Grail controls Th2 cell development by targeting STAT6 for degradation

T helper (Th)-2 cells are the major players in allergic asthma; however, the mechanisms that control Th2-mediated inflammation are poorly understood. Here we find that enhanced expression of Grail, an E3 ubiquitin ligase, in Th2 cells depends on IL-4-signaling components, Stat6 and Gata3 that bind to and transactivate the Grail promoter. Grail-deficiency in T cells leads to increased expression of Th2 effector cytokines in vitro and in vivo and Grail deficient mice are more susceptible to allergic asthma. Mechanistically, the enhanced effector function of Grail-deficient Th2 cells is mediated by increased expression of Stat6 and IL-4 receptor α-chain. Grail interacts with Stat6 and targets it for ubiquitination and degradation. Thus, our results indicate that Grail plays a critical role in controlling Th2 development through a negative feedback loop.

Recent evidence has suggested that E3 ubiquitin ligases including Cbl-b, Itch and Grail are crucial regulators of T cell activation 19 . Grail is a type I transmembrane protein localized to the endosomal compartment whose expression is associated with T cell anergy induction 20 . Recently we reported that Grail KO mice were resistant to immune tolerance induction in vitro and in vivo 21 . In addition to tolerant T cells, Grail mRNA is upregulated during normal T cell activation, suggesting that Grail function might not be restricted to T cell anergy [21][22] . Grail expression has also been linked to hyporesponsive Th2 cells in a model of chronic murine schistosomiasis 23 . In fact, overexpression of Grail in T cell hybridomas dramatically limits production of Th2 essential cytokines such as IL-2 and IL-4 20 , suggesting the role of Grail in controlling Th2 programming.
In the current study, high expression of Grail in in vitro differentiated Th2 cells compared to other T helper lineages is selectively induced upon IL-4 stimulation in a time dependent manner and depends on Th2-specific factors Stat6 and Gata3 that bind to and transactivate the Grail promoter. Grail deficiency in T cells leads to enhanced Th2 development in vitro and in vivo; Grail KO mice are more susceptible to allergic asthma. Both naïve T cells and Th2-polarized cells from Grail KO mice exhibit increased Stat6 transcription factor expression. Moreover, Grail interacts with Stat6 and promotes Stat6 ubiquitination and degradation. Our results suggest an important link between the Th2 specific expression of Grail and its role in control of Th2 development and Th2-mediated pathogenesis and immunity.

Regulation of Grail expression in T helper 2 cells
Although it is known that Grail is expressed in anergic cells generated both in vivo and in vitro, its expression is also upregulated during T cell activation [21][22] . To further dissect the role of Grail in T helper cell activation and differentiation, FACS-sorted naïve CD4 + CD25 − CD62L hi CD44 lo T cells were differentiated in vitro under Th0, Th1, Th2, and Th17 polarizing conditions and analyzed for Grail mRNA expression. Consistent with a previous study which showed upregulation of Grail in Th2 cells during chronic schistosomiasis 23 , Grail mRNA level was significantly increased in Th2 cells compared to other T helper subsets (Fig. 1a), suggesting a possible role of Grail in Th2 cell development.
We next checked whether this increase is mediated by active transcriptional machinery in the Grail promoter region. Acetyl histoneH3 lysine9/14(AcH3) and histone H3 lysine 4 methylation (H3k4) are typically associated with active transcription while H3 lysine 27 methylation (H3k27) is mainly associated with gene silencing [24][25] . Naïve CD4 + T cells differentiated under Th0 and Th2 conditions in vitro were assessed for abundance of AcH3, and H3k4) in the Th2 cells compared to Th0 cells (Fig. 1b), suggesting a role for IL-4 signaling in regulation of chromatin modification in the Grail locus. The IL-4 gene promoter region was analyzed as positive control ( Supplementary Fig. 1). To further dissect the role of IL-4 in Grail regulation, we analyzed Grail expression in naïve CD4 + T cells activated with anti-CD3/28 and treated with IL-4 or anti-IL-4 for 24, 48 or 72 hrs. This analysis showed that Grail was expressed as early as 24 hours after T cell activation and IL-4 was essential for this induction, because blockade of IL-4 in Th0 condition decreased Grail expression (Fig. 1c). Grail expression was further increased in a time dependent manner and correlated with the level of il4 expression. The above results suggest that Grail is highly expressed in Th2 cells in an IL-4 dependent manner.
As Grail expression is significantly increased upon IL-4 treatment, we assessed whether its expression is regulated by Stat6, a transcription factor which promotes Th2 differentiation in an IL-4-dependent manner 26 . Naïve CD4 + T cells from WT and Stat6 KO mice were cultured under Th2 conditions for four days and then assessed for Grail mRNA expression (Fig. 2a). Absence of Stat6 led to a significantly decreased expression of Grail in Th2 cells. Similarly, we found significantly low mRNA level of Grail in Stat6 deficient memory CD4 + T cells compared to WT cells ( Supplementary Fig. 2a). To determine the exact role of Stat6 in regulating Grail expression, we assessed the status of histone modifications at the Grail promoter locus in WT and Stat6 deficient Th2 cells by ChIP analysis. As expected Stat6 deficient Th2 cells showed decreased active histone modifications (AcH3 and H3k4) and increased abundance of inactive H3k27 modifications in the Grail promoter locus (Fig. 2b).
To check if this effect is mediated by direct binding of Stat6 to the Grail promoter locus, we performed ChIP assay with Stat6 and control antibodies (Fig. 2c). Stat6 binding was enriched in the Th2 cells compared to Th0 cells in the Grail promoter. Apart from Stat6, we also checked binding of Gata3, a transcription factor downstream of Stat6 and also IRF4 and Jun B transcription factors which are known to play important functions in Th2 cell differentiation 14,16 (Fig. 2c). Similar to Stat6, Gata3 showed increased binding to the Grail promoter in Th2 cells compared to Th0 cells, whereas we observed moderate binding of IRF4 and JunB to Grail promoter locus which was not Th2 cell specific (Fig. 2c). Binding of the above factors was also analyzed at various control loci ( Supplementary Fig.2b). The above factors were then over-expressed by retroviral transduction into naïve CD4 + T lymphocytes. Upon over-expression, both Stat6 and Gata3 facilitated a significant increase in Grail expression whereas IRF4 and JunB did not (Fig. 2d, upper panel). Expression of il4 was also checked to validate our overexpression system (Fig. 2d, lower panel).
To further determine whether the binding of Stat6 and Gata3 to the Grail promoter resulted in its transactivation, we performed dual luciferase reporter assay by overexpression of the above factors along with the Grail promoter construct in EL-4 cells. As expected both Stat6 and Gata3 transactivated the Grail promoter (Fig. 2e). To further assess whether Gata3 is required for Stat6 activity, we used Grail promoter with a mutated Gata3 binding site (GrailP-mu) (Fig. 2e). We found that endogenous activity of Grail promoter was significantly lower when Gata3 site was mutated, suggesting that Gata3 is required for Grail expression. However Stat6 is still able to transactivate the GrailP-mu. Overall, these data suggest that Stat6 and Gata3 could independently regulate Grail expression. To further confirm these observations, we overexpressed Stat6 and Gata3 in both WT and Stat6 deficient cells. We found that over-expression of either of these two factors can rescue Grail expression in Stat6 deficient cells (Fig. 2f). We also checked the expression of Il4, Stat6 and Gata3 to validate our overexpression system ( Supplementary Fig. 2c). Together these findings indicate that Grail expression is induced by IL-4 and both Stat6 and Gata3 are sufficient to induce Grail.

Enhanced Th2 response by Grail-deficient T cells in vitro
The enhanced Grail expression in Th2 cells led us to further check the role of Grail in Th2 cell development. Naive CD4 + T cells from WT and Grail KO mice were activated with anti-CD3 and anti-CD28 in the presence or absence of IL-4 (Fig. 3a). Enhanced expression level of the Th2-specific factors was detected in both IL-4-treated and untreated Grail-deficient cells compared to WT cells. Similarly, in vitro differentiated Grail deficient Th0 and Th2 cells showed increased expression of Th2 specific factors (IL-4, IL-5, IL-13 and Gata3) compared to WT cells ( Fig. 3b and c). As expected Grail-deficient Th2 cells showed increased active histone modifications (AcH3 and H3k4) in the IL-4 and Gata3 promoter locus and decreased abundance of inactive H3k27 modifications ( Supplementary Fig. 3, right panel), suggesting that Grail deficient cells more profoundly acquire a Th2 phenotype. between WT and Grail deficient cells. Interestingly, Grail deficiency in naïve T cells already led to a slight enhancement in IL-4R expression, which was significantly enhanced 24 hours after activation and peaked at 48 hours ( Fig. 3d-e). In addition, analysis of IL-4Ril4R promoter locus in naïve Grail deficient CD4 + T cells showed both active and inactive histone modifications ( Supplementary Fig. 3), suggesting that in the absence of Grail, IL-4R is poised for rapid transcription. Upon Th2 differentiation il4R promoter locus still maintained a more open chromatin configuration in Grail-deficient cells compared to WT ( Supplementary Fig.3).

IL-2 and IL-21 dependent Th2 responses in Grail KO T cells
The above results indicate the role of Grail in regulation of IL-4R expression at early stage of Th2 cell programming. In addition, we found enhanced expression of Nfatc1 in Grail deficient T cells in an IL-4-dependent manner ( Supplementary Fig. 4b). Role of NFATc1 is well known in regulation of the cytokines IL-2 and IL-21, which play a role in early Th2 development [17][18] . As expected, mRNA expression of both Il2 and Il21 was increased in Grail deficient CD4 + T cells as early as 24 hours of activation with anti-CD3/28 in an IL-4dependent manner, because IL-4 blockade diminished this difference ( Fig. 4a and Supplementary Fig. 4b). To check whether the increased IL-2 and IL-21 levels aid in the increased Th2 responses seen in Grail KO Th2 cells, we blocked both IL-2 and IL-21 expression in WT and Grail KO CD4 + T cells by respective antibodies for 48 hrs. We found that blockade of either IL-2 or IL-21 led to decreased expression of Th2 cytokines IL-4, IL-5 and IL-13 and the Il4rα more prominently in Grail KO cells compared to WT cells (Fig. 4b).
Since Grail resides in the endosome, we next treated the WT or Grail deficient T cells activated in the presence or absence of IL-4 with an endosome inhibitor Latrunculin B. mRNA analysis 48hrs post treatment showed that expression of Il4r and most Th2 specific genes were upregulated upon Latrunculin B treatment ( Fig. 4c-d), supporting the critical role of Grail in controlling Th2 development.

Grail promotes Th2 responses in vivo
To analyze the function of Grail in vivo, FACS-sorted memory CD4 + CD44 + T cells from WT and Grail KO mice were analyzed. As measured by quantitative RT-PCR (qRT-PCR), Grail deficient CD4 + CD44 + effector memory cells had significantly higher expression of major Th2 specific genes like Il4 and its receptor (Supplemtary Fig. 5a) compared to WT cells. Higher IL-4, IL-5 and IL-13 protein levels were also detected by ELISA in the Grail deficient memory cells compared to WT cells ( Supplementary Fig. 5b), supporting Grail as an essential factor in Th2 differentiation in vivo.
To further determine the role of Grail in Th2 responses in vivo, WT and Grail KO mice were immunized intraperitoneally with Ova and the adjuvant aluminum hydroxide (alum). After restimulation with Ova, T cells from Grail KO mice had significantly higher expression of IL-2, IL-4, IL-5 and IL-13 ( Fig. 5a). Similarly, intracellular staining revealed a dramatic increase in CD4 + IL-4 + cells in both the spleen and lung lymph nodes of Grail KO mice compared to wild-type mice (Fig. 5b). In addition, analysis of sorted CD4 + CD44 hi cells from immunized mice showed that Grail deficiency led to enhanced expression of Th2 specific factors (IL-4, IL-5, IL-13, and Gata3) both at mRNA and protein levels (Fig. 5c).
To check whether the increased Th2 responses in Grail KO mice are T cell intrinsic, we transferred naïve WT and Grail KO CD4 + cells into TCR-β KO mice followed by Ova in alum immunization. Consistent with above data, mice reconstituted with Grail KO CD4 + T cells showed increased expression of Th2 cytokines both at mRNA and protein levels compared to mice that received WT CD4 + T cells ( Fig. 5d-e). To further demonstrate the role of Grail in T cells, we generated mixed bone marrow chimeras by transferring a mixture of congenic CD45.1 + WT and CD45.2 + Grail-deficient bone marrow cells into sub-lethally irradiated Rag1 KO mice. Eight weeks after reconstitution, we immunized mice with Ova protein in alum. CD4 + CD44 hi Grail deficient cells exhibited enhanced Th2-specific cytokine expression compared to WT cells (Fig. 5f). The above results confirm that Grail expression in T cells is required to control Th2 programming in vivo.
As Th2 cells are pathogenic, we next subjected WT and Grail KO mice to allergic asthma using a standard protocol 21 . Histology analysis revealed that Grail deficiency resulted in significantly more inflammatory cell infiltration into the lung tissue (Fig. 6a). Consistent with histological analysis, the numbers of total cells, eosinophils, neutrophils and lymphocytes in bronchoalveolar fluid (BALF) were significantly increased in Grail KO mice compared to WT mice (Fig. 6b). In addition, we also observed increased levels of Th2 cytokines in BALF and lungs of Grail KO mice compared to their WT counterparts ( Fig. 6cd). Moreover, after restimulation ex vivo with Ova protein, spleen and lung-associated lymph node cells from Grail-KO mice produced more Th2 cytokines than did cells from WT mice ( Fig. 6e-g and Supplementary Fig. 5c), suggesting that Grail is required to control the generation of proallergic helper T cells.

Grail controls Stat6 expression in Th2 cells
The above results suggest that Grail deficiency leads to Th2-hyperresponsiveness and Th2mediated inflammation. Recently, we reported that Grail regulates CD4 + T cell activation by targeting TCR-CD3 complex via proteasome-mediated degradation 21 . Owing to its high expression in Th2 cells, first we assessed whether Grail controls Th2 cells by targeting CD3ζ. However, analysis of TCRβ down-regulation in WT and Grail KO Th2 cells upon anti-CD3 stimulation did not show difference in TCRβ levels between WT and Grail KO cells ( Supplementary Fig. 6). Next, we analyzed the protein level of Th2 transcriptional factors such as Stat6 and Gata3 in naïve WT and Grail KO CD4 + T cells either left unstimulated or stimulated with anti-CD3/28 in the presence or absence of IL-4 ( Fig. 7a and Supplementary Fig. 10a). Stat6 protein levels were increased in naïve CD4 + T cells in Grail KO mice compared to WT mice. This increase was maintained after 24hrs of anti-CD3/28 stimulation either in the presence or absence of IL-4 ( Fig. 7a and Supplementary Fig. 10a). Moreover, fully committed Th2 cells from Grail KO mice showed a more dramatic increase in Stat6 protein expression ( Fig. 7b and Supplementary Fig. 10b). In addition to Stat6, Gata3 protein expression was also significantly enhanced in Grail-deficient Th2 cells ( Fig. 7b and Supplementary Fig. 10b), whereas we did not detect enhanced Gata3 protein level at early steps of Th2 development (Supplementary Fig. 9 and 10g) ), suggesting that Grail potentially controls Gata3 expression through Stat6. To further dissect the expression and activation kinetics of Stat6 in IL-4-primed cells, we isolated naïve CD4 + T cells from WT and Grail KO mice and activated them with anti-CD3/28 and IL-4 for different time points.
Interestingly, analysis of phospho-Stat6 level revealed significant activation of Stat6 in Grail deficient T cells activated in the presence of only anti-CD3/28 compared to WT cells ( Fig.  7c and Supplementary Fig. 10c). This increase was further enhanced in the presence of IL-4. The total Stat6 levels were also increased in Grail-deficient T cells ( Fig. 7c and Supplementary Fig. 10c), suggesting that Grail may regulate Th2 programming by targeting Stat6.
To check whether Grail directly targets Stat6, we overexpressed Stat6 together with WT or Grail mutant which lacks amino acids encompassing the RING domain (amino acids 277-317) and hence the E3 ubiquitin ligase activity in HEK293T cells along with HA-tagged ubiquitin. After immunoprecipitation of Stat6 with Stat6 antibody and reprobing with HA-HRP antibodies, we observed the formation of higher molecular weight ubiquitin conjugates in the presence of WT Grail, indicating polyubiquitin chain formation ( Fig. 7d and Supplementary Fig. 10d). This ubiquitination was decreased upon lowering the concentration of WT Grail and was not present upon overexpression of the Grail mutant ( Fig. 7d and Supplementary Fig. 10d). Thus, Stat6 is a potential substrate for Grail mediated ubiquitination in Th2 cells. In further investigation, we checked the endogenous ubiquitination of Stat6 in WT and Grail deficient CD4 + T cells activated with anti-CD3/28 and IL-4 in the presence of MG132 to block any proteasome degradation. T cell lysates were then immunoprecipitated with Stat6 antibodies and immunoblotted with ubiquitin antibodies. Compared to WT cells, ubiquitination of Stat6 in Grail deficient cells was substantially reduced (Fig. 7e and Supplementary Fig. 10e), suggesting that Grail regulates Stat6 expression and function through ubiquitin-dependent proteasome degradation pathway. Moreover, immunoprecipitation experiments performed in HEK293T cells transfected with expression vectors for Stat6 and Grail-GFP revealed an interaction between Grail and Stat6 ( Fig. 7f and Supplementary Fig. 10f), thus suggesting that ubiquitination of Stat6 by Grail is mediated by their physical interaction. However we were unable to detect interaction between endogenous Grail and Stat6 proteins due to unavailability of efficient Grail antibodies. Further experiments with the endogenous Grail would be required to establish the physical interaction between Grail and Stat6 in a physiological setting.

DISCUSSION
The molecular mechanisms leading to generation of pro-allergenic Th2 cells are gradually becoming clearer. While most of the previous studies have focused on active regulators of the Th2 differentiation programme, including transcription factors like Stat6, Gata3, JunB, IRF4, c-Maf, and NFAT 7 , the mechanisms that keep Th2 cell generation and function under control remain poorly understood. In the current study, we have shown the essential role of Grail in regulating Th2 cell development and Th2-mediated pathogenic responses. Grail is preferentially expressed in Th2 cells, targets Stat6 for ubiquitination and restricts expression of Th2 cytokines. As a result, Grail deficient mice are more susceptible to Th2 dependent allergic responses like asthma.
It has been reported that Grail mRNA is upregulated during normal T cell activation even under non tolerant conditions [21][22] . As reported previously 23 , our analysis of Grail expression in in vitro differentiated different T helper subsets showed that Grail is highly expressed in Th2 cells, suggesting the potential role of Th2 cytokines in regulation of Grail expression (Fig. 1a). We tested a panel of cytokines that are known to promote Th2 development including IL-2, IL-4 and IL-21 1,[17][18]26 and found that Grail expression selectively depends on the presence of IL-4 ( Supplementary Fig.8a). Consistent with that, upon IL-4 blockade, T cells stimulated with ant-CD3 and CD28 failed to upregulate Grail expression (Fig. 1c). Moreover, we observed correlation between Grail and Il4 expression ( Fig. 1c and Supplementary Fig. 7) which suggests that Grail could serve as a marker of proallergic cells where IL-4 signaling determines Grail expression. In fact, Stat6 and Gata3, major Th2-specific transcription factors 7 , bind to and transactivate the Grail promoter (Fig.  2e) and are necessary and sufficient to induce Grail as evident from overexpression experiments (Fig. 2d). Higher Grail expression was also detected in Th9 cells ( Supplementary Fig.8b). This could be mediated by the IL-4-IL-4R-Stat6 signaling pathway which is important for the development of Th9 cells as well [27][28] .
In addition to Stat6 and Gata3, transcription factors like JunB and IRF4 are also known to be involved in the Th2 developmental process; however they did not contribute to Th2-specific Grail expression (Fig. 2 c-d). Previously, it has been proposed that NFATc1 has the potential to induce Grail in anergic T cells 29 . However, blockade of NFATc1 activity by CsA during Th2 development did not affect Grail expression and we did not detect transactivation of Grail promoter activity by overexpression of NFATc1 ( Supplementary Fig. 2d-e). It is also possible that NFATc1 could cooperate with Stat6 and Gata3 to enhance and stabilize Grail expression in Th2 cells.
Since Grail is induced in Th2 cells by IL-4 signaling, Grail may be involved in differentiation of Th2 cells. Indeed, Grail-deficiency in CD4 + T cells enhanced Th2-biased differentiation in vitro and in vivo (Fig. 3, 5-6). Interestingly Il4r expression was also enhanced in the Grail deficient T cells 24 hours after IL-4 treatment and peaked at 48 hours, suggesting the role of Grail in early stages of Th2 cell programming by regulating Il4r expression (Fig. 3d and e and Supplementary Fig. 4a). Moreover, analysis of naïve CD4 + T cells from Grail deficient mice revealed that the IL-4R promoter region is enriched in both permissive H3K4me3 and the restrictive H3K27me3 modifications (Supplementary Fig. 3). This bivalent chromatin could allow minimal IL-4R expression in naïve cells but nonetheless make it poised for rapid transcriptional activation in response to subsequent IL-4 signaling favoring Th2 differentiation. IL-2 and IL-21 are known to aid in early events of Th2 cell differentiation 17 . We found that Grail deficient CD4 + T cells expressed substantially high amounts of IL-2 and IL-21 compared to WT cells 24 hours after IL-4 treatment (Fig.4a). In addition, these cytokines facilitate the rapid expression of Il4r and Th2 cytokines in Grail deficient CD4 + T cells. Our studies for the first time indicate a critical role of Grail in controlling of Il4r expression and Th2 programming.
Our previous studies as well as others indicate that Grail negatively regulates T cell responses by targeting components of TCR signaling pathway for degradation [21][22] . However, analysis of TCRβ down-regulation in WT and Grail deficient Th2 cells upon anti-CD3 stimulation did not show difference in TCRβ levels between WT and Grail KO cells ( Supplementary Fig. 6), indicating that TCR signaling is not responsible for the profound Th2 phenotype in Grail-deficient cells. Next, we analyzed the protein level of Th2 transcriptional factors such as Stat6 and Gata3 in naïve and IL-4 treated WT and Grail KO CD4 + T cells. We found increased Stat6 protein level in naïve Grail KO CD4 + T cells compared to WT cells and this increase was maintained in Grail KO cells upon IL-4 stimulation ( Fig.7a-b and Supplementary Fig. 10a-b). We further determined that Grail interacts with Stat6 ( Fig. 7f and Supplementary Fig. 10f) and the down-regulation of elevated Stat6 levels by Grail is dependent on E3 ligase activity and mediated by proteasome-mediated degradation (Fig. 7d-e and Supplementary Fig. 10d-e). This result suggests that Grail, located in endosomal compartment, may target Stat6 via ubiquitination and proteasome-mediated degradation. Increased level of Stat6 could explain why Grail deficient cells can spontaneously acquire a Th2 phenotype. In fact, we saw significantly enhanced Gata3 expression in fully polarized Grail deficient Th2 cells compared to WT cells ( Fig. 7b and Supplementary Fig. 10b). The augmented total Stat6, phospho-Stat6 and Gata3 levels in the absence of Grail could further lead to increase in the transcription of Th2 signature cytokines by direct binding to their promoters or by regulating the chromatin accessibility to other trans-activating factors. We also found increased abundance of active histone markers (AcH3 and H3k4) and decreased levels of suppressive markers (H3k27) in the il4, il4R and Gata3 promoter locus in Grail KO Th2 cells (Supplementary Fig. 3). Stat6 is known to influence the expression of an array of genes by regulating their chromatin structure 30 . Further investigation is needed to check whether Grail directly targets and influences the expression and functional status of chromatin modifiers involved in Th2 gene expression.
In summary, our studies for the first time indicate the Th2 specific expression of Grail and its critical role in controlling Th2 responses. Grail expression is mainly mediated by IL-4 and IL-4 signaling components. It controls Th2 responses by targeting Stat6 transcription factor for degradation in activated Th2 cells. Thus, the results suggest Grail as a potential marker of allergic inflammation and provide a molecular mechanism explaining the generation of peripheral Th2 tolerance, which has implications in the prevention and treatment of allergic diseases. In addition to Grail, other E3 ubiquitin ligases such as Itch has been implicated in regulation of Th2 development by targeting JunB 31 . These E3 ligases could potentially cooperate with each other to play a synergistic role in promoting Th2 cell tolerance. Future studies in mice deficient in both Grail and Itch will potentially have therapeutic implications for allergic diseases.

Mice and cell lines
Grail KO mice were generated as described before 21 . OT II TCR transgenic, TCR-β KO, Rag1 KO, Stat6 KO and C57BL/6J mice were obtained from Jackson laboratory. All mice were housed in the SPF animal facility at M.D. Anderson Cancer Center and the animal experiments were performed using protocols approved by Institutional Animal Care and Use Committee. The EL4 (murine lymphoma) and HEK293T (human fibroblast) cell lines were purchased from ATCC and maintained in DMEM medium supplemented with 10% fetal bovine serum and antibiotics.

Retroviral transduction
Retroviral transduction was performed as described previously 21 . Briefly, naive CD4 + CD25 − CD62L hi CD44 lo T cells from OT-II mice were sorted by flow cytometry and activated with Ova peptide and irradiated WT splenic antigen-presenting cells (APCs). Twenty-four hours after activation, cells were infected by retroviruses expressing Stat6, Gata3, IRF4 or JunB or control empty vector (containing only IRES-GFP). Four days after infection, GFP + cells were flow cytometry-sorted and restimulated with anti-CD3 for 4 hrs, and mRNA expression were analyzed by qRT-PCR analysis. For over expression experiments in Fig. 2f and Supplementary Fig. 2b naive CD4 + CD25 − CD62L hi CD44 lo T cells were sorted by flow cytometry from C57BL/6J (WT) and Stat6 KO mice by flow cytometry and activated with anti-CD3 and anti-CD28 for 24hrs prior to infection.

Immunoblot (IB) analysis and ubiquitination assay
Western blot analysis and ubiquitination assay was performed as described previously 21 . Briefly, whole T cell lysates were prepared by lysing cells in triton lysis buffer. The amounts of protein were determined by Bio-Rad protein assay to ensure equal protein loading for immunoblot analysis with antibodies to Stat6, phospho-Stat6 and Gata3 (Santa Cruz Biotechnology, 1:1000 dilution) and beta-Actin (Pierce, 1:1000 dilution).
For coimmunoprecipitation experiments, HEK293T cells were transfected in 6-well plates with retroviral expression vectors containing Stat6 and Grail-GFP. Stat6 protein in the cell lysates was immunoprecipitated using anti-Stat6 antibody (5μg/sample), followed by detection of Grail by IB using anti-GFP antibody (Santa Cruz Biotechnology, 1:500 dilution).

Ovalbumin (Ova) Immunization
Male WT (C57BL/6J) and Grail KO mice (6-8 weeks old, n=5per group) were immunized with Ova protein (1.0 mg/ml) emulsified in aluminum hydroxide (Alum) intraperitoneally. Seven days after immunization, mice were sacrificed and analyzed individually. Spleen cells from Ova-immunized mice were stimulated in 96-well plates as triplicates with or without different concentrations of Ova protein. IL-2 production was determined 24 hours after T cell activation by ELISA. Effector cytokines were analyzed 3 days later by ELISA (PharMingen).

Adoptive transfer and mixed bone marrow chimera studies
Adoptive transfer and bone marrow chimera experiments were done as described previously 40 . Breifly, FACS sorted naïve CD4 + T cells from 8 week old male WT or Grail KO mice were intravenously transferred into 6-8 week old male TCR-β KO mice (10 × 10 6 cells/mouse, n=4 per group). The recipient mice were immunized i.p. with Ova in alum 24 hrs post transfer. Seven days after immunization, spleen cells from the recipient mice were isolated and CD4 + CD44 hi effector memory cells were sorted for analysis.
To generate mixed bone marrow chimera, T cell-depleted bone marrow cells were obtained from 6-8 weeks old male B6.SJL (CD45.1) or Grail KO mice and mixed 1:1 ratio before being transferred into irradiated male (6-8 weeks old, n=10) Rag1 KO mice (10 × 10 6 cells/ mouse, 750 rad). Eight weeks later, the recipient mice were immunized with Ova in alum and analyzed as above.

Asthma induction
Asthma induction was done as described previously 21,41 . Breifly, male WT (C57BL/6J) and Grail KO mice (6-8 weeks old,n=5per group) were immunized twice intraperitoneally at 2 weeks interval with 0.2 ml saline containing 100 μg Ova in Alum. On day 14 mice were intranasally challenged with Ova followed by three more challenges at day 26, 27 and 28. 24 hours post the last challenge mice were sacrificed and the BALF, lungs, spleens and the lung lymph nodes were harvested for further analysis.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.     naïve CD4 + T cells from WT and Grail KO mice were intravenously transferred into TCR-β KO mice (5 million cells per mouse,n=4 per group) followed by intraperitoneal injection with Ova in alum. Seven days later WT and Grail KO CD4 + CD44 + cells were sorted and effector cytokine levels were measured by qRT-PCR analysis (d) and ELISA (e). (f) Mixedbone marrow chimeric mice (n = 10) were generated by co-transfer of 10 million cells at a ratio of 1:1 WT (CD45.2) and Grail KO (CD45.1) bone marrow cells into Rag1 KO mice. 8 weeks later, mice were immunized with Ova in alum. One week later and WT and Grail KO CD4 + CD44 + cells were sorted and effector cytokine levels were measured by ELISA. The results shown are mean ± SEM. P values: *<0.05, **<0.01, ***<0.001, ****<0.0001, Student's t-test was performed to detect between-group differences.. The data are representative of at least three independent experiments with consistent results.  subjected to immunoprecipitation (IP) using anti-Stat6 antibody. The blot was probed with anti-HA-HRP and re-probed for Stat6 expression. (e) CD4 + T cells from WT and Grail KO mice were activated with anti-CD3 and IL-4 along with MG132 (proteasome inhibitor) for 6 hrs. The cell lysates were then immunoprecipitated using anti-Stat6 antibody and the blot was probed with anti-ubiquitin or anti-Stat6 antibodies. Relative western blotting signals were calculated as a ratio of ubiquitinated and non-ubiquitinated Stat6. (f) HEK293T cells were either left untransfected or transfected with vectors encoding Stat6, Grail-GFP or both. The lysates were subjected to IP using anti-Stat6 antibody. The blot was probed with anti-GFP antibody (upper panel) and reprobed with anti-Stat6 antibody (middle panel) as a control. The WCL was immunoblotted with anti-GFP antibody (lower panel) for checking antibody efficacy. The results shown in the figures are representative blots of at least three independent experiments. P values: *<0.05, Student's t-test was performed to detect between-group differences..