Smad3 Differentially Regulates the Induction of Regulatory and Inflammatory T Cell Differentiation*

Transforming growth factor β (TGF-β) is a crucial cytokine with pleiotropic functions on immune cells. In CD4+ T cells, TGF-β is required for induction of both regulatory T and Th17 cells. However, the molecular mechanism underlying this differential T cell fate decision remains unclear. In this study, we have evaluated the role of Smad3 in the development of Th17 and regulatory T cells. Smad3 was found to be dispensable for natural regulatory T cell function. However, induction of Foxp3 expression by TGF-β in naive T cells was significantly reduced in the absence of this molecule. On the contrary, Smad3 deficiency led to enhanced Th17 differentiation in vitro and in vivo. Moreover, Smad3 was found to interact with retinoid acid receptor-related orphan receptor γt (RORγt) and decrease its transcriptional activity. These results demonstrate that Smad3 is differentially involved in the reciprocal regulatory and inflammatory T cell generation.


Transforming growth factor ␤ (TGF-␤) is a crucial cytokine with pleiotropic functions on immune cells. In CD4 ؉ T cells, TGF-␤ is required for induction of both regulatory T and Th17
cells. However, the molecular mechanism underlying this differential T cell fate decision remains unclear. In this study, we have evaluated the role of Smad3 in the development of Th17 and regulatory T cells. Smad3 was found to be dispensable for natural regulatory T cell function. However, induction of Foxp3 expression by TGF-␤ in naive T cells was significantly reduced in the absence of this molecule. On the contrary, Smad3 deficiency led to enhanced Th17 differentiation in vitro and in vivo. Moreover, Smad3 was found to interact with retinoid acid receptor-related orphan receptor ␥t (ROR␥t) and decrease its transcriptional activity. These results demonstrate that Smad3 is differentially involved in the reciprocal regulatory and inflammatory T cell generation.
Naive CD4 ϩ T helper (Th) 5 cells, upon activation, differentiate into effector cells that are characterized by their distinct cytokine production and immune regulatory functions. Under the influence of TGF-␤, Foxp3 can be induced in activated T cells, leading to the generation of inducible regulatory T cells (iTregs) (1). In addition, TGF-␤ is also involved in the differentiation of Th17 cells, which require TGF-␤ and IL-6 or IL-21 (2,3). Thus, there is not only functional antagonism but also reciprocal regulation in the generation of Th17 and iTreg cells (4,5).
The signaling mechanism by which TGF-␤ regulates iTreg and Th17 differentiation has not been clear. We found that inhibition of TGF-␤ receptor I (TGF-␤RI) activity blocked both iTreg and Th17 differentiation (5). Moreover, deletion of Smad4 in T cells resulted in a partial defect in iTreg cell development without affecting Th17 differentiation (5). Whether distinct signaling components of TGF-␤ receptor differentially regulate iTreg and Th17 cell development has not been understood. In the present study, we have determined the role of Smad3 in the induction of regulatory T cells and Th17 cells. Although Smad3 is required for optimal induction of iTreg cells, it appears to negatively regulate Th17 cell differentiation, possibly by direct binding to ROR␥t. These results thus contribute to our understanding of the molecular antagonism of Th17 and regulatory T cells genetic programs.

EXPERIMENTAL PROCEDURES
Mice-C57BL/6 and Rag1-deficient mice were purchased from The Jackson Laboratory. Smad3 knock-out (KO) mice were kindly provided by Dr. Xiao-Fan Wang (10). Mice were housed in the specific pathogen-free animal facility at M. D. Anderson Cancer Center, and the animal experiments were performed at the age of 6 -10 weeks using protocols approved by the Institutional Animal Care and Use Committee.
Quantitative Real-time PCR-Total RNA was prepared from T cells using TRIzol reagent (Invitrogen). cDNA was synthesized using Superscript reverse transcriptase and oligo(dT) primers (Invitrogen), and gene expression was examined with a Bio-Rad iCycler optical system using iQ TM SYBR green realtime PCR kit (Bio-Rad Laboratories, Inc.). The data were normalized to Actb reference. The primers for IL-17, IL-17F, IL-21, IL-22, CCR6, CCL20, IL-23R, ROR␣, ROR␥t, IRF4, AHR, and ␤-actin were previously described (5,11,13). MOG Immunization-Female Rag1 KO mice were reconstituted with T cell-depleted bone marrow cells from Smad3 KO or wild-type (WT) littermates. 8 weeks later, mice were immunized subcutaneously at the dorsal flanks with 150 g of MOG 35-55 peptide emulsified in CFA. 7 days later, cells from spleens and draining lymph nodes of the immunized mice were isolated and restimulated with MOG for 3 days, and cytokine production was determined in the culture supernatant by ELISA.
RORE Reporter Assay-ROR␥t (5), Smad3 2SD, and TGF-␤RI T202D (12) were cloned into bicistronic retroviral vector pGFP-RV provided by Dr. Ken Murphy at Washington University (14) that contains IRES-regulated GFP. 293 T cells were co-transfected with 3 g of the (RORE)3-Luc reporter in the presence or absence of the indicated pGFP vectors. Cells were incubated for 16 h with complete medium and then for 24 h with 0.5% fetal bovine serum-containing medium. Then luciferase activity was analyzed with a Dual-Luciferase kit (Promega). Transfection efficiency was normalized by Renilla-luciferase activity.
Co-immunoprecipitation-Expression vectors encoding Myc-Smad3, FLAG-ROR␥t, and His-TGF-␤RI T202D were transfected into HEK293T cells. After 48 h, immunoprecipitation was performed using anti-FLAG-M2 antibody (Sigma-Aldrich) as described previously (5). Equivalent amounts of protein from whole cell lysates or immunoprecipitates were analyzed by Western blot using anti-FLAG-M2 or anti-c-Myc.

RESULTS AND DISCUSSION
Smad3 Regulates Foxp3 Induction in Naive T Cells-To better understand the role of the TGF-␤ signaling pathway in the induction of Th17 and Treg cell differentiation, we utilized mice deficient in Smad3 (10). These mice exhibit normal CD4 ϩ and CD8 ϩ T cell levels in vivo (data not shown). Similar to mice with a deletion of Smad4 in T cells (5), Smad3 KO mice exhibited normal numbers of CD4 ϩ CD25 ϩ Foxp3 ϩ natural Treg (nTreg) cells in spleen, peripheral lymph nodes, and mesenteric lymph nodes (Fig. 1, A and B). However, Smad3-deficient mice also displayed a significant decrease of Treg cells in thymus. When their functions were assessed, peripheral nTregs from Smad3-deficient mice were as suppressive as WT nTreg cells (Fig. 1C). Thus, these results indicate that Smad3 is not required for nTreg cell suppressive activity.
We then examined whether Smad3 is necessary for the induction of iTreg cells. Naive T cells were isolated from Smad3 KO or WT mice by FACS sorting and stimulated with platebound anti-CD3 and anti-CD28 in the presence of TGF␤. Smad3-deficient naive T cells had a profound defect on Foxp3 expression upon iTreg induction (Fig. 1, D and E). Moreover, induction of Treg-associated genes such as GPR83 and Ecm1 were also affected in Smad3-deficient T cells when compared with WT counterparts (Fig. 1E). Thus, these results indicate that Smad3 regulates the induction of Foxp3 and iTreg-associated genes.
Increased Th17 Cell Differentiation in the Absence of Smad3-Because TGF-␤ also regulates the differentiation of Th17 cells, we addressed the role of Smad3 in the generation of Th17 cells. When naive cells were activated in the presence of TGF-␤ and IL-6, enhanced IL-17-producing cells were detected in Smad3deficient cells (Fig. 2A). Gene expression profile analysis by real-time RT-PCR indicated an increase not only in IL-17A but also in IL-17F, CCR6, and CCL20 mRNA levels in Smad3-deficient cells when compared with WT counterparts (Fig. 2B and data not shown). Interestingly, no difference was observed in IL-21 or IL-22 expression, supporting that they are not regulated by TGF-␤. Furthermore, when cells were restimulated with anti-CD3, enhanced IL-17 and IL-17F but not IL-21 cyto- kine production was observed (data not shown). Moreover, a slight increase in ROR␣ and a decrease in AHR and IRF4, but no significant change in ROR␥t, were detected in Smad3-deficient Th17 cells when compared with WT cells (Fig. 2B).
We have recently demonstrated that IL-1 in combination with IL-6 and IL-23 is able to initiate Th17 genetic programming. However, this Th17-polarizing condition still requires endogenous, low levels of TGF-␤ (11). To evaluate whether Smad3 deficiency would affect Th17 differentiation under low TGF-␤ concentrations, naive T cells were stimulated with anti-CD3 and anti-CD28 in the presence of two Th17-polarizing conditions. Interestingly, although TGF-␤ and IL-6 stimulation led to increased Th17 differentiation in Smad3-deficient cells, the combination of IL-1, IL-6, and IL-23 stimulation induced similar numbers of IL-17-producing cells in both WT and KO T cells (Fig. 2, A and B), suggesting that Smad3 is required in the presence of high doses of TGF-␤ during Th17 differentiation. To assess that hypothesis, naive T cells from either WT or KO mice were stimulated with increasing concentrations of TGF-␤ in the presence of IL-6 and neutralizing antibodies against IFN-␥ and IL-4. At low TGF-␤ concentrations, similarly low levels of IL-17-producing cells were observed between WT and KO cells. However, with increasing concentrations of TGF-␤, Smad3-deficient cells had higher numbers of IL-17producing cells when compared with WT counterpart (Fig.  2C). Interestingly, enhanced IL-17-producing cells were observed in Smad3-deficient T cells at TGF-␤ concentrations that were not sufficient to induce Foxp3 expression ( Fig. 2C and data not shown), suggesting that the inhibitory role of Smad3 on Th17 cells was not dependent on Foxp3 induction.
Smad3 Directly Binds to and Decreases ROR␥t Transcriptional Activity-Because Smad3-deficient T cells exhibited enhanced capability to differentiate into IL-17-producing T cells independent of Foxp3 gene induction and given that ROR␥t levels were not affected in Smad3 KO T cells when compared with WT T cells, we next analyzed the regulation of ROR␥t function by Smad3. For that purpose, HEK293T cells were transfected with an RORE luciferase reporter vector (5) in the presence or absence of ROR␥t with or without a constitutively active Smad3-expressing vector. Although ROR␥t alone induced luciferase activity, co-expression of increasing concentrations of Smad3 significantly reduced its activity (Fig. 3A).
Given that Smad3 is able to inhibit ROR␥t transcriptional activity, we next examined the interaction of Smad3 with ROR␥t using co-immunoprecipitation. We found that Smad3 was able to bind ROR␥t when co-expressed in HEK293T cells, and this binding was enhanced upon co-expression of a constitutively active form of rat TGF-␤RI (TGF-␤RI T202D) (12) (Fig. 3B), suggesting that phosphorylation of Smad3 might enhance its affinity to ROR␥t.
Smad3 Deficiency Enhances the Generation of Inflammatory T Cells in Vivo-To analyze the role of Smad3 in Th17 cell generation in vivo, Rag1 KO mice were reconstituted with bone marrow cells from WT or Smad3 KO mice. After 8 weeks, mice were immunized with MOG peptide emulsified in CFA. 7 days later, splenocytes and draining lymph node cells were harvested from the immunized mice and restimulated with MOG peptide for 3 days. After MOG immunization, enhanced IL-17, IL-17F, and IL-22 production was detected in Rag1 KO mice reconstituted with Smad3deficient bone marrow cells when compared with those with WT counterparts (Fig. 4). On the other hand, the expression of IFN-␥ was not significantly different between the two groups of animals. Taken together, these results indicate that Smad3 deficiency enhanced Th17 differentiation in vitro and in vivo.
In summary, we have investigated the role of Smad3 in the generation of iTreg and Th17 cells. Smad3 deficiency resulted in defective Foxp3 induction but enhanced Th17 cell generation in vitro and in vivo. Smad3 was found to be part of a protein complex with ROR␥t, leading to the inhibition of ROR␥t transcriptional activity. Smad3 thus differentially regulates iTreg and Th17 cell differentiation. These results may be beneficial in our further understanding of the reciprocal regulation of these two cell lineages, allowing for the development of better approaches to design immunotherapies to target each cell type individually.  Bone marrow cells from Smad3 WT or KO mice were intravenously transferred to sublethally irradiated RAG1 KO mice (four mice per group). After 8 weeks, the recipient mice were immunized subcutaneously with 150 g of MOG 35-55 peptide emulsified in CFA. 7 days later, lymphoid cells from spleens were isolated and restimulated with MOG peptide for 3 days. Cytokine production was measured from cell-free supernatants by ELISA. Error bars indicate S.E.