In this paper we show that when naïve T cells are activated in the presence of both IL-4 and TGF-β, Foxp3 expression is inhibited and a subset of T cells is generated that selectively produces IL-10 and IL-9. Although IL-10-producing cells are known to have regulatory properties, the IL-9–IL-10-producing cells described here do not regulate but induce tissue inflammation.
IL-4 is involved in TH2 T cell differentiation and it is well described that IL-4 receptor signals are transduced by the transcription factors STAT6 and GATA-3. To date no lineage specific transcription factor(s) have been identified for IL-10-producing TR1 cells. It has been suggested that GATA-3 may participate in the transcriptional regulation of IL-10. However, our data demonstrate that IL-9–IL-10-producing cells induced in presence of TGF-β and IL-4, did not express GATA-3 suggesting that the presence of TGF-β could antagonize the induction of GATA-3 yet these cells continued to produce IL-10.
STAT proteins play an important role in T cell differentiation 29. STAT6 is involved in the TH2 differentiation and a STAT1 binding site has been found in the IL-10 promoter 29,30. In this study, we demonstrate that the inhibition of TGF-β induced Foxp3+ by IL-4 and generation of the IL-9–IL-10-producing T cells was at least partially reversed in a STAT6 deficient and GATA-3-deficient mice, suggesting that STAT6-GATA-3 may play an important role in the generation of IL-9–IL-10-producing cells. However, an alternate interpretation of these data is that in the absence of IL-4 signaling in the STAT6-deficient and GATA-3-deficient mice, there is predominant an unopposed induction of Foxp3 which prevents the differentiation of T cells to produce IL-9 and IL-10. The lack of GATA-3 expression in IL-9–IL-10-producing cells induced by TGF-β plus IL-4 would partly support this interpretation.
In addition to IL-10, IL-9 is produced by the T cells differentiated in the presence of TGF-β plus IL-4, suggesting that TGF-β is not simply scaling back of TH2 responses, but rather TGF-β and IL-4 together are inducing a new gene program that induces IL-9 production. Whether the combined effects of TGF-β and IL-4 induces another transcription factor not associated with either with TH2 or Treg cells has not been addressed. Interestingly, Foxp3 and GATA-3 physically associate with each other; this finding is perhaps reminiscent of the interaction between Foxp3 with ROR-γt, which reciprocally regulates induction of Treg or TH-17 cells 12,31. Binding of Foxp3 to GATA-3 may regulate the molecular mechanism by which TGF-β and IL-4 mutually inhibit the generation of Treg and TH2 cells. However, this mutual regulation results in the production of IL-9 by the differentiating T cells. During the course of our studies, two other groups reported that Foxp3 and GATA-3 physically associate with each other 14,15. Our results confirm these findings and show that Foxp3 does not only interact with GATA-3 but also inhibits the ability of GATA-3 to transactivate the promoter of one of the target TH2 genes, Il5.
We previously showed that TGF-β together with IL-27 can induce and expand IL-10-producing cells that co-produce IFN-γ ••• show strong immunoregulatory properties•16. IL-10 has been shown to be a potent anti-inflammatory cytokine which inhibits TH1, TH-17 and TH2 mediated immune responses 32–34. The significance of IL-10 in regulating immune responses has been demonstrated in human infectious diseases such as tuberculosis, malaria, leishmaniasis and schistosomiasis 35–37. Besides T cells, IL-10 can be produced by numerous cell types including macrophages, DCs, B cells and NK cells. IL-10 production from T cells plays a major role in down regulating T cell expansion during antigen-specific immune responses. Although Tr1 cells produce high amounts of IL-10, they can also co-produce variable amounts of IFN-γ and are able to suppress in vitro T cell responses in a Foxp3-independent manner. These findings have led to a view that IL-10 produced by the T cells have a major role in suppressing inflammation. Here we have shown that IL-10-producing cells can be generated in large numbers when naïve T cells are cultured in the presence of IL-4 plus TGF-β; however these T cells also produce IL-9, do not have regulatory properties and fail to control inflammation when transferred in vivo. The production of IL-10 and TGF-β is associated with the maintenance of peripheral tolerance by Treg cells25. In vivo, the absence of IL-10 exacerbates airway inflammation 38. These data argue that production of IL-10 by T cells may not always have suppressive functions and that the context in which IL-10 is produced by T cells may be equally important in determining whether IL-10 producing T cells are anti- or pro-inflammatory.
It is known that IL-10 plays an important role in regulating tissue inflammation in the gut. More specifically, it has been shown that IL-10 produced by the Treg cells is essential for this in vivo suppression, since IL-10 deficient Treg cells cannot cure T cell-transfer-induced colitis 27,39. In this study, the IL-10-producing T cells generated in presence of TGF-β and IL-4 induce colitis and when co-transferred with effector T cells, not only not prevent the development of the disease but also induce peripheral neuritis, a disease not induced by effector T cells (CD4+CD45RBhi) alone. These data strongly suggest that the IL-9+IL-10+Foxp3− T cells, generated in the presence of TGF-β and IL-4, do not have suppressive functions and therefore represent a unique population of IL-10-producing T cells that promote inflammation. It is not clear at this stage whether IL-9, IL-10 or both cytokines are playing a role in inducing tissue inflammation, but we believe that IL-9 produced by these T cells may be a key factor in promoting their pro-inflammatory activity. This is consistent with the observation that IL-9 in the lung promotes TH2-mediated allergic pulmonary disease characterized by high level of TH2-cytokines 40,41. We also have found that IL-9 receptors are expressed on the effector T cells (data not shown) and these cells therefore have the potential to respond to IL-9 and differentiate in the presence of IL-9+IL-10+ cells. Since IL-9 belongs to the IL-2, IL-4, IL-7, IL-15 and IL-21 cytokine family and utilizes the common γ chain for function, we believe that IL-9 produced by T cells stimulated by TGF-β and IL-4 may function as a growth and differentiation factor for other effector T cells. This, we believe, is one of the explanations why IL-9+IL-10+Foxp3− T cells are not able to control inflammatory responses but instead synergize with effector cells to potentiate their pro-inflammatory activity. Consistent with this are data from the RAG-1-deficient recipients of a mixture of effector T cells and IL-9+IL-10+ T cells showing that the effector T cells produce IL-17, which is not normally observed if effector cells alone are transferred into the RAG-1-deficient mice.
In addition to IL-9 receptor expression on effector T cells, our preliminary data indicate that IL-9 receptor is also expressed on natural-occurring Treg cells. Therefore IL-9 could exert its effects on both effector T cells and Treg cells. Whether IL-9 inhibits or interferes with the suppressive functions of regulatory cells has not been analyzed. However, IL-9 is also produced by Treg cells themselves 42 which recruits mast cells to the tissue niche and regulates effector T cells during graft rejection. Neutralization of IL-9 in vivo under this setting abrogates Treg function and promotes graft rejection. In our experiments we have tested the effects of IL-9–IL-10 producing T cells in vivo in the RAG-1-deficient mice, in the absence of Treg cells; under these experimental conditions, IL-9 produced by the IL-9–IL-10 producing T cells may act only on the effector T cells and potentiate their pro-inflammatory functions and thus induce more severe and unusual tissue inflammation (e.g., peripheral neuritis), which is not normally observed with the transfer of effector T cells alone.
We have identified a novel subset of T cells generated in presence of TGF-β and IL-4 that produces both IL-9 and IL-10, does not suppress T cell responses and promotes inflammation. Thus, depending on the context in which IL-10 is being produced by T cells, the T cells can inhibit inflammation and protect against autoimmunity or they can promote inflammation and mediate tissue destruction.
C57BL/6, Balb/c, RAG-1-deficient (Rag−/−) and STAT6 deficient (Stat6−/−) mice were obtained from Jackson Laboratories. Foxp3-GFP mice12, IL-4 transgenic mice43 and IL-10.GFP reporter mice 22 have been described previously. GATA-3-conditional-deficient mice (Gata3flox/flox) and IL-10.GFP mice were provided respectively by Dr. I-Chen Ho (Boston, MA) and Dr. R. Flavell (New Haven, CT). Mice were housed in conventional, pathogen-free facilities at Harvard Institute of Medicine. All experiments were performed in accordance with guidelines prescribed by the standing committee of animals at Harvard Medical School.
For T cell differentiation and proliferation, sorted naïve CD4+CD62L+Foxp3-GFP−, CD4+CD62L+GATA-3-KO (cre+) or CD4+CD62L+STAT6KO.Foxp3-GFP− T cells were cultured with irradiated syngenic APCs in the presence of either TGF-β (3 ng/ml) or IL-4 (20 ng/ml) or both. For suppression assays, 2.0 × 104 sorted Foxp3+ Treg cells from wild-type or STAT6-deficient (STAT6KO) mice were co-cultured in triplicate wells at a 1:1 ratio with either naïve wild-type CD4+ T cell or naïve STAT6KO CD4+ T cells (T cell responders) in the presence of soluble anti-CD3 (1 μg/ml) and irradiated syngenic APCs. When indicated anti-IL-9 (10 μg/ml, BD Biosciences) or anti-IL-10 (10 μg/ml, BD Biosciences) was added to the culture. Cells were pulsed with 1 μCi/well of [3H]-thymidine for the last 16 h of incubation and [3H]-thymidine incorporation was measured using a microbeta liquid scintillation counter (PerkinElmer).
Cells were re-stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml, Sigma), ionomycin (1 μg/ml, Sigma), and GolgiStop (1 μl/1.5ml, BD Bioscience) at 37 °C in 10% CO2 for 4 h followed by surface and intracellular staining for indicated cytokines according to the manufacturer’s (BD Bioscience) instruction. Cells were analyzed on a flow cytometer (BD Biosciences).
C57BL/6, RAG-1-deficient mice were injected with sorted CD4+Foxp3-GFP− CD45RB hi T cell sub populations in PBS as described before44,45. Mice received CD4+Foxp3-GFP−CD45RBhi T cell alone or in combination with CD4+CD62L+ T cells from IL-10.GFP mice stimulated with TGF-β and IL-4, or CD4+Foxp3+ naturally occurring Treg cells. Mice were examined for their weight loss for 9 weeks. They were then sacrificed and samples of colonic lymphoid tissues as well as brain and spinal cord were fixed in neutral buffered formalin.
Routinely processed, paraffin-embedded samples of small and large intestine and other tissues were stained with hematoxylin and eosin (H&E). Sections of brain and spinal cord were stained with Luxol fast blue-H&E stain. The presence and severity of colitis and the presence of brain and spinal cord inflammation were evaluated in a blinded manner.
The severity of colitis was graded semiquantitatively from 0 to 3 for the four following criteria: degree of epithelial hyperplasia and goblet cell depletion; leukocyte infiltration in the lamina propria; area of tissue affected; and the presence of markers of severe inflammation such as crypt abscesses, submucosal inflammation, and ulcers. Scores for each criterion were added to give an overall inflammation score for each sample of 0–12. The total colonic score was calculated as the average of the individual scores from the sections of proximal colon, mid-colon, and distal colon 28.
Culture supernatant were collected at 48 hours to determine the cytokine production by either ELISA or cytometric bead array (BD Bioscience). RNA was extracted after 48–96 hours of in vitro stimulation using RNAeasy columns (Qiagen) and subject to quantitative RT-PCR according to manufacturer’s (Applied Biosystem) instructions.
Total CD4+ T cells from GATA-3.conditional KO or wild type were transduced with a MSCV-based retroviral vector (RV-GFP) expressing CRE or Foxp3 respectively. RV-GFP is a bicistronic vector encoding for EGFP which expression monitors the expression of the transgene. The retroviral particles were produced as previously described by co-transfecting the plasmids encoding for gag-pol, the ecotropic envelope and the retroviral vector, using Fugene (Roche) into the HEK-293 cell line. Retroviral supernatant was harvested 48 to 72 hours post-transfection and centrifuged prior to use. Freshly isolated, magnetic-activated cell sorting (MACS)-purified CD4+ T cells were activated by using plate-bound anti-CD3 and anti-CD28 in the presence of mIL-4 (10 ng/ml, R&D Systems) and anti-mIL12 (10 μg/ml, Pharmingen) for TH2 differentiation. After 24 hours of activation, retroviral supernatant was added to the cells together with polybrene (8 μg/ml). The plates were centrifuged at 2000 RPM for 45 minutes and put back at 37 °C. TGF-β (3 ng/ml) was added when indicated to induce Foxp3 expression. After 48 h of incubation at 37 °C, the GFP+ cells were purified by flow cytometry and expanded for an additional 48–72 h in the presence of IL-2.
HEK-293 cell lines were transiently co-transfected with Foxp3-renilla and GATA3-, Foxp1- or STAT6-Flag tagged. Fugene-6 was used for the transfection according to the manufacturer’s instructions (Roche). Cell extracts were prepared (Promega) and immunoprecipation was performed using anti-Flag coated beads (Sigma). Foxp3-renilla expression was assessed by detection of the luminescence.
Jurkat cells were transfected using a Bio Rad electroporator (260 V); 5 × 106 cells in 0.4 ml RPMI were transfected with 5 μg reporter plasmid (Il5 promoter-luciferase) and 20 μg expression plasmid. Luciferase assays were performed after activating the cells 3 hours with PMA-ionomycin stimulation as previously described.
We thank D. Kozoriz, R. Chandwaskar, and D. Lee for cell sorting and technical assistance. This work was supported by grants from National Institutes of Health (R01AI073542-01 to M.O. and 1R01NS045937-01, 2R01NS35685-06, 2R37NS30843-11, 1R01A144880-03, 2P01A139671-07, 1P01NS38037-04 and 1R01NS046414 to V.K.K.) and National Multiple Sclerosis Society (RG-2571-D-9 to V.K.K. and RG-3882-A-1 to M.O.), and the Juvenile Diabetes Research Foundation Center for Immunological Tolerance at Harvard Medical School. V.K.K. is a recipient of the Javits Neuroscience Investigator Award from the National Institutes of Health. A.A. and V.D. are supported by a post-doctoral fellowship from National Multiple Sclerosis Society, New York.