Transcriptional control of IL-2 sensing by Foxo1 dictates neonatal Treg homeostasis

Transcriptional control of IL-2 sensing by Foxo1 dictates neonatal Treg homeostasis | 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 Transcriptional control of IL-2 sensing by Foxo1 dictates neonatal Treg homeostasis Léa Giraud, Aurélie Durand, Charlotte Guillou, Nelly Bonilla, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7824708/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 Regulatory T (Treg) cells are essential for immune tolerance and include thymic- and peripherally-derived subsets. Among thymic-derived Treg cells, those generated perinatally are phenotypically and functionally distinct from adult cells, with superior capacity to prevent autoimmunity. However, the mechanisms controlling their peripheral seeding remain unclear. Here, we show that the transcription factor Foxo1 is critical for neonatal Treg cell homeostasis rather than for the generation of thymic- or peripherally-derived subsets. Using Foxo1-deficient mice, we demonstrate that Foxo1 deficiency profoundly impairs seeding and expansion of neonatal Tregs in secondary lymphoid organs, disrupting the overall Treg cell compartment. This defect is linked to reduced IL-2 receptor β-chain (IL-2Rβ/CD122) expression, diminished STAT5 activation, and impaired IL-2 responsiveness. Mechanistically, Foxo1 directly binds the Il2rb promoter, fine-tuning its transcription. These findings establish Foxo1 as a central regulator of IL-2-mediated signaling and neonatal Treg cell homeostasis, ensuring proper subset heterogeneity, and long-term immune tolerance. One Sentence Summary : Foxo1 safeguards neonatal Treg cell homeostasis by directly controlling IL-2 receptor β-chain expression, thereby ensuring proper IL-2 responsiveness, peripheral seeding, and expansion of this critical subset. Immunology Neonatal Treg cells Foxo1 homeostasis IL-2 immune tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Regulatory CD4 T (Treg) cells are the primary mediators of peripheral tolerance under physiological conditions. In the periphery, Treg cells consist of two main subsets: thymic Treg (tTreg) cells, which are naturally produced in the thymus through two distinct developmental programs involving Foxp3 - CD25 + and CD25 - Foxp3 lo Treg cell progenitors (TregP) (1, 2) , and peripheral Treg (pTreg) cells, which acquire a similar phenotype and function after activation of naive CD4 T (CD4 T N ) cells in secondary lymphoid organs (SLOs) in the presence of TGFβ (3) . An additional level of complexity has recently emerged from the observation that the age at which tTreg cells are generated contributes to the heterogeneity of this compartment (4) . Indeed, it was reported that tTreg cells generated during the perinatal period stably persists in adult mice and has a crucial role in maintaining self-tolerance. Phenotypically, adult and perinatally-generated tTreg cells are characterized by their high expression of Helios and Nrp-1, whereas pTreg cells lack these markers (5, 6) . Perinatally generated tTreg (hereafter referred to as nnTregs) cells exhibit a more activated phenotype than their adult generated counterparts and express high levels of both Fgl2 and PD-1 proteins (4) . tTreg and pTreg cell subsets are thought to play complementary roles in maintaining immune tolerance (4, 7, 8) . While tTreg cells prevent autoimmunity by maintaining tolerance to self-tissue-derived antigens, pTreg cells are thought to mediate tolerance to non-self-antigens, such as food or fetal antigens and commensal microbes (9, 10) . However, at least in non-autoimmune-prone mice, neither tTreg cell-deficient mice, such as those with a T-cell specific deletion of the Satb1 TF (11) , nor pTreg cell-deficient mice, such as CNS1-deficient mice (9, 10, 12) develop overt multi-organ pathology comparable to the fatal autoimmunity characterizing Foxp3-deficient scurfy mice. This suggests effective compensatory mechanisms between these two Treg cell subsets (8) . Importantly, the signals driving the generation and the homeostasis of these Treg cell subsets can vary. Indeed, while the generation of tTreg cells (at least in adult mice) is dictated by TCR and IL-2 signaling pathways, TGFβ signaling (via Smad activation and binding to the Foxp3 CNS1 enhancer) is crucial for pTreg cell differentiation (12–14) . Once generated, Treg cell homeostasis is tightly maintained by various signals, primarily involving TCR and cytokine signaling (15–19) . While most Treg cells from SLOs rely heavily on interleukin-2 (IL-2) and IL-2R signaling (involving IL-2Rα, IL-2Rβ, IL-2Rγ, and STAT5) for their homeostasis (18, 20, 21) , certain Treg cell subsets depend on IL-7 or IL-33 for their maintenance (19, 22) . Nevertheless, other factors remain to be identified as regulators of Treg cell homeostasis in general, and perinatal tTreg cell homeostasis in particular, whose roles appear to extend well beyond this period. Indeed, apart from Aire (4) , which promotes immunological tolerance by inducing the expression of peripheral-tissue antigens in thymic medullary epithelial cells and has been shown to be crucial in perinatal Treg cell generation, there are no clues concerning the control of the homeostasis of this major Treg cell subset. Transcription factors (TFs) of the forkhead box O (Foxo) family, Foxo1 and Foxo3, have been implicated in multiple key biological processes in T cells such as T-cell trafficking and survival of naïve T cells (23–26) as well as in differentiation into effector T cells (27–30) . However, T-cell activation leads to the relocation of Foxo TFs to the cytosol (31) , inhibiting their transcriptional activity, making their exact roles challenging to determine. More than a bona fide transcription factors, Foxo proteins emerged as pioneer factors for gene activation modulating active chromatin states through their ability to bind condensed chromatin structures and to promote directly or indirectly chromatin opening (32) . T-cell specific Foxo1-deficient mice only develop a mild autoimmune disease, characterized by slight mononuclear cell infiltration in non-lymphoid organs at 1 year of age (24, 33) . Beyond its established role in the function of Treg cells (34) , Foxo1 has been implicated in a contradictory manner in the generation of tTreg and pTreg cells (24, 33) . Indeed, while Foxo1 has been reported as either dispensable (34) or required (33) for tTreg cell generation in the thymus, its major role in TGFβ-dependent differentiation of in vitro -induced Treg (iTreg) and pTreg cells is more widely accepted (33, 34) . In light of the growing complexity of Treg cell heterogeneity, our current understanding of the role of Foxo1 TF in Treg cell biology, particularly in the generation and homeostasis of these ontogenetically distinct Treg cell subsets, appears incomplete. In this study, we revisit its role in vivo and show that Foxo1 TF plays a key role in the establishment of the perinatal Treg cell compartment, particularly in the intense proliferation of these cells in neonatal SLOs, by modulating their sensitivity to IL-2. RESULTS CNS1-dependent pTreg cells prevent autoimmunity in Foxo1 TKO mice To address the role of CNS1-dependent pTreg cells in the maintenance of immunological tolerance in these Foxo1-deficient mice, we generated double-deficient mice (CNS1 KO Foxo1 f/f CD4-Cre + Foxp3 GFP - CNS1 KO Foxo1 TKO ), by crossing Foxo1 TKO mice with CNS1 KO (CNS1 KO Foxp3 GFP - CNS1 KO ) mice. WT control littermates (Foxo1 f/f CD4-Cre - Foxp3 GFP - Foxo1 Ctrl ), Foxo1 TKO , CNS1 KO , and CNS1 KO Foxo1 TKO mice were followed until 12 weeks of age. Surprisingly, while Foxo1 TKO and CNS1 KO mice, as expected, only develop mild manifestations of autoimmunity affecting the intestine (both colon and small intestine) and/or the lung in a few individual animals, double-deficient mice lose weight and exhibit a more severe disease characterized by pronounced cell infiltrates and tissue disorganization in most animals in the same tissues (Fig. 1A-B, and fig. S1A). Semi-supervised analysis of mesenteric Lymph node (mLN) CD4 T cells from 12-week-old WT control littermates, Foxo1 TKO , CNS1 KO , and CNS1 KO Foxo1 TKO mice by flow cytometry identified clusters of naïve (T N ), effector (T Eff ) and Treg cells, characterized by their coexpression of several surface molecules (CD44, CD25 and Nrp1) and key transcription factors (Tbet, Gata3, Rorγt and Foxp3) (Fig. 1C). Consistent with the autoimmune disease affecting the intestine of CNS1 KO Foxo1 TKO mice, separation by genotype revealed significant changes in cluster distribution, with a progressive enrichment of CD4 T Eff cells from WT to double-deficient mice. While CNS1 KO mice maintain a near-normal CD4 T cell compartment, combining CNS1 deletion with the Foxo1 TKO background further exacerbates the expected loss of T N cells to the benefit of Treg and CD4 T Eff cells observed in CNS1 KO Foxo1 TKO mice (Fig. 1D). Notably, cluster distribution among Foxp3 - conventional CD4 T (Tconv) cells also shifts between Foxo1 TKO and CNS1 KO Foxo1 TKO mice (Fig. 1D). Specifically, the percentages of CD4 T Eff cells among Tconv cells and absolute numbers of CD4 T Eff cells significantly increase in CNS1 KO Foxo1 TKO compared to Foxo1 TKO mice (Fig. 1E, and fig. S1B-E). Finally, analysis of absolute numbers and cluster distribution within CD4 T Eff cells reveals an enrichment and accumulation of Tbet- and RORγt-expressing cells (c1_ and c2_CD4_T Eff clusters respectively) in CNS1 KO Foxo1 TKO compared to Foxo1 TKO mice (Fig. 1F). Taken together, these data suggest that a CNS1-dependent subset of Treg cells mitigates both the clinical manifestations and the dysregulation of conventional T cells in Foxo1 TKO mice. To further validate the contribution of CNS1-dependent Treg cells to the peripheral Treg pool in Foxo1 TKO mice, we generated heterozygous Foxp3 CNS1KO/WT females carrying one CNS1 KO allele and one CNS1 WT allele both in Foxo1 Ctrl and Foxo1 TKO backgrounds. Owing to random X-chromosome inactivation, half of the CD4 T N cells in these mice express either allele, thereby retaining or lacking the ability to differentiate into CNS1-dependent pTreg cells (Fig. 1G, and fig. S1F, H). In both genetic backgrounds, GFP expression was linked either with the CNS1 KO allele (as test group) or with the CNS1 WT allele (as control group). GFP expression in Treg cells revealed the contribution of CNS1-dependent pTreg cells to the Treg pool in SLOs (Fig. 1H). In 4-weeks old control background mice, their contribution remained minimal. However, in Foxo1 -deficient mice, CNS1-dependent pTreg cells contributed significantly more to the overall Treg cell compartment (Fig. 1I, and fig. S1F, G). This indicates that CNS-dependent pTreg cells may play a compensatory role for the deficit in Treg cells when Foxo1 is lost. Notably, CNS1-dependent pTreg cells contributed only marginally to the Treg cell compartment in both control and Foxo1 -deficient perinates (1-week old) (fig. S1H, I), consistent with the proposed role of CNS1 in promoting pTreg cell induction at weaning (35) . pTreg cells are normally generated in Foxo1 mice Because CNS1-dependent pTreg cells are critical in immune tolerance in Foxo1 TKO mice, we further investigate the role of Foxo1 in both iTreg and pTreg cell generation. We first analyzed Treg subsets in the SLOs of 4-week-old Foxo1 Ctrl and Foxo1 TKO mice. Using semi-supervised flow cytometry analysis of peripheral lymph node (pLN) CD4 + T cells from 4-week-old Foxo1 TKO mice and WT control littermates, we identified 4 clusters of Treg cells based on the coexpression of molecules known to distinguish activated and resting Treg cell subsets (Ly-6C, CD103, CD69, CD25, and Ki67) and to discriminate tTreg from pTreg cells (Nrp1 and Helios) (Fig. 2A): Three clusters (c1, c2, c3) of tTreg-like cells expressing Nrp1 with varying levels of CD25, Ly-6C, CD69, and Helios, and a fourth cluster (c4) of pTreg-like cells lacking both Nrp1 and Helios expression (Fig. 2A and fig. S2A). Genotype-based separation revealed global changes in the distribution of Tconv and Treg cells in Foxo1 TKO mice compared to Foxo1 Ctrl mice, with a significant reduction in both the frequency of Treg cells among CD4 + T cells and their absolute numbers in pLNs (Fig. 2B, C), but not in the spleen (fig. S2B, C). Interestingly, the distribution of Treg cell clusters was differentially altered in Foxo1 TKO mice, in both organs (Fig. 2B and fig. S2D, E). Indeed, we observed an almost complete loss of c2 Treg cells, together with a significant increase in proportions in the c4 cluster in both organs and in the c1 cluster specifically in pLNs of Foxo1 TKO mice (fig. S2D, F). When analyzing absolute cell numbers, we observed a marked reduction in the three Nrp1 + tTreg-like clusters (c1, c2, and c3) in Foxo1 TKO mice compared to Foxo1 Ctrl mice, while numbers of Nrp1 - pTreg-like (c4) cells were unaffected and even slightly increased (Fig. 2D and fig. S2E). This near-complete loss of Nrp1 + tTreg-like cells in SLOs from Foxo1 TKO mice (Fig. 2E) is cell-intrinsic, as demonstrated by mixed bone marrow chimeras in which lethally irradiated mice were reconstituted with a mixture of Foxo1 Ctrl and Foxo1 TKO bone marrow cells (Fig. 2F). These observations could reflect a role for Foxo1 in the development or maintenance of tTreg-like cells, while pTreg cell generation might not depend on Foxo1. Foxo1-deficient CD4 T N cells have been described as defective in converting into iTreg cells (24, 33) . It is long known that IL-4 but also IFN-γ inhibit Foxp3 induction in iTreg polarization assays (36, 37) and we recently demonstrated that Foxo1-deficient stimulated CD4 T N cells heavily produced these 2 interleukins, even in the absence of T H 1 or T H 2 polarizing interleukins (38) . We therefore reassessed the impact of Foxo1 deletion on the iTreg cell differentiation potential in the presence or absence of IL-4-blocking and/or IFN-γ-blocking antibodies (Fig. 2G). Consistent with previous findings (33) , we confirmed that, in the absence of IL-4- and IFN-γ-blocking antibodies, CD4 T N cells from Foxo1 TKO mice hardly differentiate into iTreg cells (Fig. 2G). However, blocking both IFN-γ and IL-4 effectively restored their ability to convert into iTreg cells (Fig. 2G). This lack of a substantial defect in the ability of Foxo1-deficient CD4 T N cells to differentiate into iTreg cells in vitro was further validated in vivo in adoptive transfer experiments (Fig. 2H). CD4 T N thymocytes from 1-month-old Foxo1 Ctrl or Foxo1 TKO CD45.1 mice were transferred into CD45.2 recipient mice of the same age and genotype, and their fate was analyzed 2 months later. In this setting, both WT and Foxo1-deficient CD4 T N cells efficiently converted into pTreg cells with Foxo1-deficient CD4 T N cells differentiating even more in Foxp3-expressing than their WT counterparts most likely due to differences in homeostatic signals received in the respective hosts (Fig. 2H). Altogether, both our phenotypic data and our in vitro and in vivo conversion assays strongly support a marginal role of Foxo1 in the differentiation of CD4 T N cells into pTreg cells. Foxo1 deletion affects tTreg cell recirculation but not their generation in the thymus We then evaluated the role of Foxo1 in tTreg cell generation. Conventional CD4 T cells (Tconv), CD25 + TregP, and Foxp3 lo TregP (1, 2) were analyzed, together with CD25 + tTreg cells, which were further subdivided based on CD73 surface expression (39) into neo-tTreg (CD73 - ) and recirculating-tTreg (CD73 + ) cells. While the percentage of Foxp3 lo TregP was slightly increased in 1-week-old but not in 1-month-old Foxo1 TKO compared to age-matched Foxo1 Ctrl mice (fig. S3A), the absolute numbers of Tconv, CD25 + and Foxp3 lo TregPs as well as neo-tTreg cells were comparable in the thymus of both 1-week-old (Fig. 3A) and 1-month-old Foxo1 Ctrl or Foxo1 TKO mice (fig. S3B). Strikingly, explaining the quantitative tTreg cell defect initially published (33) , proportions and absolute numbers of recirculating-tTreg cells were greatly reduced in Foxo1 TKO compared to Foxo1 Ctrl mice (Fig. 3B and fig. S3B). To confirm these observations, we generated mixed bone marrow (BM) chimeras by co-transferring Foxo1 Ctrl (CD45.1 + ) BM cells together with BM cells (CD45.2 + ) isolated from Foxo1 TKO mice into lethally irradiated T-cell deficient CD3ε recipient mice. In this competitive setting, both the normal generation of neo-tTreg cells from Foxo1 KO precursor cells (Fig. 3C) and the defective recirculation of Foxo1 KO Treg cells within the thymus (Fig. 3D) were reproduced. These results indicate that Foxo1 is critical for the recirculation of Treg cells to the thymus, while being largely dispensable for their initial thymic development. As Foxo1 appeared to have only a limited impact on neo-tTreg cell generation, we next investigated whether the apparent loss of Nrp1 + tTreg-like cells in SLOs from Foxo1 TKO mice could result from defective cell maturation and/or thymic export. Semi-mature (SM), Mature 1 (M1) and Mature 2 (M2) maturation stages of CD4 single positive (SP) thymocytes were defined based on MHC class I (MHCI), CD24 and CD69 expression (Fig. 3E), as previously described (40) . Consistent with their position along tTreg cell differentiation trajectory, the proportion of SM cells progressively decreased from CD4 Tconv to CD25 + TregP cells and Foxp3 lo TregP cells, and was virtually absent among neo-tTreg cells. Overall, the analysis of neo-tTreg cells and their precursors did not reveal any major maturation defects in Foxo1 TKO mice (Fig. 3E), suggesting either that Foxo1 is not essential for tTreg cell production or that residual Foxo1 protein expression after gene deletion at the CD4 + CD8 + double-positive stage is sufficient to sustain this process. Thymic export of CD4 Tconv and Treg cells was assessed in SLOs 16 h after intra-thymic injection of fluorescein. Consistent with their altered expression of the homing molecules CD62L and CCR7 (23, 41) , the numbers of FITC + CD4 Tconv and Treg cells were significantly lower in the LNs of Foxo1 TKO mice compared to Foxo1 Ctrl mice (Fig. 3F). However, this analysis did not reveal any major defect of the ability of Foxo1 TKO CD4 Tconv and Treg cells to reach the spleen, nor did it reveal a greater defect in the colonization of LNs by Foxo1 KO Treg cells compared to Foxo1 KO Tconv cells. Both populations exhibited an almost 7-fold reduction in LN colonization compared to their Foxo1 WT counterparts (Fig. 3F). When pooled LNs and spleen FITC + CD4 Tconv or Treg cells were analyzed, this reduction amounted to 2.4-fold and 2.7-fold, respectively (fig. S3C). Thus, the lack of Nrp1 + tTreg-like cells in SLOs from young Foxo1 TKO mice (Fig. 2A-E) cannot be attributed to a role of Foxo1 in the early development, maturation, or thymic export of tTreg cells. Foxo1 controls perinatally generated tTreg cell seedings into SLOs Because tTreg cells effectively differentiate, mature and reach the periphery (Fig. 3) but fail to accumulate in SLOs of young Foxo1 TKO mice (Fig. 2A-E), we hypothesized that Foxo1 might play a critical role in their seeding in SLOs during the perinatal period. A semi-supervised flow cytometry analysis of spleen CD4 + T cells identified distinct clusters of CD4 Tconv and Treg cells in 1-week-old Foxo1 Ctrl and Foxo1 TKO perinates (Fig. 4A). Among these, two clusters were associated with Treg cells, the c1 cluster corresponding to resting-like Treg cells expressing low-levels of CD25, Nrp1, CD44, and Helios compared to the c2 cluster expressing high-levels of these proteins (Fig. 4A). Genotype-based separation revealed significant changes in the distribution of Tconv and Treg cells in Foxo1 TKO mice compared to Foxo1 Ctrl mice. Specifically, Foxo1 TKO mice showed a marked reduction in the percentage of Treg cells among CD4 T cells as well as in their absolute numbers (Fig. 4B, C). Furthermore, the distribution of Treg cell clusters was drastically altered in Foxo1 TKO mice, with a 2-fold reduction in the proportion of c2 Treg cells among Treg cells compared to their Foxo1 Ctrl counterparts (Fig. 4D). Notably, while the absolute numbers of c1 resting-like Treg cells remained unchanged in the spleens of Foxo1 TKO perinates, the absolute numbers of c2 Treg cells—which represent 80% of Treg cells in the spleen of Foxo1 Ctrl perinates—were reduced by tenfold in Foxo1 TKO perinates (Fig. 4D). Further analysis of the c2 Treg cell cluster revealed that, consistent with its activated phenotype, it corresponds to a proliferation-prone neonatal Treg cell subset, as evidenced by the high proportion of Ki67 + cells within this cluster (Fig. 4E). Notably, the few cells belonging to this cluster in Foxo1 TKO perinates displayed significantly lower levels of Ki67 + cells compared to their WT counterparts (Fig. 4F). This proliferative defect in Treg cells from Foxo1 TKO perinates was further substantiated by cell cycle analysis combining Ki67 staining with DNA content measurement (Fig. 4G, fig. S4A). Of note, in young adult mice, Foxo1-deficient Treg cells do not display any proliferation defect (fig. S4B). Altogether, these findings underscore a pivotal role for Foxo1 in sustaining Treg cell proliferation specifically within perinatal SLOs. Foxo1 Sets IL-2 Sensitivity by Controlling IL-2 Receptor Beta Chain Expression in Developing tTreg Cells Because Foxo1 is critical for the expansion of perinatal tTreg cells in SLOs when reaching the periphery, we investigated whether Foxo1 regulates specific transcriptional programs during tTreg differentiation that could account for their defective homeostasis. To this end, we reanalyzed previously published chromatin accessibility data (11) obtained by ATAC-seq from thymic immature CD4SP, CD25 + TregP, Foxp3 lo TregP and tTreg cells to determine the kinetics of chromatin opening during tTreg cell differentiation. Additionally, we leveraged Foxo1 ChIPseq data (42) to infer Foxo1 DNA-binding motif and perform Foxo1 footprint analysis throughout this process. Finally, we integrated these findings with RNA-seq data comparing immature CD4SP and tTreg cells (11) (Fig. 5A). Chromatin accessibility profiling identified six distinct dynamic clusters corresponding to regions that either progressively, transiently, or rapidly closed (closing clusters, c.c1 and c.c2; 43.2%) or opened (56.8%, opening clusters, o.c1-4) upon ImmCD4SP differentiation into tTreg cells (Fig. 5B). To assess the contribution of Foxo1 to these chromatin dynamics, we identified a Foxo1 consensus motif (fig. S5A) from recently published Foxo1 ChIP-seq data (42) and used it to infer global and Foxo1-associated footprints throughout tTreg cell differentiation (fig. S5B). Global footprint analysis revealed that both the consensus and ChIP-seq-predicted Foxo1 motifs clustered with transcription factors that bind chromatin sequentially along the tTreg differentiation trajectory, with maximal occupancy in immCD4SP cells and later in Foxp3 lo TregP (fig. S5B). As expected, histograms of mean normalized Tn5 insertion frequencies around Foxo1 motifs classified as “bound” showed a depletion of insertions at the motif center, with a gradual increase toward the flanking regions (fig. S5C). Consistent with its reported pioneer activity and its preferential nuclear localization, the average footprint profile around this motif exhibited only modest variation across populations (fig. S5C-D, left). In contrast, population-specific analyses uncovered discrete chromatin regions that either lost or gained Foxo1 footprints at defined stages of tTreg differentiation (fig. S5C-D, right). Although closing and opening chromatin regions were globally balanced during tTreg cell differentiation, Foxo1 footprint–associated regions were significantly enriched among opening clusters, particularly o.c1 and o.c3 (Fig. 5C). Notably, the opening of these regions followed the progressive increase in Foxo1 footprint scores (Fig. 5D). Conversely, few Foxo1 footprint–associated regions that closed during differentiation exhibited a concomitant decrease in Foxo1 footprint scores, suggesting that Foxo1 predominantly acts as a chromatin-opening factor during tTreg cell differentiation (Fig. 5D). Consistently, genes linked to Foxo1 footprints within opening chromatin regions were upregulated in tTreg cells, whereas those associated with closing regions remained largely unchanged (Fig. 5E). To further substantiate Foxo1 activity during thymic development of tTreg cells, we integrated and reanalyzed two scRNA-seq datasets generated from either sorted thymic Treg precursors and progenitors, as well as mature and recirculating tTreg cells (43) or purified Treg cells (44) (fig. S6A). This analysis, enabled us to perform Gene Regulatory Network inference using SCENIC (45) thereby identifying transcription factor activities across the tTreg cell differentiation trajectory (fig. S6B). Consistently, regulons governed by STAT5a peaked at the CD25 + TregP stage, whereas those controlled by both Foxo1 and Foxp3 gradually increased from the transitional Treg stage to mature neo-Treg cells (fig. S6C). Strikingly, Gene Set Enrichment Analysis (GSEA) of these dynamic chromatin regions, Foxo1-bound sites, and differentially expressed genes revealed a strong enrichment for IL-2/STAT5 signaling–related genes (Fig. 5F), the majority of which displayed concomitant chromatin opening and transcriptional upregulation during the transition from immCD4SP to tTreg cells (Fig. 5G). Notably, among these tTreg cell signature genes - defined by the presence of Foxo1 ChIP-seq peaks together with dynamic Foxo1 footprints associated with increasing chromatin accessibility throughout tTreg cell differentiation - included genes encoding IL-10, ST2, 4-1BB, TNFR2, CTLA-4, and HOPX. Importantly, the gene encoding the IL-2 receptor beta chain (Il2rb), a key regulator of tTreg cell generation, was also part of this set of Foxo1-regulated genes (Fig. 5G). Consistent with this observation, time-course analysis of ATAC-seq, Foxo1 ChIP-seq and footprinting data at the Il2rb locus demonstrated a progressive increase in chromatin accessibility and Foxo1 footprint depth at a Foxo1-bound site within the Il2rb promoter during tTreg cell differentiation (Fig. 5H). Importantly, these findings were corroborated in vivo at the protein level. IL2Rβ expression on the surface of tTreg cells and their immediate Foxp3 lo Treg precursors was significantly reduced in Foxo1 TKO mice (Fig. 5I). To further support the role of Foxo1 as a transcription factor regulating IL2Rβ expression during tTreg cell differentiation, we generated an in vitro two-step tTreg cell generation assay using WT thymic CD4SP T cells (46) , and compared IL2Rβ expression in the presence or absence of a Foxo1 inhibitor (Fig. 5J). Strikingly, Foxp3 + cells generated in the presence of the inhibitor exhibited markedly reduced surface IL-2Rβ expression (Fig. 5J). Lastly, consistent with the reduced IL-2Rb expression, we found, in CD25-expressing TregP and neo-tTreg cells analyzed ex vivo , that STAT5 phosphorylation/activation is impaired when Foxo1 is lost, indicating a weakened IL-2R pathway (Fig. 5K). Notably, this reduced IL-2Rβ expression in Foxo1-deficient Treg cells persisted in the SLOs of perinates (fig. S6D). Altogether, Foxo1 appears to regulate IL-2 receptor β chain expression on developing tTreg cells (Fig. 5) and Foxo1 TKO perinates exhibit a drastic reduction in nnTreg cell numbers, particularly within the c2 cluster, which is characterized by high expression of both the IL-2 receptor α chain and Nrp1 (Fig. 4). IL-2 signaling shapes nnTreg cell compartment in the periphery To determine the role of IL-2 in Treg cell seeding into SLOs in Foxo1 Ctrl and Foxo1 TKO perinates, we performed a semi-supervised flow cytometry analysis of spleen CD4 + T cells from pooled Foxo1 Ctrl (treated or not with IL-2 or anti-IL-2 blocking antibodies) and Foxo1 TKO perinates (treated or not with IL-2). We identified the same CD4 Tconv and Treg cell clusters as previously described (Fig. 4A) in 1-week-old perinates (Fig. 6A). While IL-2 treatment significantly increased the proportion of nnTreg cells among CD4⁺ T cells in both Foxo1 Ctrl and Foxo1 TKO mice, absolute numbers were not significantly affected (Fig. 6B and fig. S7A, B). Conversely, IL-2 blockade in Foxo1 Ctrl perinates markedly reduced both the frequency and absolute number of nnTreg cells (Fig. 6B, C and fig. S7A). Crucially, and in line with the nnTreg cell composition of Foxo1 TKO perinates, IL-2 modulation also altered the distribution between the c1 and c2 nnTreg cell subsets. Indeed, blocking IL-2 in Foxo1 Ctrl perinates drastically reduced both percentages of c2 Treg cells among nnTreg cells and absolute numbers of this subset (Fig. 6C). Conversely, increasing IL-2 availability in Foxo1 TKO perinates significantly increased both percentages of c2 Treg cells among nnTreg cells and their absolute number (Fig. 6D). Consistent with their low expression of the IL-2R α chain of the IL-2 receptor, numbers of c1 nnTreg cells were largely insensitive to IL-2 modulation (Fig. 6C, D). Together, these data indicate that IL-2 availability selectively controls the establishment of the nnTreg cell compartment in the periphery by sustaining the expansion of the IL-2–responsive c2 subset. This IL-2–dependent bias recapitulates the nnTreg cell imbalance observed in Foxo1-deficient perinates, thereby positioning IL-2 signaling downstream of Foxo1 as a critical determinant of early Treg cell seeding into SLOs. DISCUSSION Our study highlights the pivotal role of Foxo1 in maintaining Treg cell homeostasis. In contrast to previous reports suggesting that Foxo1 broadly orchestrates the differentiation of both thymic-derived (tTreg) and peripheral (pTreg) cell subsets (33, 34) , our findings support a more refined model. Rather than functioning as a universal driver of tTreg or pTreg cell differentiation, Foxo1 appears to be specifically required for sustaining tTreg homeostasis, acting primarily by enhancing IL-2 responsiveness during the perinatal period and supporting the expansion of newly generated neonatal Tregs (nnTregs). Our findings reconcile discrepancies from previous studies on the role of Foxo1 in Treg cell homeostasis and further reveal previously unrecognized functions of this transcription factor. In tTreg cells, it was previously reported that Foxo1 TKO mice exhibit a significant reduction in tTreg cell numbers at 3–8 weeks of age compared with Foxo1 Ctrl mice (33) . At that time, however, the capacity of Treg cells to recirculate to the thymus and modulate tTreg cell production was not fully appreciated (39) . Our results demonstrate that Foxo1 intrinsically regulates this recirculation process, providing a potential explanation for the apparent reduction in tTreg cells observed in Foxo1 TKO mice. By 4 weeks of age, recirculating Treg cells account for approximately one quarter of total thymic Treg cells in Foxo1 Ctrl mice, whereas they are virtually absent in Foxo1 TKO mice (fig. S3B). The molecular mechanisms underlying Treg cell recirculation to the thymus remain incompletely understood. While integrin α4–VCAM-1 interactions (47) and chemokine receptors such as CXCR4 and CCR6 (48, 49) have been proposed to be involved in this process, CCR7 has instead been suggested to act as a negative regulator (50) . In light of the profound impairment of thymic re-entry observed in Foxo1 TKO mice, and given the established role of Foxo1 in regulating CCR7, CD62L, and integrin α4 expression (all of which are downregulated in its absence (24) ), our findings support a key contribution of integrin α4 and suggest CD62L as an additional mediator of Treg cell recirculation, whereas a dominant role for CCR7 appears unlikely. A major caveat in interpreting the phenotype of Foxo1 TKO mice lies in the early-onset inflammation that arises in these animals (23, 24) . This inflammation, characterized by elevated inflammatory cytokine levels including IL-2, may secondarily alter both peripheral Treg cell homeostasis and their thymic development. Indeed, systemic inflammation is known to profoundly affect Treg cell proliferation, phenotype, and stability in secondary lymphoid organs, and can similarly influence tTreg cell development in the thymus (1, 2, 51–53) . A key strength of our study is that, in addition to analyses in young adult (4-week-old) and adult (12-week-old) mice, we also investigated the perinatal period by analyzing 1-week-old mice. At this early stage, recirculating tTreg cells, which accumulate rapidly after 3–4 weeks in the thymus of Foxo1 Ctrl mice, remain marginal, and Foxo1 TKO mice show little to no evidence of inflammation. Therefore, the differences observed in Treg cell numbers and phenotype at this early time point are most likely to reflect direct, cell-intrinsic consequences of Foxo1 deficiency rather than indirect effects of systemic inflammation. Although we find that Foxo1 appears dispensable for the development of both tTreg and pTreg cells (Fig. 2H and Fig. 3A-E), its role in modulating the IL-2 sensitivity of nnTreg cells and supporting their initial expansion may profoundly impact both the homeostasis of the overall Treg cell compartment and the microenvironment governing their generation and maintenance. CNS1-dependent pTreg cells have been shown to limit the late onset of allergic and asthma-like inflammation in mucosal tissues (10) and to contribute to maternal-fetal tolerance during pregnancy (9) . Our study broadens the functional scope of this extrathymically induced Treg cell subset by demonstrating its capacity to mitigate autoimmunity in genetically predisposed contexts. Specifically, we show that Foxo1 TKO CNS1 KO mice develop a markedly more severe disease than Foxo1 TKO mice, characterized by a significant weight loss, an exacerbated infiltration of mucosal tissues such as the gut and lungs, and a widespread activation of conventional T cells (Fig. 1A-G and S1A-F). Furthermore, while the loss of CNS1-dependent pTreg cells has minimal impact on Treg cell numbers and only marginally alters the overall Treg cell composition in a WT background (as observed in heterozygous Foxp3 CNS1KO/WT females, likely due to compensation by thymic-derived Treg cells), this subset appears to occupy a substantial fraction of the Treg cell niche in the secondary lymphoid organs of Foxo1 TKO mice (Fig. 1G-I, and fig. S1F, G). Such a role for pTreg cells in maintaining immune tolerance in Foxo1 TKO mice was unexpected, as Foxo1-deficient CD4 T N cells were initially reported to be severely impaired in their ability to differentiate into iTreg cells in response to TGF-β (33) . However, in those early studies, the cells used in the in vitro polarization assays were not bona fide CD4 T N (CD44 lo CD25 - Foxp3-GFP - ) cells, but rather a broader population of conventional CD4 T cells (CD4 + CD25 - CD69 - ), which likely included memory-like CD4 T cells. Moreover, neither IL-4 nor IFN-γ were neutralized during the assays. Our recent data (38) demonstrate that upon stimulation, Foxo1-deficient CD4 T N cells produce high levels of both cytokines, which may strongly interfere with iTreg cell polarization as previously described (36, 54) . In particular, IFN-γ not only inhibits Foxp3 induction during iTreg cell polarization, but also actively promotes iTreg cell instability, thereby preventing the establishment and maintenance of a stable regulatory phenotype (55) . In neonates, the first mature thymocytes that migrate to the periphery encounter a lymphoid environment devoid of preexisting T cells and therefore undergo vigorous proliferation in response to TCR engagement by self-peptide/self-MHC ligands (56–58) . In addition, the proportion of Treg cells in neonates is lower than in adult mice (59–61) , which may limit their capacity to effectively suppress effector cells. Furthermore, the peripheral T cell compartment in neonates is dominated by recent thymic emigrants (RTEs) (62) , which exhibit phenotypic and functional properties distinct from their adult counterparts (63, 64) , including an enhanced ability to produce IL-2 (64) . Consistent with this, neonatal secondary lymphoid organs show higher levels of IL-2 production by CD4 and CD8 T cells compared to those of adults (65) . This unique immunological context is therefore likely to play a critical role in shaping the nnTreg cell compartment. Our results strongly support a model in which Foxo1 regulates the initial seeding and expansion of perinatally generated tTreg cells into SLOs, primarily by modulating IL-2Rβ expression on developing tTreg cells (Fig. 5). Reduced surface expression of IL-2Rβ in Foxo1-deficient neo-tTreg cells is associated with diminished STAT5 phosphorylation compared to their wild-type counterparts (Fig. 5I, J and K). Consequently, impaired IL-2 sensing limits the seeding, activation, and proliferation of nnTreg cells within perinatal SLOs (Fig. 4E, G and fig. S4). Mechanistically, the role of Foxo1 in promoting IL-2Rβ expression may rely not only on its direct activity as a transcription factor but also on its potential pioneer function in opening chromatin to facilitate the binding of additional regulators (32) . Indeed, Il2rb transcription has been shown to depend on multiple transcription factors, including Ets1, Gabp, Sp1, Egr1, and Stat5 (66–68) . Thus, Foxo1 binding to the Il2rb promoter region (Fig. 5H) could either directly initiate Il2rb transcription during the transition from CD25 + TregP to Foxp3 lo TregP cells, or alternatively render the locus accessible to other transcription factors. The latter possibility is particularly appealing, given that Stat5 phosphorylation, indicative of its activation, is already detected at the CD25⁺ TregP stage, making it a strong candidate for cooperating with Foxo1 (Fig. 5K). A link between Foxo1 and IL-2Rβ expression has previously been reported in CD4 T cells (69) . However, in that study, Newton et al. did not examine the consequences of Foxo1 deficiency but studied the effects of constitutive Foxo1 activation through expression of a non-phosphorylatable Foxo1A3 mutant. Interestingly, despite these opposite manipulations of Foxo1 activity, both settings led to reduced surface IL-2Rβ expression. In the case of Foxo1A3, this reduction occurred independently of Il2rb transcription and was attributed instead to impaired receptor recycling via the endocytic–lysosomal pathway (69) . Together, these findings suggest that Foxo1 fine-tunes IL-2Rβ signaling at multiple levels, integrating both transcriptional and post-transcriptional mechanisms. Such multilayered regulation of IL-2 sensitivity may be particularly critical during the perinatal window, when tTreg cells must efficiently sense IL-2 to undergo expansion and establish a stable pool in SLOs (Fig. 6 and Fig. S7). By modulating IL-2Rβ expression dynamics, Foxo1 ensures that developing tTreg cells reach the activation threshold required for their survival and proliferation, while preventing aberrant activation or loss of lineage stability. This mechanism may thus represent a key safeguard for the establishment of robust Treg-mediated tolerance early in life. Beyond the superior suppressive capacity of nnTreg cells, their repertoire enriched in self-reactive specificities compared to Treg cells generated later in life (4) , and their critical role in preventing autoimmunity during adulthood (4, 70) , a defining characteristic of nnTreg cells is their remarkable ability to colonize peripheral tissues (71) . A major strength of our study lies in the use of Foxo1 TKO mice, which allow a comprehensive assessment of Foxo1's role not only in the generation of distinct Treg cell subsets but also in their early seeding and homeostasis within SLOs during the perinatal window. However, elucidating the full pathophysiological impact of Foxo1 deficiency is complicated by the pleiotropic roles of this transcription factor in both Treg and conventional T cell biology. Foxo1 orchestrates T cell trafficking into lymphoid and non-lymphoid tissues (23) , prevents conventional T cell exhaustion (41) , and promotes several core suppressive mechanisms in Treg cells (33, 34) . Our findings reveal that disruption of Foxo1 activity profoundly impairs Treg cell seeding and expansion during the perinatal window, with potential long-term consequences for immune homeostasis. Future studies should investigate how early-life perturbations in Foxo1 signaling or Treg cell seeding influence the establishment of immune tolerance and tissue homeostasis later in life. MATERIALS AND METHODS Mice One to 12-weeks-old mice were used for experiments unless otherwise indicated. C57BL/6 CD3ε KO (72) , C57BL/6 Foxp3 -GFP Foxo1 Ctrl and Foxo1 TKO CD45.1 or CD45.2 (23, 27) . C57BL/6 Foxp3-GFP CNS1 KO mice were generated using the CRISPR/Cas9 system in collaboration with the MOUSTIC genome engineering platform (Cochin Institute, Paris, France). Three pairs of gRNAs targeting flanking regions of the CNS1 enhancer within the Foxp3 locus were co-injected into C57BL/6 Foxp3-GFP zygotes: • gRNA pair 1: 5′-GAAGACATACACCACCACGG-3′ and 3′-AATTTTGCATAGAGAGATCA-5′ • gRNA pair 2: 5′-TAGATTACTCTTTTCTTGTG-3′ and 3′-CTACCATCCACGAGTCGTGT-5′ • gRNA pair 3: 5′-CGGCGGGCAATCACTTGCTT-3′ and 3′-TACTGTCGCTGTAAAGTTCA-5′ Founder animals were genotyped and validated by PCR and Sanger sequencing using the following primers: 5′-GGGGAAAATAAAGTGACTGG-3′ and 5′-ACAAGGTCTCACTCTATAG-3′. Founders carrying a confirmed 506 bp deletion within the CNS1 region were selected and backcrossed to C57BL/6 Foxp3-GFP mice for at least three generations to reduce potential off-target effects. Finally, C57BL/6 Foxp3-GFP Foxo1 TKO CNS1 KO were generated by crossing our C57BL/6 Foxp3-GFP CNS1 KO mice with C57BL/6 Foxp3-GFP Foxo1 TKO mice. All mice were maintained in our own animal facilities, under specific pathogen–free (SPF) condition. All procedures performed were approved by the ethics committee for animal experimentation (n°APAFIS #20630, #22356) and validated by the “Ministère de l’Enseignement Supérieur de la Recherche et de l'Innovation”. Sample sizes were chosen to assure reproducibility of the experiments and in accordance with the 3R rules of animal ethics regulation. Cell suspensions Adult mice were euthanized by cervical dislocation, neonates by decapitation. Peripheral lymph nodes (pLNs: pooled cervical, axillary, brachial, inguinal), mesenteric LNs (mLNs), spleen, and thymus were harvested, homogenized, and passed through a nylon cell strainer (BD Falcon). Cells were resuspended in RPMI 1640 GlutaMAX with 10% FCS (Biochrom) for adoptive transfer or culture, or in PBS with 5% FCS and 0.1% NaN3 (Interchim) for flow cytometry. Flow cytometry Cell surface and intracellular staining Cell suspensions were collected as described in “cell suspension” item and dispensed into 96-well round-bottom microtiter plates (Greiner Bioscience; 6 × 106 cells/well). Surface staining was performed as described (16) . Antibodies (Abs) are listed in Supplementary Table S1. Briefly, cells were incubated on ice, for 15 minutes per step, with Abs in 5% FCS (Eurobio Scientific), 0.1% NaN3 (Sigma-Aldrich) in PBS. Each cell staining reaction was preceded by Fc receptor blocking (anti-CD16/32, 2.4G2, BioXcell) and viability staining (LeaDead, Life Technologies). The Foxp3 Staining Buffer Set (eBioscience) was used for Foxp3, Ki67, Hoechst, Helios, Tbet, Gata3, and Rorγt intracellular staining. To assess pSTAT5 levels ex vivo, cells were immediately fixed in 4% PFA for 5 min at 37°C. Cells were then washed and permeabilized by adding ice-cold 100% methanol to a final concentration of 90% methanol and incubated for at least 30 min at -20°C. After extensive washing, the cells were stained overnight at 4°C with surface and intracellular antibodies. For Cell suspensions from heterozygote mice (GFP+/GFP-), surface staining was performed as previously described. Cells were then fixed with 2% PFA for 10 min at room temperature, washed and stained for Foxp3 expression using Foxp3 Staining Buffer Set (eBioscience). Analysis Data were acquired on a BD LSRFortessa™ cytometer (BD Biosciences) at the Cochin CYBIO facility. List-mode data files were analyzed using FlowJo software V10_10 (BD Biosciences). For unsupervised flow cytometry analysis “FCS files” were imported into R (v4.4.1) and processed using relevant packages including “flowCore, flowWorkspace, FlowSOM, Harmony, umap2,” and “ggplot2”. Briefly, “FCS files” were imported, logicle-transformed, and harmonized to ensure comparability across samples. Metadata such as “genotype”, “batch”, and “tube ID” were integrated into the expression matrix to enable accurate annotation and stratification of cellular subsets. To control for sampling bias, cells were randomly downsampled in a balanced manner across genotypes and conditions. Batch effects were corrected using the “Harmony” algorithm. Dimensionality reduction was performed using supervised UMAP (sUMAP) approaches. Clustering of cells was achieved using the “FlowSOM“ algorithm on the batch-corrected and transformed data. Identified clusters were grouped into meta-clusters based on phenotypic marker expression and manually annotated. Relative frequencies of each cell subset were computed per sample and represented as pie charts stratified by genotype and batch. Heatmaps were generated to visualize average marker expression across meta-clusters, providing a phenotypic signature of each T cell population. Final processed datasets and clustering results were exported in CSV format and subsequently used for graphical representation and statistical analysis using GraphPad Prism. IL2 and anti-IL2 treatment Neonatal mice received intraperitoneal injections of 40,000 IU/g recombinant human IL-2 (Novartis) or anti-IL-2 antibodies (S4B6 and JES6; BioXCell) according to the following schedule: IL-2 on postnatal days 1, 4, 6; anti-IL-2 at 12.5 µg/g on day 1, and 25 µg/g on days 4 and 6. Bone Marrow Chimeras CD3εKO mice were lethally irradiated (9.5 Gy) and reconstituted via intravenous injection with 5 × 10⁶ T cell–depleted bone marrow (BM) cells (80% Foxo1TKO CD45.2 + 20% Foxo1Ctrl CD45.1). T cell depletion used anti-CD4 and anti-CD8 antibodies followed by anti-rat IgG magnetic beads (Dynal Biotech). Intrathymic injection of FITC 10 µl of FITC (5 mg/ml in PBS, Sigma-Aldrich) was injected into each thymic lobe under ultrasound guidance using a Vevo 2100 high resolution ultrasound machine (Visualsonics, Toronto, Canada) equipped with a 40 MHz probe (MS-550) and an integrated injection stand. During this procedure, mice were anaesthetised with isoflurane (3% isoflurane in air for induction and maintained at 1.5 and positioned supine on a temperature-controlled platform with continuous monitoring of ECG, body temperature, and respiratory rate. Then the thoracic region was depilated. To ensure adequate control of the volume injected, a Hamilton syringe (1705TLL) connected to a 19 mm 27G needle was used. The Visualsonics system was then used to guide the needle (using B-mode imaging) into the targeted part of the thymus and ensure that the injection was carried out correctly. 16h post-injection the thymus, spleen and LNs (pLNs and mLNs pooled) were recovered. Cell sorting and adoptive transfer of CD4 T cells CD4 T cells were purified from LNs (pooled superficial cervical, axillary, brachial, inguinal, and mesenteric LNs) or thymus of C57BL/6 Foxp3-GFP mice by incubating cell suspensions on ice for 20 minutes with a mixture of anti-CD8 (53 − 6.7, Abs obtened from hybridoma supernatant), anti-CD11b (Mac-1, Biolegend), anti-Ter119 (TER-119, Biolegend),, and anti-CD19 (1D3, BioXcell or Biolegend) and then with magnetic beads coupled to anti-rat immunoglobulins (Dynal Biotech). Purified CD4 T cells were then labeled with PE-Cy7-conjugated anti-CD44, PE-conjugated anti-CD25, anti-NK1.1, anti-TCRγ/δ, anti-CD11c, anti-CD11b, anti-CD19. For immature thymic CD4SP, PE-conjugated anti-CD69 and BV510-conjugated anti-CD4 were added. Naïve CD4 T (CD4 TN) cells were flow cytometry sorted as GFP − Lin− (CD25 − NK1.1 − TCRγ/δ − CD11c − CD11b − CD19−) CD44−/lo and immature thymic CD4SP sorted as CD4 + GFP − Lin− (CD25 − NK1.1 − TCRγ/δ − CD11c − CD11b − CD19 − CD69-) CD44−/lo using a FACSAria III flow cytometer (BD Biosciences). In some experiments CD4 TN cells were injected i.v. into recipient mice. In vitro Treg cell polarization assays From peripheral CD4 TN cells Flow cytometry–sorted CD4 TN cells were stimulated for 4 days with immobilized anti-CD3 (145-2C11, 4 µg/mL, Biolegend) and anti-CD28 (37.51, 4 µg/mL, Biolegend) in presence of recombinant human IL2 (13ng/mL, R&D Systems) ± blocking antibodies (anti-IL4 (10 µg/mL, Biolegend) and/or anti-IFNγ (10 µg/mL, Biolegend)) and graded concentrations of recombinant mouse TGFα (Miltenyi Biotech). From CD4SP thymocytes Flow cytometry–sorted immature CD4SP thymocytes (CD4 + Foxp3-GFP- Lin- CD44−/lo) from the thymus of C57BL/6 Foxp3-GFP mice were pre-incubated for 1 hour at 37°C with Foxo1 inhibitor or not (AS1842856, Tebubio, 85 nM final) and stimulated for 13 hours with immobilized anti-CD3 (145-2C11, 4 µg/mL, Biolegend) and anti-CD28 (3.7.51, 4 µg/mL, Biolegend), in the presence of 5 µg/mL each of anti-mouse IL-2 antibodies (JES6 and S4B6, BioXcell). Cells were then washed, transferred to uncoated V-bottom plates, and cultured with anti-IL-2 Abs (5 µg/mL, BioXcell) and in the presence or not of Foxo1 inhibitor for 3 hours. Recombinant human IL-2 (50 U/mL) was then added, and the cells were incubated for an additional 7 hours. Finally, cells were washed and rested for 12 hours with 5 µg/mL each of anti-mouse IL-2 Abs and in the presence or not of Foxo1 inhibitor. Cells were analyzed for Foxp3 induction by flow cytometry. Histology analysis: Organs were fixed in 4% formaldehyde (ROTI®Histofix, Carl Roth) for 24 h, transferred to 70% ethanol, embedded in paraffin, sectioned, and stained with HES (at the Cochin HISTIM facility). Imaging was performed with PerkinElmer Lamina scanner and analyzed using CaseViewer. Tissues were scored for inflammatory infiltration on a 0–4 scale as previously described (73) . ChIP-, RNA- and ATAC-seq analyses ChIP-seq (GEO: GSE183315) (42) , RNA-seq and ATAC-seq datasets (DRA003955, DRA004738, DRA005202) (11) , were preprocessed with standard pipelines. Preprocessing of raw sequencing data Raw FASTQ files from ChIP-seq, ATAC-seq, and RNA-seq experiments were processed using standardized pipelines as follows: ChIP-seq Quality control was performed with FastQC (v0.12.1). Adapter trimming and removal of low-quality bases (Q < 20) were done using Trim Galore (v0.6.10). Cleaned paired-end reads were aligned to the Mus musculus mm10 reference genome using Bowtie2 (v2.4.4) with the “-sensitive” preset. SAM files were converted to BAM format, sorted, indexed, and PCR duplicates were removed using Samtools (v1.18). Then uniquely mapped reads (MAPQ ≥ 20) were retained, and those overlapping ENCODE mm10 blacklisted regions were excluded using Bedtools (v2.30.0). Identification of signal-enriched regions (peaks) was performed with MACS2 (v2.2.7.1) in BAMPE mode (paired reading), with a “q” threshold value < 0.05 and the “-call-summits” option activated. Reproducible peaks across replicates were identified using the IDR framework (v2.0.4.2), applying a global IDR threshold of 0.05. Normalized signal coverage files (BigWig format) were generated using bamCoverage (deepTools v3.5.4) with RPGC normalization. Input-subtracted ChIP signal tracks were computed using bigwigCompare, and overall data quality was summarized using MultiQC (v1.9). FRiP scores (Fraction of Reads in Peaks) were calculated using featureCounts (Subread v2.0.6). RNA-seq FastQC (v0.12.1) was used for quality assessment. Adapter trimming and removal of bases with Phred score < 20 and minimum read length of 30 bp were performed with Trim Galore (v0.6.10). Reads were aligned to Mus musculus GRCm39 genome using Bowtie2 (v2.4.4) with the “--very-sensitive” preset. BAM files were sorted and indexed with Samtools (v1.18). Gene-level quantification was performed using featureCounts (Subread v2.0.6) with Ensembl annotation release 110, complemented by transcript-level quantification via RSEM (v1.3.2). ATAC-seq Adapter trimming and quality filtering were performed with fastp (v0.23.2). Trimmed reads were aligned to the reference genome (mm10) using Bowtie2 v2.5.1, and SAM files were converted to sorted BAM using samtools (v1.18). Low quality-reads, duplicates, and mitochondrial aligments were removed. Tn5 insertion sites were adjusted by strand-specific shifting (+ 4 bp/- 5 bp MACS2 (v2.2.7.1) was used for peak calling with parameters “--nomodel --shift − 100 --extsize 200”. Signal tracks were generated as BigWig files using deepTools bamCoverage (v3.5.1) with RPKM normalization. Peaks were annotated to genomic features and nearest genes using ChIPseeker (v1.38.0). Integration of ChIP-, RNA- and ATAC-seq data To identify Foxo1 transcription factor binding dynamics during tTreg cell differentiation, de novo motif analysis was performed on reproducible Foxo1 ChIP-seq peaks using HOMER (v4.11) with default settings and matched background sequences. Motifs with hypergeometric p-values < 1e-10 were retained, and the Foxo1 position weight matrix was exported. ATAC-seq data were processed with TOBIAS (v0.13.2) for footprinting analysis. BAM files were bias-corrected with ATACorrect, and footprint scores calculated using FootprintScores. Foxo1 motif instances from HOMER were scanned within accessible chromatin, and BindDetect identified Foxo1 binding sites with footprint scores > 0.2. Differential footprinting and chromatin accessibility between differentiation stages were assessed with TOBIAS (74) , applying an FDR threshold of 0.05. Peaks containing predicted Foxo1 binding sites were annotated to nearest genes using ChIPseeker. These gene lists were integrated with RNA-seq differential expression results obtained from DESeq2 (v1.36.0), considering genes with adjusted p-value 1 as significant. Single cell RNA-seq (re)analysis Data processing Publicly available scRNA-seq data from Owen et al. (43) and Borelli et al. (44) were reanalyzed. Raw gene-cell count matrices were processed in R (v4.2.0) using Seurat (v4.3.0). Low-quality cells and doublets were removed according to standard QC metrics (mitochondrial content, number of detected features, and UMI counts). Data were normalized and scaled using Seurat default functions. Dimension reduction was performed by principal component analysis (PCA), and non-linear embedding with UMAP was computed on the top principal components. Batch effects between samples were corrected using Harmony. Cells were clustered using a graph-based approach with Seurat’s FindClusters function, and cluster identities were assigned based on canonical marker gene expression. Cell type annotation Clusters corresponding to thymic T cell developmental stages and regulatory T cell precursors were manually annotated according to established markers. The following populations were defined: proliferating DN3, pre-selection DP, post-selection DP, proliferating thymocytes, immature CD4SP, CD25 + Treg precursors, transitional Tregs, Foxp3 lo Treg precursors, thymic neo-Tregs, and recirculating Tregs. Annotation was validated by visualization of known marker genes using Nebulosa and violin plots. Gene regulatory network inference To reconstruct transcription factor–target interactions, we applied the pySCENIC (v0.12.1) workflow (45) . Gene regulatory networks were first inferred using GRNBoost2, and regulon activity was quantified at the single-cell level with AUCell. AUCell scores were added to the Seurat object metadata, allowing visualization of regulon activity distributions across annotated populations. Both continuous AUCell scores and binarized activity matrices were generated. Regulon analysis and visualization Mean regulon activity per cell type was calculated and scaled (z-score) across clusters. The most variable regulons per cell type were identified, and selected regulons of biological interest (including Foxo1 , Stat5a , Foxp3 , Gata3 , Ikzf2 , Runx1 ) were visualized using ComplexHeatmap. Binary activity matrices were also generated with AUCell thresholds and represented as heatmaps annotated by cluster identity. UMAP embeddings were generated with SCpubr (75) , including cluster-level color coding and density plots. Violin plots of regulon activities were produced with SCpubr using boxplot overlays. Regulon heatmaps (continuous and binary activity) were produced with ComplexHeatmap and circlize. Statistical analysis: Data are presented as mean ± SEM. Differences between two groups were assessed by unpaired or paired Student’s t test. Multiple groups were analyzed by one-way ANOVA with Fisher’s LSD test. p < 0.05 was considered statistically significant. Declarations Acknowledgments: We greatly acknowledge the Cochin Cytometry and Immunobiology (CYBIO), MOUSET’IC core facility and Cochin Animal Core facilities for their technological support. Léa Giraud is supported during the fourth year of her Ph.D. by a fellowship from the “Ligue contre le Cancer”. Funding: This work was supported by grants from the “Ligue contre le Cancer”, the “Association pour la Recherche contre le Cancer” and the “Agence nationale de la recherche” (ANR-25-CE14-2889) Author contributions: Conceptualization: LG, CC, BL, CA Methodology: LG, AD, SC, CC, BM, BL, CA Validation: LG, BL, CA Formal analysis: LG, AD, BM, BL, CA Investigation: LG, AD, CG, NB, SC, AL, CC, BM, CA Data Curation: LG, CA Writing - Original Draft: LG, CA Writing - Review & Editing: LG, CC, BM, BL, CA Visualization: LG, CA Supervision: BM, BL, CA Project administration: BL, CA Funding acquisition: BM, BL, CA References D. L. Owen, S. A. Mahmud, L. E. Sjaastad, J. B. Williams, J. A. Spanier, D. R. Simeonov, R. Ruscher, W. Huang, I. Proekt, C. N. Miller, C. Hekim, J. C. Jeschke, P. Aggarwal, U. Broeckel, R. S. LaRue, C. M. Henzler, M.-L. Alegre, M. S. Anderson, A. August, A. Marson, Y. Zheng, C. B. Williams, M. A. Farrar, Thymic regulatory T cells arise via two distinct developmental programs. 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Twelve-week-old Foxp3-GFP C57BL/6 mice of the indicated genotypes were analyzed: Foxo1 Ctrl (white), CNS1 KO (green), Foxo1 TKO (purple), and Foxo1 TKO CNS1 KO (yellow). Each dot represents one mouse; horizontal bars indicate mean values. (A) Histopathological infiltration scores in liver, pancreas, stomach and salivary glands. (B, D) UMAPs of semi-supervised clustering of CD4 T cells and heatmaps of marker gene expression in peripheral lymph nodes (pLNs) (B) and spleen (D). (C, E) Genotype-specific sUMAPs, with CD4 T cell cluster distributions shown as stacked area plots and pie charts in pLNs (C) and spleen (E). (F, H) Breeding scheme for generating heterozygous female mice. (G, I) Frequency of GFP + Treg cells in the indicated organs of representative mice aged 4 weeks (G) or 1 week (I). Statistical significance was determined by one-way ANOVA with Fisher’s LSD test (A, B; *p < 0.03, **p < 0.002, ****p < 0.0001; ns, not significant) or by unpaired t test (H, J; *p < 0.05; ns, not significant). figureS2.jpg fig. S2. Altered composition of splenic Treg cells in Foxo1 TKO mice. Foxp3-GFP C57BL/6 mice of the indicated genotypes were analyzed. Each dot represents one mouse; horizontal bars indicate mean values. (A) UMAP of CD4 T cells from spleens of 4-week-old Foxo1 Ctrl and Foxo1 TKO mice, with accompanying heatmap of marker expression across clusters. (B) UMAPs by genotype (left) and pie charts showing the proportions of Treg cells and conventional CD4 T cells (Tconv) among CD4 T cells and the distribution of Treg cell clusters (right). (C) Frequency of Treg cells among splenic CD4 T cells and absolute numbers of Treg cells. (D–E) Frequency among Treg cells (D) and absolute numbers (E) of individual splenic Treg cell clusters. (F) Frequency of Treg cell clusters among Treg cells in peripheral lymph nodes (pLNs). Statistical significance was determined by unpaired t test (**p < 0.01, ****p < 0.0001; ns, not significant). FigureS3.jpg fig. S3. Foxo1 is essential for tTreg cell recirculation, but dispensable for their generation. (A) Frequencies of the indicated CD4SP subsets in the thymus of 1-week-old Foxo1 Ctrl and Foxo1 TKO mice. (B) Representative CD73 expression histogram of tTreg cells (CD4SP TCRβ + Foxp3 + CD25 + ) from the thymus of 4-week-old Foxo1 Ctrl and Foxo1 TKO mice (left). Quantification of the proportions of indicated CD4SP subsets (middle left), of recirculating tTreg cells (Foxp3 + CD25 + CD73 hi ) among total tTreg cells (middle right), and of absolute cell numbers (right) in the indicated genotypes. (C) Absolute numbers of FITC + Tconv and Treg cells recovered from secondary lymphoid organs (SLOs) in representative mice following intrathymic FITC labeling. Each dot represents an individual mouse. Statistical significance was determined using unpaired t-test (*** p<0.001, **** p<0.0001, ns: not significant). figureS4OK.jpg fig. S4. Age-dependent alterations in Treg cell proliferation in Foxo1 TKO mice. (A–B) Quantification (Absolute numbers) of cycling (Ki67 + /Hoechst + ) Treg cells in the indicated organs from representative mice at 1 week (A) and 4 weeks of age (B). Each dot represents an individual mouse. Statistical significance was assessed by unpaired t-test (* p<0.05, ** p<0.01, *** p<0.001). fig.S5.jpg fig S5. Foxo1 regulates chromatin accessibility and IL-2/STAT5 signaling in developing tTreg cells. (A) Motif enrichment analysis (Homer) of ChIP-seq peaks identifies both known (Foxo1 motif) and de novo inferred (Homer motif) Foxo1 consensus motifs. (B) Unsupervised clustering of Footprint scores across thymic CD4 T cell subsets: immature CD4SP thymocytes (1), CD25 + Treg precursors (2), Foxp3 lo Treg precursors (3), and mature tTregs (4). The heatmap displays normalized Footprint scores (Z-score). The mean Z-score of each footprint cluster is shown across cell subsets. (C) Left, de novo inferred Foxo1 motif and mean normalized Tn5 insertion signal around this motif, calculated with TOBIAS from ATAC-seq samples. Middle, Venn diagram showing Foxo1-bound ATAC peaks identified in each thymic CD4 T cell subset. Right, the mean normalized Tn5 insertion signal around the de novo inferred Foxo1 motif was calculated with TOBIAS from subset-specific Foxo1-bound ATAC-seq peaks. (D) Distribution of Foxo1-bound ATAC peaks across thymic CD4 T cell subsets. Heatmaps of ATAC-seq signal at Foxo1-bound peaks. Average ATAC signal was computed ±3 kb around the center of population-specific (Right) or all Foxo1-bound peaks (Left) and visualized with a copper color scale. fig.S6.jpg fig. S6. Foxo1 shapes transcriptional programs and IL-2/STAT5 signaling in developing tTreg cells. (A) UMAP projection showing the single-cell distribution of thymic T cells, based on bioinformatic analysis used to define cell clusters (43) . (B) Regulon activities across thymic T cell subsets were inferred using SCENIC. (C) Violin plots displaying the distribution of inferred mFoxo1, mSTAT5a and mFoxp3 regulon activities. (D) Quantification of IL-2Rβ surface expression (MFI) in splenic Treg cells from 1-week-old Foxo1 Ctrl and Foxo1 TKO mice. Each dot represents an individual mouse (unpaired t-test, ****P < 0.0001). fig.S7.jpg fig. S7. IL-2 signaling regulates the size of the total nnTreg cell pool. (A–B) Proportion of Treg cells among CD4 T cells and absolute numbers of Treg cells in Foxo1 Ctrl (A) or Foxo1 TKO (B) mice treated with PBS, IL-2, or anti-IL-2 blocking antibodies on days 1, 4, and 6 of life and analyzed at day 7. Each dot represents an individual mouse (unpaired t test; *p < 0.05, **p < 0.01, ****p < 0.0001; ns, not significant). SupplementaryMaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7824708","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":561927280,"identity":"5162689c-0432-46ef-bd76-86fedbd3a362","order_by":0,"name":"Léa Giraud","email":"","orcid":"","institution":"Institut Cochin, Université de Paris Cité, CNRS (UMR 8104), INSERM (U1016), Paris, France","correspondingAuthor":false,"prefix":"","firstName":"Léa","middleName":"","lastName":"Giraud","suffix":""},{"id":561927281,"identity":"a18e4c40-249d-456c-b792-612a4f1b39e2","order_by":1,"name":"Aurélie 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12:29:29","extension":"html","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":158886,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/7ced103b7d1a689be6bb3375.html"},{"id":98766066,"identity":"ed7d1627-13b3-4e17-99a3-f5abd0205fc0","added_by":"auto","created_at":"2025-12-22 10:14:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2142427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCNS1-dependent pTreg cells prevent autoimmunity in Foxo1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e Twelve-week-old Foxp3-GFP C57BL/6 mice of the indicated genotypes were analyzed: Foxo1\u003csup\u003eCtrl\u003c/sup\u003e (white), CNS1\u003csup\u003eKO\u003c/sup\u003e (green), Foxo1\u003csup\u003eTKO\u003c/sup\u003e (purple), and Foxo1\u003csup\u003eTKO\u003c/sup\u003e CNS1\u003csup\u003eKO\u003c/sup\u003e (yellow). Each dot represents one mouse; horizontal bars indicate mean values. (\u003cstrong\u003eA\u003c/strong\u003e) Body weight by genotype and sex, with females (left) and males (right). (\u003cstrong\u003eB\u003c/strong\u003e) Histopathological scores of lymphocytic infiltration in pancreas (Pa), lung (Lu), colon (Co), liver (Li), small intestine (SI), and stomach (St); darker shading indicates higher scores. Representative H\u0026amp;E staining and individual infiltration scores for SI, colon, and lung. (\u003cstrong\u003eC\u003c/strong\u003e) Representative UMAP of CD4 T cell clusters from mesenteric lymph nodes (mLNs), with heatmap of marker expression. (\u003cstrong\u003eD\u003c/strong\u003e) Genotype-specific sUMAPs and distribution of CD4 T cell clusters shown as stacked area plots and pie charts. (\u003cstrong\u003eE\u003c/strong\u003e) Frequency among conventional CD4 T cells (Tconv, Foxp3\u003csup\u003e-\u003c/sup\u003e) and absolute number of CD4 T\u003csub\u003eEff\u003c/sub\u003e cells. (\u003cstrong\u003eF\u003c/strong\u003e) Frequency of cluster 2 CD4 T\u003csub\u003eEff\u003c/sub\u003e cells among CD4 T\u003csub\u003eEff\u003c/sub\u003e cells and absolute number of cluster 1 and 2 CD4 T\u003csub\u003eEff\u003c/sub\u003e cells. (\u003cstrong\u003eG\u003c/strong\u003e) Breeding scheme for generating heterozygous females. (\u003cstrong\u003eH\u003c/strong\u003e) Representative Foxp3/CD25 plots of mLN CD4 T cells and GFP histograms of Treg cells. (\u003cstrong\u003eI\u003c/strong\u003e) Frequency of GFP\u003csup\u003e+\u003c/sup\u003e Treg cells in the indicated heterozygous mice.\u003cstrong\u003e \u003c/strong\u003eStatistical significance was determined by one-way ANOVA with Fisher’s LSD test (A–G; *p \u0026lt; 0.03, **p \u0026lt; 0.002, ***p \u0026lt; 0.0002, ****p \u0026lt; 0.0001; ns, not significant) or by unpaired t test (J; *p \u0026lt; 0.05; ns, not significant).\u003c/p\u003e","description":"","filename":"Figure1def.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/63cb5edf55c8783b396a788e.jpg"},{"id":98766068,"identity":"40ee2e64-1d75-407d-af36-d1b6870b6776","added_by":"auto","created_at":"2025-12-22 10:14:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1545945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNormal generation of pTreg cells in Foxo1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e Foxp3-GFP C57BL/6 mice of the indicated genotypes were analyzed. Each dot represents one mouse; horizontal bars indicate mean values. (\u003cstrong\u003eA\u003c/strong\u003e) UMAP of CD4 T cells from peripheral lymph nodes (pLNs) of 4-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, with accompanying heatmap of marker expression across clusters. (\u003cstrong\u003eB\u003c/strong\u003e) UMAPs by genotype (left) and pie charts showing the proportions of Treg cells and conventional CD4 T cells (Tconv) among CD4 T cells and the distribution of Treg cell clusters (right). (\u003cstrong\u003eC\u003c/strong\u003e) Frequency of Treg cells among CD4 T cells and absolute numbers of Treg cells in pLNs. (\u003cstrong\u003eD\u003c/strong\u003e) Absolute numbers of individual Treg cell clusters in pLNs. (\u003cstrong\u003eE\u003c/strong\u003e) Representative flow cytometry plots of Treg cells from pLNs, stained for Nrp1 and Helios. (\u003cstrong\u003eF\u003c/strong\u003e) Mixed bone marrow chimera experiment. T cell–depleted CD45.2\u003csup\u003e+\u003c/sup\u003e bone marrow cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice were co-transferred with T cell–depleted CD45.1\u003csup\u003e+\u003c/sup\u003e bone marrow cells from Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice into lethally irradiated CD3ε-deficient recipients. Analysis was performed 40 days post-transfer. Diagram of the experimental design (left), Nrp1 histograms of splenic Treg cells (middle), and frequency of Nrp1\u003csup\u003e+\u003c/sup\u003e Tregs among total Treg cells (right). (\u003cstrong\u003eG\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e iTreg differentiation assay. Naïve CD4 T cells (CD4 T\u003csub\u003eN\u003c/sub\u003e) from pLNs and mLNs of Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice were cultured under iTreg-polarizing conditions. Diagram of the assay (left). Representative experiment showing Foxp3-GFP induction after 4 days in response to graded TGF-β concentrations with or without neutralizing antibodies against IL-4 or IFN-γ (middle), representative flow cytometry plots (right), and quantification. (\u003cstrong\u003eH\u003c/strong\u003e) \u003cem\u003eIn vivo\u003c/em\u003e conversion assay. 3 x10\u003csup\u003e6\u003c/sup\u003e Naïve CD4 thymocytes (CD4 T\u003csub\u003eN\u003c/sub\u003e) from 1-month-old CD45.1\u003csup\u003e+\u003c/sup\u003e Foxo1\u003csup\u003eCtrl\u003c/sup\u003e or Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice were transferred into age-matched CD45.2\u003csup\u003e+\u003c/sup\u003e hosts. Diagram of the experimental design (left), gating strategy (middle), and frequency and numbers of donor-derived CD45.1\u003csup\u003e+\u003c/sup\u003e Treg cells in the indicated organs (right). Statistical significance was determined by unpaired t test (C, D, H; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001; ns, not significant) or by one-way ANOVA with Fisher’s LSD test (G; ****p \u0026lt; 0.0001; ns, not significant).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/cf13b49b24fcb5debb7c175e.jpg"},{"id":98766076,"identity":"c500313f-b984-4357-a3c9-25bf44282ffe","added_by":"auto","created_at":"2025-12-22 10:14:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1224158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFoxo1 is essential for tTreg cell recirculation but dispensable for their generation.\u003c/strong\u003e Foxp3-GFP C57BL/6 mice of the indicated genotypes were analyzed. Each dot represents one mouse; horizontal bars indicate mean values. (\u003cstrong\u003eA\u003c/strong\u003e) Representative Foxp3/CD25 plots of thymic CD4 T cells from 1-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (left), and absolute numbers of CD4 Tconv (Foxp3\u003csup\u003e-\u003c/sup\u003e CD25\u003csup\u003e-\u003c/sup\u003e), CD25\u003csup\u003e+\u003c/sup\u003e Treg precursors (TregP; Foxp3\u003csup\u003e-\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e), Foxp3\u003csup\u003elo\u003c/sup\u003e TregP (Foxp3\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e-\u003c/sup\u003e), and neo-tTregs (Foxp3\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e) (right). (\u003cstrong\u003eB\u003c/strong\u003e) Absolute numbers of recirculating tTreg cells (Foxp3\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e CD73\u003csup\u003ehi\u003c/sup\u003e) in the thymus of Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice between 1 and 4 weeks of age. (\u003cstrong\u003eC\u003c/strong\u003e) Experimental setup of mixed bone marrow chimeras: T cell–depleted bone marrow cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e (CD45.2) and Foxo1\u003csup\u003eCtrl\u003c/sup\u003e (CD45.1) mice were co-transferred into lethally irradiated CD3ε\u003csup\u003e-/-\u003c/sup\u003e recipients (left). Representative Foxp3/CD25 plots of thymic CD4SP T cells and CD73 histograms of tTregs at day 40 post-transfer (middle). Frequencies of the indicated populations among CD4SP T cells (right). (\u003cstrong\u003eD\u003c/strong\u003e) Frequency of recirculating tTreg cells (Foxp3\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e CD73\u003csup\u003ehi\u003c/sup\u003e) among CD4SP T cells in the thymus of chimeric mice. (\u003cstrong\u003eE\u003c/strong\u003e) Maturation stage analysis of CD4SP thymocytes: gating strategy to define semi-mature (SM), mature 1 (M1), and mature 2 (M2) subsets (left), and proportions of these subsets according to genotype and cell type (right). (\u003cstrong\u003eF\u003c/strong\u003e) Intrathymic FITC labeling assay: 1-month-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice were injected intrathymically with FITC and analyzed 16 h later. Diagram of the experimental design (left), representative FITC/Foxp3 plots of CD4\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e-\u003c/sup\u003e TCRβ\u003csup\u003e+\u003c/sup\u003e T cells in the indicated organs (middle), and absolute numbers of FITC\u003csup\u003e+\u003c/sup\u003e Tconv and Treg cells (right). Statistical significance was determined by unpaired t test (A–C, F; **p \u0026lt; 0.01, ****p \u0026lt; 0.0001; ns, not significant) or paired t test (D; **p \u0026lt; 0.01; n = 4).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/2cb7b4571a2844edcfbcee5f.jpg"},{"id":98779406,"identity":"41add34e-6b0c-45e5-878a-ee3e5be20af7","added_by":"auto","created_at":"2025-12-22 12:30:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":427806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFoxo1 is essential for the perinatal seeding and expansion of tTreg cells in secondary lymphoid organs (SLOs).\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative UMAP showing clusters identified by unsupervised analysis of splenic CD4 T cells from 1-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice, with corresponding marker expression profiles shown in the heatmap. (\u003cstrong\u003eB\u003c/strong\u003e) UMAPs of splenic CD4 T cells from 1-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, with cluster distributions illustrated by pie charts. (\u003cstrong\u003eC\u003c/strong\u003e) Proportion and absolute numbers of splenic Tregs in 1-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice. (\u003cstrong\u003eD\u003c/strong\u003e) Frequency of cluster 2 Treg cells among Treg cells (left) and absolute numbers of cluster 1 and 2 Treg cells (right) in the indicated genotypes. (\u003cstrong\u003eE\u003c/strong\u003e) Histogram of Ki67 fluorescence intensity (left) and proportion of Ki67\u003csup\u003e+\u003c/sup\u003e cells within splenic Treg cell clusters from 1-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice (right). (\u003cstrong\u003eF\u003c/strong\u003e) Comparative analysis of Ki67\u003csup\u003e+\u003c/sup\u003e Treg cell clusters between Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice. (\u003cstrong\u003eG\u003c/strong\u003e) Representative Ki67/Hoechst dot plots with cycling gates for splenic Treg cells from 1-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice (left), and absolute numbers of cycling Treg cells in the indicated genotypes (right). Each dot represents an individual mouse. Statistical significance was assessed by unpaired t-test (C–D, F–G; ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001, ns: not significant) and paired t-test (E; **** p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/02d17f78526e16caa1f93a69.jpg"},{"id":98779370,"identity":"f24d3ac7-6d42-48bc-8751-fed7e25d1c7d","added_by":"auto","created_at":"2025-12-22 12:30:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2028911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFoxo1 regulates chromatin accessibility and IL-2/STAT5 signaling in developing tTreg cells. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Overview of the integrative multi-omics strategy combining ATAC-seq, ChIP-seq, motif analysis, and footprinting to dissect transcriptional regulation across Treg developmental stages. (\u003cstrong\u003eB\u003c/strong\u003e) Unsupervised clustering of ATAC-seq peaks across thymic CD4 T cell subsets: immature CD4SP thymocytes (ImmCD4SP, 1), CD25\u003csup\u003e+\u003c/sup\u003e Treg precursors (CD25\u003csup\u003e+\u003c/sup\u003e TregP, 2), Foxp3\u003csup\u003elo\u003c/sup\u003e Treg precursors (Foxp3\u003csup\u003elo\u003c/sup\u003e TregP, 3), and tTregs (4). The heatmap displays normalized chromatin accessibility (Z-score). The mean Z-score of each ATAC cluster is shown across cell subsets. (\u003cstrong\u003eC\u003c/strong\u003e) \u003cstrong\u003eLeft\u003c/strong\u003e, pie chart showing the proportion of all variable ATAC peaks contributed by each cluster. \u003cstrong\u003eRight\u003c/strong\u003e, pie chart depicting the distribution of peaks containing a Foxo1-associated footprint (Foxo1_FP), quantified within each ATAC cluster. (\u003cstrong\u003eD\u003c/strong\u003e) Footprint intensities were normalized on a per-peak basis to the ImmCD4SP stage to quantify relative accessibility changes across the Foxo1-dependent developmental trajectory. Peaks predicted to bind Foxo1 (HOMER Best-Guess, red) and all other accessible sites (black) were analyzed separately for each ATAC-defined cluster (o.c1–o.c4). For each cluster, thin red traces represent individual Foxo1-predicted peaks, and bold lines indicate mean trends. (\u003cstrong\u003eE\u003c/strong\u003e) Volcano plot of RNA expression changes between ImmCD4SP and tTregs. Genes associated with specific ATAC/Foxo1/Homer FP peaks in clusters o.c1, o.c2, o.c3, and c.c1 are highlighted in color (blue-green, turquoise, light green, magenta, respectively). Background genes are shown in light grey. Point size represents the number of shared Foxo1/Homer FP peaks in the promoter. Thresholds for significance were set at |log2 fold change| \u0026gt; 1 and adjusted p-value \u0026lt; 0.001. Selected genes of interest (Ctla4, Il2rb, Klf2, S1pr1) are labeled. (\u003cstrong\u003eF\u003c/strong\u003e) Hallmark pathway enrichment analysis of genes associated with Foxo1/Homer FP peaks with significant RNA changes (padj \u0026lt; 0.05). Each point represents a pathway, with x-axis showing fold enrichment, y-axis showing -log10(adjusted p-value), point size proportional to the number of genes in the pathway, and point color reflecting fold enrichment. The horizontal dashed line indicates the significance threshold (padj = 0.05). (\u003cstrong\u003eG\u003c/strong\u003e) Correlation between chromatin accessibility and RNA expression for genes involved in the IL2/STAT5 signaling Hallmark pathway. Each point represents a gene with Foxo1/Homer FP peaks. The x-axis shows the RNA log2 fold change between tTregs and immCD4SP, and the y-axis shows the average ATAC fold change across cell types. Point fill reflects -log10(min p-value) from ATAC data, and point size reflects -log10(adjusted p-value) from RNA data. Genes of interest are labeled. Horizontal and vertical dashed lines indicate zero fold change thresholds. (\u003cstrong\u003eH\u003c/strong\u003e) Top: Genome browser tracks of the Il2rb locus showing ATAC-seq signal across populations, Foxo1 ChIP-seq peaks, and candidate cis-regulatory elements (blue: CTCF-bound regions, red: promoter-like sequences, yellow: enhancer-like sequences). Gapdh locus is shown as a control for ATAC-seq, and Klf2 as a control for Foxo1 ChIP-seq. Shared Foxo1-ATAC peaks are indicated. Bottom: Zoomed view of the Il2rb locus showing overlaid ATAC-seq signal and corrected footprint signal (observed/expected insertions) across populations. Mean ATAC-seq signal and footprint score for shared_peak_b are shown. (\u003cstrong\u003eI\u003c/strong\u003e) Representative histogram and quantification of IL-2Rβ surface expression (MFI) in the four populations from Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (n = 4 per group). (\u003cstrong\u003eJ\u003c/strong\u003e) IL-2Rβ expression at the surface of in vitro induced tTreg-like cells generated in the presence or absence of a Foxo1 inhibitor (Foxo1_i, AS1842856). Experimental scheme, representative Foxp3 expression in CD4 T cells, and representative IL-2Rβ expression on Foxp3+ cells are shown. Quantification of IL-2Rβ surface expression (MFI) is presented. (\u003cstrong\u003eK\u003c/strong\u003e) Quantification of pSTAT5 MFI in the four populations. ****P \u0026lt; 0.0001; ns, not significant (unpaired t-test).\u003c/p\u003e","description":"","filename":"Figure5def.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/2c772dab136c0f6f06d28ccb.jpg"},{"id":98779245,"identity":"0f22f196-2b37-4b75-a83f-fdc4fcf49d80","added_by":"auto","created_at":"2025-12-22 12:30:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":392240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-2 shapes the peripheral nnTreg cell pool.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative UMAP of splenic CD4SP cells from one-week-old untreated Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice, with corresponding marker expression profiles shown in the heatmap. (\u003cstrong\u003eB\u003c/strong\u003e) Representative UMAPs of splenic CD4SP cells from one-week-old Foxo1\u003csup\u003eTKO\u003c/sup\u003e and Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice treated with PBS, IL-2, or anti-IL-2 blocking antibodies on days 1, 4, and 6 of life, overlaid onto the UMAP of untreated mice (left). Cluster proportions are shown as pie charts (right). (\u003cstrong\u003eC\u003c/strong\u003e–\u003cstrong\u003eD\u003c/strong\u003e) Proportion of cluster 2 Treg cells among Treg cells (\u003cstrong\u003eC\u003c/strong\u003e) and absolute numbers of cluster 1 and 2 Treg cells (\u003cstrong\u003eD\u003c/strong\u003e) in Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice across treatment conditions. Each dot represents an individual mouse (unpaired t test; **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001; ns, not significant).\u003c/p\u003e","description":"","filename":"Figure6def.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/0b5a05942782a51bc534087c.jpg"},{"id":98783732,"identity":"482a6451-1466-45a9-863d-85dc61d6a999","added_by":"auto","created_at":"2025-12-22 12:42:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12304507,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/99640be3-b28b-4be9-bcaf-af06b2df9638.pdf"},{"id":98766064,"identity":"8f312aa1-364a-44f0-9cdb-7cdb12e9e41f","added_by":"auto","created_at":"2025-12-22 10:14:15","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1749155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003efig. S1. CNS1-dependent pTreg cells prevent autoimmunity in Foxo1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e Twelve-week-old Foxp3-GFP C57BL/6 mice of the indicated genotypes were analyzed: Foxo1\u003csup\u003eCtrl\u003c/sup\u003e (white), CNS1\u003csup\u003eKO\u003c/sup\u003e (green), Foxo1\u003csup\u003eTKO\u003c/sup\u003e (purple), and Foxo1\u003csup\u003eTKO\u003c/sup\u003e CNS1\u003csup\u003eKO\u003c/sup\u003e (yellow). Each dot represents one mouse; horizontal bars indicate mean values. (\u003cstrong\u003eA\u003c/strong\u003e) Histopathological infiltration scores in liver, pancreas, stomach and salivary glands. (\u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e) UMAPs of semi-supervised clustering of CD4 T cells and heatmaps of marker gene expression in peripheral lymph nodes (pLNs) (\u003cstrong\u003eB\u003c/strong\u003e) and spleen (\u003cstrong\u003eD\u003c/strong\u003e). (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e) Genotype-specific sUMAPs, with CD4 T cell cluster distributions shown as stacked area plots and pie charts in pLNs (\u003cstrong\u003eC\u003c/strong\u003e) and spleen (\u003cstrong\u003eE\u003c/strong\u003e). (\u003cstrong\u003eF, H\u003c/strong\u003e) Breeding scheme for generating heterozygous female mice. (\u003cstrong\u003eG\u003c/strong\u003e, \u003cstrong\u003eI\u003c/strong\u003e) Frequency of GFP\u003csup\u003e+\u003c/sup\u003e Treg cells in the indicated organs of representative mice aged 4 weeks (\u003cstrong\u003eG\u003c/strong\u003e) or 1 week (\u003cstrong\u003eI\u003c/strong\u003e). Statistical significance was determined by one-way ANOVA with Fisher’s LSD test (A, B; *p \u0026lt; 0.03, **p \u0026lt; 0.002, ****p \u0026lt; 0.0001; ns, not significant) or by unpaired t test (H, J; *p \u0026lt; 0.05; ns, not significant).\u003c/p\u003e","description":"","filename":"FigureS1def.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/aa2ab576211ee5379e8caaa0.jpg"},{"id":98780691,"identity":"6f5f3780-2265-4638-9200-b192c0f245c4","added_by":"auto","created_at":"2025-12-22 12:31:34","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":679479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003efig. S2. Altered composition of splenic Treg cells in Foxo1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e Foxp3-GFP C57BL/6 mice of the indicated genotypes were analyzed. Each dot represents one mouse; horizontal bars indicate mean values. (\u003cstrong\u003eA\u003c/strong\u003e) UMAP of CD4 T cells from spleens of 4-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, with accompanying heatmap of marker expression across clusters. (\u003cstrong\u003eB\u003c/strong\u003e) UMAPs by genotype (left) and pie charts showing the proportions of Treg cells and conventional CD4 T cells (Tconv) among CD4 T cells and the distribution of Treg cell clusters (right). (\u003cstrong\u003eC\u003c/strong\u003e) Frequency of Treg cells among splenic CD4 T cells and absolute numbers of Treg cells. (\u003cstrong\u003eD\u003c/strong\u003e–\u003cstrong\u003eE\u003c/strong\u003e) Frequency among Treg cells (\u003cstrong\u003eD\u003c/strong\u003e) and absolute numbers (\u003cstrong\u003eE\u003c/strong\u003e) of individual splenic Treg cell clusters. (\u003cstrong\u003eF\u003c/strong\u003e) Frequency of Treg cell clusters among Treg cells in peripheral lymph nodes (pLNs). Statistical significance was determined by unpaired t test (**p \u0026lt; 0.01, ****p \u0026lt; 0.0001; ns, not significant).\u003c/p\u003e","description":"","filename":"figureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/780295b86a4d0b67917d51a3.jpg"},{"id":98778623,"identity":"3b8185ed-231d-4021-8d2a-6cd64b268d31","added_by":"auto","created_at":"2025-12-22 12:29:28","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":469700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003efig. S3. Foxo1 is essential for tTreg cell recirculation, but dispensable for their generation.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Frequencies of the indicated CD4SP subsets in the thymus of 1-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice. (\u003cstrong\u003eB\u003c/strong\u003e) Representative CD73 expression histogram of tTreg cells (CD4SP TCRβ\u003csup\u003e+\u003c/sup\u003e Foxp3\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e) from the thymus of 4-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (left). Quantification of the proportions of indicated CD4SP subsets (middle left), of recirculating tTreg cells (Foxp3\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e CD73\u003csup\u003ehi\u003c/sup\u003e) among total tTreg cells (middle right), and of absolute cell numbers (right) in the indicated genotypes. (\u003cstrong\u003eC\u003c/strong\u003e) Absolute numbers of FITC\u003csup\u003e+\u003c/sup\u003e Tconv and Treg cells recovered from secondary lymphoid organs (SLOs) in representative mice following intrathymic FITC labeling. Each dot represents an individual mouse. Statistical significance was determined using unpaired t-test (*** p\u0026lt;0.001, **** p\u0026lt;0.0001, ns: not significant).\u003c/p\u003e","description":"","filename":"FigureS3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/661019dc3974e0b7f52ea7ff.jpg"},{"id":98766074,"identity":"155cde1b-07cc-4d57-85a7-eaf27cc9c7b0","added_by":"auto","created_at":"2025-12-22 10:14:16","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":197633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003efig. S4. Age-dependent alterations in Treg cell proliferation in Foxo1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e–\u003cstrong\u003eB\u003c/strong\u003e) Quantification (Absolute numbers) of cycling (Ki67\u003csup\u003e+\u003c/sup\u003e/Hoechst\u003csup\u003e+\u003c/sup\u003e) Treg cells in the indicated organs from representative mice at 1 week (\u003cstrong\u003eA\u003c/strong\u003e) and 4 weeks of age (\u003cstrong\u003eB\u003c/strong\u003e). Each dot represents an individual mouse. Statistical significance was assessed by unpaired t-test (* p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"figureS4OK.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/ad6d727bd33ccb8cf0a378d9.jpg"},{"id":98766077,"identity":"c60fa228-0fc2-49da-9af3-96382690d77e","added_by":"auto","created_at":"2025-12-22 10:14:16","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1393103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003efig S5. Foxo1 regulates chromatin accessibility and IL-2/STAT5 signaling in developing tTreg cells. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Motif enrichment analysis (Homer) of ChIP-seq peaks identifies both known (Foxo1 motif) and de novo inferred (Homer motif) Foxo1 consensus motifs. (\u003cstrong\u003eB\u003c/strong\u003e) Unsupervised clustering of Footprint scores across thymic CD4 T cell subsets: immature CD4SP thymocytes (1), CD25\u003csup\u003e+\u003c/sup\u003e Treg precursors (2), Foxp3\u003csup\u003elo\u003c/sup\u003e Treg precursors (3), and mature tTregs (4). The heatmap displays normalized Footprint scores (Z-score). The mean Z-score of each footprint cluster is shown across cell subsets. (\u003cstrong\u003eC\u003c/strong\u003e) \u003cstrong\u003eLeft\u003c/strong\u003e, \u003cem\u003ede novo\u003c/em\u003e inferred Foxo1 motif and mean normalized Tn5 insertion signal around this motif, calculated with TOBIAS from ATAC-seq samples. \u003cstrong\u003eMiddle\u003c/strong\u003e, Venn diagram showing Foxo1-bound ATAC peaks identified in each thymic CD4 T cell subset. \u003cstrong\u003eRight\u003c/strong\u003e, the mean normalized Tn5 insertion signal around the \u003cem\u003ede novo\u003c/em\u003e inferred Foxo1 motif was calculated with TOBIAS from subset-specific Foxo1-bound ATAC-seq peaks. (\u003cstrong\u003eD\u003c/strong\u003e) Distribution of Foxo1-bound ATAC peaks across thymic CD4 T cell subsets. Heatmaps of ATAC-seq signal at Foxo1-bound peaks. Average ATAC signal was computed ±3 kb around the center of population-specific (\u003cstrong\u003eRight\u003c/strong\u003e) or all Foxo1-bound peaks (\u003cstrong\u003eLeft\u003c/strong\u003e) and visualized with a copper color scale.\u003c/p\u003e","description":"","filename":"fig.S5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/b923822645689c73fe834609.jpg"},{"id":98766080,"identity":"17481e17-ea97-408b-b8a4-77aeb25a2374","added_by":"auto","created_at":"2025-12-22 10:14:16","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1625372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003efig. S6. Foxo1 shapes transcriptional programs and IL-2/STAT5 signaling in developing tTreg cells.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) UMAP projection showing the single-cell distribution of thymic T cells, based on bioinformatic analysis used to define cell clusters \u003cem\u003e(43)\u003c/em\u003e. (\u003cstrong\u003eB\u003c/strong\u003e) Regulon activities across thymic T cell subsets were inferred using SCENIC. (\u003cstrong\u003eC\u003c/strong\u003e) Violin plots displaying the distribution of inferred mFoxo1, mSTAT5a and mFoxp3 regulon activities. (D) Quantification of IL-2Rβ surface expression (MFI) in splenic Treg cells from 1-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice. Each dot represents an individual mouse (unpaired t-test, ****P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"fig.S6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/dcfadafcce97b69eb3738c9f.jpg"},{"id":98766081,"identity":"2a245a67-5c71-4a0e-9caf-4acf4cf9687a","added_by":"auto","created_at":"2025-12-22 10:14:16","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":285364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003efig. S7. IL-2 signaling regulates the size of the total nnTreg cell pool.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e–\u003cstrong\u003eB\u003c/strong\u003e) Proportion of Treg cells among CD4 T cells and absolute numbers of Treg cells in Foxo1\u003csup\u003eCtrl\u003c/sup\u003e (\u003cstrong\u003eA\u003c/strong\u003e) or Foxo1\u003csup\u003eTKO\u003c/sup\u003e (\u003cstrong\u003eB\u003c/strong\u003e) mice treated with PBS, IL-2, or anti-IL-2 blocking antibodies on days 1, 4, and 6 of life and analyzed at day 7. Each dot represents an individual mouse (unpaired t test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001; ns, not significant).\u003c/p\u003e","description":"","filename":"fig.S7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/71afaf01ebb9bfeb46984878.jpg"},{"id":98766079,"identity":"f8e40a15-3d2f-4af4-b9f2-8150ac3cf8e7","added_by":"auto","created_at":"2025-12-22 10:14:16","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":23487,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7824708/v1/963c41913e910704d5bb99b4.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eTranscriptional control of IL-2 sensing by Foxo1 dictates neonatal Treg homeostasis\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRegulatory CD4 T (Treg) cells are the primary mediators of peripheral tolerance under physiological conditions. In the periphery, Treg cells consist of two main subsets: thymic Treg (tTreg) cells, which are naturally produced in the thymus through two distinct developmental programs involving Foxp3\u003csup\u003e-\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e and CD25\u003csup\u003e-\u003c/sup\u003e Foxp3\u003csup\u003elo\u003c/sup\u003e Treg cell progenitors (TregP) \u003cem\u003e(1, 2)\u003c/em\u003e, and peripheral Treg (pTreg) cells, which acquire a similar phenotype and function after activation of naive CD4 T (CD4 T\u003csub\u003eN\u003c/sub\u003e) cells in secondary lymphoid organs (SLOs) in the presence of TGFβ \u003cem\u003e(3)\u003c/em\u003e. An additional level of complexity has recently emerged from the observation that the age at which tTreg cells are generated contributes to the heterogeneity of this compartment \u003cem\u003e(4)\u003c/em\u003e. Indeed, it was reported that tTreg cells generated during the perinatal period stably persists in adult mice and has a crucial role in maintaining self-tolerance. Phenotypically, adult and perinatally-generated tTreg cells are characterized by their high expression of Helios and Nrp-1, whereas pTreg cells lack these markers \u003cem\u003e(5, 6)\u003c/em\u003e. Perinatally generated tTreg (hereafter referred to as nnTregs) cells exhibit a more activated phenotype than their adult generated counterparts and express high levels of both Fgl2 and PD-1 proteins \u003cem\u003e(4)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003etTreg and pTreg cell subsets are thought to play complementary roles in maintaining immune tolerance \u003cem\u003e(4, 7, 8)\u003c/em\u003e. While tTreg cells prevent autoimmunity by maintaining tolerance to self-tissue-derived antigens, pTreg cells are thought to mediate tolerance to non-self-antigens, such as food or fetal antigens and commensal microbes \u003cem\u003e(9, 10)\u003c/em\u003e. However, at least in non-autoimmune-prone mice, neither tTreg cell-deficient mice, such as those with a T-cell specific deletion of the Satb1 TF \u003cem\u003e(11)\u003c/em\u003e, nor pTreg cell-deficient mice, such as CNS1-deficient mice \u003cem\u003e(9, 10, 12)\u003c/em\u003e develop overt multi-organ pathology comparable to the fatal autoimmunity characterizing Foxp3-deficient scurfy mice. This suggests effective compensatory mechanisms between these two Treg cell subsets \u003cem\u003e(8)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eImportantly, the signals driving the generation and the homeostasis of these Treg cell subsets can vary. Indeed, while the generation of tTreg cells (at least in adult mice) is dictated by TCR and IL-2 signaling pathways, TGFβ signaling (via Smad activation and binding to the Foxp3 CNS1 enhancer) is crucial for pTreg cell differentiation \u003cem\u003e(12\u0026ndash;14)\u003c/em\u003e. Once generated, Treg cell homeostasis is tightly maintained by various signals, primarily involving TCR and cytokine signaling \u003cem\u003e(15\u0026ndash;19)\u003c/em\u003e. While most Treg cells from SLOs rely heavily on interleukin-2 (IL-2) and IL-2R signaling (involving IL-2Rα, IL-2Rβ, IL-2Rγ, and STAT5) for their homeostasis \u003cem\u003e(18, 20, 21)\u003c/em\u003e, certain Treg cell subsets depend on IL-7 or IL-33 for their maintenance \u003cem\u003e(19, 22)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eNevertheless, other factors remain to be identified as regulators of Treg cell homeostasis in general, and perinatal tTreg cell homeostasis in particular, whose roles appear to extend well beyond this period. Indeed, apart from Aire \u003cem\u003e(4)\u003c/em\u003e, which promotes immunological tolerance by inducing the expression of peripheral-tissue antigens in thymic medullary epithelial cells and has been shown to be crucial in perinatal Treg cell generation, there are no clues concerning the control of the homeostasis of this major Treg cell subset.\u003c/p\u003e \u003cp\u003eTranscription factors (TFs) of the forkhead box O (Foxo) family, Foxo1 and Foxo3, have been implicated in multiple key biological processes in T cells such as T-cell trafficking and survival of na\u0026iuml;ve T cells \u003cem\u003e(23\u0026ndash;26)\u003c/em\u003e as well as in differentiation into effector T cells \u003cem\u003e(27\u0026ndash;30)\u003c/em\u003e. However, T-cell activation leads to the relocation of Foxo TFs to the cytosol \u003cem\u003e(31)\u003c/em\u003e, inhibiting their transcriptional activity, making their exact roles challenging to determine. More than a \u003cem\u003ebona fide\u003c/em\u003e transcription factors, Foxo proteins emerged as pioneer factors for gene activation modulating active chromatin states through their ability to bind condensed chromatin structures and to promote directly or indirectly chromatin opening \u003cem\u003e(32)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eT-cell specific Foxo1-deficient mice only develop a mild autoimmune disease, characterized by slight mononuclear cell infiltration in non-lymphoid organs at 1 year of age \u003cem\u003e(24, 33)\u003c/em\u003e. Beyond its established role in the function of Treg cells \u003cem\u003e(34)\u003c/em\u003e, Foxo1 has been implicated in a contradictory manner in the generation of tTreg and pTreg cells \u003cem\u003e(24, 33)\u003c/em\u003e. Indeed, while Foxo1 has been reported as either dispensable \u003cem\u003e(34)\u003c/em\u003e or required \u003cem\u003e(33)\u003c/em\u003e for tTreg cell generation in the thymus, its major role in TGFβ-dependent differentiation of \u003cem\u003ein vitro\u003c/em\u003e-induced Treg (iTreg) and pTreg cells is more widely accepted \u003cem\u003e(33, 34)\u003c/em\u003e. In light of the growing complexity of Treg cell heterogeneity, our current understanding of the role of Foxo1 TF in Treg cell biology, particularly in the generation and homeostasis of these ontogenetically distinct Treg cell subsets, appears incomplete.\u003c/p\u003e \u003cp\u003eIn this study, we revisit its role \u003cem\u003ein vivo\u003c/em\u003e and show that Foxo1 TF plays a key role in the establishment of the perinatal Treg cell compartment, particularly in the intense proliferation of these cells in neonatal SLOs, by modulating their sensitivity to IL-2.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCNS1-dependent pTreg cells prevent autoimmunity in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eTo address the role of CNS1-dependent pTreg cells in the maintenance of immunological tolerance in these Foxo1-deficient mice, we generated double-deficient mice (CNS1\u003csup\u003eKO\u003c/sup\u003e Foxo1\u003csup\u003ef/f\u003c/sup\u003e CD4-Cre\u003csup\u003e+\u003c/sup\u003e Foxp3\u003csup\u003eGFP\u003c/sup\u003e - CNS1\u003csup\u003eKO\u003c/sup\u003e Foxo1\u003csup\u003eTKO\u003c/sup\u003e), by crossing Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice with CNS1\u003csup\u003eKO\u003c/sup\u003e (CNS1\u003csup\u003eKO\u003c/sup\u003e Foxp3\u003csup\u003eGFP\u003c/sup\u003e - CNS1\u003csup\u003eKO\u003c/sup\u003e) mice. WT control littermates (Foxo1\u003csup\u003ef/f\u003c/sup\u003e CD4-Cre\u003csup\u003e-\u003c/sup\u003e Foxp3\u003csup\u003eGFP\u003c/sup\u003e - Foxo1\u003csup\u003eCtrl\u003c/sup\u003e), Foxo1\u003csup\u003eTKO\u003c/sup\u003e, CNS1\u003csup\u003eKO\u003c/sup\u003e, and CNS1\u003csup\u003eKO\u003c/sup\u003e Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice were followed until 12 weeks of age. Surprisingly, while Foxo1\u003csup\u003eTKO\u003c/sup\u003e and CNS1\u003csup\u003eKO\u003c/sup\u003e mice, as expected, only develop mild manifestations of autoimmunity affecting the intestine (both colon and small intestine) and/or the lung in a few individual animals, double-deficient mice lose weight and exhibit a more severe disease characterized by pronounced cell infiltrates and tissue disorganization in most animals in the same tissues (Fig.\u0026nbsp;1A-B, and fig. S1A).\u003c/p\u003e \u003cp\u003eSemi-supervised analysis of mesenteric Lymph node (mLN) CD4 T cells from 12-week-old WT control littermates, Foxo1\u003csup\u003eTKO\u003c/sup\u003e, CNS1\u003csup\u003eKO\u003c/sup\u003e, and CNS1\u003csup\u003eKO\u003c/sup\u003e Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice by flow cytometry identified clusters of na\u0026iuml;ve (T\u003csub\u003eN\u003c/sub\u003e), effector (T\u003csub\u003eEff\u003c/sub\u003e) and Treg cells, characterized by their coexpression of several surface molecules (CD44, CD25 and Nrp1) and key transcription factors (Tbet, Gata3, Rorγt and Foxp3) (Fig.\u0026nbsp;1C). Consistent with the autoimmune disease affecting the intestine of CNS1\u003csup\u003eKO\u003c/sup\u003e Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, separation by genotype revealed significant changes in cluster distribution, with a progressive enrichment of CD4 T\u003csub\u003eEff\u003c/sub\u003e cells from WT to double-deficient mice. While CNS1\u003csup\u003eKO\u003c/sup\u003e mice maintain a near-normal CD4 T cell compartment, combining CNS1 deletion with the Foxo1\u003csup\u003eTKO\u003c/sup\u003e background further exacerbates the expected loss of T\u003csub\u003eN\u003c/sub\u003e cells to the benefit of Treg and CD4 T\u003csub\u003eEff\u003c/sub\u003e cells observed in CNS1\u003csup\u003eKO\u003c/sup\u003e Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig.\u0026nbsp;1D). Notably, cluster distribution among Foxp3\u003csup\u003e-\u003c/sup\u003e conventional CD4 T (Tconv) cells also shifts between Foxo1\u003csup\u003eTKO\u003c/sup\u003e and CNS1\u003csup\u003eKO\u003c/sup\u003e Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig.\u0026nbsp;1D). Specifically, the percentages of CD4 T\u003csub\u003eEff\u003c/sub\u003e cells among Tconv cells and absolute numbers of CD4 T\u003csub\u003eEff\u003c/sub\u003e cells significantly increase in CNS1\u003csup\u003eKO\u003c/sup\u003e Foxo1\u003csup\u003eTKO\u003c/sup\u003e compared to Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig.\u0026nbsp;1E, and fig. S1B-E). Finally, analysis of absolute numbers and cluster distribution within CD4 T\u003csub\u003eEff\u003c/sub\u003e cells reveals an enrichment and accumulation of Tbet- and RORγt-expressing cells (c1_ and c2_CD4_T\u003csub\u003eEff\u003c/sub\u003e clusters respectively) in CNS1\u003csup\u003eKO\u003c/sup\u003e Foxo1\u003csup\u003eTKO\u003c/sup\u003e compared to Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig.\u0026nbsp;1F). Taken together, these data suggest that a CNS1-dependent subset of Treg cells mitigates both the clinical manifestations and the dysregulation of conventional T cells in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eTo further validate the contribution of CNS1-dependent Treg cells to the peripheral Treg pool in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, we generated heterozygous Foxp3\u003csup\u003eCNS1KO/WT\u003c/sup\u003e females carrying one CNS1\u003csup\u003eKO\u003c/sup\u003e allele and one CNS1\u003csup\u003eWT\u003c/sup\u003e allele both in Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e backgrounds. Owing to random X-chromosome inactivation, half of the CD4 T\u003csub\u003eN\u003c/sub\u003e cells in these mice express either allele, thereby retaining or lacking the ability to differentiate into CNS1-dependent pTreg cells (Fig.\u0026nbsp;1G, and fig. S1F, H). In both genetic backgrounds, GFP expression was linked either with the CNS1\u003csup\u003eKO\u003c/sup\u003e allele (as test group) or with the CNS1\u003csup\u003eWT\u003c/sup\u003e allele (as control group). GFP expression in Treg cells revealed the contribution of CNS1-dependent pTreg cells to the Treg pool in SLOs (Fig.\u0026nbsp;1H). In 4-weeks old control background mice, their contribution remained minimal. However, in \u003cem\u003eFoxo1\u003c/em\u003e-deficient mice, CNS1-dependent pTreg cells contributed significantly more to the overall Treg cell compartment (Fig.\u0026nbsp;1I, and fig. S1F, G). This indicates that CNS-dependent pTreg cells may play a compensatory role for the deficit in Treg cells when Foxo1 is lost. Notably, CNS1-dependent pTreg cells contributed only marginally to the Treg cell compartment in both control and \u003cem\u003eFoxo1\u003c/em\u003e-deficient perinates (1-week old) (fig. S1H, I), consistent with the proposed role of CNS1 in promoting pTreg cell induction at weaning \u003cem\u003e(35)\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003epTreg cells are normally generated in Foxo1 mice\u003c/h3\u003e\n\u003cp\u003eBecause CNS1-dependent pTreg cells are critical in immune tolerance in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, we further investigate the role of Foxo1 in both iTreg and pTreg cell generation. We first analyzed Treg subsets in the SLOs of 4-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice. Using semi-supervised flow cytometry analysis of peripheral lymph node (pLN) CD4\u003csup\u003e+\u003c/sup\u003e T cells from 4-week-old Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice and WT control littermates, we identified 4 clusters of Treg cells based on the coexpression of molecules known to distinguish activated and resting Treg cell subsets (Ly-6C, CD103, CD69, CD25, and Ki67) and to discriminate tTreg from pTreg cells (Nrp1 and Helios) (Fig.\u0026nbsp;2A): Three clusters (c1, c2, c3) of tTreg-like cells expressing Nrp1 with varying levels of CD25, Ly-6C, CD69, and Helios, and a fourth cluster (c4) of pTreg-like cells lacking both Nrp1 and Helios expression (Fig.\u0026nbsp;2A and fig. S2A). Genotype-based separation revealed global changes in the distribution of Tconv and Treg cells in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice compared to Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice, with a significant reduction in both the frequency of Treg cells among CD4\u003csup\u003e+\u003c/sup\u003e T cells and their absolute numbers in pLNs (Fig.\u0026nbsp;2B, C), but not in the spleen (fig. S2B, C). Interestingly, the distribution of Treg cell clusters was differentially altered in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, in both organs (Fig.\u0026nbsp;2B and fig. S2D, E). Indeed, we observed an almost complete loss of c2 Treg cells, together with a significant increase in proportions in the c4 cluster in both organs and in the c1 cluster specifically in pLNs of Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (fig. S2D, F). When analyzing absolute cell numbers, we observed a marked reduction in the three Nrp1\u003csup\u003e+\u003c/sup\u003e tTreg-like clusters (c1, c2, and c3) in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice compared to Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice, while numbers of Nrp1\u003csup\u003e-\u003c/sup\u003e pTreg-like (c4) cells were unaffected and even slightly increased (Fig.\u0026nbsp;2D and fig. S2E). This near-complete loss of Nrp1\u003csup\u003e+\u003c/sup\u003e tTreg-like cells in SLOs from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig.\u0026nbsp;2E) is cell-intrinsic, as demonstrated by mixed bone marrow chimeras in which lethally irradiated mice were reconstituted with a mixture of Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e bone marrow cells (Fig.\u0026nbsp;2F). These observations could reflect a role for Foxo1 in the development or maintenance of tTreg-like cells, while pTreg cell generation might not depend on Foxo1.\u003c/p\u003e \u003cp\u003eFoxo1-deficient CD4 T\u003csub\u003eN\u003c/sub\u003e cells have been described as defective in converting into iTreg cells \u003cem\u003e(24, 33)\u003c/em\u003e. It is long known that IL-4 but also IFN-γ inhibit Foxp3 induction in iTreg polarization assays \u003cem\u003e(36, 37)\u003c/em\u003e and we recently demonstrated that Foxo1-deficient stimulated CD4 T\u003csub\u003eN\u003c/sub\u003e cells heavily produced these 2 interleukins, even in the absence of T\u003csub\u003eH\u003c/sub\u003e1 or T\u003csub\u003eH\u003c/sub\u003e2 polarizing interleukins \u003cem\u003e(38)\u003c/em\u003e. We therefore reassessed the impact of Foxo1 deletion on the iTreg cell differentiation potential in the presence or absence of IL-4-blocking and/or IFN-γ-blocking antibodies (Fig.\u0026nbsp;2G). Consistent with previous findings \u003cem\u003e(33)\u003c/em\u003e, we confirmed that, in the absence of IL-4- and IFN-γ-blocking antibodies, CD4 T\u003csub\u003eN\u003c/sub\u003e cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice hardly differentiate into iTreg cells (Fig.\u0026nbsp;2G). However, blocking both IFN-γ and IL-4 effectively restored their ability to convert into iTreg cells (Fig.\u0026nbsp;2G). This lack of a substantial defect in the ability of Foxo1-deficient CD4 T\u003csub\u003eN\u003c/sub\u003e cells to differentiate into iTreg cells \u003cem\u003ein vitro\u003c/em\u003e was further validated \u003cem\u003ein vivo\u003c/em\u003e in adoptive transfer experiments (Fig.\u0026nbsp;2H). CD4 T\u003csub\u003eN\u003c/sub\u003e thymocytes from 1-month-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e or Foxo1\u003csup\u003eTKO\u003c/sup\u003e CD45.1 mice were transferred into CD45.2 recipient mice of the same age and genotype, and their fate was analyzed 2 months later. In this setting, both WT and Foxo1-deficient CD4 T\u003csub\u003eN\u003c/sub\u003e cells efficiently converted into pTreg cells with Foxo1-deficient CD4 T\u003csub\u003eN\u003c/sub\u003e cells differentiating even more in Foxp3-expressing than their WT counterparts most likely due to differences in homeostatic signals received in the respective hosts (Fig.\u0026nbsp;2H).\u003c/p\u003e \u003cp\u003eAltogether, both our phenotypic data and our \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e conversion assays strongly support a marginal role of Foxo1 in the differentiation of CD4 T\u003csub\u003eN\u003c/sub\u003e cells into pTreg cells.\u003c/p\u003e\n\u003ch3\u003eFoxo1 deletion affects tTreg cell recirculation but not their generation in the thymus\u003c/h3\u003e\n\u003cp\u003eWe then evaluated the role of Foxo1 in tTreg cell generation. Conventional CD4 T cells (Tconv), CD25\u003csup\u003e+\u003c/sup\u003e TregP, and Foxp3\u003csup\u003elo\u003c/sup\u003e TregP \u003cem\u003e(1, 2)\u003c/em\u003e were analyzed, together with CD25\u003csup\u003e+\u003c/sup\u003e tTreg cells, which were further subdivided based on CD73 surface expression \u003cem\u003e(39)\u003c/em\u003e into neo-tTreg (CD73\u003csup\u003e-\u003c/sup\u003e) and recirculating-tTreg (CD73\u003csup\u003e+\u003c/sup\u003e) cells.\u003c/p\u003e \u003cp\u003eWhile the percentage of Foxp3\u003csup\u003elo\u003c/sup\u003e TregP was slightly increased in 1-week-old but not in 1-month-old Foxo1\u003csup\u003eTKO\u003c/sup\u003e compared to age-matched Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice (fig. S3A), the absolute numbers of Tconv, CD25\u003csup\u003e+\u003c/sup\u003e and Foxp3\u003csup\u003elo\u003c/sup\u003e TregPs as well as neo-tTreg cells were comparable in the thymus of both 1-week-old (Fig.\u0026nbsp;3A) and 1-month-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e or Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (fig. S3B). Strikingly, explaining the quantitative tTreg cell defect initially published \u003cem\u003e(33)\u003c/em\u003e, proportions and absolute numbers of recirculating-tTreg cells were greatly reduced in Foxo1\u003csup\u003eTKO\u003c/sup\u003e compared to Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice (Fig.\u0026nbsp;3B and fig. S3B). To confirm these observations, we generated mixed bone marrow (BM) chimeras by co-transferring Foxo1\u003csup\u003eCtrl\u003c/sup\u003e (CD45.1\u003csup\u003e+\u003c/sup\u003e) BM cells together with BM cells (CD45.2\u003csup\u003e+\u003c/sup\u003e) isolated from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice into lethally irradiated T-cell deficient CD3ε recipient mice. In this competitive setting, both the normal generation of neo-tTreg cells from Foxo1\u003csup\u003eKO\u003c/sup\u003e precursor cells (Fig.\u0026nbsp;3C) and the defective recirculation of Foxo1\u003csup\u003eKO\u003c/sup\u003e Treg cells within the thymus (Fig.\u0026nbsp;3D) were reproduced. These results indicate that Foxo1 is critical for the recirculation of Treg cells to the thymus, while being largely dispensable for their initial thymic development.\u003c/p\u003e \u003cp\u003eAs Foxo1 appeared to have only a limited impact on neo-tTreg cell generation, we next investigated whether the apparent loss of Nrp1\u003csup\u003e+\u003c/sup\u003e tTreg-like cells in SLOs from Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice could result from defective cell maturation and/or thymic export. Semi-mature (SM), Mature 1 (M1) and Mature 2 (M2) maturation stages of CD4 single positive (SP) thymocytes were defined based on MHC class I (MHCI), CD24 and CD69 expression (Fig.\u0026nbsp;3E), as previously described \u003cem\u003e(40)\u003c/em\u003e. Consistent with their position along tTreg cell differentiation trajectory, the proportion of SM cells progressively decreased from CD4 Tconv to CD25\u003csup\u003e+\u003c/sup\u003e TregP cells and Foxp3\u003csup\u003elo\u003c/sup\u003e TregP cells, and was virtually absent among neo-tTreg cells. Overall, the analysis of neo-tTreg cells and their precursors did not reveal any major maturation defects in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig.\u0026nbsp;3E), suggesting either that Foxo1 is not essential for tTreg cell production or that residual Foxo1 protein expression after gene deletion at the CD4\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e double-positive stage is sufficient to sustain this process. Thymic export of CD4 Tconv and Treg cells was assessed in SLOs 16 h after intra-thymic injection of fluorescein. Consistent with their altered expression of the homing molecules CD62L and CCR7 \u003cem\u003e(23, 41)\u003c/em\u003e, the numbers of FITC\u003csup\u003e+\u003c/sup\u003e CD4 Tconv and Treg cells were significantly lower in the LNs of Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice compared to Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice (Fig.\u0026nbsp;3F). However, this analysis did not reveal any major defect of the ability of Foxo1\u003csup\u003eTKO\u003c/sup\u003e CD4 Tconv and Treg cells to reach the spleen, nor did it reveal a greater defect in the colonization of LNs by Foxo1\u003csup\u003eKO\u003c/sup\u003e Treg cells compared to Foxo1\u003csup\u003eKO\u003c/sup\u003e Tconv cells. Both populations exhibited an almost 7-fold reduction in LN colonization compared to their Foxo1\u003csup\u003eWT\u003c/sup\u003e counterparts (Fig.\u0026nbsp;3F). When pooled LNs and spleen FITC\u003csup\u003e+\u003c/sup\u003e CD4 Tconv or Treg cells were analyzed, this reduction amounted to 2.4-fold and 2.7-fold, respectively (fig. S3C).\u003c/p\u003e \u003cp\u003eThus, the lack of Nrp1\u003csup\u003e+\u003c/sup\u003e tTreg-like cells in SLOs from young Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig.\u0026nbsp;2A-E) cannot be attributed to a role of Foxo1 in the early development, maturation, or thymic export of tTreg cells.\u003c/p\u003e\n\u003ch3\u003eFoxo1 controls perinatally generated tTreg cell seedings into SLOs\u003c/h3\u003e\n\u003cp\u003eBecause tTreg cells effectively differentiate, mature and reach the periphery (Fig.\u0026nbsp;3) but fail to accumulate in SLOs of young Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig.\u0026nbsp;2A-E), we hypothesized that Foxo1 might play a critical role in their seeding in SLOs during the perinatal period. A semi-supervised flow cytometry analysis of spleen CD4\u003csup\u003e+\u003c/sup\u003e T cells identified distinct clusters of CD4 Tconv and Treg cells in 1-week-old Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e perinates (Fig.\u0026nbsp;4A). Among these, two clusters were associated with Treg cells, the c1 cluster corresponding to resting-like Treg cells expressing low-levels of CD25, Nrp1, CD44, and Helios compared to the c2 cluster expressing high-levels of these proteins (Fig.\u0026nbsp;4A). Genotype-based separation revealed significant changes in the distribution of Tconv and Treg cells in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice compared to Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice. Specifically, Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice showed a marked reduction in the percentage of Treg cells among CD4 T cells as well as in their absolute numbers (Fig.\u0026nbsp;4B, C). Furthermore, the distribution of Treg cell clusters was drastically altered in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, with a 2-fold reduction in the proportion of c2 Treg cells among Treg cells compared to their Foxo1\u003csup\u003eCtrl\u003c/sup\u003e counterparts (Fig.\u0026nbsp;4D). Notably, while the absolute numbers of c1 resting-like Treg cells remained unchanged in the spleens of Foxo1\u003csup\u003eTKO\u003c/sup\u003e perinates, the absolute numbers of c2 Treg cells\u0026mdash;which represent 80% of Treg cells in the spleen of Foxo1\u003csup\u003eCtrl\u003c/sup\u003e perinates\u0026mdash;were reduced by tenfold in Foxo1\u003csup\u003eTKO\u003c/sup\u003e perinates (Fig.\u0026nbsp;4D). Further analysis of the c2 Treg cell cluster revealed that, consistent with its activated phenotype, it corresponds to a proliferation-prone neonatal Treg cell subset, as evidenced by the high proportion of Ki67\u003csup\u003e+\u003c/sup\u003e cells within this cluster (Fig.\u0026nbsp;4E). Notably, the few cells belonging to this cluster in Foxo1\u003csup\u003eTKO\u003c/sup\u003e perinates displayed significantly lower levels of Ki67\u003csup\u003e+\u003c/sup\u003e cells compared to their WT counterparts (Fig.\u0026nbsp;4F). This proliferative defect in Treg cells from Foxo1\u003csup\u003eTKO\u003c/sup\u003e perinates was further substantiated by cell cycle analysis combining Ki67 staining with DNA content measurement (Fig.\u0026nbsp;4G, fig. S4A). Of note, in young adult mice, Foxo1-deficient Treg cells do not display any proliferation defect (fig. S4B).\u003c/p\u003e \u003cp\u003eAltogether, these findings underscore a pivotal role for Foxo1 in sustaining Treg cell proliferation specifically within perinatal SLOs.\u003c/p\u003e\n\u003ch3\u003eFoxo1 Sets IL-2 Sensitivity by Controlling IL-2 Receptor Beta Chain Expression in Developing tTreg Cells\u003c/h3\u003e\n\u003cp\u003eBecause Foxo1 is critical for the expansion of perinatal tTreg cells in SLOs when reaching the periphery, we investigated whether Foxo1 regulates specific transcriptional programs during tTreg differentiation that could account for their defective homeostasis. To this end, we reanalyzed previously published chromatin accessibility data \u003cem\u003e(11)\u003c/em\u003e obtained by ATAC-seq from thymic immature CD4SP, CD25\u003csup\u003e+\u003c/sup\u003e TregP, Foxp3\u003csup\u003elo\u003c/sup\u003e TregP and tTreg cells to determine the kinetics of chromatin opening during tTreg cell differentiation. Additionally, we leveraged Foxo1 ChIPseq data \u003cem\u003e(42)\u003c/em\u003e to infer Foxo1 DNA-binding motif and perform Foxo1 footprint analysis throughout this process. Finally, we integrated these findings with RNA-seq data comparing immature CD4SP and tTreg cells \u003cem\u003e(11)\u003c/em\u003e (Fig.\u0026nbsp;5A).\u003c/p\u003e \u003cp\u003eChromatin accessibility profiling identified six distinct dynamic clusters corresponding to regions that either progressively, transiently, or rapidly closed (closing clusters, c.c1 and c.c2; 43.2%) or opened (56.8%, opening clusters, o.c1-4) upon ImmCD4SP differentiation into tTreg cells (Fig.\u0026nbsp;5B). To assess the contribution of Foxo1 to these chromatin dynamics, we identified a Foxo1 consensus motif (fig. S5A) from recently published Foxo1 ChIP-seq data \u003cem\u003e(42)\u003c/em\u003e and used it to infer global and Foxo1-associated footprints throughout tTreg cell differentiation (fig. S5B). Global footprint analysis revealed that both the consensus and ChIP-seq-predicted Foxo1 motifs clustered with transcription factors that bind chromatin sequentially along the tTreg differentiation trajectory, with maximal occupancy in immCD4SP cells and later in Foxp3\u003csup\u003elo\u003c/sup\u003e TregP (fig. S5B).\u003c/p\u003e \u003cp\u003eAs expected, histograms of mean normalized Tn5 insertion frequencies around Foxo1 motifs classified as \u0026ldquo;bound\u0026rdquo; showed a depletion of insertions at the motif center, with a gradual increase toward the flanking regions (fig. S5C). Consistent with its reported pioneer activity and its preferential nuclear localization, the average footprint profile around this motif exhibited only modest variation across populations (fig. S5C-D, left). In contrast, population-specific analyses uncovered discrete chromatin regions that either lost or gained Foxo1 footprints at defined stages of tTreg differentiation (fig. S5C-D, right).\u003c/p\u003e \u003cp\u003eAlthough closing and opening chromatin regions were globally balanced during tTreg cell differentiation, Foxo1 footprint\u0026ndash;associated regions were significantly enriched among opening clusters, particularly o.c1 and o.c3 (Fig.\u0026nbsp;5C). Notably, the opening of these regions followed the progressive increase in Foxo1 footprint scores (Fig.\u0026nbsp;5D). Conversely, few Foxo1 footprint\u0026ndash;associated regions that closed during differentiation exhibited a concomitant decrease in Foxo1 footprint scores, suggesting that Foxo1 predominantly acts as a chromatin-opening factor during tTreg cell differentiation (Fig.\u0026nbsp;5D). Consistently, genes linked to Foxo1 footprints within opening chromatin regions were upregulated in tTreg cells, whereas those associated with closing regions remained largely unchanged (Fig.\u0026nbsp;5E).\u003c/p\u003e \u003cp\u003eTo further substantiate Foxo1 activity during thymic development of tTreg cells, we integrated and reanalyzed two scRNA-seq datasets generated from either sorted thymic Treg precursors and progenitors, as well as mature and recirculating tTreg cells \u003cem\u003e(43)\u003c/em\u003e or purified Treg cells \u003cem\u003e(44)\u003c/em\u003e (fig. S6A). This analysis, enabled us to perform Gene Regulatory Network inference using SCENIC \u003cem\u003e(45)\u003c/em\u003e thereby identifying transcription factor activities across the tTreg cell differentiation trajectory (fig. S6B). Consistently, regulons governed by STAT5a peaked at the CD25\u003csup\u003e+\u003c/sup\u003e TregP stage, whereas those controlled by both Foxo1 and Foxp3 gradually increased from the transitional Treg stage to mature neo-Treg cells (fig. S6C).\u003c/p\u003e \u003cp\u003eStrikingly, Gene Set Enrichment Analysis (GSEA) of these dynamic chromatin regions, Foxo1-bound sites, and differentially expressed genes revealed a strong enrichment for IL-2/STAT5 signaling\u0026ndash;related genes (Fig.\u0026nbsp;5F), the majority of which displayed concomitant chromatin opening and transcriptional upregulation during the transition from immCD4SP to tTreg cells (Fig.\u0026nbsp;5G). Notably, among these tTreg cell signature genes - defined by the presence of Foxo1 ChIP-seq peaks together with dynamic Foxo1 footprints associated with increasing chromatin accessibility throughout tTreg cell differentiation - included genes encoding IL-10, ST2, 4-1BB, TNFR2, CTLA-4, and HOPX. Importantly, the gene encoding the IL-2 receptor beta chain (Il2rb), a key regulator of tTreg cell generation, was also part of this set of Foxo1-regulated genes (Fig.\u0026nbsp;5G).\u003c/p\u003e \u003cp\u003eConsistent with this observation, time-course analysis of ATAC-seq, Foxo1 ChIP-seq and footprinting data at the \u003cem\u003eIl2rb\u003c/em\u003e locus demonstrated a progressive increase in chromatin accessibility and Foxo1 footprint depth at a Foxo1-bound site within the \u003cem\u003eIl2rb\u003c/em\u003e promoter during tTreg cell differentiation (Fig.\u0026nbsp;5H).\u003c/p\u003e \u003cp\u003eImportantly, these findings were corroborated \u003cem\u003ein vivo\u003c/em\u003e at the protein level. IL2Rβ expression on the surface of tTreg cells and their immediate Foxp3\u003csup\u003elo\u003c/sup\u003e Treg precursors was significantly reduced in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig.\u0026nbsp;5I). To further support the role of Foxo1 as a transcription factor regulating IL2Rβ expression during tTreg cell differentiation, we generated an \u003cem\u003ein vitro\u003c/em\u003e two-step tTreg cell generation assay using WT thymic CD4SP T cells \u003cem\u003e(46)\u003c/em\u003e, and compared IL2Rβ expression in the presence or absence of a Foxo1 inhibitor (Fig.\u0026nbsp;5J). Strikingly, Foxp3\u003csup\u003e+\u003c/sup\u003e cells generated in the presence of the inhibitor exhibited markedly reduced surface IL-2Rβ expression (Fig.\u0026nbsp;5J).\u003c/p\u003e \u003cp\u003eLastly, consistent with the reduced IL-2Rb expression, we found, in CD25-expressing TregP and neo-tTreg cells analyzed \u003cem\u003eex vivo\u003c/em\u003e, that STAT5 phosphorylation/activation is impaired when Foxo1 is lost, indicating a weakened IL-2R pathway (Fig.\u0026nbsp;5K). Notably, this reduced IL-2Rβ expression in Foxo1-deficient Treg cells persisted in the SLOs of perinates (fig. S6D).\u003c/p\u003e \u003cp\u003eAltogether, Foxo1 appears to regulate IL-2 receptor β chain expression on developing tTreg cells (Fig.\u0026nbsp;5) and Foxo1\u003csup\u003eTKO\u003c/sup\u003e perinates exhibit a drastic reduction in nnTreg cell numbers, particularly within the c2 cluster, which is characterized by high expression of both the IL-2 receptor α chain and Nrp1 (Fig.\u0026nbsp;4).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIL-2 signaling shapes nnTreg cell compartment in the periphery\u003c/h2\u003e \u003cp\u003eTo determine the role of IL-2 in Treg cell seeding into SLOs in Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e perinates, we performed a semi-supervised flow cytometry analysis of spleen CD4\u003csup\u003e+\u003c/sup\u003e T cells from pooled Foxo1\u003csup\u003eCtrl\u003c/sup\u003e (treated or not with IL-2 or anti-IL-2 blocking antibodies) and Foxo1\u003csup\u003eTKO\u003c/sup\u003e perinates (treated or not with IL-2). We identified the same CD4 Tconv and Treg cell clusters as previously described (Fig.\u0026nbsp;4A) in 1-week-old perinates (Fig.\u0026nbsp;6A). While IL-2 treatment significantly increased the proportion of nnTreg cells among CD4⁺ T cells in both Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, absolute numbers were not significantly affected (Fig.\u0026nbsp;6B and fig. S7A, B). Conversely, IL-2 blockade in Foxo1\u003csup\u003eCtrl\u003c/sup\u003e perinates markedly reduced both the frequency and absolute number of nnTreg cells (Fig.\u0026nbsp;6B, C and fig. S7A).\u003c/p\u003e \u003cp\u003eCrucially, and in line with the nnTreg cell composition of Foxo1\u003csup\u003eTKO\u003c/sup\u003e perinates, IL-2 modulation also altered the distribution between the c1 and c2 nnTreg cell subsets. Indeed, blocking IL-2 in Foxo1\u003csup\u003eCtrl\u003c/sup\u003e perinates drastically reduced both percentages of c2 Treg cells among nnTreg cells and absolute numbers of this subset (Fig.\u0026nbsp;6C). Conversely, increasing IL-2 availability in Foxo1\u003csup\u003eTKO\u003c/sup\u003e perinates significantly increased both percentages of c2 Treg cells among nnTreg cells and their absolute number (Fig.\u0026nbsp;6D). Consistent with their low expression of the IL-2R α chain of the IL-2 receptor, numbers of c1 nnTreg cells were largely insensitive to IL-2 modulation (Fig.\u0026nbsp;6C, D).\u003c/p\u003e \u003cp\u003eTogether, these data indicate that IL-2 availability selectively controls the establishment of the nnTreg cell compartment in the periphery by sustaining the expansion of the IL-2\u0026ndash;responsive c2 subset. This IL-2\u0026ndash;dependent bias recapitulates the nnTreg cell imbalance observed in Foxo1-deficient perinates, thereby positioning IL-2 signaling downstream of Foxo1 as a critical determinant of early Treg cell seeding into SLOs.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study highlights the pivotal role of Foxo1 in maintaining Treg cell homeostasis. In contrast to previous reports suggesting that Foxo1 broadly orchestrates the differentiation of both thymic-derived (tTreg) and peripheral (pTreg) cell subsets \u003cem\u003e(33, 34)\u003c/em\u003e, our findings support a more refined model. Rather than functioning as a universal driver of tTreg or pTreg cell differentiation, Foxo1 appears to be specifically required for sustaining tTreg homeostasis, acting primarily by enhancing IL-2 responsiveness during the perinatal period and supporting the expansion of newly generated neonatal Tregs (nnTregs).\u003c/p\u003e \u003cp\u003eOur findings reconcile discrepancies from previous studies on the role of Foxo1 in Treg cell homeostasis and further reveal previously unrecognized functions of this transcription factor. In tTreg cells, it was previously reported that Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice exhibit a significant reduction in tTreg cell numbers at 3\u0026ndash;8 weeks of age compared with Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice \u003cem\u003e(33)\u003c/em\u003e. At that time, however, the capacity of Treg cells to recirculate to the thymus and modulate tTreg cell production was not fully appreciated \u003cem\u003e(39)\u003c/em\u003e. Our results demonstrate that Foxo1 intrinsically regulates this recirculation process, providing a potential explanation for the apparent reduction in tTreg cells observed in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice. By 4 weeks of age, recirculating Treg cells account for approximately one quarter of total thymic Treg cells in Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice, whereas they are virtually absent in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (fig. S3B). The molecular mechanisms underlying Treg cell recirculation to the thymus remain incompletely understood. While integrin α4\u0026ndash;VCAM-1 interactions \u003cem\u003e(47)\u003c/em\u003e and chemokine receptors such as CXCR4 and CCR6 \u003cem\u003e(48, 49)\u003c/em\u003e have been proposed to be involved in this process, CCR7 has instead been suggested to act as a negative regulator \u003cem\u003e(50)\u003c/em\u003e. In light of the profound impairment of thymic re-entry observed in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, and given the established role of Foxo1 in regulating CCR7, CD62L, and integrin α4 expression (all of which are downregulated in its absence \u003cem\u003e(24)\u003c/em\u003e), our findings support a key contribution of integrin α4 and suggest CD62L as an additional mediator of Treg cell recirculation, whereas a dominant role for CCR7 appears unlikely.\u003c/p\u003e \u003cp\u003eA major caveat in interpreting the phenotype of Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice lies in the early-onset inflammation that arises in these animals \u003cem\u003e(23, 24)\u003c/em\u003e. This inflammation, characterized by elevated inflammatory cytokine levels including IL-2, may secondarily alter both peripheral Treg cell homeostasis and their thymic development. Indeed, systemic inflammation is known to profoundly affect Treg cell proliferation, phenotype, and stability in secondary lymphoid organs, and can similarly influence tTreg cell development in the thymus \u003cem\u003e(1, 2, 51\u0026ndash;53)\u003c/em\u003e. A key strength of our study is that, in addition to analyses in young adult (4-week-old) and adult (12-week-old) mice, we also investigated the perinatal period by analyzing 1-week-old mice. At this early stage, recirculating tTreg cells, which accumulate rapidly after 3\u0026ndash;4 weeks in the thymus of Foxo1\u003csup\u003eCtrl\u003c/sup\u003e mice, remain marginal, and Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice show little to no evidence of inflammation. Therefore, the differences observed in Treg cell numbers and phenotype at this early time point are most likely to reflect direct, cell-intrinsic consequences of Foxo1 deficiency rather than indirect effects of systemic inflammation.\u003c/p\u003e \u003cp\u003eAlthough we find that Foxo1 appears dispensable for the development of both tTreg and pTreg cells (Fig.\u0026nbsp;2H and Fig.\u0026nbsp;3A-E), its role in modulating the IL-2 sensitivity of nnTreg cells and supporting their initial expansion may profoundly impact both the homeostasis of the overall Treg cell compartment and the microenvironment governing their generation and maintenance. CNS1-dependent pTreg cells have been shown to limit the late onset of allergic and asthma-like inflammation in mucosal tissues \u003cem\u003e(10)\u003c/em\u003e and to contribute to maternal-fetal tolerance during pregnancy \u003cem\u003e(9)\u003c/em\u003e. Our study broadens the functional scope of this extrathymically induced Treg cell subset by demonstrating its capacity to mitigate autoimmunity in genetically predisposed contexts. Specifically, we show that Foxo1\u003csup\u003eTKO\u003c/sup\u003e CNS1\u003csup\u003eKO\u003c/sup\u003e mice develop a markedly more severe disease than Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, characterized by a significant weight loss, an exacerbated infiltration of mucosal tissues such as the gut and lungs, and a widespread activation of conventional T cells (Fig.\u0026nbsp;1A-G and S1A-F). Furthermore, while the loss of CNS1-dependent pTreg cells has minimal impact on Treg cell numbers and only marginally alters the overall Treg cell composition in a WT background (as observed in heterozygous Foxp3\u003csup\u003eCNS1KO/WT\u003c/sup\u003e females, likely due to compensation by thymic-derived Treg cells), this subset appears to occupy a substantial fraction of the Treg cell niche in the secondary lymphoid organs of Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice (Fig.\u0026nbsp;1G-I, and fig. S1F, G).\u003c/p\u003e \u003cp\u003eSuch a role for pTreg cells in maintaining immune tolerance in Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice was unexpected, as Foxo1-deficient CD4 T\u003csub\u003eN\u003c/sub\u003e cells were initially reported to be severely impaired in their ability to differentiate into iTreg cells in response to TGF-β \u003cem\u003e(33)\u003c/em\u003e. However, in those early studies, the cells used in the \u003cem\u003ein vitro\u003c/em\u003e polarization assays were not \u003cem\u003ebona fide\u003c/em\u003e CD4 T\u003csub\u003eN\u003c/sub\u003e (CD44\u003csup\u003elo\u003c/sup\u003e CD25\u003csup\u003e-\u003c/sup\u003e Foxp3-GFP\u003csup\u003e-\u003c/sup\u003e) cells, but rather a broader population of conventional CD4 T cells (CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e-\u003c/sup\u003e CD69\u003csup\u003e-\u003c/sup\u003e), which likely included memory-like CD4 T cells. Moreover, neither IL-4 nor IFN-γ were neutralized during the assays. Our recent data \u003cem\u003e(38)\u003c/em\u003e demonstrate that upon stimulation, Foxo1-deficient CD4 T\u003csub\u003eN\u003c/sub\u003e cells produce high levels of both cytokines, which may strongly interfere with iTreg cell polarization as previously described \u003cem\u003e(36, 54)\u003c/em\u003e. In particular, IFN-γ not only inhibits Foxp3 induction during iTreg cell polarization, but also actively promotes iTreg cell instability, thereby preventing the establishment and maintenance of a stable regulatory phenotype \u003cem\u003e(55)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn neonates, the first mature thymocytes that migrate to the periphery encounter a lymphoid environment devoid of preexisting T cells and therefore undergo vigorous proliferation in response to TCR engagement by self-peptide/self-MHC ligands \u003cem\u003e(56\u0026ndash;58)\u003c/em\u003e. In addition, the proportion of Treg cells in neonates is lower than in adult mice \u003cem\u003e(59\u0026ndash;61)\u003c/em\u003e, which may limit their capacity to effectively suppress effector cells. Furthermore, the peripheral T cell compartment in neonates is dominated by recent thymic emigrants (RTEs) \u003cem\u003e(62)\u003c/em\u003e, which exhibit phenotypic and functional properties distinct from their adult counterparts \u003cem\u003e(63, 64)\u003c/em\u003e, including an enhanced ability to produce IL-2 \u003cem\u003e(64)\u003c/em\u003e. Consistent with this, neonatal secondary lymphoid organs show higher levels of IL-2 production by CD4 and CD8 T cells compared to those of adults \u003cem\u003e(65)\u003c/em\u003e. This unique immunological context is therefore likely to play a critical role in shaping the nnTreg cell compartment. Our results strongly support a model in which Foxo1 regulates the initial seeding and expansion of perinatally generated tTreg cells into SLOs, primarily by modulating IL-2Rβ expression on developing tTreg cells (Fig.\u0026nbsp;5). Reduced surface expression of IL-2Rβ in Foxo1-deficient neo-tTreg cells is associated with diminished STAT5 phosphorylation compared to their wild-type counterparts (Fig.\u0026nbsp;5I, J and K). Consequently, impaired IL-2 sensing limits the seeding, activation, and proliferation of nnTreg cells within perinatal SLOs (Fig.\u0026nbsp;4E, G and fig. S4). Mechanistically, the role of Foxo1 in promoting IL-2Rβ expression may rely not only on its direct activity as a transcription factor but also on its potential pioneer function in opening chromatin to facilitate the binding of additional regulators \u003cem\u003e(32)\u003c/em\u003e. Indeed, \u003cem\u003eIl2rb\u003c/em\u003e transcription has been shown to depend on multiple transcription factors, including Ets1, Gabp, Sp1, Egr1, and Stat5 \u003cem\u003e(66\u0026ndash;68)\u003c/em\u003e. Thus, Foxo1 binding to the \u003cem\u003eIl2rb\u003c/em\u003e promoter region (Fig.\u0026nbsp;5H) could either directly initiate \u003cem\u003eIl2rb\u003c/em\u003e transcription during the transition from CD25\u003csup\u003e+\u003c/sup\u003e TregP to Foxp3\u003csup\u003elo\u003c/sup\u003e TregP cells, or alternatively render the locus accessible to other transcription factors. The latter possibility is particularly appealing, given that Stat5 phosphorylation, indicative of its activation, is already detected at the CD25⁺ TregP stage, making it a strong candidate for cooperating with Foxo1 (Fig.\u0026nbsp;5K).\u003c/p\u003e \u003cp\u003eA link between Foxo1 and IL-2Rβ expression has previously been reported in CD4 T cells \u003cem\u003e(69)\u003c/em\u003e. However, in that study, Newton \u003cem\u003eet al.\u003c/em\u003e did not examine the consequences of Foxo1 deficiency but studied the effects of constitutive Foxo1 activation through expression of a non-phosphorylatable Foxo1A3 mutant. Interestingly, despite these opposite manipulations of Foxo1 activity, both settings led to reduced surface IL-2Rβ expression. In the case of Foxo1A3, this reduction occurred independently of \u003cem\u003eIl2rb\u003c/em\u003e transcription and was attributed instead to impaired receptor recycling via the endocytic\u0026ndash;lysosomal pathway \u003cem\u003e(69)\u003c/em\u003e. Together, these findings suggest that Foxo1 fine-tunes IL-2Rβ signaling at multiple levels, integrating both transcriptional and post-transcriptional mechanisms. Such multilayered regulation of IL-2 sensitivity may be particularly critical during the perinatal window, when tTreg cells must efficiently sense IL-2 to undergo expansion and establish a stable pool in SLOs (Fig.\u0026nbsp;6 and Fig. S7). By modulating IL-2Rβ expression dynamics, Foxo1 ensures that developing tTreg cells reach the activation threshold required for their survival and proliferation, while preventing aberrant activation or loss of lineage stability. This mechanism may thus represent a key safeguard for the establishment of robust Treg-mediated tolerance early in life.\u003c/p\u003e \u003cp\u003eBeyond the superior suppressive capacity of nnTreg cells, their repertoire enriched in self-reactive specificities compared to Treg cells generated later in life \u003cem\u003e(4)\u003c/em\u003e, and their critical role in preventing autoimmunity during adulthood \u003cem\u003e(4, 70)\u003c/em\u003e, a defining characteristic of nnTreg cells is their remarkable ability to colonize peripheral tissues \u003cem\u003e(71)\u003c/em\u003e. A major strength of our study lies in the use of Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice, which allow a comprehensive assessment of Foxo1's role not only in the generation of distinct Treg cell subsets but also in their early seeding and homeostasis within SLOs during the perinatal window. However, elucidating the full pathophysiological impact of Foxo1 deficiency is complicated by the pleiotropic roles of this transcription factor in both Treg and conventional T cell biology. Foxo1 orchestrates T cell trafficking into lymphoid and non-lymphoid tissues \u003cem\u003e(23)\u003c/em\u003e, prevents conventional T cell exhaustion \u003cem\u003e(41)\u003c/em\u003e, and promotes several core suppressive mechanisms in Treg cells \u003cem\u003e(33, 34)\u003c/em\u003e. Our findings reveal that disruption of Foxo1 activity profoundly impairs Treg cell seeding and expansion during the perinatal window, with potential long-term consequences for immune homeostasis. Future studies should investigate how early-life perturbations in Foxo1 signaling or Treg cell seeding influence the establishment of immune tolerance and tissue homeostasis later in life.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eMice\u003c/h2\u003e\n \u003cp\u003eOne to 12-weeks-old mice were used for experiments unless otherwise indicated. C57BL/6 CD3\u0026epsilon;\u003csup\u003eKO\u003c/sup\u003e \u003cem\u003e(72)\u003c/em\u003e, C57BL/6 Foxp3 -GFP Foxo1\u003csup\u003eCtrl\u003c/sup\u003e and Foxo1\u003csup\u003eTKO\u003c/sup\u003e CD45.1 or CD45.2 \u003cem\u003e(23, 27)\u003c/em\u003e. C57BL/6 Foxp3-GFP CNS1\u003csup\u003eKO\u003c/sup\u003e mice were generated using the CRISPR/Cas9 system in collaboration with the MOUSTIC genome engineering platform (Cochin Institute, Paris, France). Three pairs of gRNAs targeting flanking regions of the CNS1 enhancer within the Foxp3 locus were co-injected into C57BL/6 Foxp3-GFP zygotes:\u003c/p\u003e\n \u003cp\u003e\u0026bull; gRNA pair 1: 5\u0026prime;-GAAGACATACACCACCACGG-3\u0026prime; and 3\u0026prime;-AATTTTGCATAGAGAGATCA-5\u0026prime;\u003c/p\u003e\n \u003cp\u003e\u0026bull; gRNA pair 2: 5\u0026prime;-TAGATTACTCTTTTCTTGTG-3\u0026prime; and 3\u0026prime;-CTACCATCCACGAGTCGTGT-5\u0026prime;\u003c/p\u003e\n \u003cp\u003e\u0026bull; gRNA pair 3: 5\u0026prime;-CGGCGGGCAATCACTTGCTT-3\u0026prime; and 3\u0026prime;-TACTGTCGCTGTAAAGTTCA-5\u0026prime;\u003c/p\u003e\n\n\u003c/div\u003e \u003cp\u003eFounder animals were genotyped and validated by PCR and Sanger sequencing using the following primers: 5\u0026prime;-GGGGAAAATAAAGTGACTGG-3\u0026prime; and 5\u0026prime;-ACAAGGTCTCACTCTATAG-3\u0026prime;. Founders carrying a confirmed 506 bp deletion within the CNS1 region were selected and backcrossed to C57BL/6 Foxp3-GFP mice for at least three generations to reduce potential off-target effects. Finally, C57BL/6 Foxp3-GFP Foxo1\u003csup\u003eTKO\u003c/sup\u003e CNS1\u003csup\u003eKO\u003c/sup\u003e were generated by crossing our C57BL/6 Foxp3-GFP CNS1\u003csup\u003eKO\u003c/sup\u003e mice with C57BL/6 Foxp3-GFP Foxo1\u003csup\u003eTKO\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eAll mice were maintained in our own animal facilities, under specific pathogen\u0026ndash;free (SPF) condition. All procedures performed were approved by the ethics committee for animal experimentation (n\u0026deg;APAFIS #20630, #22356) and validated by the \u0026ldquo;Minist\u0026egrave;re de l\u0026rsquo;Enseignement Sup\u0026eacute;rieur de la Recherche et de l'Innovation\u0026rdquo;. Sample sizes were chosen to assure reproducibility of the experiments and in accordance with the 3R rules of animal ethics regulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell suspensions\u003c/h2\u003e \u003cp\u003eAdult mice were euthanized by cervical dislocation, neonates by decapitation. Peripheral lymph nodes (pLNs: pooled cervical, axillary, brachial, inguinal), mesenteric LNs (mLNs), spleen, and thymus were harvested, homogenized, and passed through a nylon cell strainer (BD Falcon). Cells were resuspended in RPMI 1640 GlutaMAX with 10% FCS (Biochrom) for adoptive transfer or culture, or in PBS with 5% FCS and 0.1% NaN3 (Interchim) for flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eCell surface and intracellular staining\u003c/h2\u003e \u003cp\u003eCell suspensions were collected as described in \u0026ldquo;cell suspension\u0026rdquo; item and dispensed into 96-well round-bottom microtiter plates (Greiner Bioscience; 6 \u0026times; 106 cells/well). Surface staining was performed as described \u003cem\u003e(16)\u003c/em\u003e. Antibodies (Abs) are listed in Supplementary Table S1. Briefly, cells were incubated on ice, for 15 minutes per step, with Abs in 5% FCS (Eurobio Scientific), 0.1% NaN3 (Sigma-Aldrich) in PBS. Each cell staining reaction was preceded by Fc receptor blocking (anti-CD16/32, 2.4G2, BioXcell) and viability staining (LeaDead, Life Technologies). The Foxp3 Staining Buffer Set (eBioscience) was used for Foxp3, Ki67, Hoechst, Helios, Tbet, Gata3, and Rorγt intracellular staining. To assess pSTAT5 levels ex vivo, cells were immediately fixed in 4% PFA for 5 min at 37\u0026deg;C. Cells were then washed and permeabilized by adding ice-cold 100% methanol to a final concentration of 90% methanol and incubated for at least 30 min at -20\u0026deg;C. After extensive washing, the cells were stained overnight at 4\u0026deg;C with surface and intracellular antibodies.\u003c/p\u003e \u003cp\u003eFor Cell suspensions from heterozygote mice (GFP+/GFP-), surface staining was performed as previously described. Cells were then fixed with 2% PFA for 10 min at room temperature, washed and stained for Foxp3 expression using Foxp3 Staining Buffer Set (eBioscience).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis\u003c/h2\u003e \u003cp\u003eData were acquired on a BD LSRFortessa\u0026trade; cytometer (BD Biosciences) at the Cochin CYBIO facility. List-mode data files were analyzed using FlowJo software V10_10 (BD Biosciences). For unsupervised flow cytometry analysis \u0026ldquo;FCS files\u0026rdquo; were imported into R (v4.4.1) and processed using relevant packages including \u0026ldquo;flowCore, flowWorkspace, FlowSOM, Harmony, umap2,\u0026rdquo; and \u0026ldquo;ggplot2\u0026rdquo;. Briefly, \u0026ldquo;FCS files\u0026rdquo; were imported, logicle-transformed, and harmonized to ensure comparability across samples. Metadata such as \u0026ldquo;genotype\u0026rdquo;, \u0026ldquo;batch\u0026rdquo;, and \u0026ldquo;tube ID\u0026rdquo; were integrated into the expression matrix to enable accurate annotation and stratification of cellular subsets. To control for sampling bias, cells were randomly downsampled in a balanced manner across genotypes and conditions. Batch effects were corrected using the \u0026ldquo;Harmony\u0026rdquo; algorithm. Dimensionality reduction was performed using supervised UMAP (sUMAP) approaches. Clustering of cells was achieved using the \u0026ldquo;FlowSOM\u0026ldquo; algorithm on the batch-corrected and transformed data. Identified clusters were grouped into meta-clusters based on phenotypic marker expression and manually annotated. Relative frequencies of each cell subset were computed per sample and represented as pie charts stratified by genotype and batch. Heatmaps were generated to visualize average marker expression across meta-clusters, providing a phenotypic signature of each T cell population. Final processed datasets and clustering results were exported in CSV format and subsequently used for graphical representation and statistical analysis using GraphPad Prism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIL2 and anti-IL2 treatment\u003c/h2\u003e \u003cp\u003eNeonatal mice received intraperitoneal injections of 40,000 IU/g recombinant human IL-2 (Novartis) or anti-IL-2 antibodies (S4B6 and JES6; BioXCell) according to the following schedule: IL-2 on postnatal days 1, 4, 6; anti-IL-2 at 12.5 \u0026micro;g/g on day 1, and 25 \u0026micro;g/g on days 4 and 6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBone Marrow Chimeras\u003c/h2\u003e \u003cp\u003eCD3εKO mice were lethally irradiated (9.5 Gy) and reconstituted via intravenous injection with 5 \u0026times; 10⁶ T cell\u0026ndash;depleted bone marrow (BM) cells (80% Foxo1TKO CD45.2\u0026thinsp;+\u0026thinsp;20% Foxo1Ctrl CD45.1). T cell depletion used anti-CD4 and anti-CD8 antibodies followed by anti-rat IgG magnetic beads (Dynal Biotech).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIntrathymic injection of FITC\u003c/h2\u003e \u003cp\u003e10 \u0026micro;l of FITC (5 mg/ml in PBS, Sigma-Aldrich) was injected into each thymic lobe under ultrasound guidance using a Vevo 2100 high resolution ultrasound machine (Visualsonics, Toronto, Canada) equipped with a 40 MHz probe (MS-550) and an integrated injection stand. During this procedure, mice were anaesthetised with isoflurane (3% isoflurane in air for induction and maintained at 1.5 and positioned supine on a temperature-controlled platform with continuous monitoring of ECG, body temperature, and respiratory rate. Then the thoracic region was depilated. To ensure adequate control of the volume injected, a Hamilton syringe (1705TLL) connected to a 19 mm 27G needle was used. The Visualsonics system was then used to guide the needle (using B-mode imaging) into the targeted part of the thymus and ensure that the injection was carried out correctly. 16h post-injection the thymus, spleen and LNs (pLNs and mLNs pooled) were recovered.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCell sorting and adoptive transfer of CD4 T cells\u003c/h2\u003e \u003cp\u003eCD4 T cells were purified from LNs (pooled superficial cervical, axillary, brachial, inguinal, and mesenteric LNs) or thymus of C57BL/6 Foxp3-GFP mice by incubating cell suspensions on ice for 20 minutes with a mixture of anti-CD8 (53\u0026thinsp;\u0026minus;\u0026thinsp;6.7, Abs obtened from hybridoma supernatant), anti-CD11b (Mac-1, Biolegend), anti-Ter119 (TER-119, Biolegend),, and anti-CD19 (1D3, BioXcell or Biolegend) and then with magnetic beads coupled to anti-rat immunoglobulins (Dynal Biotech). Purified CD4 T cells were then labeled with PE-Cy7-conjugated anti-CD44, PE-conjugated anti-CD25, anti-NK1.1, anti-TCRγ/δ, anti-CD11c, anti-CD11b, anti-CD19. For immature thymic CD4SP, PE-conjugated anti-CD69 and BV510-conjugated anti-CD4 were added. Na\u0026iuml;ve CD4 T (CD4 TN) cells were flow cytometry sorted as GFP\u0026thinsp;\u0026minus;\u0026thinsp;Lin\u0026minus; (CD25\u0026thinsp;\u0026minus;\u0026thinsp;NK1.1\u0026thinsp;\u0026minus;\u0026thinsp;TCRγ/δ\u0026thinsp;\u0026minus;\u0026thinsp;CD11c\u0026thinsp;\u0026minus;\u0026thinsp;CD11b\u0026thinsp;\u0026minus;\u0026thinsp;CD19\u0026minus;) CD44\u0026minus;/lo and immature thymic CD4SP sorted as CD4\u0026thinsp;+\u0026thinsp;GFP\u0026thinsp;\u0026minus;\u0026thinsp;Lin\u0026minus; (CD25\u0026thinsp;\u0026minus;\u0026thinsp;NK1.1\u0026thinsp;\u0026minus;\u0026thinsp;TCRγ/δ\u0026thinsp;\u0026minus;\u0026thinsp;CD11c\u0026thinsp;\u0026minus;\u0026thinsp;CD11b\u0026thinsp;\u0026minus;\u0026thinsp;CD19\u0026thinsp;\u0026minus;\u0026thinsp;CD69-) CD44\u0026minus;/lo using a FACSAria III flow cytometer (BD Biosciences). In some experiments CD4 TN cells were injected i.v. into recipient mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro Treg cell polarization assays\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003eFrom peripheral CD4 TN cells\u003c/h2\u003e \u003cp\u003eFlow cytometry\u0026ndash;sorted CD4 TN cells were stimulated for 4 days with immobilized anti-CD3 (145-2C11, 4 \u0026micro;g/mL, Biolegend) and anti-CD28 (37.51, 4 \u0026micro;g/mL, Biolegend) in presence of recombinant human IL2 (13ng/mL, R\u0026amp;D Systems)\u0026thinsp;\u0026plusmn;\u0026thinsp;blocking antibodies (anti-IL4 (10 \u0026micro;g/mL, Biolegend) and/or anti-IFNγ (10 \u0026micro;g/mL, Biolegend)) and graded concentrations of recombinant mouse TGFα (Miltenyi Biotech).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFrom CD4SP thymocytes\u003c/h2\u003e \u003cp\u003eFlow cytometry\u0026ndash;sorted immature CD4SP thymocytes (CD4\u0026thinsp;+\u0026thinsp;Foxp3-GFP- Lin- CD44\u0026minus;/lo) from the thymus of C57BL/6 Foxp3-GFP mice were pre-incubated for 1 hour at 37\u0026deg;C with Foxo1 inhibitor or not (AS1842856, Tebubio, 85 nM final) and stimulated for 13 hours with immobilized anti-CD3 (145-2C11, 4 \u0026micro;g/mL, Biolegend) and anti-CD28 (3.7.51, 4 \u0026micro;g/mL, Biolegend), in the presence of 5 \u0026micro;g/mL each of anti-mouse IL-2 antibodies (JES6 and S4B6, BioXcell). Cells were then washed, transferred to uncoated V-bottom plates, and cultured with anti-IL-2 Abs (5 \u0026micro;g/mL, BioXcell) and in the presence or not of Foxo1 inhibitor for 3 hours. Recombinant human IL-2 (50 U/mL) was then added, and the cells were incubated for an additional 7 hours. Finally, cells were washed and rested for 12 hours with 5 \u0026micro;g/mL each of anti-mouse IL-2 Abs and in the presence or not of Foxo1 inhibitor. Cells were analyzed for Foxp3 induction by flow cytometry.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eHistology analysis:\u003c/h2\u003e \u003cp\u003eOrgans were fixed in 4% formaldehyde (ROTI\u0026reg;Histofix, Carl Roth) for 24 h, transferred to 70% ethanol, embedded in paraffin, sectioned, and stained with HES (at the Cochin HISTIM facility). Imaging was performed with PerkinElmer Lamina scanner and analyzed using CaseViewer. Tissues were scored for inflammatory infiltration on a 0\u0026ndash;4 scale as previously described \u003cem\u003e(73)\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eChIP-, RNA- and ATAC-seq analyses\u003c/h2\u003e \u003cp\u003eChIP-seq (GEO: GSE183315) \u003cem\u003e(42)\u003c/em\u003e, RNA-seq and ATAC-seq datasets (DRA003955, DRA004738, DRA005202) \u003cem\u003e(11)\u003c/em\u003e, were preprocessed with standard pipelines.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003ePreprocessing of raw sequencing data\u003c/h2\u003e \u003cp\u003eRaw FASTQ files from ChIP-seq, ATAC-seq, and RNA-seq experiments were processed using standardized pipelines as follows:\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eChIP-seq\u003c/strong\u003e \u003cp\u003eQuality control was performed with FastQC (v0.12.1). Adapter trimming and removal of low-quality bases (Q\u0026thinsp;\u0026lt;\u0026thinsp;20) were done using Trim Galore (v0.6.10). Cleaned paired-end reads were aligned to the Mus musculus mm10 reference genome using Bowtie2 (v2.4.4) with the \u0026ldquo;-sensitive\u0026rdquo; preset. SAM files were converted to BAM format, sorted, indexed, and PCR duplicates were removed using Samtools (v1.18). Then uniquely mapped reads (MAPQ\u0026thinsp;\u0026ge;\u0026thinsp;20) were retained, and those overlapping ENCODE mm10 blacklisted regions were excluded using Bedtools (v2.30.0). Identification of signal-enriched regions (peaks) was performed with MACS2 (v2.2.7.1) in BAMPE mode (paired reading), with a \u0026ldquo;q\u0026rdquo; threshold value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and the \u0026ldquo;-call-summits\u0026rdquo; option activated. Reproducible peaks across replicates were identified using the IDR framework (v2.0.4.2), applying a global IDR threshold of 0.05. Normalized signal coverage files (BigWig format) were generated using bamCoverage (deepTools v3.5.4) with RPGC normalization. Input-subtracted ChIP signal tracks were computed using bigwigCompare, and overall data quality was summarized using MultiQC (v1.9). FRiP scores (Fraction of Reads in Peaks) were calculated using featureCounts (Subread v2.0.6).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRNA-seq\u003c/strong\u003e \u003cp\u003eFastQC (v0.12.1) was used for quality assessment. Adapter trimming and removal of bases with Phred score\u0026thinsp;\u0026lt;\u0026thinsp;20 and minimum read length of 30 bp were performed with Trim Galore (v0.6.10). Reads were aligned to Mus musculus GRCm39 genome using Bowtie2 (v2.4.4) with the \u0026ldquo;--very-sensitive\u0026rdquo; preset. BAM files were sorted and indexed with Samtools (v1.18). Gene-level quantification was performed using featureCounts (Subread v2.0.6) with Ensembl annotation release 110, complemented by transcript-level quantification via RSEM (v1.3.2).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eATAC-seq\u003c/strong\u003e \u003cp\u003eAdapter trimming and quality filtering were performed with fastp (v0.23.2). Trimmed reads were aligned to the reference genome (mm10) using Bowtie2 v2.5.1, and SAM files were converted to sorted BAM using samtools (v1.18). Low quality-reads, duplicates, and mitochondrial aligments were removed. Tn5 insertion sites were adjusted by strand-specific shifting (+\u0026thinsp;4 bp/- 5 bp MACS2 (v2.2.7.1) was used for peak calling with parameters \u0026ldquo;--nomodel --shift \u0026minus;\u0026thinsp;100 --extsize 200\u0026rdquo;. Signal tracks were generated as BigWig files using deepTools bamCoverage (v3.5.1) with RPKM normalization. Peaks were annotated to genomic features and nearest genes using ChIPseeker (v1.38.0).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eIntegration of ChIP-, RNA- and ATAC-seq data\u003c/h2\u003e \u003cp\u003eTo identify Foxo1 transcription factor binding dynamics during tTreg cell differentiation, de novo motif analysis was performed on reproducible Foxo1 ChIP-seq peaks using HOMER (v4.11) with default settings and matched background sequences. Motifs with hypergeometric p-values\u0026thinsp;\u0026lt;\u0026thinsp;1e-10 were retained, and the Foxo1 position weight matrix was exported.\u003c/p\u003e \u003cp\u003eATAC-seq data were processed with TOBIAS (v0.13.2) for footprinting analysis. BAM files were bias-corrected with ATACorrect, and footprint scores calculated using FootprintScores. Foxo1 motif instances from HOMER were scanned within accessible chromatin, and BindDetect identified Foxo1 binding sites with footprint scores\u0026thinsp;\u0026gt;\u0026thinsp;0.2. Differential footprinting and chromatin accessibility between differentiation stages were assessed with TOBIAS \u003cem\u003e(74)\u003c/em\u003e, applying an FDR threshold of 0.05.\u003c/p\u003e \u003cp\u003ePeaks containing predicted Foxo1 binding sites were annotated to nearest genes using ChIPseeker. These gene lists were integrated with RNA-seq differential expression results obtained from DESeq2 (v1.36.0), considering genes with adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2 fold change| \u0026gt; 1 as significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSingle cell RNA-seq (re)analysis\u003c/h2\u003e \u003cdiv id=\"Sec28\" class=\"Section4\"\u003e \u003ch2\u003eData processing\u003c/h2\u003e \u003cp\u003ePublicly available scRNA-seq data from Owen et al. \u003cem\u003e(43)\u003c/em\u003e and Borelli et al. \u003cem\u003e(44)\u003c/em\u003e were reanalyzed. Raw gene-cell count matrices were processed in R (v4.2.0) using Seurat (v4.3.0). Low-quality cells and doublets were removed according to standard QC metrics (mitochondrial content, number of detected features, and UMI counts). Data were normalized and scaled using Seurat default functions. Dimension reduction was performed by principal component analysis (PCA), and non-linear embedding with UMAP was computed on the top principal components. Batch effects between samples were corrected using Harmony. Cells were clustered using a graph-based approach with Seurat\u0026rsquo;s FindClusters function, and cluster identities were assigned based on canonical marker gene expression.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eCell type annotation\u003c/h2\u003e \u003cp\u003eClusters corresponding to thymic T cell developmental stages and regulatory T cell precursors were manually annotated according to established markers. The following populations were defined: proliferating DN3, pre-selection DP, post-selection DP, proliferating thymocytes, immature CD4SP, CD25\u003csup\u003e+\u003c/sup\u003e Treg precursors, transitional Tregs, Foxp3\u003csup\u003elo\u003c/sup\u003e Treg precursors, thymic neo-Tregs, and recirculating Tregs. Annotation was validated by visualization of known marker genes using Nebulosa and violin plots.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGene regulatory network inference\u003c/h3\u003e\n\u003cp\u003eTo reconstruct transcription factor\u0026ndash;target interactions, we applied the pySCENIC (v0.12.1) workflow \u003cem\u003e(45)\u003c/em\u003e. Gene regulatory networks were first inferred using GRNBoost2, and regulon activity was quantified at the single-cell level with AUCell. AUCell scores were added to the Seurat object metadata, allowing visualization of regulon activity distributions across annotated populations. Both continuous AUCell scores and binarized activity matrices were generated.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eRegulon analysis and visualization\u003c/h2\u003e \u003cp\u003eMean regulon activity per cell type was calculated and scaled (z-score) across clusters. The most variable regulons per cell type were identified, and selected regulons of biological interest (including \u003cem\u003eFoxo1\u003c/em\u003e, \u003cem\u003eStat5a\u003c/em\u003e, \u003cem\u003eFoxp3\u003c/em\u003e, \u003cem\u003eGata3\u003c/em\u003e, \u003cem\u003eIkzf2\u003c/em\u003e, \u003cem\u003eRunx1\u003c/em\u003e) were visualized using ComplexHeatmap. Binary activity matrices were also generated with AUCell thresholds and represented as heatmaps annotated by cluster identity.\u003c/p\u003e \u003cp\u003eUMAP embeddings were generated with SCpubr \u003cem\u003e(75)\u003c/em\u003e, including cluster-level color coding and density plots. Violin plots of regulon activities were produced with SCpubr using boxplot overlays. Regulon heatmaps (continuous and binary activity) were produced with ComplexHeatmap and circlize.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis:\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Differences between two groups were assessed by unpaired or paired Student\u0026rsquo;s t test. Multiple groups were analyzed by one-way ANOVA with Fisher\u0026rsquo;s LSD test. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe greatly acknowledge the Cochin Cytometry and Immunobiology (CYBIO), MOUSET\u0026rsquo;IC core facility and Cochin Animal Core facilities for their technological support. L\u0026eacute;a Giraud is supported during the fourth year of her Ph.D. by a fellowship from the \u0026ldquo;Ligue contre le Cancer\u0026rdquo;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the \u0026ldquo;Ligue contre le Cancer\u0026rdquo;, the \u0026ldquo;Association pour la Recherche contre le Cancer\u0026rdquo; and the \u0026ldquo;Agence nationale de la recherche\u0026rdquo; (ANR-25-CE14-2889)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization: LG, CC, BL, CA\u003c/p\u003e\n\u003cp\u003eMethodology: LG, AD, SC, CC, BM, BL, CA\u003c/p\u003e\n\u003cp\u003eValidation: LG, BL, CA\u003c/p\u003e\n\u003cp\u003eFormal analysis: LG, AD, BM, BL, CA\u003c/p\u003e\n\u003cp\u003eInvestigation: LG, AD, CG, NB, SC, AL, CC, BM, CA\u003c/p\u003e\n\u003cp\u003eData Curation: LG, CA\u003c/p\u003e\n\u003cp\u003eWriting - Original Draft: LG, CA\u003c/p\u003e\n\u003cp\u003eWriting - Review \u0026amp; Editing: LG, CC, BM, BL, CA\u003c/p\u003e\n\u003cp\u003eVisualization: LG, CA\u003c/p\u003e\n\u003cp\u003eSupervision: BM, BL, CA\u003c/p\u003e\n\u003cp\u003eProject administration: BL, CA\u003c/p\u003e\n\u003cp\u003eFunding acquisition: BM, BL, CA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eD. 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Blanco-Carmona, Generating publication ready visualizations for Single Cell transcriptomics using SCpubr, 2022.02.28.482303 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"763c9026-0d55-48e9-b65b-621855adf942","identifier":"10.13039/501100004099","name":"Ligue Contre le Cancer","awardNumber":"TDYF25933","order_by":0},{"identity":"3d454aac-992e-4ff4-a549-5cd6f268971d","identifier":"10.13039/501100001665","name":"Agence Nationale de la Recherche","awardNumber":"ANR-25-CE14-2889","order_by":1}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Institut Cochin","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":"Neonatal Treg cells, Foxo1, homeostasis, IL-2, immune tolerance","lastPublishedDoi":"10.21203/rs.3.rs-7824708/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7824708/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRegulatory T (Treg) cells are essential for immune tolerance and include thymic- and peripherally-derived subsets. Among thymic-derived Treg cells, those generated perinatally are phenotypically and functionally distinct from adult cells, with superior capacity to prevent autoimmunity. However, the mechanisms controlling their peripheral seeding remain unclear. Here, we show that the transcription factor Foxo1 is critical for neonatal Treg cell homeostasis rather than for the generation of thymic- or peripherally-derived subsets. Using Foxo1-deficient mice, we demonstrate that Foxo1 deficiency profoundly impairs seeding and expansion of neonatal Tregs in secondary lymphoid organs, disrupting the overall Treg cell compartment. This defect is linked to reduced IL-2 receptor β-chain (IL-2Rβ/CD122) expression, diminished STAT5 activation, and impaired IL-2 responsiveness. Mechanistically, Foxo1 directly binds the \u003cem\u003eIl2rb\u003c/em\u003e promoter, fine-tuning its transcription. These findings establish Foxo1 as a central regulator of IL-2-mediated signaling and neonatal Treg cell homeostasis, ensuring proper subset heterogeneity, and long-term immune tolerance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOne Sentence Summary\u003c/strong\u003e: Foxo1 safeguards neonatal Treg cell homeostasis by directly controlling IL-2 receptor β-chain expression, thereby ensuring proper IL-2 responsiveness, peripheral seeding, and expansion of this critical subset.\u003c/p\u003e","manuscriptTitle":"Transcriptional control of IL-2 sensing by Foxo1 dictates neonatal Treg homeostasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 10:14:11","doi":"10.21203/rs.3.rs-7824708/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":"2950a5d1-3858-4486-82f7-721292808281","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59838979,"name":"Immunology"}],"tags":[],"updatedAt":"2025-12-22T10:14:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 10:14:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7824708","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7824708","identity":"rs-7824708","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}