Article

Foxo3 Factor Drives Pathogenic T Helper 1 Differentiation by Inducing the Expression of Eomes

Graphical Abstract Authors Caroline Stienne, Michae¨ l F. Michieletto, Mehdi Benamar, ..., Stephen M. Hedrick, Abdelhadi Saoudi, Anne S. Dejean

Correspondence [email protected]

In Brief The role of Foxo3 in effector CD4+ T cells has not been addressed. Stienne et al. demonstrate that Foxo3 expression in CD4+ T cells controls the expression of Eomes and that the Foxo3-Eomes axis is required to achieve the complete specialized program of CD4+ T cell differentiation and development of autoimmunity.

Highlights Accession Numbers d Strength of TCR signal controls Foxo3 expression in effector GSE86287 CD4+ T cells d Foxo3 drives pathogenic Th1 cell differentiation through direct regulation of Eomes d Foxo3 and Eomes act independently of T-bet for pathogenic Th1 cell differentiation d Foxo3 controls the susceptibility to neuroinflammation

Stienne et al., 2016, Immunity 45, 774–787 October 18, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.immuni.2016.09.010 Immunity Article

Foxo3 Drives Pathogenic T Helper 1 Differentiation by Inducing the Expression of Eomes

Caroline Stienne,1,2,3,7 Michae¨ l F. Michieletto,1,2,3,7 Mehdi Benamar,1,2,3 Nade` ge Carrie´ ,4 Isabelle Bernard,1,2,3 Xuan-Hung Nguyen,1,2,3 Yannick Lippi,5 Fanny Duguet,1,2,3 Roland S. Liblau,1,2,3 Stephen M. Hedrick,6 Abdelhadi Saoudi,1,2,3 and Anne S. Dejean1,2,3,8,* 1UMR Inserm, U1043, Toulouse 31300, France 2UMR CNRS, U5282, Toulouse 31300, France 3Universite´ de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse 31300, France 4UGM 4127, Oncopole, Toulouse 31059, France 5Toxalim (Research Centre in Food Toxicology), Universite´ de Toulouse, INRA, ENVT, INP-Purpan, UPS, Toulouse 31024, France 6Molecular Biology Section, Division of Biological Sciences and Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0377, USA 7Co-first author 8Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2016.09.010

SUMMARY All of these activities are altered by phosphorylation, acetyla- tion, methylation, and ubiquitination, and these post-transla- The transcription factor Foxo3 plays a crucial role in tional modifications influence Foxo intracellular localization, myeloid cell function but its role in lymphoid cells turnover, transactivation, and transcriptional specificity (Zhao remains poorly defined. Here, we have shown that et al., 2011). Foxo3 expression was increased after T cell Foxo TFs, through their role in the control of cell cycle pro- engagement and played a specific role in the po- gression and , were first described as tumor suppres- larization of CD4+ T cells toward pathogenic T helper sor . Nonetheless, numerous studies have revealed that Foxo1 and Foxo3 also play fundamental roles in physiologic 1 (Th1) cells producing interferon-g (IFN-g) and gran- and pathologic immune responses (Dejean et al., 2011; Hedrick, ulocyte monocyte colony stimulating factor (GM- 2009; Hedrick et al., 2012; Ouyang and Li, 2011). Because of the CSF). Consequently, Foxo3-deficient mice exhibited similarity between their DNA-binding domains, all Foxo factors reduced susceptibility to experimental autoimmune can in principle bind to related sequences and therefore should encephalomyelitis. At the molecular level, we identi- regulate the same target genes. Experiments using mice defi- fied Eomes as a direct target gene for Foxo3 in cient for a single Foxo isoform, however, clearly demonstrate CD4+ T cells and we have shown that lentiviral-based that Foxo1 and Foxo3 have independent physiological functions overexpression of Eomes in Foxo3-deficient CD4+ in the immune system, suggesting that Foxo functions could be T cells restored both IFN-g and GM-CSF produc- closely linked to their distinct cell type-specific expression pat- tion. Thus, the Foxo3-Eomes pathway is central to terns (Dejean et al., 2011; Hedrick, 2009). achieve the complete specialized gene program Foxo1 is abundantly expressed in lymphoid cells, where it has been shown to regulate many features of lymphocyte homeo- required for pathogenic Th1 cell differentiation and stasis including survival, homing, and differentiation. Indeed, development of neuroinflammation. Foxo1 has critical functions in B cell development, homing, class-switch recombination, and somatic hypermutation (Amin and Schlissel, 2008; Dengler et al., 2008). Foxo1 also regulates INTRODUCTION both naive and memory T cell survival and trafficking (Kerdiles et al., 2009; Kim et al., 2013; Ouyang et al., 2009, 2010), The Foxo (Forkhead Box class O) family of transcription fac- thymic regulatory T (tTreg) and peripheral regulatory T (pTreg) tors (TFs) governs processes such as cellular proliferation, cell development and function (Kerdiles et al., 2010; Merkenschl- apoptosis, energy metabolism, autophagy, and stress resis- ager and von Boehmer, 2010; Ouyang et al., 2010, 2012), as well tance in response to changes in the abundance of nutrients as T helper 1 (Th1), Th17, and T follicular helper (Tfh) cell differ- and growth factors (Eijkelenboom and Burgering, 2013). Foxo entiation (Kerdiles et al., 2010; Laine´ et al., 2015; Merkenschlager can act as either transcriptional activators or repressors and von Boehmer, 2010; Oestreich et al., 2012; Ouyang et al., upon their high-affinity binding to the consensus sequence 2012; Stone et al., 2015). So far, no specific role for Foxo1 has 50-GTAAA(T/C)AA-30, known as the Daf-16 family member-bind- been assigned in immune cells other than lymphocytes. ing element (Obsil and Obsilova, 2011). In addition, Foxo factors Foxo3 is the main isoform expressed in the myeloid compart- can bind and modulate other TFs (van der Vos and Coffer, 2011). ment. Our previous study has shown that Foxo3 is a key

774 Immunity 45, 774–787, October 18, 2016 ª 2016 Elsevier Inc. A 100 Foxo3-/- 200 * suppressor of inflammatory cytokine production by dendritic cells WT CD4 CD62L+ CD44- (DCs) and macrophages (Dejean et al., 2009). These results are 80 WT CD4 CD62L- CD44+ 150 60 consistent with a non-coding polymorphism in human FOXO3 100 that limits inflammatory monocyte responses resulting in milder 40 + Max (%) 50 WT CD4 T cells Crohn’s disease and rheumatoid arthritis but more severe malaria 20 CD62L+ CD44- - + (Lee et al., 2013). The role played by Foxo3 in T cells is less well 0 Foxo3 (relative MFI) 0 CD62L CD44 / / 3 4 5 01010 10 defined. Using Foxo1À ÀFoxo3À À mice, studies have demon- B Foxo3 600 strated that Foxo1 and Foxo3 cooperatively control the develop- 100 * + * ment and function of Foxp3 Treg cells (Kerdiles et al., 2010; 80 Ouyang et al., 2010). Others have shown that Foxo3 limits the Foxo3-/- 400 60 + WT NS expansion of memory CD8 T cells during acute or chronic viral 40 WT (0.125µg/ml) infection (Sullivan et al., 2012a, 2012b). To date, however, the pre- Max (%) µ 200 WT (0.25 g/ml) + 20 WT (2 µg/ml) cise role of Foxo3 in effector CD4 T cells has not been addressed. Foxo3(relative MFI) 0 In this study, we show that the expression of Foxo3 was 3 4 5 0 01010 10 .5 1 2 + 0 increased in CD4 T cells after activation and correlated with .1250.25 Foxo3 0 Anti-CD3 (µg/ml) T cell receptor (TCR) signaling strength. To address the rele- 1000 C 100 * vance of this upregulation, we analyzed the impact of Foxo3 + 80 800 * deficiency on CD4 T cell effector functions and found that + Foxo3-/- 600 Foxo3 drives Eomes-dependent differentiation of IFN-g GM- 60 + WT NS CSF pathogenic Th1 cells and that this pathway is needed for 40 WT 18h 400 Max (%) WT 36h the development of central nervous system inflammation. 20 WT 72h 200 Foxo3 (relative MFI) 0 3 4 5 0 RESULTS 01010 10 8 6 2 1 3 7 Foxo3 Hours TCR Triggering Leads to Increased Expression of Foxo3 Foxo3-/- WT WT + D in CD4 T Cells (10 µg/mL) (1 µg/mL) (10 µg/mL) + + In vivo, activated (CD62LÀCD44 ) CD4 T cells were found to exhibit a 3-fold increase in Foxo3 expression when compared DIC + + to naive (CD62L CD44À) CD4 T cells (Figure 1A). We therefore addressed whether CD4+ T cell activation had an impact on the expression of Foxo3. Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAbs and analyzed for Foxo3 expression. Dapi T cell receptor (TCR) triggering resulted in a dose-dependent upregulation of Foxo3 in CD4+ T cells (Figure 1B), with increased expression over time (Figure 1C), whereas CD28-induced costi- mulation did not influence Foxo3 expression (Figure S1A). A Foxo3 dose-dependent upregulation of Foxo3 was also recorded when OT-II CD4+ T cells were stimulated with antigen-presenting

cell (APC) loaded with increasing doses of OVA323–339 peptide, confirming that TCR-dependent signal intensity regulated Merge Foxo3 expression in activated CD4+ T cells (Figure S1B). To determine key signaling events inducing Foxo3 expression upon stimulation, we next activated CD4+ T cells with anti-CD3 mAbs in the presence of a series of inhibitors that block specific WTFoxo3-/- WT Foxo3-/- E pathways downstream of TCR. We found that inhibition of pro- Anti-CD3 (µg/ml) 1101011010 tein kinase C (PKCs) prevented Foxo3 upregulation whereas in- PLC-γ hibition of ERK, p38, or JNK kinase pathways had no effect Foxo3 (C) Foxo3 expression by naive WT CD4+ T cells stimulated with anti-CD3 mAbs TFIID (2 mg/mL) for 18, 36, or 72 hr (n = 4 mice per genotype). Mean and SEM of the relative MFI of Foxo3 expression was calculated by subtracting the WT MFI / Nucleus Cytoplasm from the Foxo3À À MFI. (D) Immunofluorescence staining of Foxo3 in naive CD4+ T cell from WT or + / Figure 1. Increased Foxo3 Expression in CD4 T Cells after TCR Foxo3À À mice stimulated in vitro with the indicated dose of anti-CD3 mAbs for Engagement 48 hr (scale bars represent 10 mm). + (A) Foxo3 expression by naive CD62L CD44À (white bars) and acti- (E) Immunoblot analysis of Foxo3, PLC-g, and TFIID expression in nuclear and + + + / vated CD62LÀCD44 (dark gray bars) WT CD4 T cells (n = 7 mice per cytoplasmic fractions of naive CD4 T cells from WT or Foxo3À À mice stim- genotype). ulated in vitro as in (D). (B) Foxo3 expression by naive WT CD4+ T cells stimulated in vitro with the Data are representative of three independent experiments. Error bars, SEM; indicated dose of anti-CD3 mAbs (n = 4 mice per genotype). p values (Mann-Whitney U test). See also Figure S1.

Immunity 45, 774–787, October 18, 2016 775 WT B WT Figure 2. Foxo3 Deficiency Impaired Patho- A -/- Foxo3-/- Foxo3 genic Th1 Cell Differentiation -/- WT Foxo3 35 / 30 * * (A) IFN-g production by WT or Foxo3À À naive 5 5 30 10 10 * CD4+ T cells stimulated with anti-CD3 (0.5 g/mL) 15 8 25 m 4 10 104 20 * 20 under non-polarizing condition for 36 hr. Fre- (Percent)

* 3 3 (Percent) 10 10 γ quency of IFN- produced by WT (black circles) or 15 g γ 10 / + 2 2 10 Foxo3À À (open circles) CD4 T cells stimulated 10 10 IFN- IFN- 5 with anti-CD3 Abs for 36 hr (n = 5 mice per CD4 0 0

3 4 5 0 010 10 10 0103 104 105 0 genotype). 0 1 2 36 72 0.5 IFN-γ 0.25 (B) Frequency of IFN- production by WT (black µ Hours g Anti-CD3( g/ml) / + WT bars) or Foxo3À À (open bars) CD4 T cells stimu- WT -/- -/- -/- D Foxo3 C WT Foxo3 Foxo3 15 lated with anti-CD3 mAbs (2 mg/mL) for the indi- 9 * 5 5 * cated time (n = 5 mice per genotype). 10 10 4 / 9 * * (C) GM-CSF production by WT or Foxo3À À naive 4 10 4 10 6 10 CD4+ T cells stimulated as in (A) (n = 5 mice per 3 103 10 genotype). 3 5 2 102 10 (D) Frequency of GM-CSF production by WT (black CD4 GM-CSF (Percent) / + 0 0 0 GM-CSF (Percent) 0 bars) or Foxo3À À (open bars) CD4 T cells stimu- 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 0 .5 1 2 36 72 0 lated as in (B) (n = 4 mice per genotype). GM-CSF 0.25 Anti-CD3(µg/ml) Hours (E) T-bet expression by WT (black circles) or / + WT WT Foxo3-/- Foxo3À À (open circles) CD4 T cells stimulated as E -/- F WT Foxo3 Foxo3-/- 6000 100 100 * in (A) (n = 5 mice per genotype). 5 10 105 * * * 80 (F) Frequency and MFI of T-bet expression by WT 75 48 75 / + 4 4 4000 10 10 (black bars) or Foxo3À À (open bars) CD4 T cells * 60

103 3 50 stimulated as in (B) (n = 5 mice per genotype). 10 40 * 2000 + 2 T-bet (MFI) (G and H) Frequency and MFI of IFN-g expression 10 102 25

T-bet (Percent) T-bet (Percent) 20 / +

CD4 by WT (black bars) or Foxo3À À (open bars) CD4 0 0 3 4 5 0 0 0 010 10 10 0103 104 105 T cells stimulated with anti-CD3 mAbs in Th1 cell- 0 5 1 2 36 72 36 72 T-bet 0.5 0.2 Hours polarizing conditions for 36 hr (n = 5 mice per ge- µ Hours Anti-CD3( g/ml) notype) (G) or stimulated with 2 g/mL of anti-CD3 WT m -/- G Th1 cells Th1 cells WT Foxo3-/- H Foxo3 mAbs in Th1 cell polarizing conditions for 36 or Anti-CD3 (0.5µg/ml) Anti-CD3 (2µg/ml) 72 hr (n = 5 mice per genotype) (H). 60 * 1750 5000 * * (I and J) Frequency and MFI of T-bet expression by Isotype 1500 100 100 4000 + -/- 1250 naive CD4 T from WT (black bars) or Foxo3-defi- Foxo3 40 80 80 * 1000 * 3000 (MFI) WT (MFI) cient mice (open bars) (I stimulated as in G; J

60

60 (Percent) γ 750 γ 2000 γ stimulated as in H) (n = 5 mice per genotype). 40 40 20 Max (%) 500 * IFN- IFN-

IFN- 1000 Data are representative of three independent 20 20 250

0 experiments. Error bars, SEM; p values (Mann- 03 3 4 5 3 3 4 5 0 0 -10 0 10 10 10 -10 0 10 10 10 0 0.5 2 0.5 2 36 72 IFN-γ IFN-γ Whitney U test). See also Figures S2 and S3. Anti-CD3(µg/ml) Anti-CD3(µg/ml) Hours

Foxo3-/- WT I Th1 cells Th1 cells WT J Foxo3-/- Anti-CD3 (0.5µg/ml) Anti-CD3 (2µg/ml) 90 2000 3000 *

100 * 100 Isotype 1500 80 60 2000 Foxo3 expression and nuclear accumula- 80 Foxo3-/- * 60 1000 + 60 WT tion in activated CD4 T cells. 40 40 30 1000 Max (%) T-bet (MFI) 500 T-bet (MFI) 20 20 T-bet (Percent) + 0 Foxo3 Deficiency Impairs CD4 T 0 3 4 5 3 4 5 0 10 10 10 0 0 0 0 10 10 10 T-bet 0.5 2 0.5 2 36 72 Cell Differentiation T-bet Anti-CD3(µg/ml) Anti-CD3(µg/ml) Hours To better understand the significance of enhanced Foxo3 expression in effector (Figure S1C). In agreement, stimulation with phorbol 12-myris- CD4+ T cells, in vitro experiments were performed in which naive / + tate 13-acetate (PMA) alone was able to induce Foxo3 expres- Foxo3À À or WT CD4 T cells were stimulated under neutral con- sion whereas ionomycin did not (Figure S1D). To dissect the ditions with increasing concentrations of anti-CD3 mAbs. Under pathway downstream of PKC, we used inhibitors of NF-kB and those culture conditions, the frequencies of IFN-g-(Figures 2A the NFAT transcription factor and showed that TCR-induced and 2B) and GM-CSF- (Figures 2C and 2D) secreting cells in / + Foxo3 expression was NF-kB dependent (Figure S1E). Taken Foxo3À À CD4 T cells were reduced by half of that observed together, these data suggest that PKCs and NF-kB pathways in WT CD4+ T cells after either 36 or 72 hr of culture whereas sur- downstream of TCR positively regulate Foxo3 expression in vival, proliferation, or IL-2, IL-13, IL-4, and TNF production were CD4+ T cells. unaffected (Figures S2A and S2B) and the production of IL-10 Because activation of Foxo3 was correlated with its sub- and IL-17 was undetectable (data not shown). This decreased cellular localization, immunofluorescence staining and sub- frequency of IFN-g- and GM-CSF-positive cells was also cellular fractionation combined with immunoblot analysis were observed when cells were stimulated with both anti-CD3 and performed. Foxo3 was almost entirely localized in the nucleus anti-CD28 mAbs, indicating that a co-stimulatory signal was of activated CD4+ T cells (Figures 1D and 1E). Altogether, our not sufficient to restore cytokine production by Foxo3-deficient data show that TCR-dependent signal intensity correlates with cells (Figure S2C). In addition, a delayed and diminished

776 Immunity 45, 774–787, October 18, 2016 expression of T-bet, the ‘‘master regulator’’ of Th1 cell differen- and 72 hr, correlating with the expression of Foxo3 (Figure 3F). / + tiation (Szabo et al., 2000), was observed in Foxo3À À CD4 We next assessed Eomes expression in other Th cell subsets. T cells upon TCR engagement (Figures 2E and 2F). Decreased In Th1 cell-polarizing conditions, Foxo3 also controlled Eomes IFN-g production associated with a Foxo3 deficiency was also expression (Figure 3G). Nevertheless, Eomes expression is found under Th1 cell-polarizing conditions (Figure 2G) whereas IL-12 independent (Figure 3H) and its expression rose between proliferation and survival were not affected (Figure S2E). More- 36 and 72 hr as observed for Th0 cells (Figure 3I). Finally, Eomes over, the Foxo3 deficiency not only decreased the frequency of expression was low in Th17 and Treg cells as compared to Th0 IFN-g+ cells but also impacted the overall amount of IFN-g pro- cells (Figure S4F). These results collectively show that Foxo3 duced on a per-cell basis, as demonstrated by the decreased expression is required for TCR-induced Eomes expression in / + + MFI of IFN-g expressed by Foxo3À À CD4 T cells after either CD4 T cells. 36 or 72 hr of culture (Figure 2H). The frequency of T-bet-ex- / + + pressing cells was equivalent in both Foxo3À À and WT CD4 Foxo3 Indirectly Controls Ifng and Csf2 in CD4 T Cells Th1 cells (Figure 2I); however, Foxo3 deficiency was also asso- through the Regulation of Eomes Expression ciated with decreased T-bet MFI in Th1 cells (Figure 2J). Because Foxo3 expression was highly increased in CD4+ T cells / + We next assessed the ability of Foxo3À À CD4 T cell to differ- expressing Eomes (Figure 4A), we hypothesized that Foxo3 entiate into different Th cell lineages when stimulated in polar- might directly control Eomes transcription. To assess this possi- izing conditions. We showed that Foxo3 deficiency did not bility, we first performed in silico analysis to identify conserved impact Th2, Th17, or Foxp3 Treg cell differentiation (Figure S3A). Foxo-binding sites (FBSs) in mouse and human EOMES loci. / + In particular, Foxo3À À CD4 T cells were fully able to differen- We found three putative FBSs: one (FBS1) located in the pro- tiate into Foxp3+ pTreg cells induced by transforming growth moter of Eomes gene (chr9: 118,478,419) and the other two factor (TGF-b) signaling (Figure S3B) or suboptimal TCR activa- (FBS2 and FBS3) positioned downstream of the 30 UTR of Eomes tion (Figure S3C; Li et al., 2013a). Moreover, we showed that (chr9: 118,487,803), in a region enriched in transcription factor / tTreg cells from Foxo3À À mice were as suppressive as WT tTreg binding sites that might therefore represent a putative 30 UTR cells (Figure S3D). Collectively, these results show that Foxo3 enhancer region (p30UTR-E) (Figure 4B). To determine whether promotes TCR-induced production of IFN-g and GM-CSF and Foxo3 can directly bind within the Eomes locus, we conducted has no notable impact on Th2, Th17, or Treg cell differentiation. chromatin immuno-precipitation experiments using primer sets designed to amplify regions located at each identified FBS. We Foxo3 Is Required for TCR-Induced Eomes Expression found that Foxo3 could bind to the FBS1, although binding by CD4+ T Cells was more pronounced for FBS2 and FBS3 (Figure 4C). To understand the molecular mechanisms whereby Foxo3 con- To address whether these FBS regions are involved in the trols CD4+ T cell differentiation, unbiased analysis of genes regulation of Eomes expression, we conducted luciferase re- differentially expressed in Foxo3-deficient versus Foxo3-suffi- porter assays. HEK293T cells were transfected with a reporter cient CD4+ T cells was achieved using both resting and activated plasmid in which a 1 Kb fragment located upstream of the human CD4+ T cells obtained after 12 or 24 hr of stimulation with anti- promoter region of EOMES was cloned into the pGL3-Basic vec- / CD3 mAbs. When comparing unstimulated WT and Foxo3À À tor (pEomes_luc) (Li et al., 2013b). Cells were co-transfected CD4+ T cells, only five transcripts showed greater than 2-fold with plasmids coding for different forms of V5-tagged-FOXO3: change, suggesting that Foxo3 plays minimal role in resting the constitutively active form of FOXO3 (FOXO3TM) (Brunet CD4+ T cells (Figure S4A). This number increased upon TCR et al., 1999), the Nt fragment from FOXO3TM used as domi- engagement suggesting that Foxo3 is mainly active after TCR nant-negative (FOXO3-A32A253-Nt) (Charvet et al., 2003), stimulation (FDR % 0.05) (Figures 3A and S4B). Three main or the active FOXO3TM mutated in the DNA binding domain networks were impacted by Foxo3 deletion among which (FOXO3TM-H212R). Transfection of FOXO3TM induced a the ‘‘IFN-g and IFN-g response’’ was the most dysregulated 2-fold increase in luciferase activity, whereas the transfection pathway (Figure 3B). The second network was enriched for of FOXO3-A32A253-Nt had no impact (Figure 4D). To assess metabolic functional categories, confirming the role of Foxo3 whether the 30 UTR region is involved for EOMES expression, in the regulation of cellular metabolism (Figure S4C). The third an 81 bp fragment of the p30UTR-E region containing the two identified cluster was enriched in genes involved in ‘‘immune putative FBSs was sub-cloned into the pEomes-luc vector cell trafficking,’’ suggesting that Foxo3 might have a role in (pEomes_p30UTR-E_luc). Using this construct, we found a T cell migration and homing (Figure S4D). 6-fold increased luciferase activity in the presence of FOXO3TM, Among all dysregulated genes, Eomes was the second (T12h) whereas the mutant FOXO3TM-H212R failed to affect luciferase / + and first (T24h) most suppressed gene in Foxo3À À CD4 T cells. activity, indicating that FOXO3 bound directly to the FBS in Analyses by RT-qPCR and flow cytometry confirmed that the p30UTR-E_region of EOMES (Figure 4E). Altogether, these + Foxo3-deficient CD4 T cells exhibited a decreased expression results show that FOXO3 binds to FBSs present in the 30 UTR of Eomes after activation (Figures 3C and 3D). Although Eomes region of EOMES and that EOMES is a direct transcriptional expression is lower in CD4+ T cells than in CD8+ T cells, target gene of FOXO3. its expression increases after activation (Figure S4E). Indeed, We next assessed whether Eomes expression was also linked TCR-dependent signal intensity controlled Eomes expression in to GM-CSF and IFN-g secretion in CD4+ T cells. Intracellular CD4+ T cells, and this expression was largely Foxo3 dependent staining showed that the expression of Eomes was higher + + + + (Figure 3E). Eomes expression by CD4 T cells was detected in GM-CSF IFN-g cells as compared to GM-CSFÀIFN-g or + after 18 hr of stimulation and rose substantially between 36 GM-CSFÀIFN-gÀ (Figure 4F). Moreover, when naive CD4

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m Prss12 m /Lo niC3mb ne h cell Th1 under mAbs anti-CD3 of g/mL /Lo niC3mb n L1 n=4 = (n IL-12 and mAbs anti-CD3 of g/mL

+ 0 0.5 Phyhd1 el ntmltd(0 rstimulated or (T0) unstimulated cells T

Foxo3 WT Cdc25b 1 Vipr1 % Foxo3 WT Dapl1 C .5adfl hne>2o 2). < or 2 > change fold and 0.05 iueS4 Figure -/- Gbp11 m Relative expression /Lat-D Asadthe and mAbs anti-CD3 g/mL Sell 0.04 0.06 0.08 0.10 0.00 0.02

-/- Klf2 Pde2a

Eomes S1pr1 Foxo3 WT . ** Chi3l7 Il7r -2 -1 0 1 2 -/-

Log(FC Foxo3-/--WT) m /Lof g/mL g and A B Figure 4. Eomes Is a Direct Target Gene of Foxo3 100 Foxo3-/- 1200 ** E1 E3 E5 EOMES (A) Foxo3 expression gated on Eomes+ (dark gray) - pEOMES 3’UTR-E 80 WT Eomes + 900 + E2 and EomesÀ (light gray) WT CD4 T cells stimu- 60 WT Eomes E4 E6 600 CACTTCAAGGAAAAAAGAAGA lated with 2 mg/mL of anti-CD3 mAbs (n = 6–7 mice 40 TAAACAAAACATCCCCACCCCCGCAT Max (%) Max FBS1 per genotype). Foxo3 (MFI) FBS2 300 20 GGCGGAGAATGTAAACAAACCGGGCGCCGAGCCCTT (B) Schematic structure of the EOMES gene, the GATCTTTATTTAAATAGAGGCTTGTTTATCGGTGTCATC 0 0 3 4 5 arrow represents transcriptional start site of a 0 10 10 10 FBS3 WT Eomes- Foxo3 WT Eomes+ gene, the black boxes represent exon position (E1 to E6), the positions and sequences of the putative C D pEOMES_luc E pEOMES_p3’UTR-E _luc Forkhead-binding sites (FBS) are highlighted in gray. 7 7 ** IgG FOXO3TM (C) Chromatin immunoprecipitation analysis of FOXO3TM-H212R ** 0.012 Foxo3 6 6 + FOXO3-A32A253-Nter ** Foxo3 binding to the Eomes locus in purified CD4 0.010 5 5 T cells stimulated for 24 hr with 2 mg/mL anti-CD3 mAbs. Results are expressed as percentage of 0.008 4 ** 4 ** input. 0.006 3 * 3 (D and E) HEK293 T cells were co-transfected with

Input(%) 0.004 2 2 reporter plasmids containing the human promoter region of EOMES cloned into the pGL3-Basic Relative luciferase activity luciferase Relative 0.002 1 activity luciferase Relative 1 vector (pEOMES_luc) (D) or (E) plasmids contain- 0.000 0 0 ing the human promoter region of EOMES with the 0 0 0 0 50 50 50 50 50 50 100300 10030 10030 100300 10030 FBS1 FBS2 FBS3 3105 Mock 10030 Mock 30 UTR region containing the two putative FBSs Plasmid (ng) Plasmid (ng) (pEOMES-p30UTR-E_luc) together with plasmids coding for different forms of FOXO3: the consti- tutively active FOXO3a mutant (FOXO3TM, black F Unstimulated IFN-γ+GM-CSF- G IFN-γ GM-CSF bars), the constitutively active FOXO3TM mutated IFN-γ -GM-CSF- IFN-γ+GM-CSF+ 8000 60 * * * for the DNA binding domain (FOXO3TM-H212R, 700 ** * 600 6000 * gray bars), the constitutive active FOXO3TM 40 500 ** deleted for the transactivation domain (FOXO3- 400 4000 pg/ml 300 * 20 A32A253-Nter, dashed bars), or empty vector Max (%) Max 200 2000 fl/fl + Eomes (MFI) 100 Eomes -Cd4-cre (n=4) (Mock). All luciferase activities were normalized fl/fl - 0 0 0 Eomes -Cd4-cre (n=4) to the expression of the co-transfected Renilla 1410 Eomes 1410 Anti-CD3 (µg/ml) Anti-CD3 (µg/ml) luciferase.

-/- (F) Eomes expression gated on IFN-gÀGM-CSFÀ -/- Foxo3 H Foxo3 + + + +LV-GFP +LV-Eomes or IFN-g GM-CSFÀ and IFN-g GM-CSF pro- ducing CD4+ T cells stimulated with 2 g/mL anti- 0.5 10 m CD3 mAbs under non-polarizing condition (n = 5 IFN-γ IFN-γ GM-CSF I GM-CSF mice per genotype). 15 40 40 (G) Naive CD4+ T cells purified from Eomesfl/fl- CD4 *** ** * + fl/fl Cd4-cre (black bars) or Eomes -Cd4-creÀ Eomes Eomes 30 30 10 (open bars) were stimulated with anti-CD3 mAbs 9 3 13 3 30 28 20 20 and the secretion of IFN-g and GM-CSF was percent Percent 5 percent analyzed by ELISA in the supernatant after 3 days 10 10 of culture (n = 4 mice per genotype). 10 2 3 0 (H) IFN-g and GM-CSF expression in naive GM-CSF 0 0 / + - Foxo3À À CD4 T cells transduced with lentiviral IFN-γ T-LV Eomes T-LV Eomes+ particles expressing the GFP alone (LV-GFP) or Eomes and the GFP (LV-Eomes) and gated on either GFP-transduced (LV-GFP, left), non-transduced (LV-Eomes, middle), or Eomes-transduced (LV-Eomes, right) CD4+ T cells. + + + + / + (I) Frequency of IFN-g , IFN-g GM-CSF , or GM-CSF cells among Foxo3À À CD4 T cells either non-transduced (LV-EomesÀ, open dots) or transduced (LV-Eomes+, gray dots) (n = 11 mice, from 3 independent experiments). Data are representative of at least three independent experiments or two independent experiments (C). Error bars, SEM; p values (Mann-Whitney U test). See also Figure S5.

T cells purified from mice with a T cell-specific deletion of Eomes that Eomes transduction of Foxo3-deficient T cells restored the (Eomesfl/flCd4-cre) were stimulated in vitro with increased con- expression of both IFN-g and GM-CSF (Figures 4H and 4I). centration of anti-CD3 mAbs, both GM-CSF and IFN-g secretion This finding supports the notion that Foxo3 indirectly regulates were reduced in Eomesfl/flCd4-cre+CD4+ T cells as compared Ifng and Csf2 in CD4+ T cells through the regulation of Eomes fl/fl to Eomes Cd4-creÀ cells (Figure 4G), whereas the prolifera- expression. tion and survival were similar (data not shown). Therefore, the In addition, we address whether Eomes directly controls Ifng decreased Eomes expression associated with Foxo3 deficiency and Csf2 expression. We performed an in silico analysis and might explain the defect GM-CSF and IFN-g secretion in found six highly conserved noncoding sequences enriched in / + Foxo3À ÀCD4 T cells. To address this issue directly, we tested DNaseI hypersensitivity sites and putative transcription factor whether lentiviral-based overexpression of Eomes could over- binding sites positioned downstream of the 30 UTR of CSF2. come the defect in IFN-g and GM-CSF production. We showed Next, luciferase reporter assays were performed by coupling

Immunity 45, 774–787, October 18, 2016 779 these elements to the proximal CSF2 promoter. Using this tech- mates were immunized with MOG35-55 peptide emulsified in nique, we were unable to demonstrate a direct regulation of CFA. Although the incidence of EAE disease and the mean day / CSF2 by EOMES (Figure S5A). In contrast, the same technique of onset were similar, Foxo3À À mice developed a much less revealed that EOMES, but not FOXO3, directly transactivates severe disease than their wild-type counterparts (Figure 6A). the promoter of IFNG (Figure S5B). Moreover, we showed that Comparable results were obtained when male mice were used, Foxo3 was unable to transactivate the Ifng locus (Figure S5C). showing that there was no gender bias (Figure S6A). To assess Altogether, these data support the concept that the Eomes- whether this decreased EAE severity was the consequence of + / Foxo3 axis is required for the polarization of effector CD4 a bias in the TCR repertoire, Foxo3À À mice were crossed to T cells into IFN-g- and GM-CSF-producing cells. 2D2 TCR transgenic mice in which the CD4+ T cell population ex- presses an I-Ab-restricted TCR specific for the immune-domi-

Eomes Acts Independently of T-bet for GM-CSF nant MOG35-55 peptide (Bettelli et al., 2003). Foxo3 deficiency Regulation in CD4+ T Cells in 2D2 mice also led to a reduction of disease severity (Figure 6B). Foxo3 deficiency affects both T-bet and Eomes expression by To exclude the implication of Foxo3 expression by the resident + / CD4 T cells, so we next wondered whether Eomes and T-bet cells of the CNS, passive EAE was induced in Foxo3À À and could be co-regulated and to what extent diminished GM-CSF WT recipients by transfer of WT MOG-specific CD4+ T cells and IFN-g secretion resulted from decreased T-bet expression differentiated in vitro into encephalitogenic Th1 and Th17 cells. in Foxo3-deficient CD4+ T cells. A time course analysis showed The analysis of clinical scores showed that the incidence and that Eomes expression precedes that of T-bet and the defect in severity of EAE induced was similar between the two genotypes Eomes and IFN-g preceded the reduction of T-bet expression in (Figure S6B), thereby excluding any implication of Foxo3 expres- / + Foxo3À À CD4 T cells, suggesting that initial production of IFN-g sion in the target organ. by CD4+ T cell might be Eomes dependent but T-bet indepen- Additional experiments were conducted to decipher the dent (Figure 5A). In this regard, previous studies demonstrate relative contribution of Foxo3 in T cells versus APCs during that the first wave of IFN-g is T-bet independent and causes EAE. The impact of a Foxo3 deficiency in non-T cells was as- + the autocrine induction of T-bet (Schulz et al., 2009). Therefore, sessed by transferring WT MOG35-55-specific 2D2 CD4 / / the decreased T-bet expression in Foxo3À À cells might be due T cells into Foxo3-deficient or -sufficient Rag2À À mice. Mice to the decreased Eomes-dependent IFN-g secretion. were next immunized and disease severity was evaluated. / + To test this hypothesis, WT and Foxo3À À CD4 T cells were Both groups of mice developed EAE with similar incidence, ki- stimulated in the presence of neutralizing anti-IFNg monoclonal netics, and severity (Figure 6C). These data point to a minimal antibody (mAb) to prevent T-bet induction by IFN-g. Upon role of Foxo3 in non-T cells during EAE development. Further- IFN-g neutralization, a clear reduction of T-bet expression was more, EAE experiments were next conducted on genetically observed, leading to similar expression of T-bet in both WT engineered mice harboring a T cell-specific deletion of Foxo3 / + fl/fl fl/fl + and Foxo3À À CD4 T cells (Figure 5B). These results establish (Foxo3 -Cd4-cre). Foxo3 -Cd4-cre mice developed disease that Foxo3 has no direct impact on T-bet expression and further with a reduced incidence and severity as compared to fl/fl indicate that decreased T-bet resulted from decreased IFN-g Foxo3 -Cd4-creÀ control mice, demonstrating that Foxo3 / + secretion by Foxo3À À CD4 T cells. We also showed that the controls the susceptibility to EAE in a T cell-intrinsic manner expression of Eomes was independent of the signaling pathway (Figure 6D). Altogether, these results reveal that Foxo3 expres- downstream of IFN-g since the expression of Eomes was not sion in CD4+ T cells plays a critical role in the susceptibility to affected by blocking IFN-g (Figure 5C). CNS inflammation. We next analyzed cytokine secretion in presence of blocking anti-IFN-g antibody. Although suppressing the IFN-g autocrine Foxo3 Drives the Differentiation of Pathogenic IFN-g+ effect strongly impacted the production of IFN-g, it had no effect and GM-CSF+ CD4+ T Cells during EAE on GM-CSF production. Yet, GM-CSF secretion was dimin- We next assessed whether the outcome of EAE in Foxo3-defi- / ished in Foxo3À À cells whereas T-bet expression remained cient mice was accompanied by differences in polarization of unchanged (Figures 5D and S5D). These results further support both peripheral and CNS-infiltrating CD4+ T cells. In agreement the notion that the Foxo3-Eomes pathway, but not T-bet, is crit- with our results obtained in vitro, MOG-specific Foxo3-deficient ical for GM-CSF regulation. Similar results were obtained in CD4+ T cells produced lower amounts of the effector cytokines T cells overexpressing Eomes after lentiviral transduction. Under IFN-g and GM-CSF whereas the production of IL-17, TNF, and conditions in which the IFN-g was blocked and expression of other cytokines was not affected (Figures 7A and S7A). Intracel- T-bet was low, Eomes overexpression still resulted in increased lular staining was performed to identify which Th cell subset was IFN-g and GM-CSF expression (Figures 5E and 5F). These data impacted by Foxo3 deficiency. We observed a large decrease in + + + further demonstrate that Eomes can act independently of T-bet the proportion of both IFN-g GM-CSFÀ and IFN-g GM-CSF + + to control IFN-g and GM-CSF secretion. CD4 T cells. The frequency of IL-17 GM-CSFÀ cells was not impacted, whereas Foxo3-deficient CD4+ T cell exhibited a slight Foxo3 Controls the Severity to Neuroinflammation decreased frequency of IL-17+GM-CSF+ cells (Figure 7B). The We next addressed the in vivo relevance of the Eomes-Foxo3 frequency of Foxp3 Treg cells was unaffected in immunized pathway by assessing whether Foxo3 deficiency modifies the Foxo3-deficient mice (Figure 7C). susceptibility to experimental autoimmune encephalomyelitis To address whether this defective CD4+ T cell differentiation (EAE), a well-characterized mouse model for multiple sclerosis was also observed in CNS-infiltrating leukocytes, mononuclear / / (MS). Hence, Foxo3À À female mice and their wild-type litter- infiltrating cells from the spinal cord and brain of Foxo3À À and

780 Immunity 45, 774–787, October 18, 2016 A WT Foxo3-/- 18h 36h 72h 18h 36h 72h

5 5 5 5 5 5 10 16 4 10 40 6 10 57 14 10 7 1 10 21 2.5 10 32 6 4 4 4 4 4 10 10 104 10 10 10

3 3 3 3 3 10 10 103 10 10 10

2 2 2 2 2 10 10 102 10 10 10

Eomes Eomes 0 0 80 1 0 54 1 0 26 2 89 2 0 75 1 0 60 2 3 4 5 3 4 5 3 4 5 3 4 5 3 4 5 01010 10 0103 104 105 01010 10 01010 10 01010 10 01010 10 IFN-γ IFN-γ

5 5 5 10 10 5 5 5 3 0.5 10 30 5 63 18 10 3 0.5 10 20 3 10 37 6 4 4 4 10 10 4 4 4 10 10 10 10

3 3 3 10 10 3 3 3 10 10 10 10

2 2 2 2 T-bet T-bet 10 10 2 2 10 10 10 10 0 0 91 5 0 61 3 16 2 0 93 3 0 75 2 0 53 3 3 4 5 3 4 5 3 4 5 01010 10 01010 10 3 4 5 3 4 5 3 4 5 01010 10 01010 10 01010 10 01010 10 IFN-γ IFN-γ 5 5 5 5 5 10 10 10 10 11 1 105 15 29 10 5 65 6 2 7 15 8 28 4 4 4 4 4 10 10 10 10 104 10

3 3 3 3 3 10 10 10 10 103 10

2 2 2 Eomes 2 2 Eomes 10 10 10 10 102 10 0 0 0 0 0 0 86 2 44 12 9 21 88 4 65 12 42 22 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 0102 103 104 105 010 10 10 10 T-bet T-bet

B C D +IgG +Anti-IFN-γ 4.5 5.5 5.5 4.5 T-bet T-bet Eomes WT 1600 * 80 * 50 ** * 60 1200 40 8.5 1.5 -/- WT Foxo3 30

40 MFI 800 ns Percent

Max (%) 1.2 2.5 1.5

20 GM-CSF 1.5 WT 20 ns 400

+Anti-IFN-γ Eomes (percent)10 0 0 0 γ -γ γ - Foxo3-/- N IgG IgG IgG i-IF 4.5 -/- +Anti-IFN-γ ti-IFN t 2 Foxo3 An Anti-IFN- An -/- WT Foxo3 IFN-γ T-bet

-/- -/- E Foxo3 Foxo3 F +LV-GFP +LV-Eomes T-bet GM-CSF IFN-γ ns 25 ∗ 12 5000 ∗ ∗

CD4 4000 20 10 8 3000 ns 15 ∗ 6 Percent 2000 10 Percent 4 T-bet (MFI) T-bet 1000 5 2

CD4 0 0 0 γ -γ γ G N- G Ig IgG IFN Ig +Anti-IFN-γ +Anti-IFN-γ +Anti-IFN-γ i- Anti-IF Ant Anti-IFN-

-/- -/- Eomes T-bet Foxo3 -LV-GFP Foxo3 -LV-Eomes

Figure 5. Eomes Acts Independently of T-bet for GM-CSF Regulation in CD4+ T Cells / + (A) Kinetics of T-bet, Eomes, and IFN-g expression in naive WT or Foxo3À À CD4 T cells stimulated with 2 mg/mL of anti-CD3 mAbs for 18, 36, or 72 hr (n = 4 mice per genotype). / + (B and C) T-bet expression (B) or Eomes expression (C) in naive WT (black bars/histograms) or Foxo3À À (white bars/histograms) CD4 T cells stimulated with anti- CD3 mAbs in the absence or presence of anti-IFN-g blocking mAbs (n = 4–5 mice per group). + + + + / + (D) Frequency of IFN-g , IFN-g GM-CSF , and GM-CSF producing cells in naive WT or Foxo3À À CD4 T cells cultured in the absence or presence of anti-IFN-g neutralizing mAbs. / + (E) Eomes and T-bet expression in naive WT or Foxo3À À CD4 T cells transduced with either control (LV-GFP) or Eomes (LV-EOMES) expressing lentiviral particles in presence or absence of anti-IFN-g mAbs. / + (F) Frequency of GM-CSF- and IFN-g-producing cells in naive Foxo3À À CD4 T cells transduced either with control (LV-GFP, open bars/dots) or Eomes (LV- EOMES, gray bars/dots) in presence or absence of anti-IFN-g mAbs. Data are representative of at least three independent experiments. Error bars, SEM; p values (Mann-Whitney U test). See also Figure S5.

Immunity 45, 774–787, October 18, 2016 781 A Foxo3-/- (n=14) WT (n=14)

100 5 *** 80 *** 80 4 60 60 3 40 40 2 Clinical scores Clinical 20 1 20 Incidence (percent) WT -/- 0 0 score cumulative Mean 0 Foxo3 0102030 0102030 DPI

B 2D2-Foxo3-/- (n=6) 2D2- WT (n=6) 100 3 * 50 * 80 40 2 60 30 40 1 20 20 Incidence (percent)

Clinical scores Clinical 10 2D2- WT 0 0 -/- Mean cumulative score cumulative Mean 0 2D2-Foxo3 0102030 10 20 30 40 DPI

C WT 2D2 injected into Rag2-/--Foxo3-/- (n=7) WT 2D2 injected into Rag2-/-- Foxo3+/+ (n=6)

ns 100 60 5

80 4 40 60 3

40 2 20 Clinical scores Clinical 20 Incidence (percent) 1 Rag2-/--Foxo3+/+ -/- -/- 0 0 score cumulative Mean 0 Rag2 -Foxo3 05101520 0 5101520 DPI D Foxo3fl/fl-CD4-cre+ (n=11) Foxo3fl/fl-CD4-cre- (n=14)

100 20 2.0 ** ** 80 1.5 15 60 1.0 10 40 Clinical scores Clinical Incidence (percent) 20 0.5 5 Foxo3fl/fl-CD4-cre-

Mean cumulative score cumulative Mean fl/fl + 0 0.0 0 Foxo3 -CD4-cre 01020300102030

Figure 6. Total Foxo3-Deficient Mice and Mice with a T Cell-Specific Deletion of Foxo3 Are Less Susceptible to EAE / (A) Foxo3À À (open circles, bars) and WT littermate (black circles, bars) mice were immunized with 50 mg of peptide MOG35-55 emulsified in CFA at day 0 and 200 ng of pertussis toxin was injected i.v. on day 0 and day 2 (n = 14 mice per genotype). / (B) 2D2-Foxo3À À (open circles, bars) or 2D2-WT (black circles, bars) were injected i.v. with 150 ng of pertussis toxin at day 0 (n = 6 mice per genotype). / / / +/+ 4 + 6 (C) Rag2À ÀFoxo3À À (opencircles, bars)or Rag2À ÀFoxo3 (blackcircles, bars) mice were injected i.v.with 23 10 2D2-WT naive CD4 T cells mixed with 43 10 WT + CD4 T cells. Mice were then immunized with 50 mg of peptide MOG35-55 emulsified in CFA and injected i.v. with 100 ng of pertussis toxin (n = 6–7 mice per genotype). fl/fl + fl/fl (D) Foxo3 -Cd4-cre (open circles, bars) or Foxo3 -Cd4-creÀ (black circles, bars) littermate controls were immunized as in (A). Incidence and mean cumulative clinical scores are shown (n = 11–14 per genotype). Incidence, clinical scores, and mean with SEM of cumulative clinical scores were calculated. Error bars, SEM; p values (Mann-Whitney U test); p values for clinical scores (two-way ANOVA). Data are representative of at least three independent experiments. See also Figure S6.

WT littermate mice were isolated and characterized by flow to the brain at the expense of the spinal cord (Figures S7B–S7D). cytometry. Analysis of T cell distribution in brain versus spinal As for their peripheral counterparts, Foxo3-deficient CD4+ T cells cord showed that Foxo3-deficient T cells migrated preferentially from the brain and spinal cord exhibited a decreased capacity

782 Immunity 45, 774–787, October 18, 2016 A IFN-γ GM-CSF IL-17

3500 6000 ** * 10000 8000 2500 4000 ** * 6000

pg/ml 1500 4000 2000 500 2000 Foxo3-/- 0 0 WT 010100 010100 010100 MOG35-55 (µg/ml) MOG35-55 (µg/ml) MOG35-55 (µg/ml)

B IFN-γ + IFN-γ + IL-17 + IL-17 + C GM-CSF− GM-CSF + GM-CSF − GM-CSF + ns 30 6 ** 1.5 0.9 15 ** **5 ** 20 4 1.0 0.6 10 3 10 2 0.5 0.3 5 Frequency

1 Foxp3 (percent) Foxo3-/- 0 0 0.0 0.0 0 WT 010100 010100 010100 010100

MOG35-55 (µg/ml) MOG35-55 (µg/ml) MOG35-55 (µg/ml) MOG35-55 (µg/ml)

D IFN-γ GM-CSF IL-17 E ns 20 14000 * 500 ** 7000 12000 6000 400 15 10000 5000 8000 * 300 4000 10 pg/ml 6000 200 3000 4000 2000 5 fl/fl + 100 (percent) Foxp3 Foxo3 -CD4-cre 2000 1000 Foxo3fl/fl-CD4-cre− 0 0 0 0 010100 010100 010100

MOG35-55 (µg/ml) MOG35-55 (µg/ml) MOG35-55 (µg/ml)

Figure 7. Foxo3 Deficiency in T Cells Is Associated with Reduced Differentiation of IFN-g and GM-CSF Pathogenic CD4+ T Cells during EAE / (A) Foxo3À À (open circles, n = 8) and WT littermate (black circles, n = 8) mice were immunized with 50 mg of peptide MOG35-55 emulsified in CFA. At day 9 post- + immunization, CD4 T cells were purified from spleens and restimulated in vitro with WT APC and MOG35-55 peptide. The secretion of IFN-g, GM-CSF, and IL-17 was analyzed by ELISA in the supernatant after 3 days of culture (n = 4 mice per genotype). + (B) Frequency of IFN-g-, GM-CSF-, and IL-17-producing CD4 T cells was determined by intracellular staining after overnight restimulation with MOG35-55 peptide (n = 8 mice per genotype). + / (C) The expression of Foxp3 by splenic CD4 T cells from immunized WT and Foxo3À À mice was assessed by intracellular staining (n = 8 mice per genotype). fl/fl + fl/fl (D) Foxo3 -Cd4-cre or Foxo3 -Cd4-creÀ littermate controls were immunized with 100 mg of peptide MOG35-55 emulsified in CFA. At day 9 post-immunization, splenocytes were restimulated in vitro with MOG35-55 peptide and IFN-g, GM-CSF, and IL-17 secretion was analyzed by ELISA (n = 9 mice per genotype). + fl/fl + fl/fl (E) The expression of Foxp3 by splenic CD4 T cells from immunized Foxo3 -Cd4-cre or Foxo3 -Cd4-creÀ mice was assessed (n = 8 mice per genotype). Data are representative of at least two independent experiments. Error bars, SEM; p values (Mann-Whitney U test). See also Figure S7. to secrete IFN-g and GM-CSF (Figures S7C–S7E). The propor- encephalitogenic CD4+ T cell differentiation and susceptibility tion of CNS-infiltrating Foxp3+CD4+ T cells was not altered by to CNS autoimmunity. Foxo3 deficiency (Figure S7F). / + As described in total Foxo3À À mice, MOG-specific CD4 DISCUSSION T cells from Foxo3fl/fl-Cd4-cre+ exhibited decreased secretion of IFN-g and GM-CSF whereas IL-17 secretion was unchanged Up to now, the role of Foxo3 in CD4+ T cell has been unappreci- (Figure 7D). Again, the frequency of Foxp3 Treg cells was unal- ated, mainly because of its low expression in lymphoid cells and tered in both periphery and CNS (Figure 7E and S7G). Altogether, also because of the dominant role of Foxo1. The present study these results reveal the T cell-intrinsic control of Foxo3 on showed that TCR engagement results in increased expression

Immunity 45, 774–787, October 18, 2016 783 of Foxo3 in CD4+ T cells and that this increase correlates with Foxo3-deficient cells led to reduction of IFN-g and disrupted TCR signaling strength. Moreover, this increased Foxo3 expres- the positive feedback loop by which IFN-g supports T-bet sion has a functional impact on CD4+ T cells. Foxo3 deletion in expression. Indeed, our results demonstrated that neither primary CD4+ T cells specifically impaired their ability to secrete Eomes nor Foxo3 were able to directly regulate T-bet expres- IFN-g and GM-CSF. Importantly, microarray analyses showed sion. Moreover, inhibition of the IFN-g autocrine loop had no ef- that decreased expression of genes involved in the IFN-g fect on GM-CSF secretion, further demonstrating that the pathway was not associated with global defect of CD4+ T cell Foxo3-Eomes pathway, but not T-bet, is critical for GM-CSF activation or changes in expression of genes from Th2, Th17, regulation (O’Connor et al., 2013). or Treg cell programs, further demonstrating that Foxo3 plays Uncontrolled CD4+ T cell polarization may have pathological a specific role in the polarization of pathogenic CD4+ T cells. consequences and lead to autoimmune diseases. We showed These results are consistent with our in vitro and in vivo results that Foxo3 deficiency diminished disease severity and that this showing that, after anti-CD3 stimulation or immunization with phenotype is T cell intrinsic and correlated with the reduced abil- + / + MOG35-55 peptide, CD4 T cells from Foxo3À À mice showed a ity of Foxo3-deficient CD4 T cells to differentiate into IFN-g- and decreased production of IFN-g and GM-CSF whereas the ability GM-CSF-producing CD4+ T cells. IFN-g, IL-17, and GM-CSF are of these cells to secrete IL-17, type 2 cytokines, or IL-10 was not the main effector cytokines in the pathophysiology of both EAE affected. We therefore conclude that Foxo3 deficiency is not and MS (Codarri et al., 2010; Goverman, 2009; Korn et al., associated with a general defect in CD4+ T cell activation but 2009). In immunized Foxo3-deficient animals, the frequency of rather impacts Th cell polarization by specifically disturbing the MOG-specific Th17 cells was unaffected, excluding the involve- production of both IFN-g and GM-CSF. ment of Th17 cells in the observed phenotype. Decreased IFN-g Several studies show that Foxo factors are crucial for Foxp3 production by Foxo3-deficient CD4+ T cells may impact T cell Treg cell development and function (Kerdiles et al., 2010; distribution within the CNS. Indeed, Foxo3-deficient T cells Ouyang et al., 2010, 2012). We demonstrated here that the migrated preferentially to the brain rather than spinal cord. These Treg cell program is not altered in Foxo3-deficient cells and results are consistent with studies showing that the Th17-Th1 that Foxo3-deficient Treg cells are as suppressive as WT Treg cell ratio of infiltrating T cells determines the topography of cells. Moreover, Foxo3 deficiency did not impact the proportion CNS inflammation (Goverman, 2009; Stromnes et al., 2008). of peripheral or CNS-infiltrating Foxp3 Treg cells during EAE. However, we can not exclude that Foxo3 might have a direct Therefore, Foxo3 is not necessary for development, differentia- role in T cell migration and homing since microarray analysis tion, migration, or function of Foxp3 Treg cells. showed that Foxo3-deficient CD4+ T cells exhibited increased Analysis of the molecular mechanism underlying these phe- expression of Klf2, S1pr1, and Sell and decreased expression notypes revealed that Foxo3 induces expression of the TF of Ccr8. Eomes. We showed that Eomes expression is controlled by Perhaps most importantly, we showed that Foxo3 deficiency TCR signaling strength and correlates with the dynamics of also impacted the ability of CD4+ T cell to produce GM-CSF, a Foxo3 expression in CD4+ T cells, supporting the notion that key factor in the effector phase of EAE (McQualter et al., 2001; Foxo3 might regulate Eomes in CD4+ T cells. Eomes was Ponomarev et al., 2007). Both Th1 and Th17 cells can secrete indeed a direct target gene of Foxo3 in CD4+ T cells. Transac- GM-CSF during EAE (Codarri et al., 2011). However, a recent + tivation of Eomes by Foxo3 was dependent upon a 30 UTR study showed that GM-CSF Th cells might represent a unique distal region containing two FBSs and may correspond to an Th cell lineage distinct from that of Th1 and Th17 cells (Hern- enhancer region. Accordingly, the analysis of Foxo3 genome- dler-Brandstetter and Flavell, 2014; Sheng et al., 2014). The fac- wide binding profile showed that this TF acts as a transcrip- tors regulating Csf2 expression remain to be defined (Croxford tional activator, regulating target through et al., 2015). Here, we have shown that GM-CSF-producing transcription initiation by binding preferentially to enhancer CD4+ T cells exhibited high and sustained expression of Eomes regions with increased conservation (Eijkelenboom et al., and that low Eomes expression impaired the differentiation of 2013a, 2013b). GM-CSF-producing cells. These data suggest the implication In CD4+ T cells, most of the described roles for Eomes are of this TF in the gene program of GM-CSF-secreting CD4+ redundant with T-bet (Steiner et al., 2011; Suto et al., 2006; T cells. In agreement, recent transcriptomic studies showed Yang et al., 2008). Here, we have provided information on the that Eomes is among the genes that are specifically expressed critical role of Eomes, independent of T-bet, in CD4+ T cell polar- by the GM-CSF+ Th cell lineage (Sheng et al., 2014). The role ization. Overexpression of Eomes overcame the defect in IFN-g of this T-box transcription factor in CNS neuroinflammation and GM-CSF production by Foxo3-deficient CD4+ T cells, has recently been demonstrated. Indeed, mice harboring a supporting the notion that Eomes is involved in Ifng and Csf-2 T cell-specific deletion of Eomes developed EAE with reduced regulation in CD4+ T cells. Moreover, under conditions in which severity, a similar phenotype as Foxo3-deficient mice (Raveney T-bet upregulation was blocked, Eomes overexpression still et al., 2015). Moreover, EOMES has been identified as a suscep- resulted in increased IFN-g and GM-CSF expression. These tibility gene in MS (Parnell et al., 2014; Patsopoulos et al., 2011). results are in agreement with data showing that Eomes is In addition, an increased proportion of Eomes+CD4+ T cells has responsible for the T bet-independent production of IFN-g in been reported in patients with secondary progressive MS as T-bet-deficient or GATA3-deficient CD4+ T cells (Yagi et al., compared to relapsing remitting MS or healthy controls and 2010; Yang et al., 2008). Therefore, the Foxo3-Eomes axis is these cells accumulate in the CSF from MS patients, further sup- part of the signaling events responsible for the first wave of porting the role of this transcription factor in CNS inflammation in IFN-g. As a consequence, decreased Eomes expression by humans (Raveney et al., 2015).

784 Immunity 45, 774–787, October 18, 2016 EXPERIMENTAL PROCEDURES p24-well plates coated overnight with 40 mg/mL of RetroNectin (TAKRA). 18 hr after activation, the medium was replaced by OptiMEM medium contain- Mice ing lentiviral particles (LV-EOMES or LV-GFP). Anti-CD28 and IL-2 were added / fl/fl Foxo3À À (Dejean et al., 2009), 2D2 (Bettelli et al., 2003), Eomes Cd4-cre (Zhu (10 UI/mL and 2 mg/mL, respectively). Cells were then centrifuged (3,000 rpm) fl/fl et al., 2010), Foxo3 Cd4-cre (Paik et al., 2007), and C57BL/6 mice were main- for 1 hr at 32C and incubated overnight at 37C. The next day, supernatant was tained in the breeding facility of PreCREFRE (Toulouse UMS06) under SPF replaced by complete RPMI medium supplemented with IL-2 (10 UI/mL) and conditions. All animal procedures were conducted in accordance with institu- anti-CD28 (2 mg/mL). 72 hr after transduction, infected cells were then activated tional guidelines on Animal Experimentation and were under a French Ministry with PMA plus ionomycin (0.5 mg/mL each) for 4 hr plus Golgiplug (1/1,000). of Agriculture license. Cells were then stained and analyzed by flow cytometry (FACS LSRII).

Experimental Autoimmune Encephalomyelitis Statistical Analysis To induce active EAE, mice were immunized with 50 mg of MOG35-55 peptide p values were determined by Mann-Whitney tests. p values < 0.05 were (Polypeptide) emulsified with Complete Freund Adjuvant (CFA) containing considered statistically significant (***p < 0.001, **p < 0.005, *p < 0.01). All error 2 mg/mL of Mycobacterium tuberculosis (Difco). 200 ng/mL of pertussis toxin bars represent the SEM. For EAE clinical scores, p values were determined by fl/fl (COGER) was given at day 0 and day 2 after immunization. For Foxo3 -Cd4- two-way ANOVA (***p < 0.001, **p < 0.005, *p < 0.01). cre, 100 mg of MOG35-55 peptide was used. Clinical score were evaluated on a five-stage scale from 0 to 5. ACCESSION NUMBERS CD4+ T Cell Purification, Stimulation, and Flow Cytometry Naive CD62L+CD4+ T cells were obtained by negative selection of total CD4+ Microarray data and experimental details are available in the Gene Expression T cells (Dynal) and positive selection by CD62L+ beads (Myltenyi). Naive CD4+ Omnibus (GEO) database (accession GSE86287). T cells were stimulated with anti-CD3 antibody (Biolegend) with or without anti-CD28 (BD Biosciences) in non-polarizing condition or with IL-12 and SUPPLEMENTAL INFORMATION IL-2 (R&D) for Th1 cell-polarizing condition. Cytokines and transcription factor expression were measured by intracellular staining using the ‘‘Foxp3 staining Supplemental Information includes seven figures, one table, and Supple- buffer’’ (Ebioscience). Antibodies were all purchased from Ebioscience, BD mental Experimental Procedures and can be found with this article online at PharMingen, or Cell Signaling for anti-Foxo3 mAbs (clone 75D8). All samples http://dx.doi.org/10.1016/j.immuni.2016.09.010. were acquired and analyzed with the LSR II flow cytometer (Becton Dickinson) and FlowJo software (TreeStar). AUTHOR CONTRIBUTIONS

Microarray Gene Expression Study C.S. and M.F.M. performed experiments and data analysis and helped to write + Gene expression analysis was performed on purified naive CD4 T cells from the manuscript, and M.B., N.C., and I.B. performed experiments and data / Foxo3À À (n = 3–4) or WT (n = 4) littermate controls either unstimulated (T0) or analysis and helped with in vivo experiments. X.-H.N. performed experiments stimulated with 2 mg/mL of anti-CD3 mAbs for 12 (T12) or 24 (T24) hours at related to luciferase constructs. Y.L. performed microarray data analysis and the GeT facility (Ge´ noToul, Ge´ nopole Toulouse Midi-Pyre´ ne´ es) using Agilent design. S.M.H. provided mice and helped to write the manuscript. F.D. helped Sureprint G3 Mouse microarrays (8x60K, design 028005) according to the with in vitro experiment. R.S.L. and A.S. gave advice for experiment proced- manufacturer’s instructions. ures and helped to write the manuscript. A.S.D. performed and oversaw research, designed experiments, and wrote the manuscript. Chromatin Immunoprecipitation CD4+ T cells were stimulated with anti-CD3 (2 mg/mL) and anti-CD28 (1 mg/mL) ACKNOWLEDGMENTS mAbs for 24 hr. Foxo3 ChIP experiments were performed using iDeal ChIP- Seq Kit for Transcription Factors (Diagenode, C01010055) with some modifi- We would like to thank Dr. B. Li (Institut Pasteur of Shanghai, China) for cations. In brief, cells were fixed with 1% PFA during 15 min and then glycine providing us the pGL3-Eomes-Luc reporter construct; Dr. C. Charvet (Institut (0.250 mM) was added. Cells were then lysed with manufacturer’s buffers and Cochin, Paris, France) for FOXO3 plasmids; A. Thouard and Dr. M. Szelechow- sonicated with 15 cycles of 30 s ON/60 s OFF using a bioruptor pico. Sonicated ski for advice in lentiviral production; Dr. V. Adoue and Dr. O. Joffre for their chromatin was incubated overnight at 4C either with 5 mg of anti-Foxo3 help on ChIP experiments; Dr. S. Kassem for her expertise on EAE experi- antibody (Santa Cruz cat# sc-11351X) or an IgG control. Chromatin was ments; Dr. L.T. Mars for his help with funding; and Dr. D. Dunia, Dr. N. Fazilleau, then washed and eluted using manufacturer’s recommendations. For ChIP and Dr. R. Lesourne for their critical comments on the manuscript. We would analysis, qPCR was performed using SyberGreen Master mix (Roche) on a also like to thank the flow cytometry and microscopy core facility (CPTP) and 480 LightCycler in duplicate with primers listed in Table S1. Percent of input the animal house staff members for their technical assistance (UMS 06). This was calculated using the following formula: 2^(adjusted INPUT-Ct (IP))3100 work was supported financially by grants from Inserm Dotation (Nouveau-re- where adjusted INPUT = Ct INPUT – log2 (1). crute´ ), the ANR (project ANR-09-RPDOC-015-01), ARSEP Foundation (project R112195BB) and Midi-Pyre´ ne´ es Region. C.S. was the recipient of a doctoral Luciferase Assay fellowship from ARSEP Fondation (R14067BB). HEK293T cells were co-transfected both with Eomes_Luc or pEomes_ p30E_luc plasmids together with plasmids coding for different forms of Received: March 17, 2015 FOXO3 (FOXO3TM, FOXO3TM-H212R, or FOXO3-A32A253-Nter) or with an Revised: August 21, 2016 empty vector using Genejuice (Novagen). Luciferase assays were performed Accepted: September 12, 2016 with a dual luciferase assay kit (Promega, Dual-Luciferase Reporter Assay Published: October 11, 2016 System, E1910) and all luciferase activities were normalized to the expression of the co-transfected Renilla luciferase. REFERENCES Lentiviral Vector Transduction of Naive CD4+ T Cells The gene encoding eomes was synthetized and fully sequenced by Life Tech- Amin, R.H., and Schlissel, M.S. (2008). Foxo1 directly regulates the tran- nologies. The cDNA was then inserted into a pWPXLd-IRES-GFP backbone scription of recombination-activating genes during B cell development. Nat. vector using BamHI and PmeI restriction sites to make the pWPXLD-Eomes- Immunol. 9, 613–622. 6 / + IRES-GPF vector. 5 3 10 naive Foxo3À À CD4 T cells were activated with Bettelli, E., Pagany, M., Weiner, H.L., Linington, C., Sobel, R.A., and Kuchroo, anti-CD3 (3 mg/mL) plus soluble anti-CD28 (2 mg/mL) and IL-2 (10 UI/mL) in V.K. (2003). Myelin oligodendrocyte glycoprotein-specific T cell receptor

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