Foxo1 restrains naive CD4 T cell differentiation into type 1 and type 2 effector cells by limiting early cytokine production

Foxo1 restrains naive CD4 T cell differentiation into type 1 and type 2 effector cells by limiting early cytokine production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Foxo1 restrains naive CD4 T cell differentiation into type 1 and type 2 effector cells by limiting early cytokine production Léa GIRAUD, Charlotte GUILLOU, Théo LEVEL, Aurélie DURAND, Céline CHARVET, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8403997/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Over the past 15 years, Foxo1 has emerged as a key transcription factor in T cell biology, regulating survival, trafficking, and the establishment of memory responses. Its role in naive CD4 T cell differentiation into effector cells, however, appears more complex. Foxo1 has been reported to be essential for the generation of induced regulatory T cells and TH9 cells, while restraining the differentiation of TH17 and TFH lineages. Surprisingly, its contribution to TH1 and TH2 differentiation has remained poorly defined. Here, we show that memory CD4 T cells from Foxo1 TKO mice exhibited enhanced capacity to produce type 1 and type 2 cytokines ex vivo , associated with increased T-bet and GATA3 expression. Foxo1-deficient naive CD4 T cells displayed a strong bias toward TH1 and TH2 differentiation in vitro , even under nonpolarizing conditions, driven by rapid and robust production of IL-4, IL-13 and IFN-γ upon activation. Coculture experiments further revealed that Foxo1-sufficient cells benefited from cytokines produced by Foxo1-deficient cells, while competition for IFN-γ limited the differentiation of the latter. Mechanistically, IL-4 stimulation elicited increased STAT1 and STAT3 phosphorylation in Foxo1-deficient naive CD4 T cells. Pharmacological inhibition of STAT3, but not STAT1, markedly reduced their TH1/TH2 polarization capacity, although residual differentiation suggested the involvement of additional mechanisms. Immunology Helper T cells Foxo1 TH1 TH2 STAT proteins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Members of the forkhead box O (Foxo) transcription factor family are part of an evolutionary conserved pathway, first discovered downstream of insulin and insulin-like growth factor receptors 1 . In mammals, there are 4 Foxo members, namely Foxo1, Foxo3, Foxo4 and Foxo6 which share high protein homology 2 . Foxo1, Foxo3 and Foxo4 are expressed in almost all tissues whereas Foxo6 expression is mainly restricted to the central nervous system 3 and the oxidative muscle 4 . In all cell types, Foxo transcription factors are essential for many processes, such as metabolism, quiescence, cell survival and differentiation 5 . T cells mainly express Foxo1 and Foxo3 6 although a recent article has shown that Foxo4 deficiency in T cells boosts their differentiation potential toward interferon (IFN)-γ-producing cells 7 . The transcription factor Foxo1 controls many aspects of T cell physiology in mammals. First, it regulates peripheral T cell homeostasis 8 . More precisely, Foxo1 increases T cell survival by inducing IL-7 receptor expression 8 . Foxo1 is also crucial for T cell trafficking by increasing the expression of key molecules involved in their entry to and exit from lymphoid and non-lymphoid tissues such as CD62L, S1PR1 and CCR7 8,9 . Recently, it has been shown that Foxo1 limits the expression of inhibitory receptors and subsequent T cell exhaustion during aging as well as in CAR T cells 10–12 . Delpoux et al. and Zhang et al. have demonstrated that Foxo1 is required for the efficient generation of long-lasting memory CD8 T cells following viral or bacterial infections 13,14 . Indeed, Foxo1 deficiency leads to the differentiation of naive CD8 T cells into terminally differentiated senescent effector cells 15,16 . Finally, Foxo1 plays a crucial role in directing the differentiation of naive CD4 T cells into effector cells 17–21 . Naive CD4 T cells can differentiate into a broad spectrum of effector cells characterized by specific cytokine production and expression of a master transcription factor controlling this ability. For instance, type 1 T Helper (TH1) cells, which allow eradication of intracellular pathogens, are characterized by their production of IFN-γ, a potent activator of cell-mediated immunity. TH2 cells, whose generation predominantly occurs to enhance elimination of parasitic infections, are characterized by production of interleukin (IL)-4, IL-5, and IL-13. TH9 cells, characterized by their ability to produce IL-9 and IL-21, play a role in the protection from helminths. TH17 cells are characterized by IL-17 and IL-22 secretion and are described to play an important role in anti-microbial responses by orchestrating innate immune function, such as granulocyte accumulation at the inflammatory site. T Follicular Helper (TFH) cells produce IL-21 and are involved in the humoral response by triggering germinal center B cells into antibody-secreting plasma and memory B cells. Finally, induced regulatory T cells (iTreg cells) produce TGF-β and IL-10 and play a crucial role in maintaining peripheral self-tolerance and suppressing excessive immune responses deleterious to the host. Importantly, each of these effector CD4 T cell subsets is characterized by the expression of a lineage specific transcription factor (T-bet for TH1 cells, GATA-3 for TH2 cells, PU.1 for TH9, RORγt/RORα for TH17 cells, Bcl6 for TFH cells and Foxp3 for iTreg cells). Foxo1 either inhibits or enhances the differentiation potential of naive CD4 T cells into a given T helper cell lineage. Indeed, on one hand, Foxo1 inhibits TFH cell differentiation through its ability to dampen the expression of genes important for their differentiation, including Bcl6 19 . Moreover, we have demonstrated that Foxo1 is a T cell intrinsic inhibitor of the TH17 program 20 . On the other hand, several articles have reported that Foxo1 is required for TH9 cell generation 22,23 . In addition, Foxo1 deficiency in mouse T cells has been proposed to impair both the generation and suppressive function of iTreg cells 17,18,24 . Surprisingly, the involvement of Foxo1 in TH1/TH2 cell generation has not yet been studied with precision. In this paper, we show that the memory CD4 T cell compartment from the secondary lymphoid organs (SLOs) of mice lacking the expression of Foxo1 in T cells (Foxo1 TKO mice, 8 ) contains higher proportions of TH1 and TH2 effector cells. In agreement, naive CD4 T cells from Foxo1 TKO mice exhibit a higher potential for differentiating into TH1 and TH2 cells than their T cell counterparts from Wild-Type (WT) mice in vitro . Finally, we sought to identify the molecular mechanisms linking Foxo1 expression to the inhibition of TH1- and TH2 cell differentiation. RESULTS Increased proportions of types 1 and 2 effector CD4 T cells in the SLOs of Foxo1 TKO mice As in our previous articles 12,25 , throughout this manuscript, naive, memory and regulatory CD4 T cells (CD4 N , CD4 M and CD4 R cells respectively) as well as naive and memory CD8 T cells (CD8 N and CD8 M cells respectively) have been defined based on their expression of CD44 and Foxp3. Briefly, among TCR b + cells, CD4 R cells were defined as Foxp3 + CD4 + CD8 a - cells, CD4 M cells as CD44 hi Foxp3 - CD4 + CD8 a - cells and CD4 N cells as CD44 -/low Foxp3 - CD8 a - cells. CD44 expression was also used to discriminate between naive and memory CD8 T cells. The proportions and absolute numbers of CD4 M cells in the SLOs were not significantly different one week after birth in mice deficient for Foxo1 expression in T cells (CD4 cre Foxo1 fl/fl mice alias Foxo1 TKO mice) versus mice proficient for Foxo1 expression in T cells (Foxo1 fl/fl mice not expressing the Cre recombinase alias Foxo1 Ctrl mice; Fig. 1A, 1B). However, the proportions of CD4 M cells increased sharply thereafter in Foxo1 TKO mice, so that the absolute numbers of these cells were significantly higher in all the SLOs of 12-week-old Foxo1 TKO mice than in those of Foxo1 Ctrl mice of the same age (Fig. 1A, 1B). Plasma concentrations of IFN-γ (TH1) and IL-5 (TH2) were higher in 8-week-old Foxo1 TKO mice compared to age-matched Foxo1 Ctrl mice (Fig. 1C). However, these results may reflect the quantitative expansion of CD4 M cells observed in the SLOs of Foxo1 TKO mice rather than qualitative alterations in the memory CD4 T cell compartment. Of note, plasma IL-4 concentrations were not significantly increased in Foxo1 TKO mice. To distinguish between these 2 hypotheses, we then compared the ex vivo capacity of CD4 M cells from Foxo1 TKO and Foxo1 Ctrl mice to produce type 1 (IFN-g) versus type 2 (IL-13) cytokines (Fig. 1D-F). The proportion of IFN-g producing CD4 M cells was increased in all SLOs of Foxo1 TKO compared to Foxo1 Ctrl mice, while the percentage of IL-13 + cells among CD4 M cells was increased only in pLNs (Fig. 1D, 1E). Interestingly, in pLNs for IL-13 and in all SLOs for IFN-g, cytokine + CD4 M cells produced larger amounts of these cytokines in Foxo1 TKO mice than in WT mice (Fig. 1F). Consistent with their enhanced production of type 1 and type 2 cytokines, a higher proportion of CD4 M cells from Foxo1 TKO mice expressed T-bet and GATA3. Moreover, cells expressing these transcription factors displayed higher expression levels than their counterparts from Foxo1 Ctrl mice (Fig. 1G–I). Interestingly, although the proportions of CD8 M producing IFN-g or expressing T-bet were only modestly increased in Foxo1 TKO mice compared to Foxo1 Ctrl mice, the relative Mean Fluorescence Intensity (MFI) of these parameters among positive cells was markedly and significantly increased (Fig. S1A-C). CD4 N cells from Foxo1 TKO mice are prone to differentiate into types 1 and 2 effector T cells We hypothesized that the over-representation of TH1 and TH2 effector cells among peripheral CD4 M cells from Foxo1 TKO mice might result from an increased propensity of Foxo1-deficient CD4 N cells to differentiate into these lineages. To test this, we purified CD4 N cells from Foxo1 TKO and Foxo1 Ctrl mice and polarized them in vitro toward TH1 (Fig. 2A-D) or TH2 (Fig. 2E-H) effector cells. CD4 N cells from Foxo1 TKO mice were markedly more efficient than their counterparts from Foxo1 Ctrl mice at differentiating into IFN-g- (Fig. 2B, 2C) or IL-13- (Fig. 2F, 2G) producing cells. Notably, upon activation, Foxo1-deficient CD4 N cells not only exhibited increased frequencies of type 1 and type 2 cytokine producing cells but also produced higher cytokine amounts than their Foxo1 Ctrl counterparts (Fig. 2D, 2H). Surprisingly, even in the absence of polarizing cytokine (IL-12 for TH1 cells and IL-4 for TH2 cells), Foxo1-deficient CD4 N cells differentiated very efficiently into TH1 or TH2 effector cells (Fig. 2B, 2F). Indeed, on the one hand, around 30% of the progeny of Foxo1-deficient CD4 N cells acquired the ability to produce IFN-g after 4 days of activation in the absence of exogenous IL-12 (Fig. 2B, 2C). On the other hand, about 75% of the progeny of the same cells produced IL-13 upon a 5-day activation in the absence of exogenous IL-4 (Fig. 2F, 2G). CD4 N cells from Foxo1 TKO mice are thus prone to differentiate into TH1 and TH2 effector cells upon activation. Previous articles have shown that Foxo1 expression was required for the differentiation of CD4 N cells into TH9 cells 21 and iTreg cells 17,18 in vitro . Of note, in the TH9 polarization assay, an antibody neutralizing IFN-g was not added in the culture medium 22,23 and IL-4 and IFN-g were not blocked in the course of CD4 N cell differentiation into iTreg cells 17,18 . Therefore, we repeated these experiments, this time adding cytokine-neutralizing antibodies to the culture medium (Fig. S2). Interestingly, when a blocking antibody neutralizing IFN-g during the TH9 polarization assay, CD4 N cells from Foxo1 TKO mice differentiated even more efficiently than CD4 N cells from Foxo1 Ctrl mice into IL-9-producing cells (Fig. S2A-D). Neutralization of IL-4 and IFN-g almost completely restored the ability of CD4 N cells from Foxo1 TKO to differentiate into iTreg cells (Giraud et al.). However, in contrast to what we observed for TH1 and TH2 polarization, the addition of the polarizing cytokine TGF-β to the culture medium was strictly required for Foxo1-deficient CD4 N cells to differentiate into TH9 cells (Fig. S2A, S2B) or iTreg cells (Giraud et al.). CD4 N cells from Foxo1 TKO mice are biased to differentiate into TH1 and TH2 effector cells under nonpolarizing conditions To further investigate the enhanced propensity of Foxo1-deficient CD4 N to differentiate into TH1 and TH2 effector cells, we stimulated them under nonpolarizing conditions (Fig. 3A, TH0 polarization assay). After 4 days, a significant proportion of the progeny of CD4 N cells from Foxo1 TKO mice acquired the capacity to produce either IFN-g or IL-13 (Fig. 3B, 3C). Notably, very few cells produced both cytokines simultaneously. In contrast, the majority of Foxo1-deficient cells at this time-point co-expressed T-bet and GATA3 (Fig. 3D, 3E). Thus, at the level of lineage-defining transcription factors, Foxo1 deficiency enables CD4 T cells to adopt a mixed TH1/TH2 effector phenotype upon activation, although regulatory mechanisms appear to restrict individual cells from concomitantly producing both type 1 and type 2 cytokines. When neutralizing antibodies against IFN-γ and IL-4 were added to the culture medium (Fig. 3F), a fraction of Foxo1-deficient CD4 T cells still differentiated into IFN-γ– or IL-13–producing cells (Fig. 3G). Similarly, some stimulated cells continued to express T-bet or GATA3 (Fig. 3H). However, the frequencies were markedly reduced compared with cultures without cytokine neutralization, suggesting the existence of a positive feedback loop in which early IFN-γ and IL-4 production enhances the ability of Foxo1-deficient CD4 N cells to differentiate into TH1 and TH2 lineages, respectively. We next asked whether Foxo1 activity is required during TH0 polarization to restrain the differentiation of CD4 N cells into TH1 cells and TH2 cells. To address this question, we supplemented the culture medium with AS1842856, a chemical inhibitor that prevents Foxo1 from binding to DNA ( 20,26 , Fig. 3I). Blocking Foxo1 transcriptional activity during the TH0 polarization assay did not boost the ability of CD4 N cells to differentiate into TH1 or TH2 effector cells (Fig. 3J, 3K). Thus, Foxo1 activity would be required prior to CD4 N cell activation to prevent their differentiation into TH1 or TH2 effector cells under nonpolarizing (TH0) conditions. Rapid and robust IL-4 and IFN- g production by Foxo1-deficient CD4 N cells upon activation underlies their biased differentiation into TH1 and TH2 effector cells We hypothesized that CD4 N from Foxo1 TKO mice would rapidly produce type 1 and type 2 cytokines upon activation. In line with our assumption, after only 2 days of culture in the TH1 polarization assay, a substantial fraction of Foxo1-deficient CD4 T cells produced IFN-γ (Fig. 4A). Similarly, on day 2 of the TH2 polarization assay, higher proportions CD4 N cells from Foxo1T KO mice produced IL-13 (Fig. 4A, 4B) and IL-4 (Fig. S3A, S3B)." To determine whether early cytokine production by Foxo1-deficient CD4 T cells during polarization assays contributes to their increased propensity to differentiate into TH1 or TH2 effector cells, we cocultured Foxo1-deficient CD4 N cells with Foxo1-sufficient CD4 N cells (Fig. 4B). When cocultured with CD4 N cells from Foxo1 TKO mice, WT CD4 N cells gave rise to significantly higher proportions of IFN-g- and IL13-producing cells during TH1 and TH2 polarization assays, respectively, than when cultured alone (Fig. 4C). Notably, in TH1 cocultures, Foxo1-deficient cells differentiated less efficiently into TH1 effector cells than when cultured alone (Fig. 4C). Overall, these results strongly suggest that, in coculture, Foxo1-expressing CD4 N cells benefit from the early cytokine production of Foxo1-deficient cells. We hypothesized that Foxo1-deficient and Foxo1-sufficient CD4 N cells may compete for IFN-g during the TH1 polarization assay. Consistent with this hypothesis, the addition of exogenous IFN-γ to the culture medium significantly enhanced the differentiation of CD4 N cells from Foxo1 Ctrl mice into TH1 effector cells but had no effect on CD4 N cells from Foxo1 TKO mice (Fig. 4D, 4E). Conversely, neutralization of IFN-g during the polarization assay markedly impaired the capacity of Foxo1-deficient CD4 N cells to polarize into TH1 effector cells (Fig. 4F, 4G). However, this treatment completely abrogated the differentiation of WT CD4 N cells into TH1 effector cells, so that in these conditions, CD4 N cells from Foxo1 TKO mice still differentiated more efficiently than their counterparts from Foxo1 Ctrl mice. These results suggest that mechanisms other than IFN-γ overproduction contribute to the enhanced TH1 differentiation capacity of Foxo1-deficient CD4 N cells. Interestingly, neutralization of IL-13 during the TH2 polarization assays markedly reduced the capacity of Foxo1-deficient CD4 N cells to differentiate into IL-13-producing cells while not affecting WT CD4 N cells (Fig. 4H, 4I). Altogether, these results suggest that the enhanced capacity of Foxo1-deficient CD4 N cells to differentiate into TH1 and TH2 effector cells arises from ( i ) their rapid production of IFN-γ and IL-13 and ( ii ) a subsequent positive feedback loop in which these cytokines further amplify their polarization potential. Enhanced STAT3 phosphorylation in response to IL-4 may underlie the increased TH2 differentiation potential of Foxo1-deficient CD4 N Cells Differentiation of CD4 N cells to TH effector cells relies on polarizing cytokine signaling pathway, particularly on the activation of STAT transcription factors 27,28 . We assessed the phosphorylation of STAT proteins, namely STAT1, STAT3, STAT4 and STAT6, in CD4 N cells after a 15-minute culture with either IL-12 or IL-4 (Fig. 5). Surprisingly, at this time-point, IL-12 failed to induce phosphorylation of any tested STAT proteins, including STAT4, which is typically activated by IL-12 and required for efficient TH1 polarization (Fig. 5A, 5B). In contrast, IL-4, not only strongly induced phosphorylation of STAT6, the canonical mediator downstream of the IL-4 receptor, but also triggered significant phosphorylation of STAT1 and STAT3 (Fig. 5A, 5B). Interestingly, while IL-4–induced STAT6 phosphorylation was comparable between Foxo1-sufficient and Foxo1-deficient CD4 N cells, IL-4 triggered markedly stronger STAT1 and STAT3 phosphorylation in CD4 N from Foxo1 TKO mice than in their counterparts from Foxo1 Ctrl mice (Fig. 5B). We next tested whether inhibition of STAT1 (Fludarabine) or STAT3 (C188-9) influenced the ability of CD4 N from Foxo1 TKO mice to produce IFN-γ and IL-13 upon activation under nonpolarizing conditions (Fig. 5C, 5D). STAT1 inhibition induced only a modest reduction in cytokine production by Foxo1-deficient CD4 N cells and had no effect on Foxo1-sufficient CD4 N cells. In contrast, STAT3 inhibition markedly impaired the ability of CD4 N from Foxo1 TKO mice to produce IFN-γ and IL-13 upon activation. Nonetheless, even in the presence of the STAT3 inhibitor, a higher proportion of Foxo1-deficient CD4 N cells produced these cytokines compared to Foxo1-sufficien CD4 N cells (Fig. 5C, 5D), suggesting that additional mechanisms contribute to the enhanced propensity of Foxo1-deficient CD4 N cells to differentiate into TH1 and TH2 effector cells. DISCUSSION Foxo1 is a key transcription factor in T cell biology, required for T cell survival, trafficking, and the generation of efficient memory responses 8,9,13,14 . Its role in the differentiation of CD4 N cells into effector subsets, however, appears more complex. While Foxo1 has been proposed to be essential for the generation of peripheral regulatory CD4 T cells and TH9 cells, it may restrain the differentiation of CD4 N into TFH and TH17 effector cells 17–20,22,23 . In the present study, we demonstrate that Foxo1 also strongly limits the differentiation of CD4 N into TH1 and TH2 effector cells (Fig. 2). Notably, Foxo1-deficient CD4 N cells readily differentiated into TH1 and TH2 cells upon stimulation in vitro , even in the absence of exogenous polarizing cytokines (Fig. 3). Our findings support a two-step mechanism: ( i ) Foxo1-deficient CD4 N cells rapidly produce high levels of type 1 (IFN-γ) and type 2 (IL-4 and IL-13) cytokines upon activation, and ( ii ) this early cytokine burst amplifies their propensity to differentiate into TH1 and TH2 effector cells. Indeed, neutralization IFN-γ or IL-13 markedly reduced the ability of Foxo1-deficient CD4 N cells to differentiate into TH1 and TH2 cells, respectively (Fig. 4). Conversely, supplementation of the culture medium with IFN-γ enhanced the capacity of Foxo1-sufficient CD4 N cells to differentiate into TH1 effector cells (Fig. 4). These findings are consistent with previous reports showing that IFN-γ amplifies and stabilizes TH1 cell generation and phenotype 29 and that the propensity of IL-13-deficient CD4 N cells to differentiate into TH2 cells is strongly diminished 30,31 . It remains important to elucidate the molecular mechanisms underlying the rapid production of IL-4, IL-13 and IFN-γ by Foxo1-deficient CD4 N cells upon stimulation (Fig. 4). Interestingly, pharmacological inhibition of Foxo1 during the TH0 polarization assay did not recapitulate the phenotype observed with Foxo1-deficient CD4 N cells (Fig. 3). This finding suggests that the absence of Foxo1 expression may lead to a stable reprogramming of CD4 N cells, predisposing them to produce type 1 and type 2 cytokines. Beyond its transcriptional function, Foxo1 has also been implicated in chromatin remodeling in T cells. For instance, using genome-wide expression and methylation analyses, Baessler et al. demonstrated that the methylcytosine dioxygenase Tet2 interacts with Foxo1 to mediate DNA demethylation and proper expression of target genes in CD4 T cells 32 . In addition, Gray et al. showed that Foxo1 is required to limit deposition of the repressive histone mark H3K27me3 by the Polycomb Repressive Complex 2 (PRC2) although no direct interaction between Foxo1 and PRC2 subunits could be detected in co-immunoprecipitation assays 33 . Strikingly, CD4 N cells deficient for Ezh2, the catalytic subunit of PRC2, spontaneously differentiated into type 1 and type 2 cytokine-producing cells under nonpolarizing conditions, a phenotype strongly reminiscent of that observed in Foxo1-deficient CD4 N cells 34,35 . Together, these findings suggest that Foxo1 may act as an epigenetic gatekeeper, preventing premature acquisition of TH1 and TH2 effector programs in naïve CD4 N cells. Our data also point to a potential role for STAT3, as its pharmacological inhibition strongly impaired the ability of CD4 N cells from Foxo1 TKO mice to produce IFN-γ and IL-13 upon activation under nonpolarizing conditions (Fig. 5). Consistently, STAT3 was rapidly phosphorylated upon IL-4 stimulation, with significantly higher levels observed in Foxo1-deficient than in Foxo1-sufficient CD4 N cells (Fig. 5). IL-4 also induced robust STAT6 phosphorylation, the canonical STAT protein downstream of type I and type II IL-4 receptors 36,37 , but the extent of STAT6 activation did not differ between Foxo1-deficient and Foxo1-sufficient CD4 N cells (Fig. 5). Several studies have implicated STAT3 in the TH2 differentiation pathway and suggested that it may cooperate with STAT6 to promote TH2 cell development 38–40 . The marked decrease in IFN-γ–producing cells upon STAT3 inhibition is somewhat unexpected. Indeed, while STAT1 phosphorylation is well established to promote TH1 cell differentiation, most notably by directly activating T-bet expression in synergy with STAT4 29,41 , STAT3 activation, by contrast, has generally been described as exerting an inhibitory effect on this process 42 . Here, we show that Foxo1 also strongly limits the ability of CD4 N cells to differentiate into TH1 and TH2 effector cells, a feature that may largely account for previous reports describing an absolute requirement for Foxo1 in the generation of TH9 or peripheral regulatory T cells 17,18,22,23 . Indeed, our results clearly demonstrate that the impaired generation of induced regulatory T cells and TH9 cells observed in vitro in the absence of Foxo1 expression stems from the intrinsic propensity of Foxo1-deficient T cells to produce TH1- and TH2-type cytokines, such as IFN-γ and IL-4. Neutralization of IFN-γ during TH9 polarization, and of both IFN-γ and IL-4 during regulatory T cell polarization, restores the differentiation of Foxo1-deficient CD4 N cells into TH9 and induced regulatory T cells to levels comparable to, or even exceeding, those of control CD4 N cells (Fig. S2 et Giraud et al.). Collectively, these findings suggest that Foxo1 acts as a global brake on CD4 N cell differentiation, restricting their potential to polarize toward all tested effector lineages. We therefore propose that Foxo1 fine-tunes the responsiveness of CD4 N cells to their environment, and in particular to the cytokine milieu present at the time of activation. In the absence of Foxo1, naïve CD4 T cells would escape this regulation and undergo uncontrolled differentiation, potentially leading to immune responses that are no longer aligned with the nature of the initiating signals, such as pathogen type or inflammatory context. METHODS Mice. 1-week-old to 12-week-old mice were used. C57BL/6 Foxp3-GFP Foxo1 TKO and Foxo1 Ctrl CD45.1 or CD45.2 mice 8,20 and C57BL/6 CD3eKO 43 and were maintained in our own animal facilities, under specific pathogen–free (SPF) conditions. All procedures were approved by the ethics committee for animal experimentation n°34 and validated by the “Ministère de l’Enseignement Supérieur de la Recherche et de l'Innovation” with the number APAFIS≠20630-2018033016303981v5. Sample sizes were chosen to assure reproducibility of the experiments and in accordance with the 3R of animal ethics regulation. Cell suspensions. Peripheral lymph nodes (pooled cervical, axillary, brachial and inguinal lymph nodes; pLNs), mesenteric LNs (mLNs) and spleen were homogenized and passed through a nylon cell strainer (BD Falcon) in 5% FCS, 0.1% NaN3 (Interchim) in phosphate saline buffer saline (PBS) for flow cytometry. Fluorescence staining and flow cytometry. Cell suspensions were collected and dispensed into 96-well round-bottom microtiter plates (Greiner Bioscience; 6x10 6 cells/well). Surface staining was performed as described 44 . Briefly, cells were incubated on ice, for 15 min/step, with Abs in 5% FCS (Biochrom), 0.1% NaN3 (Sigma-Aldrich) in PBS. Each cell staining reaction was preceded by a 15-min incubation with a purified anti-mouse CD16/32 Ab (FcgRII/III block; 2.4G2, BioXcell). The Foxp3 Staining Buffer Set (eBioscience) was used for Foxp3 intracellular staining. For determination of intracellular cytokine production, cells were stimulated with 0.5 µg/ml PMA (Sigma-Aldrich), 0.5 µg/ml ionomycin (Sigma-Aldrich), and 10 μg/ml BrefeldinA (Sigma-Aldrich) for 2 hrs at 37°C. Cells were then stained for surface markers, fixed in 2% paraformaldehyde in PBS, and permeabilized with 0.5% saponin, followed by labeling with specific cytokine Abs. For determination of the phosphorylation of STAT proteins, cells were immediately fixed at the end of the culture with 4% PFA during 5 min at 37°C; then washed twice and permeabilized with cold methanol at -20°C for 30 min. Cells were then washed 3 times and stained overnight at 4°C for surface markers and intracellular pSTAT proteins.Multi-color immunofluorescence was analyzed using a BD-Fortessa cytometer (BD Biosciences).Data acquisition and cell sorting were performed at the Cochin CYBIO facility. Purification of CD4 N cells. CD4 T cells were purified from LNs (pooled superficial cervical, axillary, brachial, inguinal, and mesenteric LNs) of Foxo1 TKO and Foxo1 Ctrl mice by incubating cell suspensions on ice for 20 minutes with a mixture of anti-CD8 (53-6.7), anti-CD11b (Mac-1) and anti-CD19 (1D3) Abs obtained from hybridoma supernatants, and then with magnetic beads coupled to anti-rat immunoglobulins (Dynal Biotech). CD4 N cells were then flow cytometry sorted as Foxp3-GFP - , Lineage (CD25, TCRgd, CD8 b , CD11b, CD11c)-PE - , CD44-APC -/lo cells using a FACS-ARIA3 flow cytometer (BD Biosciences) at the Cochin CYBIO facility. Cell Cultures. In vitro polarization assays. 20x10 4 Flow-cytometry sorted CD4 N cells from the LNs of C57BL/6 Foxp3-GFP mice were stimulated for 4 (TH0, TH1) or 5 (TH2) days with immobilized anti-CD3e (clone 145.2C11; 4µg/ml; obtained from hybridoma supernatants) and anti-CD28 (clone 37.51; eBioscience; 4μg/ml) antibodies, in the absence (TH0) or presence of graded concentrations of exogenous IL12 (TH1, R&D) or IL-4 (TH2, R&D). 15μg/mL of a monoclonal antibody neutralizing IL-4 (TH1, BioLegend) or IFN-g (TH2, BioLegend) were added in the TH1 or TH2 polarization assays respectively. In some experiments, TH1 polarization was also done in the presence of graded concentrations of IFN-g. In some other experiments, CD4 N cells were cultured under nonpolarizing conditions in the presence or absence of a STAT1 inhibitor (fludarabine, SelleckChem) or a STA3 inhibitor (C188-9, SelleckChem). For the TH9 polarization assay, CD4 N cells were stimulated for 5 days as indicated above in the presence of graded concentrations of exogenous TGF-β (Invitrogen) and of 10ng/mL of IL-4. 15μg/mL of a monoclonal antibody neutralizing IFN-g were added in the TH9 polarization assay. 15μg/mL of a monoclonal antibody neutralizing IL-4 and of a monoclonal antibody neutralizing IFN-g in the iTreg polarization assay. Cocultures. In some experiments (Fig. 4C, Fig. S4C), 10x10 4 purified CD4 N cells from Foxo1 TKO and Foxo1 Ctrl mice were cultured together under nonpolarizing conditions (TH0) or under TH1- or TH2-polarizing conditions. Cultures for studying the phosphorylation of STAT proteins. 1x10 6 CD4T cells from Foxo1 TKO and Foxo1 Ctrl mice were purified, let to rest at 37°C for 1 hr and then cultured for 15 min in medium supplemented or not with 10ng/ml of IL-4 or 20ng/ml IL-12. Statistical analysis. Data are expressed as mean ± SEM, and the significance of differences between two series of results was assessed using the Student’s unpaired or paired t test. Values of p < 0.05 were considered as statistically significant. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Declarations Author Contributions C.A., B.M. and B.L. designed experiments. C.G. performed the experiments shown in Figures 1-3 and Figures S1-S2. LG performed experiments shown in Figures 4 and 5. T.L. performed experiments shown in Figures 4 and Figures S3. LG and B.M. made the figures. B.L. wrote the paper. Acknowledgments We greatly acknowledge M. Andrieu from the Cochin Immunobiology facility. This work was supported by a grant from the “Fondation pour la Recherche Médicale” (FRM team number EQU202103012662). Théo Level was supported by a Ph.D. fellowship from the “LaBex Who AM I?”. References Link W, Fernandez-Marcos PJ (2017) FOXO transcription factors at the interface of metabolism and cancer. Int J Cancer 141:2379–2391 Gui T, Burgering BMT (2021) FOXOs: masters of the equilibrium. FEBS J. 10.1111/febs.16221 Jacobs FMJ et al (2003) FoxO6, a novel member of the FoxO class of transcription factors with distinct shuttling dynamics. J Biol Chem 278:35959–35967 Chung SY et al (2013) FoxO6 and PGC-1α form a regulatory loop in myogenic cells. Biosci Rep 33:e00045 Xing Y-Q et al (2018) The regulation of FOXO1 and its role in disease progression. Life Sci 193:124–131 Hedrick SM, Michelini H, Doedens R, Goldrath AL, A. W., Stone E (2012) L. FOXO transcription factors throughout T cell biology. 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Sci Adv 8:eabm4982 Gray SM, Amezquita RA, Guan T, Kleinstein SH, Kaech SM (2017) Polycomb Repressive Complex 2-Mediated Chromatin Repression Guides Effector CD8 + T Cell Terminal Differentiation and Loss of Multipotency. Immunity 46:596–608 Zhang Y et al (2014) The polycomb repressive complex 2 governs life and death of peripheral T cells. Blood 124:737–749 Tumes DJ et al (2013) The polycomb protein Ezh2 regulates differentiation and plasticity of CD4(+) T helper type 1 and type 2 cells. Immunity 39:819–832 Takeda K et al (1996) Essential role of Stat6 in IL-4 signalling. Nature 380:627–630 Kaplan MH, Schindler U, Smiley ST, Grusby MJ (1996) Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4:313–319 Wills-Karp M, Finkelman FD (2008) Untangling the complex web of IL-4- and IL-13-mediated signaling pathways. Sci Signal 1:pe55 Stritesky GL et al (2011) The transcription factor STAT3 is required for T helper 2 cell development. Immunity 34:39–49 Lim H, Cho M, Choi G, Na H, Chung Y (2015) Dynamic control of Th2 cell responses by STAT3 during allergic lung inflammation in mice. Int Immunopharmacol 28:846–853 Afkarian M et al (2002) T-bet is a STAT1-induced regulator of IL-12R expression in naïve CD4 + T cells. Nat Immunol 3:549–557 Oh H-M, Yu C-R, Dambuza I, Marrero B, Egwuagu CE (2012) STAT3 protein interacts with Class O Forkhead transcription factors in the cytoplasm and regulates nuclear/cytoplasmic localization of FoxO1 and FoxO3a proteins in CD4(+) T cells. J Biol Chem 287:30436–30443 Delpoux A et al (2012) Foxp3-independent loss of regulatory CD4 + T-cell suppressive capacities induced by self-deprivation. Eur J Immunol 42:1237–1249 Delpoux A et al (2014) TCR signaling events are required for maintaining CD4 regulatory T cell numbers and suppressive capacities in the periphery. J Immunol Baltim Md 1950 193:5914–5923 Additional Declarations The authors declare no competing interests. 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07:29:46","extension":"html","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102167,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8403997/v1/281794232aa8f02f3dfb5c8a.html"},{"id":98776731,"identity":"f4aa2b4c-b731-4d37-93ba-23a8d1d1e12c","added_by":"auto","created_at":"2025-12-22 12:23:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":294630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased proportions of type 1 and type 2 effector cells among memory CD4 T cells from Foxo1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice. \u003c/strong\u003ePeripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs) and spleen of a 1- to 12-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice were recovered and stained. \u003cstrong\u003eA,\u003c/strong\u003e CD4/CD44 representative dot-plots are shown for pLN CD4 T cells. \u003cstrong\u003eB,\u003c/strong\u003e Percentages (upper panel) and absolute numbers (lower panel) of memory CD4 T cells (CD4\u003csub\u003eM\u003c/sub\u003e) in the pLNs, mLNs and spleen as a function of mouse age. \u003cstrong\u003eC,\u003c/strong\u003e Concentrations of IFN-g, IL-4 and IL-5 in the plasma of 6-8-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice. \u003cstrong\u003eD,\u003c/strong\u003e CD4/IFN-g (upper panel) and CD4/IL-13 (lower panel) representative dot-plots are shown for CD4\u003csub\u003eM\u003c/sub\u003e cells from the pLNs of 6-8-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice. \u003cstrong\u003eE,\u003c/strong\u003e Percentages of IFN-g- (upper panel) and IL-13- (lower panel) producing cells among CD4\u003csub\u003eM\u003c/sub\u003e cells. \u003cstrong\u003eF, \u003c/strong\u003eIFN-g- (upper panel) and IL-13- (lower panel) relative Mean Fluorescence Intensity (MFI) of cytokine-producing CD4\u003csub\u003eM\u003c/sub\u003e cells.\u003cstrong\u003e G,\u003c/strong\u003e CD4/T-bet (upper panel) and CD4/GATA3 (lower panel) representative dot-plots are shown for CD4\u003csub\u003eM\u003c/sub\u003e cells from the pLNs of 6-8-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice. \u003cstrong\u003eH,\u003c/strong\u003e Percentages of T-bet- (upper panel) and GATA-3- (lower panel) expressing cells among CD4\u003csub\u003eM\u003c/sub\u003e cells. \u003cstrong\u003eI, \u003c/strong\u003eT-bet- (upper panel) and GATA-3- (lower panel) relative MFI of T-bet- and GATA-3-expressing CD4\u003csub\u003eM\u003c/sub\u003e cells respectively. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. ****p \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8403997/v1/1898e2a5dcf5b5037ac362ff.png"},{"id":98739697,"identity":"7d6d1547-10f9-4968-92c5-522ee6154038","added_by":"auto","created_at":"2025-12-22 07:29:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":501838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD4\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e cells from Foxo1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice are prone to differentiate into TH1 and TH2 effector cells.\u003c/strong\u003e CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl \u003c/sup\u003emice were purified and cultured in TH1- (\u003cstrong\u003eA-D\u003c/strong\u003e) or TH2- (\u003cstrong\u003eE-H\u003c/strong\u003e) polarizing conditions. \u003cstrong\u003eA,\u003c/strong\u003e Diagram illustrating the TH1 polarization assay. \u003cstrong\u003eB,\u003c/strong\u003e CD4?/IFN-g representative dot-plots are shown for CD4 T cells at different concentrations of IL-12. \u003cstrong\u003eC,\u003c/strong\u003e A representative experiment showing the proportion of IFN-g-producing cells among CD4 T cells as a function of IL-12 concentration (left panel) and quantification for IL-12 = 20 ng/ml (right panel). \u003cstrong\u003eD,\u003c/strong\u003e IFN-g-relative MFI of IFN-g-producing CD4 cells for IL-12 = 20 ng/ml. \u003cstrong\u003eE,\u003c/strong\u003e Diagram illustrating the TH2 polarization assay. \u003cstrong\u003eF,\u003c/strong\u003e CD4?/IL-13 representative dot-plots are shown for CD4 T cells at different concentrations of IL-4. \u003cstrong\u003eG,\u003c/strong\u003e A representative experiment showing the proportion of IL-13 producing cells among CD4 T cells as a function of IL-4 concentration (left panel) and quantification for IL-4 = 10 ng/ml (right panel). \u003cstrong\u003eH,\u003c/strong\u003e IL-13-relative MFI of IL-13-producing CD4 cells for IL-4 = 10 ng/ml. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. ****p \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8403997/v1/8cb028d724e1abefe2ea1c56.png"},{"id":98739696,"identity":"6fff2947-f174-4e02-add8-58729d56d5fb","added_by":"auto","created_at":"2025-12-22 07:29:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":340610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;CD4\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e cells from Foxo1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice are biased towards TH1- and TH2 cell differentiation.\u003c/strong\u003e CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl \u003c/sup\u003emice were purified and cultured in nonpolarizing conditions (TH0, \u003cstrong\u003eA-E\u003c/strong\u003e), in the presence of anti-IFN-g and anti-IL-4 antibodies (\u003cstrong\u003eF-H\u003c/strong\u003e) or in the presence of AS, a Foxo1 chemical inhibitor. \u003cstrong\u003eA,\u003c/strong\u003e Diagram illustrating the TH0 polarization assay. \u003cstrong\u003eB,\u003c/strong\u003e IL-13/IFN-g representative dot-plots are shown for CD4 T cells. \u003cstrong\u003eC,\u003c/strong\u003e Percentages of IFN-g- (left panel) and IL-13- (right panel) producing cells among CD4 cells. \u003cstrong\u003eD,\u003c/strong\u003e GATA-3/T-bet representative dot-plots are shown for CD4 T cells. \u003cstrong\u003eE,\u003c/strong\u003e Percentages of T-bet- (left panel) and GATA3- (right panel) expressing cells among CD4 cells.\u003cstrong\u003e F,\u003c/strong\u003e Diagram illustrating the TH0 polarization assay in the presence of antibodies neutralizing IFN-g and IL-4. \u003cstrong\u003eG,\u003c/strong\u003e Percentages of IFN-g- (left panel) and IL-13- (right panel) producing cells among CD4 cells.\u003cstrong\u003e H,\u003c/strong\u003e Percentages of T-bet- (left panel) and GATA3- (right panel) expressing cells among CD4 cells. \u003cstrong\u003eI,\u003c/strong\u003e Diagram illustrating the TH0 polarization assay in the presence of AS1842856. \u003cstrong\u003eJ,\u003c/strong\u003e Percentages of IFN-g- (left panel) and IL-13- (right panel) producing cells among CD4 cells.\u003cstrong\u003e K,\u003c/strong\u003e Percentages of T-bet- (left panel) and GATA3- (right panel) expressing cells among CD4 cells. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. ****p \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8403997/v1/95e1b91db0bffeebe5cbf2eb.png"},{"id":98739702,"identity":"b3db746d-83dd-4fad-9811-79ef55073d2a","added_by":"auto","created_at":"2025-12-22 07:29:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":291459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFoxo1-deficient CD4\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e cells boost the differentation potential of WT CD4\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e cells towards TH1 and TH2 cell lineages.\u003c/strong\u003e CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl \u003c/sup\u003emice were purified and cultured in TH1- or TH2-polarizing conditions (\u003cstrong\u003eA-B\u003c/strong\u003e). \u003cstrong\u003eA,\u003c/strong\u003e CD4/IFN-g (upper panel) and CD4/IL-13 (lower panel) representative dot-plots are shown for CD4 T cells after 1 and 2 days of culture in TH1- or TH2-polarizing conditions respectively. \u003cstrong\u003eB,\u003c/strong\u003e Percentages of IFN-g- (left panel) and IL-13- (right panel) producing cells among CD4 cells in TH1 and TH2 polarization assays respectively. \u003cstrong\u003eC,\u003c/strong\u003e CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl \u003c/sup\u003emice were cultured either separately or together 4 or 5 days in TH1- or TH2-polarizing conditions, respectively. Percentages of IFN-g- (left panel) and IL-13- (right panel) producing cells among CD4 cells in TH1 and TH2 polarization assays respectively. \u003cstrong\u003eD,\u003c/strong\u003e Diagram illustrating the TH1 polarization assay in the presence of graded doses of exogenous IFN-g. \u003cstrong\u003eE,\u003c/strong\u003e Percentages of IFN-g-producing cells among CD4 cells are shown as a function of the concentration of the exogenous IFN-g added in the culture medium. At least 3 independent experiments were performed. \u003cstrong\u003eF,\u003c/strong\u003e Diagram illustrating the TH1 polarization assay in the presence or absence of an antibody neutralizing IFN-g. \u003cstrong\u003eG,\u003c/strong\u003e Percentages of IFN-g-producing cells among CD4 cells are shown. \u003cstrong\u003eH,\u003c/strong\u003e Diagram illustrating the TH2 polarization assay in the presence or absence of an antibody neutralizing IL-13. \u003cstrong\u003eI,\u003c/strong\u003e Percentages of IL-13-producing cells among CD4 cells are shown. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. ****p \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8403997/v1/4d2152bc60b0abc29a6f387b.png"},{"id":98777151,"identity":"2807e3cb-9e50-483b-b934-a3d18e50b8e1","added_by":"auto","created_at":"2025-12-22 12:25:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":260768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased STAT3 phosphorylation in Foxo1-deficient CD4\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e cells may boost their differentation potential towards TH1 and TH2 cell lineages. \u003c/strong\u003eCD4\u003csub\u003e \u003c/sub\u003eT cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl \u003c/sup\u003emice were purified and cultured for 15 min in medium supplemented or not with IL-12 or IFN-g (\u003cstrong\u003eA, B\u003c/strong\u003e). \u003cstrong\u003eA, \u003c/strong\u003erepresentative fluorescence histograms of pSTAT1, pSTAT2, pSTAT3 and pSTAT4 in WT CD4\u003csub\u003eN\u003c/sub\u003e cells after 15 minutes of culture in the indicated conditions. \u003cstrong\u003eB,\u003c/strong\u003e Relative phospho-STAT MFIs in CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eCTRL\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice in the indicated culture conditions. CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl \u003c/sup\u003emice were also cultured under nonpolarizing conditions in the presence or absence of a STAT1 inhibitor (fludarabine, \u003cstrong\u003eC\u003c/strong\u003e) or a STA3 inhibitor (C188-9, \u003cstrong\u003eD\u003c/strong\u003e). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. ****p \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8403997/v1/415fb89e336eec8cbbb3def4.png"},{"id":98785150,"identity":"adf2c4f9-3c23-4260-9c45-7c2bb9fafebb","added_by":"auto","created_at":"2025-12-22 12:43:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2537786,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8403997/v1/95fb1c8c-00c1-4c67-9fc0-f0007ab0fede.pdf"},{"id":98779519,"identity":"a7e22d24-4ae3-494a-9160-3aff33963b26","added_by":"auto","created_at":"2025-12-22 12:30:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":417913,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Files 1 to 3\u003c/p\u003e","description":"","filename":"SupplementaryFiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-8403997/v1/e42a53d55dc811f4d1822209.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eFoxo1 restrains naive CD4 T cell differentiation into type 1 and type 2 effector cells\u003c/strong\u003e \u003cstrong\u003eby limiting early cytokine production\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMembers of the forkhead box O (Foxo) transcription factor family are part of an evolutionary conserved pathway, first discovered downstream of insulin and insulin-like growth factor receptors \u003csup\u003e1\u003c/sup\u003e. In mammals, there are 4 Foxo members, namely Foxo1, Foxo3, Foxo4 and Foxo6 which share high protein homology \u003csup\u003e2\u003c/sup\u003e.\u0026nbsp;Foxo1, Foxo3 and Foxo4 are expressed in almost all tissues whereas Foxo6 expression is mainly restricted to the central nervous system\u003csup\u003e3\u003c/sup\u003e and the oxidative muscle \u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn all cell types, Foxo transcription factors are essential for many processes, such as metabolism, quiescence, cell survival and differentiation \u003csup\u003e5\u003c/sup\u003e. T cells mainly express Foxo1 and Foxo3 \u003csup\u003e6\u003c/sup\u003e although a recent article has shown that Foxo4 deficiency in T cells boosts their differentiation potential toward interferon (IFN)-γ-producing cells \u003csup\u003e7\u003c/sup\u003e. The transcription factor Foxo1 controls many aspects of T cell physiology in mammals. First, it regulates peripheral T cell homeostasis \u003csup\u003e8\u003c/sup\u003e. More precisely, Foxo1 increases T cell survival by inducing IL-7 receptor expression \u003csup\u003e8\u003c/sup\u003e. Foxo1 is also crucial for T cell trafficking by increasing the expression of key molecules involved in their entry to and exit from lymphoid and non-lymphoid tissues such as CD62L, S1PR1 and CCR7 \u003csup\u003e8,9\u003c/sup\u003e. Recently, it has been shown that Foxo1 limits the expression of inhibitory receptors and subsequent T cell exhaustion during aging as well as in CAR T cells \u003csup\u003e10–12\u003c/sup\u003e. Delpoux et al. and Zhang et al. have demonstrated that Foxo1 is required for the efficient generation of long-lasting memory CD8 T cells following viral or bacterial infections \u003csup\u003e13,14\u003c/sup\u003e. Indeed, Foxo1 deficiency leads to the differentiation of naive CD8 T cells into terminally differentiated senescent effector cells \u003csup\u003e15,16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFinally, Foxo1\u0026nbsp;plays a crucial role in directing the differentiation of naive CD4 T cells into effector cells \u003csup\u003e17–21\u003c/sup\u003e. Naive CD4 T cells can differentiate into a broad spectrum of effector cells characterized by specific cytokine production and expression of a master transcription factor controlling this ability.\u0026nbsp;For instance, type 1 T Helper (TH1) cells, which allow eradication of intracellular pathogens, are characterized by their production of IFN-γ, a potent activator of cell-mediated immunity. TH2 cells, whose generation predominantly occurs to enhance elimination of parasitic infections, are characterized by production of interleukin (IL)-4, IL-5, and IL-13. TH9 cells, characterized by their ability to produce IL-9 and IL-21, play a role in the protection from helminths. TH17 cells are characterized by IL-17 and IL-22 secretion and are described to play an important role in anti-microbial responses by orchestrating innate immune function, such as granulocyte accumulation at the inflammatory site. T Follicular Helper (TFH) cells produce IL-21 and are involved in the humoral response by triggering germinal center B cells into antibody-secreting plasma and memory B cells. Finally, induced regulatory T cells (iTreg cells) produce TGF-β and IL-10 and play a crucial role in maintaining peripheral self-tolerance and suppressing excessive immune responses deleterious to the host. Importantly, each of these effector CD4 T cell subsets is characterized by the expression of a lineage specific transcription factor (T-bet for TH1 cells, GATA-3 for TH2 cells, PU.1 for TH9, RORγt/RORα for TH17 cells, Bcl6 for TFH cells and Foxp3 for iTreg cells). Foxo1 either inhibits or enhances the differentiation potential of naive CD4 T cells into a given T helper cell lineage. Indeed, on one hand, Foxo1 inhibits TFH cell differentiation through its ability to dampen the expression of genes important for their differentiation, including Bcl6\u0026nbsp;\u003csup\u003e19\u003c/sup\u003e. Moreover, we have demonstrated that Foxo1 is a T cell intrinsic inhibitor of the TH17 program\u0026nbsp;\u003csup\u003e20\u003c/sup\u003e. On the other hand, several articles have reported that Foxo1 is required for TH9 cell generation\u0026nbsp;\u003csup\u003e22,23\u003c/sup\u003e. In addition, Foxo1 deficiency in mouse T cells has been proposed to impair both the generation and suppressive function of iTreg cells\u0026nbsp;\u003csup\u003e17,18,24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSurprisingly, the involvement of Foxo1 in TH1/TH2 cell generation has not yet been studied with precision. In this paper, we show that the memory CD4 T cell compartment from the secondary lymphoid organs (SLOs) of mice lacking the expression of Foxo1 in T cells (Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice,\u0026nbsp;\u003csup\u003e8\u003c/sup\u003e) contains higher proportions of TH1 and TH2 effector cells. In agreement, naive CD4 T cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice exhibit a higher potential for differentiating into TH1 and TH2 cells than their T cell counterparts from Wild-Type (WT) mice \u003cem\u003ein vitro\u003c/em\u003e. Finally, we sought to identify the molecular mechanisms linking Foxo1 expression to the inhibition of TH1- and TH2 cell differentiation.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eIncreased proportions of types 1 and 2 effector CD4 T cells in the SLOs of Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs in our previous articles\u0026nbsp;\u003csup\u003e12,25\u003c/sup\u003e, throughout this manuscript, naive, memory and regulatory CD4 T cells (CD4\u003csub\u003eN\u003c/sub\u003e, CD4\u003csub\u003eM\u003c/sub\u003e and CD4\u003csub\u003eR\u003c/sub\u003e cells respectively) as well as naive and memory CD8 T cells (CD8\u003csub\u003eN\u003c/sub\u003e and CD8\u003csub\u003eM\u003c/sub\u003e cells respectively) have been defined based on their expression of CD44 and Foxp3. Briefly, among TCR\u003csub\u003eb\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e cells, CD4\u003csub\u003eR\u003c/sub\u003e cells were defined as Foxp3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e CD8\u003csub\u003ea\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e cells, CD4\u003csub\u003eM\u003c/sub\u003e cells as CD44\u003csup\u003ehi\u003c/sup\u003e Foxp3\u003csup\u003e-\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e CD8\u003csub\u003ea\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e cells and CD4\u003csub\u003eN\u003c/sub\u003e cells as CD44\u003csup\u003e-/low\u003c/sup\u003e Foxp3\u003csup\u003e-\u003c/sup\u003e CD8\u003csub\u003ea\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e cells. CD44 expression was also used to discriminate between naive and memory CD8 T cells.\u003c/p\u003e\n\u003cp\u003eThe proportions and absolute numbers of CD4\u003csub\u003eM\u003c/sub\u003e cells in the SLOs were not significantly different one week after birth in mice deficient for Foxo1 expression in T cells\u0026nbsp;(CD4\u003csup\u003ecre\u003c/sup\u003e Foxo1\u003csup\u003efl/fl\u003c/sup\u003e mice alias Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice)\u0026nbsp;versus mice proficient for Foxo1 expression in T cells (Foxo1\u003csup\u003efl/fl\u003c/sup\u003e mice not expressing the Cre recombinase alias Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice; Fig. 1A, 1B). However, the proportions of CD4\u003csub\u003eM\u003c/sub\u003e cells increased sharply thereafter in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, so that the absolute numbers of these cells were significantly higher in all the SLOs of 12-week-old Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice than in those of Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice of the same age (Fig. 1A, 1B).\u003c/p\u003e\n\u003cp\u003ePlasma concentrations of IFN-γ (TH1) and IL-5 (TH2) were higher in 8-week-old Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice compared to age-matched Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice (Fig. 1C). However, these results may reflect the quantitative expansion of CD4\u003csub\u003eM\u003c/sub\u003e cells observed in the SLOs of Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice rather than qualitative alterations in the memory CD4 T cell compartment. Of note, plasma IL-4 concentrations were not significantly increased in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice. To distinguish between these 2 hypotheses, we then compared the \u003cem\u003eex vivo\u003c/em\u003e capacity of CD4\u003csub\u003eM\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice to produce type 1 (IFN-g) versus type 2 (IL-13) cytokines (Fig. 1D-F). The proportion of IFN-g\u0026nbsp;producing CD4\u003csub\u003eM\u003c/sub\u003e cells was increased in all SLOs of Foxo1\u003csup\u003eTKO\u003c/sup\u003e compared to\u0026nbsp;Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice, while the percentage of IL-13\u003csup\u003e+\u003c/sup\u003e cells among CD4\u003csub\u003eM\u003c/sub\u003e cells was increased only in pLNs (Fig. 1D, 1E). Interestingly, in pLNs for IL-13 and in all SLOs for IFN-g, cytokine\u003csup\u003e+\u003c/sup\u003e CD4\u003csub\u003eM\u003c/sub\u003e cells produced larger amounts of these cytokines in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice than in WT mice (Fig. 1F). Consistent with their enhanced production of type 1 and type 2 cytokines, a higher proportion of CD4\u003csub\u003eM\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice expressed T-bet and GATA3. Moreover, cells expressing these transcription factors displayed higher expression levels than their counterparts from Foxo1\u003csup\u003eCtrl\u0026nbsp;\u003c/sup\u003emice (Fig. 1G–I). Interestingly, although the proportions of CD8\u003csub\u003eM\u003c/sub\u003e producing IFN-g\u0026nbsp;or expressing T-bet were only modestly increased in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice compared to\u0026nbsp;Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice, the relative Mean Fluorescence Intensity (MFI) of these parameters among positive cells was markedly and significantly increased (Fig. S1A-C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice are prone to differentiate into types 1 and 2 effector T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe hypothesized that the over-representation of TH1 and TH2 effector cells among peripheral CD4\u003csub\u003eM\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice might result from an increased propensity of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into these lineages. To test this, we purified CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice and polarized them \u003cem\u003ein vitro\u003c/em\u003e toward TH1 (Fig. 2A-D) or TH2 (Fig. 2E-H) effector cells. CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice were markedly more efficient than their counterparts from Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice at differentiating into IFN-g- (Fig. 2B, 2C) or IL-13- (Fig. 2F, 2G) producing cells. Notably, upon activation, Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells not only exhibited increased frequencies of type 1 and type 2 cytokine producing cells but also produced higher cytokine amounts than their Foxo1\u003csup\u003eCtrl\u003c/sup\u003e counterparts (Fig. 2D, 2H).\u003c/p\u003e\n\u003cp\u003eSurprisingly, even in the absence of polarizing cytokine (IL-12 for TH1 cells and IL-4 for TH2 cells), Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells differentiated very efficiently into TH1 or TH2 effector cells (Fig. 2B, 2F). Indeed, on the one hand, around 30% of the progeny of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells acquired the ability to produce IFN-g\u0026nbsp;after 4 days of activation in the absence of exogenous IL-12 (Fig. 2B, 2C). On the other hand, about 75% of the progeny of the same cells produced IL-13 upon a 5-day activation in the absence of exogenous IL-4 (Fig. 2F, 2G). CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice are thus prone to differentiate into TH1 and TH2 effector cells upon activation.\u003c/p\u003e\n\u003cp\u003ePrevious articles have shown that Foxo1 expression was required for the differentiation of CD4\u003csub\u003eN\u003c/sub\u003e cells into TH9 cells\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e and iTreg cells\u0026nbsp;\u003csup\u003e17,18\u003c/sup\u003e \u003cem\u003ein vitro\u003c/em\u003e. Of note, in the TH9 polarization assay, an antibody neutralizing IFN-g\u0026nbsp;was not added in the culture medium\u0026nbsp;\u003csup\u003e22,23\u003c/sup\u003e and IL-4 and IFN-g\u0026nbsp;were not blocked in the course of CD4\u003csub\u003eN\u003c/sub\u003e cell differentiation into iTreg cells\u0026nbsp;\u003csup\u003e17,18\u003c/sup\u003e. Therefore, we repeated these experiments, this time adding cytokine-neutralizing antibodies to the culture medium (Fig. S2). Interestingly, when a blocking antibody neutralizing IFN-g\u0026nbsp;during the TH9 polarization assay, CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice differentiated even more efficiently than CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice into IL-9-producing cells (Fig. S2A-D). Neutralization of IL-4 and IFN-g\u0026nbsp;almost completely restored the ability of CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e to differentiate into iTreg cells (Giraud et al.). However, in contrast to what we observed for TH1 and TH2 polarization, the addition of the polarizing cytokine TGF-β to the culture medium was strictly required for Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into TH9 cells (Fig. S2A, S2B) or iTreg cells (Giraud et al.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice are biased to differentiate into TH1 and TH2 effector cells under nonpolarizing conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the enhanced propensity of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e to differentiate into TH1 and TH2 effector cells, we stimulated them under nonpolarizing conditions (Fig. 3A, TH0 polarization assay). After 4 days, a significant proportion of the progeny of CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice acquired the capacity to produce either IFN-g\u0026nbsp;or IL-13 (Fig. 3B, 3C). Notably, very few cells produced both cytokines simultaneously. In contrast, the majority of Foxo1-deficient cells at this time-point co-expressed T-bet and GATA3 (Fig. 3D, 3E). Thus, at the level of lineage-defining transcription factors, Foxo1 deficiency enables CD4 T cells to adopt a mixed TH1/TH2 effector phenotype upon activation, although regulatory mechanisms appear to restrict individual cells from concomitantly producing both type 1 and type 2 cytokines.\u003c/p\u003e\n\u003cp\u003eWhen neutralizing antibodies against IFN-γ and IL-4 were added to the culture medium (Fig. 3F), a fraction of Foxo1-deficient CD4 T cells still differentiated into IFN-γ– or IL-13–producing cells (Fig. 3G). Similarly, some stimulated cells continued to express T-bet or GATA3 (Fig. 3H). However, the frequencies were markedly reduced compared with cultures without cytokine neutralization, suggesting the existence of a positive feedback loop in which early IFN-γ and IL-4 production enhances the ability of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into TH1 and TH2 lineages, respectively.\u003c/p\u003e\n\u003cp\u003eWe next asked whether Foxo1 activity is required during TH0 polarization to restrain the differentiation of CD4\u003csub\u003eN\u003c/sub\u003e cells into TH1 cells and TH2 cells. To address this question, we supplemented the culture medium with AS1842856, a chemical inhibitor that prevents Foxo1 from binding to DNA (\u003csup\u003e20,26\u003c/sup\u003e, Fig. 3I). Blocking Foxo1 transcriptional activity during the TH0 polarization assay did not boost the ability of CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into TH1 or TH2 effector cells (Fig. 3J, 3K). Thus, Foxo1 activity would be required prior to CD4\u003csub\u003eN\u003c/sub\u003e cell activation to prevent their differentiation into TH1 or TH2 effector cells under nonpolarizing (TH0) conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRapid and robust IL-4 and IFN-\u003c/strong\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;production by Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells upon activation underlies their biased differentiation into TH1 and TH2 effector cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe hypothesized that CD4\u003csub\u003eN\u003c/sub\u003e from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice would rapidly produce type 1 and type 2 cytokines upon activation. In line with our assumption, after only 2 days of culture in the TH1 polarization assay, a substantial fraction of Foxo1-deficient CD4 T cells produced IFN-γ (Fig. 4A). Similarly, on day 2 of the TH2 polarization assay, higher proportions CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1T\u003csup\u003eKO\u003c/sup\u003e mice produced IL-13 (Fig. 4A, 4B) and IL-4 (Fig. S3A, S3B).\"\u003c/p\u003e\n\u003cp\u003eTo determine whether early cytokine production by Foxo1-deficient CD4 T cells during polarization assays contributes to their increased propensity to differentiate into TH1 or TH2 effector cells, we cocultured Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells with Foxo1-sufficient CD4\u003csub\u003eN\u003c/sub\u003e cells (Fig. 4B). When cocultured with CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, WT CD4\u003csub\u003eN\u003c/sub\u003e cells gave rise to significantly higher proportions of IFN-g- and IL13-producing cells during TH1 and TH2 polarization assays, respectively, than when cultured alone (Fig. 4C). Notably, in TH1 cocultures, Foxo1-deficient cells differentiated less efficiently into TH1 effector cells than when cultured alone (Fig. 4C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, these results strongly suggest that, in coculture, Foxo1-expressing CD4\u003csub\u003eN\u003c/sub\u003e cells benefit from the early cytokine production of Foxo1-deficient cells. We hypothesized that Foxo1-deficient and Foxo1-sufficient CD4\u003csub\u003eN\u003c/sub\u003e cells may compete for IFN-g\u0026nbsp;during the TH1 polarization assay. Consistent with this hypothesis, the addition of exogenous IFN-γ to the culture medium significantly enhanced the differentiation of CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice into TH1 effector cells but had no effect on CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig. 4D, 4E). Conversely, neutralization of IFN-g\u0026nbsp;during the polarization assay markedly impaired the capacity of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells to polarize into TH1 effector cells (Fig. 4F, 4G). However, this treatment completely abrogated the differentiation of WT CD4\u003csub\u003eN\u003c/sub\u003e cells into TH1 effector cells, so that in these conditions, CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice still differentiated more efficiently than their counterparts from Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice. These results suggest that mechanisms other than IFN-γ\u0026nbsp;overproduction contribute to the enhanced TH1 differentiation capacity of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells. Interestingly, neutralization of IL-13 during the TH2 polarization assays markedly reduced the capacity of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into IL-13-producing cells while not affecting WT CD4\u003csub\u003eN\u003c/sub\u003e cells (Fig. 4H, 4I). Altogether, these results suggest that the enhanced capacity of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into TH1 and TH2 effector cells arises from (\u003cstrong\u003ei\u003c/strong\u003e) their rapid production of IFN-γ\u0026nbsp;and IL-13 and (\u003cstrong\u003eii\u003c/strong\u003e) a subsequent positive feedback loop in which these cytokines further amplify their polarization potential.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnhanced STAT3 phosphorylation in response to IL-4 may underlie the increased TH2 differentiation potential of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferentiation of CD4\u003csub\u003eN\u003c/sub\u003e cells to TH effector cells relies on polarizing cytokine signaling pathway,\u0026nbsp;particularly on the activation of STAT transcription factors\u0026nbsp;\u003csup\u003e27,28\u003c/sup\u003e. We assessed the phosphorylation of STAT proteins, namely STAT1, STAT3, STAT4 and STAT6, in CD4\u003csub\u003eN\u003c/sub\u003e cells after a 15-minute culture with either IL-12 or IL-4 (Fig. 5). Surprisingly, at this time-point, IL-12 failed to induce phosphorylation of any tested STAT proteins, including STAT4, which is typically activated by IL-12 and required for efficient TH1 polarization (Fig. 5A, 5B). In contrast, IL-4, not only strongly induced phosphorylation of STAT6, the canonical mediator downstream of the IL-4 receptor, but also triggered significant phosphorylation of STAT1 and STAT3 (Fig. 5A, 5B).\u0026nbsp;Interestingly, while IL-4–induced STAT6 phosphorylation was comparable between Foxo1-sufficient and Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells, IL-4 triggered markedly stronger STAT1 and STAT3 phosphorylation in CD4\u003csub\u003eN\u0026nbsp;\u003c/sub\u003efrom Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice than in their counterparts from Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice (Fig. 5B).\u003c/p\u003e\n\u003cp\u003eWe next tested whether inhibition of STAT1 (Fludarabine) or STAT3 (C188-9) influenced the ability of CD4\u003csub\u003eN\u003c/sub\u003e from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice to produce IFN-γ and IL-13 upon activation under nonpolarizing conditions (Fig. 5C, 5D). STAT1 inhibition induced only a modest reduction in cytokine production by Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells and had no effect on Foxo1-sufficient CD4\u003csub\u003eN\u003c/sub\u003e cells. In contrast, STAT3 inhibition markedly impaired the ability of CD4\u003csub\u003eN\u003c/sub\u003e from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice to produce IFN-γ and IL-13 upon activation. Nonetheless, even in the presence of the STAT3 inhibitor, a higher proportion of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells produced these cytokines compared to Foxo1-sufficien CD4\u003csub\u003eN\u003c/sub\u003e cells (Fig. 5C, 5D), suggesting that additional mechanisms contribute to the enhanced propensity of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into TH1 and TH2 effector cells.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eFoxo1 is a key transcription factor in T cell biology, required for T cell survival, trafficking, and the generation of efficient memory responses\u0026nbsp;\u003csup\u003e8,9,13,14\u003c/sup\u003e. Its role in the differentiation of CD4\u003csub\u003eN\u003c/sub\u003e cells into effector subsets, however, appears more complex. While Foxo1 has been proposed to be essential for the generation of peripheral regulatory CD4 T cells and TH9 cells, it may restrain the differentiation of CD4\u003csub\u003eN\u003c/sub\u003e into TFH and TH17 effector cells\u0026nbsp;\u003csup\u003e17–20,22,23\u003c/sup\u003e. In the present study, we demonstrate that Foxo1 also strongly limits the differentiation of CD4\u003csub\u003eN\u003c/sub\u003e into TH1 and TH2 effector cells (Fig. 2). Notably, Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells readily differentiated into TH1 and TH2 cells upon stimulation \u003cem\u003ein vitro\u003c/em\u003e, even in the absence of exogenous polarizing cytokines (Fig. 3). Our findings support a two-step mechanism: (\u003cstrong\u003ei\u003c/strong\u003e) Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells rapidly produce high levels of type 1 (IFN-γ) and type 2 (IL-4 and IL-13) cytokines upon activation, and (\u003cstrong\u003eii\u003c/strong\u003e) this early cytokine burst amplifies their propensity to differentiate into TH1 and TH2 effector cells. Indeed, neutralization IFN-γ\u0026nbsp;or IL-13 markedly reduced the ability of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into TH1 and TH2 cells, respectively (Fig. 4). Conversely, supplementation of the culture medium with IFN-γ\u0026nbsp;enhanced the capacity of Foxo1-sufficient CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into TH1 effector cells (Fig. 4). These findings are consistent with previous reports showing that IFN-γ\u0026nbsp;amplifies and stabilizes TH1 cell generation and phenotype\u0026nbsp;\u003csup\u003e29\u003c/sup\u003e and that the propensity of IL-13-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into TH2 cells is strongly diminished\u0026nbsp;\u003csup\u003e30,31\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt remains important to elucidate the molecular mechanisms underlying the rapid production of IL-4, IL-13 and IFN-γ by Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells upon stimulation (Fig. 4). Interestingly, pharmacological inhibition of Foxo1 during the TH0 polarization assay did not recapitulate the phenotype observed with Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells (Fig. 3). This finding suggests that the absence of Foxo1 expression may lead to a stable reprogramming of CD4\u003csub\u003eN\u003c/sub\u003e cells, predisposing them to produce type 1 and type 2 cytokines. Beyond its transcriptional function, Foxo1 has also been implicated in chromatin remodeling in T cells. For instance, using genome-wide expression and methylation analyses, Baessler et al. demonstrated that the methylcytosine dioxygenase Tet2 interacts with Foxo1 to mediate DNA demethylation and proper expression of target genes in CD4 T cells\u0026nbsp;\u003csup\u003e32\u003c/sup\u003e. In addition, Gray et al. showed that Foxo1 is required to limit deposition of the repressive histone mark H3K27me3 by the Polycomb Repressive Complex 2 (PRC2) although no direct interaction between Foxo1 and PRC2 subunits could be detected in co-immunoprecipitation assays\u0026nbsp;\u003csup\u003e33\u003c/sup\u003e. Strikingly, CD4\u003csub\u003eN\u003c/sub\u003e cells deficient for Ezh2, the catalytic subunit of PRC2, spontaneously differentiated into type 1 and type 2 cytokine-producing cells under nonpolarizing conditions, a phenotype strongly reminiscent of that observed in Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells\u0026nbsp;\u003csup\u003e34,35\u003c/sup\u003e. Together, these findings suggest that Foxo1 may act as an epigenetic gatekeeper, preventing premature acquisition of TH1 and TH2 effector programs in naïve CD4\u003csub\u003eN\u003c/sub\u003e cells.\u003c/p\u003e\n\u003cp\u003eOur data also point to a potential role for STAT3, as its pharmacological inhibition strongly impaired the ability of CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice to produce IFN-γ and IL-13 upon activation under nonpolarizing conditions (Fig. 5). Consistently, STAT3 was rapidly phosphorylated upon IL-4 stimulation, with significantly higher levels observed in Foxo1-deficient than in Foxo1-sufficient CD4\u003csub\u003eN\u003c/sub\u003e cells (Fig. 5). IL-4 also induced robust STAT6 phosphorylation, the canonical STAT protein downstream of type I and type II IL-4 receptors\u0026nbsp;\u003csup\u003e36,37\u003c/sup\u003e, but the extent of STAT6 activation did not differ between Foxo1-deficient and Foxo1-sufficient CD4\u003csub\u003eN\u003c/sub\u003e cells (Fig. 5). Several studies have implicated STAT3 in the TH2 differentiation pathway and suggested that it may cooperate with STAT6 to promote TH2 cell development\u0026nbsp;\u003csup\u003e38–40\u003c/sup\u003e. The marked decrease in IFN-γ–producing cells upon STAT3 inhibition is somewhat unexpected. Indeed, while STAT1 phosphorylation is well established to promote TH1 cell differentiation, most notably by directly activating T-bet expression in synergy with STAT4 \u003csup\u003e29,41\u003c/sup\u003e, STAT3 activation, by contrast, has generally been described as exerting an inhibitory effect on this process \u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHere, we show that Foxo1 also strongly limits the ability of CD4\u003csub\u003eN\u003c/sub\u003e cells to differentiate into TH1 and TH2 effector cells, a feature that may largely account for previous reports describing an absolute requirement for Foxo1 in the generation of TH9 or peripheral regulatory T cells\u0026nbsp;\u003csup\u003e17,18,22,23\u003c/sup\u003e. Indeed, our results clearly demonstrate that the impaired generation of induced regulatory T cells and TH9 cells observed \u003cem\u003ein vitro\u003c/em\u003e in the absence of Foxo1 expression stems from the intrinsic propensity of Foxo1-deficient T cells to produce TH1- and TH2-type cytokines, such as IFN-γ and IL-4. Neutralization of IFN-γ during TH9 polarization, and of both IFN-γ and IL-4 during regulatory T cell polarization, restores the differentiation of Foxo1-deficient CD4\u003csub\u003eN\u003c/sub\u003e cells into TH9 and induced regulatory T cells to levels comparable to, or even exceeding, those of control CD4\u003csub\u003eN\u003c/sub\u003e cells (Fig. S2 et Giraud et al.). Collectively, these findings suggest that Foxo1 acts as a global brake on CD4\u003csub\u003eN\u003c/sub\u003e cell differentiation, restricting their potential to polarize toward all tested effector lineages. We therefore propose that Foxo1 fine-tunes the responsiveness of CD4\u003csub\u003eN\u003c/sub\u003e cells to their environment, and in particular to the cytokine milieu present at the time of activation. In the absence of Foxo1, naïve CD4 T cells would escape this regulation and undergo uncontrolled differentiation, potentially leading to immune responses that are no longer aligned with the nature of the initiating signals, such as pathogen type or inflammatory context.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eMice. 1-week-old to 12-week-old mice were used. C57BL/6 Foxp3-GFP Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl\u003c/sup\u003e CD45.1 or CD45.2 mice\u0026nbsp;\u003csup\u003e8,20\u003c/sup\u003e and C57BL/6 CD3eKO\u0026nbsp;\u003csup\u003e43\u003c/sup\u003e and were maintained in our own animal facilities, under specific pathogen–free (SPF) conditions. All procedures were approved by the ethics committee for animal experimentation n°34 and validated by the “Ministère de l’Enseignement Supérieur de la Recherche et de l'Innovation” with the number APAFIS≠20630-2018033016303981v5. Sample sizes were chosen to assure reproducibility of the experiments and in accordance with the 3R of animal ethics regulation.\u003c/p\u003e\n\u003cp\u003eCell suspensions.\u0026nbsp;Peripheral lymph nodes (pooled cervical, axillary, brachial and inguinal lymph nodes; pLNs), mesenteric LNs (mLNs) and spleen were homogenized and passed through a nylon cell strainer (BD Falcon) in 5% FCS, 0.1% NaN3 (Interchim) in phosphate saline buffer saline (PBS) for flow cytometry.\u003c/p\u003e\n\u003cp\u003eFluorescence staining and flow cytometry. Cell suspensions were collected and dispensed into 96-well round-bottom microtiter plates (Greiner Bioscience; 6x10\u003csup\u003e6\u003c/sup\u003e cells/well). Surface staining was performed as described\u0026nbsp;\u003csup\u003e44\u003c/sup\u003e. Briefly, cells were incubated on ice, for 15 min/step, with Abs in 5% FCS (Biochrom),\u0026nbsp;0.1% NaN3 (Sigma-Aldrich) in PBS. Each cell staining reaction was preceded by a 15-min incubation with a purified anti-mouse CD16/32 Ab (FcgRII/III block; 2.4G2, BioXcell).\u0026nbsp;The Foxp3 Staining Buffer Set (eBioscience) was used for Foxp3 intracellular staining. For determination of intracellular cytokine production, cells were stimulated with 0.5 µg/ml PMA (Sigma-Aldrich), 0.5 µg/ml ionomycin (Sigma-Aldrich), and 10 μg/ml BrefeldinA (Sigma-Aldrich) for 2 hrs at 37°C. Cells were then stained for surface markers, fixed in 2% paraformaldehyde in PBS, and permeabilized with 0.5% saponin, followed by labeling with specific cytokine Abs. For determination of the phosphorylation of STAT proteins, cells were immediately fixed at the end of the culture with 4% PFA during 5 min at 37°C; then washed twice and permeabilized with cold methanol at -20°C for 30 min. Cells were then washed 3 times and stained overnight at 4°C for surface markers and intracellular pSTAT proteins.Multi-color immunofluorescence was analyzed using a BD-Fortessa cytometer (BD Biosciences).Data acquisition and cell sorting were performed at the Cochin CYBIO facility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePurification of CD4\u003csub\u003eN\u003c/sub\u003e cells.\u0026nbsp;CD4 T cells were purified from LNs (pooled superficial cervical, axillary, brachial, inguinal, and mesenteric LNs) of Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice by incubating cell suspensions on ice for 20 minutes with a mixture of anti-CD8 (53-6.7), anti-CD11b (Mac-1) and anti-CD19 (1D3) Abs obtained from hybridoma supernatants, and then with magnetic beads coupled to anti-rat immunoglobulins (Dynal Biotech). CD4\u003csub\u003eN\u003c/sub\u003e cells were then flow cytometry sorted as Foxp3-GFP\u003csup\u003e-\u003c/sup\u003e, Lineage (CD25, TCRgd, CD8\u003csub\u003eb\u003c/sub\u003e, CD11b, CD11c)-PE\u003csup\u003e-\u003c/sup\u003e, CD44-APC\u003csup\u003e-/lo\u003c/sup\u003e cells using a FACS-ARIA3 flow cytometer (BD Biosciences) at the Cochin CYBIO facility.\u003c/p\u003e\n\u003cp\u003eCell Cultures.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro polarization assays.\u003c/em\u003e20x10\u003csup\u003e4\u003c/sup\u003e Flow-cytometry sorted CD4\u003csub\u003eN\u003c/sub\u003e cells from the LNs of C57BL/6 Foxp3-GFP mice were stimulated for 4 (TH0, TH1) or 5 (TH2) days with immobilized anti-CD3e\u0026nbsp;(clone 145.2C11; 4µg/ml; obtained from hybridoma supernatants) and anti-CD28 (clone 37.51; eBioscience; 4μg/ml) antibodies, in the absence (TH0) or presence of graded concentrations of exogenous IL12 (TH1, R\u0026amp;D) or IL-4 (TH2, R\u0026amp;D). 15μg/mL of a monoclonal antibody neutralizing IL-4 (TH1, BioLegend) or IFN-g\u0026nbsp;(TH2, BioLegend) were added in the TH1 or TH2 polarization assays respectively. In some experiments, TH1 polarization was also done in the presence of graded concentrations of IFN-g. In some other experiments, CD4\u003csub\u003eN\u003c/sub\u003e cells were cultured under nonpolarizing conditions in the presence or absence of a STAT1 inhibitor (fludarabine, SelleckChem) or a STA3 inhibitor (C188-9, SelleckChem). For the TH9 polarization assay, CD4\u003csub\u003eN\u003c/sub\u003e cells were stimulated for 5 days as indicated above in the presence of graded concentrations of exogenous TGF-β (Invitrogen) and of 10ng/mL of IL-4. 15μg/mL of a monoclonal antibody neutralizing IFN-g\u0026nbsp;were added in the TH9 polarization assay. 15μg/mL of a monoclonal antibody neutralizing IL-4 and of a monoclonal antibody neutralizing IFN-g\u0026nbsp;in the iTreg polarization assay.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCocultures.\u003c/em\u003e In some experiments (Fig. 4C, Fig. S4C), 10x10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003epurified CD4\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl\u0026nbsp;\u003c/sup\u003emice were cultured together under nonpolarizing conditions (TH0) or under TH1- or TH2-polarizing conditions.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCultures for studying the phosphorylation of STAT proteins.\u003c/em\u003e1x10\u003csup\u003e6\u003c/sup\u003e CD4T cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl\u0026nbsp;\u003c/sup\u003emice were purified, let to rest at 37°C for 1 hr and then cultured for 15 min in medium supplemented or not with 10ng/ml of IL-4 or 20ng/ml IL-12.\u003c/p\u003e\n\u003cp\u003eStatistical analysis. Data are expressed as mean ± SEM, and the significance of differences between two series of results was assessed using the Student’s unpaired or paired t test. Values of p \u0026lt; 0.05 were considered as statistically significant. (*, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.A., B.M. and B.L. designed experiments. C.G. performed the experiments shown in Figures 1-3 and Figures S1-S2. LG performed experiments shown in Figures 4 and 5. T.L. performed experiments shown in Figures 4 and Figures S3. LG and B.M. made the figures. B.L. wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe greatly acknowledge M. Andrieu from the Cochin Immunobiology facility. This work was supported by a grant from the \u0026ldquo;Fondation pour la Recherche M\u0026eacute;dicale\u0026rdquo; (FRM team number EQU202103012662). Th\u0026eacute;o Level was supported by a Ph.D. fellowship from the \u0026ldquo;LaBex Who AM I?\u0026rdquo;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLink W, Fernandez-Marcos PJ (2017) FOXO transcription factors at the interface of metabolism and cancer. Int J Cancer 141:2379\u0026ndash;2391\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGui T, Burgering BMT (2021) FOXOs: masters of the equilibrium. 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J Immunol Baltim Md 1950 193:5914\u0026ndash;5923\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"59858c12-9503-436f-8065-a5672db599e7","identifier":"10.13039/501100002915","name":"Fondation pour la Recherche Médicale","awardNumber":"EQU202103012662","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Helper T cells, Foxo1, TH1, TH2, STAT proteins","lastPublishedDoi":"10.21203/rs.3.rs-8403997/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8403997/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOver the past 15 years, Foxo1 has emerged as a key transcription factor in T cell biology, regulating survival, trafficking, and the establishment of memory responses. Its role in naive CD4 T cell differentiation into effector cells, however, appears more complex. Foxo1 has been reported to be essential for the generation of induced regulatory T cells and TH9 cells, while restraining the differentiation of TH17 and TFH lineages. Surprisingly, its contribution to TH1 and TH2 differentiation has remained poorly defined. Here, we show that memory CD4 T cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice exhibited enhanced capacity to produce type 1 and type 2 cytokines \u003cem\u003eex vivo\u003c/em\u003e, associated with increased T-bet and GATA3 expression. Foxo1-deficient naive CD4 T cells displayed a strong bias toward TH1 and TH2 differentiation \u003cem\u003ein vitro\u003c/em\u003e, even under nonpolarizing conditions, driven by rapid and robust production of IL-4, IL-13 and IFN-γ upon activation. Coculture experiments further revealed that Foxo1-sufficient cells benefited from cytokines produced by Foxo1-deficient cells, while competition for IFN-γ limited the differentiation of the latter. Mechanistically, IL-4 stimulation elicited increased STAT1 and STAT3 phosphorylation in Foxo1-deficient naive CD4 T cells. Pharmacological inhibition of STAT3, but not STAT1, markedly reduced their TH1/TH2 polarization capacity, although residual differentiation suggested the involvement of additional mechanisms.\u003c/p\u003e","manuscriptTitle":"Foxo1 restrains naive CD4 T cell differentiation into type 1 and type 2 effector cells by limiting early cytokine production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 07:29:37","doi":"10.21203/rs.3.rs-8403997/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a06f6d42-0876-43ac-8543-4bcb7d560dc5","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60045878,"name":"Immunology"}],"tags":[],"updatedAt":"2025-12-22T07:29:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 07:29:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8403997","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8403997","identity":"rs-8403997","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}