CCAR2 Dictates tTreg Instability and iTreg-Driven Dendritic Cell Tolerance via Divergent AKT/mTOR Modulation in High-Salt Microenvironments

CCAR2 Dictates tTreg Instability and iTreg-Driven Dendritic Cell Tolerance via Divergent AKT/mTOR Modulation in High-Salt Microenvironments | 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 CCAR2 Dictates tTreg Instability and iTreg-Driven Dendritic Cell Tolerance via Divergent AKT/mTOR Modulation in High-Salt Microenvironments Yating Li, Linxiao Song, Jun Yang, Jiale Tian, Xiaonan Li, Li Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6528083/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 Purpose A high-salt environment serves as a pro-inflammatory milieu that induces autoimmune responses by triggering self-reactive immune activation. While thymus-derived regulatory T cells (tTregs) exhibit significantly impaired immunosuppressive function under high-salt diet (HSD) conditions, the TGF-β-induced Treg subset (iTregs) retains full stability and functional integrity in high-salt environments. Despite these findings, endogenous salt-resistant molecular mechanisms that preserve Treg-mediated immunosuppression remain unidentified. Therefore, to address this gap, we propose to investigate the therapeutic potential of Treg cell adoptive transfer in experimental autoimmune encephalomyelitis (EAE) mouse models. By systematically analyzing the differential capacity of tTregs and iTregs to reprogram pro-inflammatory dendritic cells (DCs) into tolerogenic DCs under high-salt conditions, this study aims to identify the mechanistic distinctions that confer resistance to salt-induced inflammatory perturbations in iTregs, while tTregs remain susceptible. Methods Both Treg cell subsets generated from Foxp3-GFP mice were transferred into naïve Rag1-/- mice, GFP frequency were dynamically detected and compared within each time point. Subsequently, an EAE mouse model was established, and either iTregs or tTregs were intravenously administrated. Clinical scores were continuously recorded, while brain inflammation was evaluated using hematoxylin and eosin (H&E) staining. Additionally, brain-infiltrating Th1/Th17 cells and the presence of splenic CD11c + dendritic cells (DCs) were analyzed by flow cytometry. A DC-T co-culture assay was then conducted, followed by mechanistic studies using western blotting and FACS. Finally, CCAR2-deficient tTregs and iTregs were generated and co-cultured with DCs with or without NaCl addition. The expression of antigen-presenting molecules and the activation of the AKT/mTOR signaling pathway were then systematically evaluated. Results iTregs demonstrate superior efficacy over tTregs in alleviating brain inflammation in both EAE and high-salt diet (HSD)-exacerbated EAE. Unlike tTregs, iTregs suppress pro-inflammatory dendritic cells (DCs) and promote their conversion to an anti-inflammatory phenotype, primarily via membrane-bound TGF-β signaling rather than IL-10R signaling. This functional transformation of DCs is likely mediated by iTreg-induced inhibition of the AKT/mTOR signaling pathway. Notably, under high-salt conditions, this regulatory crosstalk appears specific to iTregs, as tTregs conversely upregulate AKT/mTOR in DCs. Furthermore, CCAR2 contributes to tTreg instability, and its knockdown restores tTreg functionality. In contrast, iTregs enhance DC tolerogenic phenotypes independently of CCAR2. Conclusion This study delineates a previously unrecognized functional dichotomy between Treg subsets, revealing that iTregs uniquely endow DC tolerance in high-salt environments through membrane-bound TGF-β-dependent suppression of AKT/mTOR signaling, whereas tTregs exacerbate DC immunogenicity via CCAR2-mediated pathway activation. By identifying CCAR2 as a critical destabilizing factor in tTregs and demonstrating the salt-resistant mechanistic signature of iTregs, our findings not only redefine microenvironment-specific regulatory paradigms in autoimmune pathogenesis but also establish iTregs as a superior therapeutic modality for inflammation-dominated disorders, particularly under metabolically stressful conditions such as high-salt exposure. Experimental autoimmune encephalomyelitis (EAE) High-salt diet IL-10 TGF-β-induced regulatory T cells (iTregs) TGF-β Thymus-derived natural regulatory T cells (tTregs) Tolerogenic DCs (tDCs) CCAR2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction CD4 + Foxp3 + regulatory T cells (Tregs) exert their immunosuppressive actions to limit tissue injury and control hyperinflammatory responses contributing to homeostasis.[ 1 , 2 ] Tregs display heterogeneity and are broadly categorized into three subsets: thymus-derived naturally occurring Treg cells (tTregs), peripherally derived Treg cells (pTregs), and TGF-β-induced Treg cells (iTregs). [ 1 ] Previous studies, including our own, have underscored both the similarities and distinctions among specific Treg subsets.[ 1 , 2 ] Despite their notable preventive role in controlling autoimmune diseases in various animal models such as experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis (CIA), dextran sodium sulfate (DSS)-induced colitis, and streptozotocin-induced type 1 diabetes (T1DM), the therapeutic efficacy of tTregs remains suboptimal.[ 1 ] Komatsu et al. reported that adoptively transferred tTregs preferentially lose Foxp3 expression and undergo trans differentiation into pathogenic T helper 17 (Th17) cells, thereby exacerbating the onset and severity of the CIA model.[ 3 ] Furthermore, other scholars, in conjunction with our findings, have reported tTregs are prone to convert into other T effector cell subsets, including IFNγ-producing (Th1), IL-17A-producing (Th17) T cells, concomitant with the Foxp3 degradation and a reduction in their immunosuppressive functionality.[ 4 ] These changes often occur in the disease-microenvironment with autoimmune abnormalities.[ 5 , 6 ] Furthermore, our studies have documented the resistance of iTregs, but not tTregs, to IL-6-driven conversion into Th17 cells. Notably, adoptive transfer of iTregs, but not tTregs, significantly mitigated bone erosion in the CIA model, attributed to the enhanced stability and functionality of iTregs compared to tTregs following cell infusion.[ 4 ] Studies have demonstrated that high-salt conditions diminish the suppressive capacity of tTreg cells.[ 7 ] Sodium chloride (NaCl) constitutes a fundamental component of daily dietary intake. Preliminary investigations have unveiled that excessive salt promotes the differentiation of Th17 cells, culminating in a highly pathogenic phenotype that exacerbates EAE.[ 8 ] High-salt intake was also demonstrated that can activate the complement system, inflammasomes, leading to salt-sensitive hypertension.[ 9 ] High salt could also induce lipid oxidation in DCs, which in turn, exacerbates high blood pressure.[ 10 ] Furthermore, high salt increases the secretion of IFNγ in tTregs, compromising their suppressive functionality and exacerbating experimental graft-versus-host disease.[ 11 ] In contrast, the results of our study revealed that, unlike tTregs, iTregs remain relatively stable and function effectively under conditions of elevated sodium chloride concentrations. Notably, high salt does not significantly change the transcriptional profiles of either iTreg-specific markers or inflammatory genes. Moreover, we in-vivo corroborated that iTregs exert substantial control over colitis progression, whereas tTregs predominantly lose their inhibitory potency. 5 We previously demonstrated that cell cycle and apoptosis regulator 2 (CCAR2, also called Deleted breast cancer 1, DBC1) functionally cooperates with Foxp3, resulting in triggering Foxp3 degradation in inflammatory cytokines stimulation. CCAR2 −/− Treg cells maintained Foxp3 expression and enhanced suppressive function when compared to WT Treg cells under TNF-α treatment.[ 12 ] EAE serves as the primary experimental model for multiple sclerosis (MS), a human inflammatory demyelinating disorder characterized by immune dysregulation and infiltration of immune cells into the central nervous system (CNS).[ 13 ] Notably, T cells play a pivotal role in the pathogenesis of EAE, wherein peripheral T cell activation by viral or other infectious antigens or superantigens leads to the production of inflammatory cytokines and facilitates their traversal across the blood-brain barrier. The severity of EAE is also correlated with the recruitment of dendritic cells (DCs) into the CNS.[ 14 ] Particularly, conventional DCs, which are highly specialized antigen-presenting cells (APCs), assume a critical role in immune activation by bridging innate and adaptive immune system.[ 15 ] Under immune homeostatic conditions, DCs patrol the CNS microenvironment, functioning as sentinels. Upon activation, these DCs adopt a pro-inflammatory phenotype and migrate to lymph nodes, thereby fostering the generation of self-reactive T cells and other immune cell subsets. Conversely, DCs also possess tolerogenic properties, contributing to the maintenance of central and peripheral tolerance as well as the resolution of ongoing immune reactions.[ 16 ] Tolerogenic DCs (tol-DCs) restrain effector T cells and promote Treg differentiation through various mechanisms, including cytokine secretion (e.g., IL-10, IL-27, and TGF-β), expression of indoleamine 2,3-dioxygenase (IDO), and regulation of extracellular levels of adenosine triphosphate (ATP) and adenosine. Treg cells possess the ability to confer tolerogenic functions upon conventional DCs, thereby suppressing Th1 and Th17 responses and ameliorating the autoimmune disease phenotype.[ 17 ] Consequently, despite numerous studies documenting the preventive roles of both tTregs and iTregs in EAE progression, uncertainties persist regarding whether both Treg subsets exert equivalent immunosuppressive effects in inhibiting EAE exacerbation induced by high dietary salt intake. Additionally, elucidating whether Treg cells modulate EAE progression through DC phenotype modulation remains an important avenue of inquiry. We demonstrated that iTregs, unlike tTregs, robustly resist high-salt-induced inflammatory conditions, effectively attenuating high-salt diet-aggravated EAE progression. This is primarily attributed to iTregs significantly reducing conventional DC frequency, downregulating antigen-presenting molecules, and upregulating LAP expression. Moreover, CCAR2 depletion stabilizes tTregs, preventing their Th17-like transformation under high-salt conditions, whereas iTregs enhance DC tolerogenic phenotypes independently of CCAR2. 2 Materials and Methods 2.1Mice Male C57BL/6J (B6) mice, 6–8 weeks old, were purchased from Veterinary Research Lanzhou Institute. B6 CCAR2 −/− and B6 Foxp3-GFP mice were kindly provided by Pro. Songguo Zheng (School of Cell and Gene Therapy, Shanghai Jiao Tong University School of Medicine, Shanghai, China.) B6 Rag1 −/− mice were purchased from Sai ye Biotechnology Co., LTD. All mice were kept under specific pathogen-free conditions of Lanzhou University Medical School. Animals were used in accordance with the regulations stipulated by the Animal Care and Use Committee of Lanzhou University. 2.2 EAE, high salt diet The anesthetized mice were subcutaneously injected on the upper and lower back with 250 µg of MOG 35 − 55 (Proteimax), fully emulsified in an equivalent volume of Complete Freund's Adjuvant (CFA, Sigma, St Louis, MO, USA) containing 4 mg/ml of heat-killed Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA). At approximately 0- and 48- hour post-immunization, intraperitoneal injections of 500 ng pertussis toxin (Alexis, San Diego, USA) were administered. Clinical scores were daily assessed, with the mean score of each mouse being noted every three days. In certain experiments, mice were provided with a high salt-rich chow containing 6% sodium chloride (ssniff, Germany; Xie-tong, Jiangsu, China) and tap water containing 1% NaCl (NaCl high) ad libitum for 2 weeks prior to EAE induction. Throughout the experimental period, no observable differences of weight change were noted (data not shown), indicating similar consumption of food and water between groups. The high-salt diet was maintained until the mice were sacrificed. 2.3 Adoptive transfer experiments C57BL/6 EAE mice were randomly assigned to receive tail intravenous injections of iTregs, tTregs (both 1 × 10 6 cells per mouse), or vehicle saline on day 4 post-immunization. To investigate the in-vivo mechanical suppression of Treg subsets, anti-IL-10 receptor or isotype-matched IgG1 antibody (0.25 mg/kg body weight), or ALK5 inhibitor (LY-364947, 0.5 mg/mouse; Sigma) were treated intraperitoneally weekly for a total of 3 injections, with the first injection occurring the day after cell infusion. In most experiments, each group consisted of 5 mice, with experiments repeated at least twice yielding similar results, and the data presented here represents one of those experiments. On day 30 after MOG injection, all mice (n = 5 mice per group) were euthanized, and cells from spleens and brains were harvested. Proportions of Treg cells, Th17 cells, and Th1 cells, or CD11c + DCs were examined by flow cytometry. 2.4 Tissue sampling and immune cell isolation At day 30, animals were euthanized using the perfusion with 4% (w/v) paraformaldehyde under deep anesthesia (10% chloralhydrate, 0.2ml/mouse, i.p.), and then perfused transcardially using cold phosphate buffered saline. Subsequently, brain, spleen and lymph nodes were collected for further processing. Specifically, spleens and lymph nodes were placed in cell strainers (70 µm) and homogenized using syringe plungers, followed by washing with in RPMI-1640 containing 2% NCS. Brain tissues were minced and then subjected to digestion using 2 mg/ml collagenase IV (Sigma Aldrich) in 1640 containing 2% NCS at 37°C with agitation for 60 minutes. Following digestion, brain tissues were filtered and subsequently separated using a 30%/70% Percoll solution (Cytiva) via centrifugation (800 × g , 20 min). Then isolated cell pellets were collected for staining and flow detection procedure. 2.5 Histology Mice in each group underwent perfusion with 4% (w/v) paraformaldehyde under deep anesthesia induced by intraperitoneal injection of 10% chloralhydrate (0.2 ml/mouse). Subsequently, brains were meticulously extracted and embedded in paraffin, and the segments were dissected and subjected to staining with hematoxylin and eosin (H&E) following established protocols, which the degree of inflammation was blindly assessed according to the published criteria.[ 18 ] Scores were in a semiquantitative fashion for inflammation graded: 0, no inflammatory cell infiltration; 1, scattered cell infiltration can be seen throughout the visual field; 2, organization of perivascular infiltrates, or mild cell infiltration into the parenchyma; 3, perivascular cuffing with extension into the adjacent tissue.[ 18 ] 2.6 tTreg isolation, iTreg generation, Treg subset conversion in vitro and in vivo Thymus-derived Tregs (tTregs) from B6 WT, B6 CCAR2 −/− or B6 Foxp3-GFP mice through gating of CD4 + CD25 − or CD4 + GFP + cells, achieving a purity of 95–99%. These cells were subsequently stimulated with anti-CD3/CD28-coated beads (Invitrogen, Carlsbad, CA) at a ratio of 1 bead: 2 cells and rhIL-2 (100 U/ml; R&D Systems, Minneapolis, MN) for 72-hour expansion. All tTregs utilized in the in-vitro experiments of this study underwent prior expansion. For iTreg cell generation, naïve CD4 + CD44 − CD62L + T cells from WT, CCAR2 −/− or Foxp3-GFP B6 mice (purity > 95%) were stimulated with anti-CD3/CD28 dynabeads (cells: beads = 5:1, Invitrogen) in the presence of rhTGF-β (2 ng/ml) and rh-IL-2 (50 U/ml) (both from R&D System) for 72 hours. Both Treg subsets were cultured in X-VIVO 15 medium (LONZA) in the presence or absence of NaCl (40 mM, Sigma-Aldrich), IL-6 (100 ng/ml, R&D System), or a combination of both. For in-vivo Treg subset conversion, primary isolated tTregs or generated iTregs from Foxp3-GFP reporter mice were further sorted into CD4 + GFP + cells with a purity of 99%. Then, a total of 0.5x10 6 purified tTregs or iTregs were intravenously administrated into Rag1 −/− mice. In some groups, tTregs or iTregs were pre-treated with 40 mM NaCl and 100 ng/ml IL-6 for 48 hours prior to sorting and adoptive transfer. Mice were euthanized on days 5 and 10, and T cells from the mesenteric lymph nodes (MLN) were stained for IFNγ and IL-17A. 2.7 Treg suppression assay in vitro Initially, both tTregs and iTregs were pre-treated with or without 40 mM NaCl and 100 ng/ml IL-6 in the presence of anti-CD3/CD28 dynabeads (5 cells per bead) for 72 hours. Subsequently, the pretreated cells were co-cultured with 5 × 10 5 naïve CD4 + CD44 − CD62L + T cells (from WT B6 mice) labeled with 2 µM CFSE (BioLegend) at various ratios as indicated. Cell mixtures were simultaneously stimulated with soluble anti-CD3 (0.5 µg/ml) for 3 days. Division index was calculated by assessing the rates and intensity of CFSE dilution using flow cytometry. 2.8 Co-culture of naive T responder cells with DC subsets Splenic CD11c + DCs were isolated from PBS-, iTreg-, tTreg-treated EAE mice at day 30 using the mouse CD11c- cell MicroBeads isolation kit (Miltenyi). For DC-mediated proliferation assay, the WT B6 naïve T cells labeled with CFSE (2 uM) were cocultured with these DC subsets in the presence of anti-CD3 (0.1 ug/ml; BD Pharmingen) stimulation for 3 days. T-cell proliferation was assessed by analyzing the CFSE dilution by FACS. For the DC-induced Treg cell differentiation assay, WT CD4 + CD44 − CD62L + naïve T cells sorted from the B6 Foxp3-GFP mice were cocultured with these DC subsets with the anti-CD3 (0.5 ug/ml) and rh-IL2 (50 U/ml) addition for 3 days. GFP expression was assessed using flow cytometry. 2.9 Co-culture of Treg cells with splenic DCs Splenic CD11c + DCs (WT B6 mice) with the purity > 90% were cultured in a medium supplemented with GM-CSF (500 U/ml) and IL-4 (200 U/ml) for 72 h. Then DCs were collected, washed, and re-seeded in 48-well plates containing RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) followed by the co-culture with WT-tTregs, WT-iTregs, NaCl-pretreated WT-tTregs and NaCl-pretreated WT-iTregs at a ratio of 5:1 (Tregs:DCs ) in the presence of anti-CD3 (0.5 µg/ml, BD Pharmingen) stimulation for 24 hours. Subsequently, DCs from each group were collected separately followed by WB examination. In additional mechanistic studies, WT or CCAR2-knockout tTregs and iTregs, including those pretreated with NaCl for 48 hours, were co-cultured with WT splenic CD11c + DCs at the same 5:1 ratio for 24 hours in the presence of anti-CD3 (0.5 µg/ml) stimulation. Similarly, DCs were then collected separately, but followed by FACS examination. 2.10 Flow-cytometric analyses Collected cells were resuspended in cold PBS containing 0.1% BSA and then stained with conjugated-antibodies or their isotype controls for CD3, CD4, and CD25 (eBioscience) at 4°C for 15–20 min. To assess Foxp3 expression, cells were first subjected to surface staining, followed by treatment with a fixation buffer (Biolegend). Subsequently, the cells were permeabilized using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and maintained at 4°C for 1 h, then were incubated with FITC-conjugated rat anti-mouse Foxp3 antibody (FJK-16s, eBioscience) for FACS detection. For intracellular staining of cytokines, cultured cells were stimulated ex vivo with PMA and ionomycin (both 0.25ug/ml; Sigma-Aldrich) for 5 hours at 37°C in the presence of brefeldin A (5 ug/ml; BioLegend) for the last 4 hours. After surface staining, cells were fixed and permeabilized using the Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) prior to intracellular staining: anti-mouse IFNγ-PE and anti-mouse IL-17A-APC (TC11-18H10). For DC staining, total splenocytes were pretreated with anti-CD16/CD32 antibodies (BD Biosciences), then stained with the surface antibodies: anti-CD11c, anti-MHCII, anti-CD80, anti-CD86, anti-LAP, anti-B7H4 (all from eBioscience). To assess p-Akt and p-P70S6K expression in DCs, surface CD11c stained cells were fixed, then permeabilized, followed by the incubation with monoclonal rabbit (Ser473, Cell Signaling Technology) against p-Akt or Phospho-p70 S6 Kinase (Thr389) Antibody (649704, BioLegend). IL-10 production in DCs was measured by stimulating total splenocytes with 10 ng/ml PMA and 100 µg/ml ionomycin for 6 hours in the presence of 5 µg/ml Monensin. After surface CD11c staining, cells were fixed, permeabilized, and then anti-mouse IL-10-APC (JES5-16E3, Biolegend) was used for intracellular staining. All flow cytometric analyses were conducted using the following isotype controls: PE Rat IgG2a, Rat IgG2k; APC Rat IgG2a, Rat IgG1; PerCP-Cy5.5 Rat IgG2b. Data were analyzed and acquired with FlowJo 10 software. 2.11 Western blotting DCs co-cultured with different Treg subsets were collected and lysed in pre-warmed RPMI 1640. Briefly, total protein was extracted from cultured CD11c + cells and transferred onto PVDF membranes, which were subsequently blocked in 5% BSA or 5% non-fat milk in TBST for 1 hour. Following this, the membranes were incubated overnight at 4°C with primary antibodies targeting phospho-Akt (Ser473), total AKT, phospho-P70S6 kinase (Thr389), total P70S6 kinase, and GAPDH, all sourced from Cell Signaling Technology (Danvers, MA). On the following day, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour. Protein bands were visualized using ECL reagent (Beyotime, Shanghai, China). 2.12 Statistical methods Statistics were conducted using GraphPad Prism software (Version 5), if not indicated elsewhere, as mean ± SEM. To assess differences between groups, two-tailed Student’s t-test was used. For comparisons across multiple groups, one-way ANOVA analysis was performed with Tukey’s post hoc tests. Statistical significance was considered as p < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant). 3 Results 3.1 iTregs and tTregs exhibit distinct functional stability and immunosuppressive capacities under high-salt condition combined with pro-inflammatory cytokine stimulation. Previous studies have reported that the inflammatory cytokine, like IL-6 drives the conversion of tTregs, rather than iTregs, into pathogenic Th17 cells.[ 19 , 20 ] Additionally, sodium chloride converts tTregs, but not iTregs, into IFNγ-producing Th1-like cells.[ 8 ] To further compare the conversion of the two Treg subsets into the Th17 or/and Th1 lineages, NaCl combined with IL-6 was used as a stimulant. CD4 + GFP + cells were sorted from thymic cells (tTregs) in B6 Foxp3-GFP mice, while iTreg cells (iTregs) were generated from splenic naïve CD4 + CD44 − CD62L + GFP − T cells using a standard protocol as previously described.[ 6 ] Initially, these Tregs were cultured in the presence of NaCl (40 mM) or IL-6 (100 ng/mL) for 3 days, and subsequently, IFNγ- or IL-17A-producing cells were examined by flow cytometry. Consistent with previous findings, the tTreg subset exhibited higher levels of IFNγ and IL-17A expression compared to the iTreg subset under 40 mM NaCl stimulation (Figure S1A, 1B). Furthermore, when exposed to IL-6, tTregs also expressed significantly more IL-17A than iTregs (Figure S1A, S1C). Interestingly, when each Treg subset was co-stimulated with both IL-6 and NaCl in the presence of anti-IFNγ, IL-17A production was robustly increased in the tTreg subset, whereas the iTreg subset remained entirely resistant to expressing IL-17A (Fig. 1A, B). Subsequently, the in-vivo stability of the two Treg subsets was compared. Both tTregs and iTregs were pretreated with IL-6 and NaCl for 48 hours and were then adoptively transferred into Rag1 −/− recipients at a dose of 0.5 × 10 6 per mouse to assess their stability under lymphopenic conditions for 10 consecutive days. GFP-expressing cells were quantified in the MLN on day 5 and day 10, respectively. As depicted in Fig. 1C and 1D, there was some loss of Foxp3 in both Treg populations, but slightly less in iTregs than in tTregs at day 5 after adoptive infusion. However, by day 10, approximately 75% of Foxp3 expression was retained in iTregs, especially in those pretreated with NaCl and IL-6. Conversely, the pretreated tTregs displayed a notable reduction in Foxp3 expression compared to untreated tTregs. Concurrently with changes in Foxp3 expression, both tTregs and pretreated tTregs exhibited robust expression of IL-17A and IFNγ, with the production of these two cytokines significantly higher in pretreated tTregs than in untreated tTregs (Fig. 1E, F). In line with the in vitro findings, no significant expression of IL-17A and IFNγ was observed in either iTregs or pretreated iTregs (Fig. 1E, F). Finally, we sought to ascertain whether the two Treg subsets retained their function faithfully when pretreated with IL-6 and NaCl. Carboxy fluorescein (CFSE)-labeled splenic CD4 + T cells from WT B6 mice were cultured with Treg subsets at various ratios of T responder and Treg cells, which were primed with or without (NaCl + IL-6) for 3 days. Upon addition of iTregs or tTregs to T responder cells, CFSE labeling revealed a marked inhibition of proliferation, demonstrating a dose-dependent inhibitory effect. Nevertheless, tTregs subjected to pretreatment displayed a marked reduction in their inhibitory function, whereas iTregs that were pretreated preserved their suppressive capacity (Fig. 1G, H). Furthermore, the suppression exerted by pretreated iTregs against T cell proliferation far surpassed that of pretreated tTregs at the indicated ratios (Fig. 1G, H). In summary, these results unequivocally demonstrate that iTreg cells are more stable and exhibit superior functionality compared to tTregs under conditions of inflammatory cytokines combined with sodium chloride. iTregs were generated from naïve CD4 + T cells of Foxp3-GFP mice following the protocol outlined in the Materials and Methods section. Subsequently, iTregs or thy-mus-sorted tTregs were stimulated with soluble anti-CD3 (2 ug/mL) and anti-CD28 (1 ug/mL) for 3 days in the presence or absence of NaCl (40 mM) (Panel A, B) or rm-IL-6 (100 ug/mL) (A, C). The expression levels of naïve IFNγ and IL-17A were determined at the same time by flow cytometry. Data are presented as mean ± SEM of three independent experiments (B, C). Statistical analysis was performed using the paired Student’s t-test when comparing only two groups, with significance denoted as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns indicating not significant. tTregs sorted from thymus expanded and activated. In parallel, iTregs were induced from naive CD4 + T cells. Both Treg cell subsets were from Foxp3-GFP mice and cultured in standard media for 72 h. Following this, cells were harvested, thoroughly washed, and then cultured in media supplemented with NaCl (40 mM) and IL-6 (100 ng/mL) for an additional 48 h, denoted as pretreated-tTregs or pretreated-iTregs. Subsequently, these cells were collected, surface-stained for CD4, and intracellularly stained for IL-17A and IFNγ, with measurements performed on CD4 + GFP- and CD4 + GFP + cells via FACS. Representative FACS plots from three independent experiments are depicted (A, B), (n = 6). In a parallel experiment, tTregs, pretreated-tTregs, iTregs, and pretreated-iTregs were transferred into Rag1-/- mice. The mice were euthanized at day 5 or day 10, and total MLN cells were collected. Assessment of Foxp3-GFP loss (C, D) and determination of the frequency of CD4 + IL-17A + and CD4 + IFNγ + cells (E, F) in the MLN from each group were compared. Representative results, presented as mean ± SEM from two distinct experiments, are il-lustrated, with statistical significance analyzed using unpaired t-tests (D, F). Furthermore, iTregs were restimulated under 40 mM NaCl for 3 days and subsequently analyzed by flow cytometry for IL-17A and IFNγ. Representative FACS data (Panel G) and data quantification (Panels G, H) are provided. The bar graph summarizes results from independent experiments (n = 3), with statistical analyses conducted using paired t tests. Notably, ns denotes not significant, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. 3.2 iTregs exhibit superior functional activity compared to tTregs in EAE-mediated brain inflammation To investigate the distinct effects of the two Treg subsets in mitigating brain inflammation, we used the murine MOG 35 − 55 -induced EAE model. Mice disease manifestation was observed at day 12 post-immunization, characterized initially by tail weakness, followed by hindlimb and/or forelimb paralysis, as previously described.[ 21 ] Intravenous transfer of Treg subsets occurred 4 days after the expression of EAE. As anticipated, both iTregs and tTregs exhibited a significant functional suppression on the clinical expression of EAE, delaying onset and ameliorating symptoms (Fig. 2A). H&E staining revealed a markedly lower level of lymphocyte infiltration in the tTreg- or iTreg-treated groups compared to the disease model group, with iTreg groups revealing a more pronounced alleviation of lymphocyte infiltration compared to tTreg groups (Fig. 2B). Notably, we observed that iTregs displayed a superior effect on restraining lymphocytic infiltration compared to tTregs, correlating with the clinical symptoms depicted in Fig. 2A. Furthermore, we analyzed the change of Th1 and Th17 cells in each group, as these subsets represent crucial pathogenic immune cells in EAE development. Flow cytometric analyses demonstrated significant suppression of Th17 cells in the spleens (Fig. 2C) and brains (Fig. 2D) of both treatment groups compared to disease model controls. Particularly, the brain -Th17 population was dramatically decreased in mice administered with iTregs compared to those administered with tTregs ( p < 0.05) (Fig. 2E, 2G), indicating a distinct therapeutic difference between iTregs and tTregs. Subsequently, the frequency of Th1 cells in the spleens and brains was significantly reduced in iTreg-treated, but not in tTreg-treated group compared to the model group (Fig. 2F, 2H). Specifically, tTreg administration only inhibited brain Th1 cell development, with less obvious effects compared to iTreg administration (Fig. 2H). Given the critical role of DC priming of T lymphocytes in EAE pathogenesis, we analyzed splenic CD11c + DCs in each group. Both tTreg and iTreg subsets decreased the frequency of splenic CD11c + DCs, with iTregs displaying superior efficacy compared to tTregs (Fig. 2I, 2J). These findings collectively support the notion that both tTregs and iTregs alleviate EAE severity, with iTregs exhibiting more potent suppressive activity, potentially involving DCs in this effect. 1×106 tTregs or iTregs, generated as described previously, were adoptively trans-ferred into B6 male mice on day 4 following MOG35-55/CFA immunization. Clinical scores of the recipient mice were recorded at various time points post-immunization (A). Histological analyses were conducted, showcasing sections of H&E staining of brain tissues from each experimental group (20x) (B). Experiments were terminated on day 30, following which cells from spleens (C) and brains (D) were harvested and stimulated with PMA, ionomycin, and BFA. Subsequently, IFNγ- and IL-17A-producing CD4 + T cells from spleens (E, F) and brains (G, H) were analyzed via flow cytometry. Data presented are representative of at least 5 mice per group. Additionally, staining of indicated CD11c + splenic DCs on day 30 was performed via flow cytometry (I, J). All data are representative of at least 3 independent experiments. Statistical analyses were conducted using one-way ANOVA, with significance denoted as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns indicating not significant. 3.3 iTregs inhibit the pro-inflammatory DC phenotype and promote tolerogenic activities in the EAE environment The immunogenicity of dendritic cells (DCs) reflects a balance of pro- and anti-inflammatory signals in EAE. Pro-inflammatory DCs aggravate EAE progress, while tolerogenic DCs play a modulatory role in restoring immune homeostasis and relieve EAE pathogenesis.[ 22 ] Subsequently, we assessed the immunophenotype and function of splenic CD11c + DCs from the three groups: PBS-treated (model-DCs, m-DCs), tTreg-treated (t-DCs), and iTreg-treated (i-DCs) EAE mice. The mean fluorescence intensity (MFI) of the co-stimulatory molecule CD80 was significantly lower only in the i-DCs compared to the m-DCs and t-DCs (Fig. 3A, 3B). Likewise, the MFI of another co-stimulatory molecule CD86 in CD11c + DCs was significantly decreased in mice treated with the iTreg subset versus the control and tTreg-treated mice. Interestingly, i-DCs expressed much lower levels of CD86 compared to t-DCs (Fig. 3A, 3C). Additionally, the MFI of the major histocompatibility class II molecule (I-Ad), as well as activation molecules such as CD40 and CD83, were also decreased in both Treg-treated groups compared to the model group, although not reaching statistical significance (data not shown) (Fig. 3A, 3B). Investigation of well-known tolerogenic DC cells also revealed that i-DCs significantly expressed higher levels of latency-associated peptide (LAP) (Fig. 3A, 3D) and intracellular IL-10 (Fig. 3E) compared to that of m-DCs, as indicated by MFI or frequency respectively. And we did not observe significant expression of the two tolerogenic markers in t-DCs, but only observed an increasing trend (Fig. 3A, 3D, 3E). These findings indicate that iTregs are more capable of inducing DC immune tolerance than tTregs, which may help to explain why the iTreg subset is more effective than tTregs in treating EAE. Based on these findings, we hypothesize that i-DCs would capably suppress T cell proliferation and promote Treg cell differentiation. As expected, when the i-DC subset was cocultured with CFSE-labeled naive T cells (CD4 + CD62L + CD44 − ), the CFSE proliferation was significantly restrained compared to that cocultured with m-DCs or t-DCs (Fig. 3F, 3G). Similarly, i-DCs also exhibited a remarkable ability to induce Treg cell development. Approximately 10% of the naïve T cells became CD25 + GFP + regulatory T cells (Fig. 3H, 3I). And this was not observed from the PBS-DCs coculture system. Although t-DCs also obviously generated a significant amount of Foxp3 + cells, i-DCs had a robustly powerful ability in the induction of naive T cells into Treg cells compared to t-DCs (Fig. 3I). Thus, these results indicate that iTreg-modified DCs are rendered hyporesponsive and have anti-inflammatory function. PBS-treated, tTreg-treated, and iTreg-treated EAE mice (n = 5 per group) were eu-thanized on day 30. Total splenocytes were collected, and DCs were stained and gated on CD11c. Expression levels of CD80, CD86, and LAP on CD11chi splenic DCs were depicted (A), with corresponding statistical graphs presented (B, C, D). The data revealed are representative of at least 3 independent experiments. Some splenocytes were stimulated with PMA, ionomycin, and monensin, followed by staining for CD11c and IL-10. The bar graph illustrates the frequency of CD11c + IL-10 + DCs (E). Additionally, PBS-treated DCs, tTreg-treated DCs, and iTreg-treated DCs were sorted from the spleens of EAE mice, then added to naive B6 T cells labeled with CFSE, and T cell proliferation was assessed by CFSE dilution (F), with statistical data indicated (G). Furthermore, a portion of these three groups of DCs was added to naive T cells (from B6 Foxp3-GFP mice) at a 1:5 ratio in the presence of anti-CD3 and rhIL-2 for 3 days, and Foxp3-GFP expression was measured using flow cytometry (H, I). All data presented are representative of at least 3 independent experiments. Statistical analyses were conducted using one-way ANOVA, with significance denoted as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns indicating not significant. 3.4 iTregs endow DCs with a tolerogenic phenotype and function mainly via TGF-β but not IL-10 signaling It has been well documented that both Treg subsets secrete soluble TGF-β and IL-10, which are crucial for exerting immunosuppressive function.[ 23 ] To further determine whether membrane-bound TGF-β and IL-10 receptors are also required for Treg-mediated suppression in EAE, we used an inhibitor of TGF-β receptor I (ALK5) and an anti-IL-10R antibody in the EAE model. EAE induction and iTreg cell infusion were performed, as depicted in Fig. 2A. Additionally, some of the mice treated with iTreg cells were injected with a neutralizing anti-IL-10R antibody (isotype-matched IgG1 antibody as its control) or ALK5 inhibitor (ALK5i). Our results revealed that iTreg infusion exerted a significant inhibitory function on the treatment of EAE, as demonstrated by a reduction in clinical scores (Fig. 4A) and histological brain inflammation (Fig. 4B). Administration of DMSO or IgG1 alone did not influence disease progression in the EAE mice compared to the model group (data not shown). IL-10 signaling blockade resulted in a statistically significant effect on the therapeutic efficacy of iTregs, as determined by clinical scores. Nevertheless, the unchanged neuroinflammation scores following anti-IL-10R blockade suggest that IL-10 signaling may play a limited role in the immunosuppressive capability of iTreg cells in the treatment of EAE (Fig. 4B). Conversely, the clinical scores and the pathological inflammation scores were both completely reversed in the iTreg + ALK5i group compared to the iTreg only group, indicating that blockade of TGF-β receptor I activity apparently impaired the protective effect of iTreg cells (Fig. 4A, 4B). These results preliminarily indicated that TGF-β and IL-10 signaling both play a role in executing suppressive ability of iTreg cells, whereas TGF-β signaling is more crucial. The presence of Th1 and Th17 cells in the brains was also examined using FACS. As expected, iTreg infusion significantly repressed the brain Th17 cell differentiation. However, co-administration of ALK5i mostly restored the Th17 frequency in the brains to EAE levels compared to the iTreg infusion group. Meanwhile, we only observed the reverse trend in the inhibition of Th17 cell frequency by iTregs treatment in the anti-IL-10R antibody administration group (Fig. 4C, 4D). Also, IFNγ-producing Th1 cells were also statistically decreased in iTreg-treated brains. Conversely, neither ALK5i nor anti-IL-10R could significantly reverse the inhibitory effect on Th1 differentiation by iTreg infusion (Fig. 4C, 4E). These observations illustrate that TGF-β signaling plays a dominant role in the immunosuppressive function of iTregs on EAE, whereas IL-10R signaling plays a partial role, if any. In line with the aforementioned findings, we examined the frequency of splenic DCs from each group of EAE mice at day 30 post-immunization. iTreg-treated mice revealed a remarkable decrease in CD11c + DCs in spleens. ALK5i administration completely diminished this phenomenon, but anti-IL-10R did not (Fig. 4F). CD11c + DCs expressed a baseline level of CD80, CD86, and LAP in splenocytes treated with PBS. DCs from iTreg-treated mice exhibited a downregulation of CD80 and CD86 (Figure S2A, S2B), along with an upregulation of LAP expression (Figure S2A, S2C). In comparison to the iTreg-treated splenocytes, co-administration of ALK5i significantly upregulated the expression of CD80 and CD86 (Figure S2A, S2B), while decreasing the expression of LAP (Figure S2A, S2C). However, statistically significant changes in these molecules in the co-administration of the anti-IL-10R group were not observed (Figure S2A-2C). Next, a series of DC-T cell co-culture experiments were conducted to assess the impact of ALK5i and anti-IL10R on the tolerogenic activity of these in vivo-modified DCs. CD11c + DCs were sorted from the spleen (splenocytes) of each EAE group on day 30. These DCs were cocultured with B6 naïve T cells using the same Treg-polarizing conditions as described in Fig. 3. As revealed in Fig. 4G, compared to DCs sorted from PBS-treated EAE mice, iTreg-DCs enabled up to 10% of naive T cells to begin expression of Foxp3. However, splenic DCs from the ALK5i co-administration group dramatically lost their tolerogenic activity, resulting in little formation of CD4 + Foxp3 + cells. However, DCs from the anti-IL10-treated group exhibited some degree of tolerogenic function, although the generation of CD4 + Foxp3 + cells were statistically lower compared to iTreg-DCs (Fig. 4G, 4H). In another approach, CFSE dilution was also assessed using the coculture system. WT B6 naive T cells cocultured with iTreg-DCs proliferated much less than those cocultured with PBS-DCs. Similarly, naive T cells with ALK5i-co-administered DCs exhibited an obvious capacity to proliferate at a level similar to that of those cultured with PBS-DCs. Conversely, anti-IL10R-co-administered DCs retained some of their immunosuppressive function, however this was not statistically significant, as revealed by the inhibition of CFSE proliferation compared to the iTreg-DCs (Fig. 4I, 4J). Taken together, these data reveal that iTregs primarily induce the formation of tolerogenic DCs via membrane-bound TGF-β signaling, rather than IL-10 signaling. iTreg cells were generated and administered, and EAE was induced as described previously. Subsequently, iTregs, ALK5i, or anti-IL-10R were administered in separate groups, with DMSO treated as the model group. The severity of EAE was assessed (A). Histological changes in the brains of mice from each group on day 30 post-immunization were assessed by removing brain sections, fixing them, and conducting H&E staining, with typical photographs displayed (B). On day 30 post-immunization, brains were harvested, and single-cell suspensions were prepared. Populations of IL-17-producing Th17 cells (C, D) and IFNγ-producing Th1 cells (C, E) were analyzed by flow cytometry, with representative flow cytometry data indicated for each group. Additionally, the frequency of splenic CD11c + DCs was examined by flow cytometry (F). Splenic CD11c + DCs were sorted from each group on day 30, and some were co-cultured with naive Foxp3-GFP T cells (G), while others were co-cultured with CFSE-labeled naive B6 T cells (I). Foxp3-GFP expression (H) and CFSE dilution (J) were assessed by flow cytometry after 3 days of culture. In vitro results presented as means ± SEM of triplicate wells from three independent experiments, with n = 5 mice per group. Statistical analyses were conducted using unpaired t-tests (D, F) and paired t-tests (H, J), with * indicating p < 0.05, ** indicating p < 0.01, *** indicating p < 0.001, and ns. indicating not significant. (PBS-DC vs. iTreg-DC), (iTreg-DC vs. (iTreg + ALK5i)-DC), (iTreg-DC vs. (iTreg + anti-IL-10R)-DC). iTreg cells were generated and administered, and EAE was induced as described previously. Subsequently, iTregs, ALK5i, or anti-IL-10R were administered in separate groups, with DMSO treated as the model group. All mice were sacrificed on day 30, and total splenocytes were collected for analysis. DCs were stained and gated on CD11c (A). The expression levels of CD80 and CD86 (A, B) as well as LAP (A, C) on CD11c + DCs were detected by FACS. Data are presented as mean ± SEM of three separate experiments, with significance denoted as * for p < 0.05, ** for p < 0.01, and ns indicating not significant. (PBS-DC vs. iTreg-DC), (iTreg-DC vs. (iTreg + ALK5i)-DC), (iTreg-DC vs. (iTreg + anti-IL-10R)-DC). 3.5 Inhibition of the AKT/mTOR pathway causes iTreg cells to gain DC tolerogenic capacity The participation of AKT/mTOR signaling pathway in the inflammatory and mature process of DCs was well demonstrated.[ 24 ] AKT Phosphorylation leads to the activation of mTOR signaling along with several downstream targets. P70S6K, one of the most important substrates of mTOR signaling, contributes to the regulation of inflammatory cytokine production in DCs, participating in their antigen presenting and proinflammatory function.[ 25 , 26 ] To investigate whether iTregs modify the immunosuppressive capacity of splenic DCs via AKT/mTOR signaling, intracellular levels of total AKT (t-AKT), phospho-AKT (p-AKT), total P70S6K (t-P70S6K), and phospho-P70S6K (p-P70S6K) in splenic DCs treated with iTregs or tTregs were compared. As measured by WB, the expression of total AKT and P70S6K was similar in DCs treated with iTregs or tTregs (Fig. 5A, 5B). However, p-AKT and p-P70S6K decreased considerably only in the iTreg-treated group, and these differences were statistically significant (Fig. 5A, 5C). Given that high salt and/or inflammatory cytokines distinctly influence the stability and function of both Treg subsets in vitro and in vivo, we immediately assessed the phenotype of DCs cocultured with the Treg subsets that had been pretreated with a combination of NaCl and IL-6. Pretreated iTregs significantly inhibited p-Akt and p-P70S6K expression on DCs compared to that of pretreated tTregs (Fig. 5A, 5E). We did not find statistical differences in t-Akt and t-P70S6K between the two groups (Fig. 5A, 5D). In sum, these findings suggest that AKT/mTOR pathway possibly plays an important role in mediating the suppressive effects of iTregs on the immunomodulatory activity of DCs. Additionally, it can be elucidated that the iTreg subset exhibits enhanced suppressive activity in facilitating the tolerogenic function of DCs in comparison to the tTreg subset. This superior suppressive capability may be achieved through the inhibition of the AKT/mTOR signaling pathway. Both subsets of Tregs were stimulated with or without NaCl at a concentration of 40 mM, in the presence of anti-CD3/CD28 microbeads (5 cells per bead) and rhIL-2 at a concentration of 50 U/ml, for a duration of 3 days. Subsequently, the cells were washed and cocultured with splenic CD11c + DCs for an additional 48 hours, at a T to DC ratio of 2:1 (A). Representative immunoblots depicting the expression levels of Akt, phosphorylated Akt (p-Akt), P70S6K, and phosphorylated P70S6K proteins are displayed (B-E). The data presented are representative of three independent experiments, with statistical significance determined using a paired t-test. Significance levels are denoted as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns indicating not significant. 3.6 iTregs, but not tTregs, have a therapeutic effect on EAE mice by reducing high-salt diet-induced brain inflammation High-salt diet exacerbates EAE progression.[ 27 ] Previous studies have identified a population of pro-inflammatory tTreg cells that are modified by high salt. These cells are characterized by the secretion of IFNγ and have been found to be dysfunctional both in vitro and in vivo.[ 28 ] However, we demonstrated that TGF-β-induced iTreg populations are highly stable and functional under high salt conditions.[ 3 ] Therefore, to determine the varying immunosuppressive capabilities of the two Treg subsets in regulating high-salt diet-fed EAE mice, we conducted an adoptive transfer study using a therapeutic regimen. As depicted in Fig. 6A, we observed that iTregs exerted a significant inhibitory function on the treatment of high salt fed EAE, leading to an amelioration of the clinical scores. However, tTregs had a significantly reduced inhibitory effect on high salt-fed EAE mice progression (Fig. 6A). Similarly, results from the detection of IL-17A + and IFNγ + of CD4 + GFP − T cells in the brains revealed that iTregs significantly reduced the frequency of Th1 and Th17 cells, whereas tTregs failed to suppress both inflammatory T effector cells (Fig. 6B, 6C). Next, as a matter of course, we examined the frequency and phenotype of splenic DCs from each group. CD11c + DCs significantly decreased in the group that received iTreg administration compared to the PBS controls. However, the administration of tTreg had only a slight impact on the frequency of splenic DCs (Fig. 6D, 6E). Meanwhile, iTregs induced a decreased expression of CD80 and CD86 on splenic DCs ( p < 0.05). In contrast, the MFI index of these two DC maturation markers returned to levels similar to those in the PBS control group after treatment with tTregs (Fig. 6F, 6G). Furthermore, LAP expression was augmented in splenic DCs in the iTreg-treated group but not in the tTreg-treated group (Fig. 6F, 6G). Taken together, based on a face-to-face comparison experiment, we validated that both Treg subsets exhibit distinct biological characteristics in a complex environment, such as a high salt environment and under inflammatory conditions. Thus, TGF-β-induced Treg cells may have some advantages in clinical application. Wild-type mice were subjected to a high-salt diet for two weeks prior to active im-munization with MOG35-55 peptide. Subsequently, both subsets of Tregs were gen-erated and administered as described previously. The mean clinical scores of EAE from each group are depicted (A). CD4 + T cells from the brains were analyzed on day 30, with flow cytometric analysis conducted to determine the frequencies of IL17A + and IFNγ + CD4 + cells in the respective mouse groups (B, C). The frequency of splenic CD11c + DCs was assessed by FACS on day 30 (D, E). Kinetic analysis of CD80, CD86, and LAP expression in splenic CD11c + cells was performed using flow cytometry (n = 5 per group and time point) (Panel F, G). Statistical analyses were conducted using one-way ANOVA, with significance indicated as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns indicating not significant. 3.7 CCAR2 deficiency maintains tTreg cell stability in high-salt condition Since the high-salt condition weakened the tTreg cell stability endowing these cells Th17-like phenotype, and had little impact on iTreg cells, we decided to evaluate CCAR2-deficient and CCAR2-expressing tTregs in the same environment (NaCl 40 mM). In this regard, thymic Treg cells (CD4 + CD25 + T cells) were MACS-sorted, and then were stimulated with anti-CD3/CD28 microbeads and IL-2 in the presence or absence of NaCl in vitro . The Foxp3 level was examined after 72 h. CCAR2 deficiency slightly promoted Foxp3 expression in tTreg cells, and this trend was also observed by the addition of salt (Fig. 7A). iTreg cells were induced from the WT or CCAR2 deficiency naïve T cells using the standard protocol either in standard media or in high-salt media for 72 h. We observed that in the absence of CCAR2, differentiation of iTreg cells was markedly enhanced. Identically, there was no significant change of iTreg cell development under high-salt conditions (Fig. 7B). To further validate whether CCAR2 deficiency in Treg cells has the potential resistance to NaCl modification, IL-17A and IFNγ expression were then assessed in these groups. High-salt condition significantly induced IFNγ expression in tTregs. However, in the absence of CCAR2, IFNγ expression in tTregs was decrease to some extent compared to WT tTregs. More importantly, high salt failed to induce IFNγ expression in CCAR2 deficient tTregs (Fig. 7C, E). Similar to IFNγ, high-salt condition also significantly induced IL-17A expression in tTregs. However, CCAR2 deficiency significantly abolished the effect of high salt, as we found a distinct reduction in the frequency of IL-17A + tTregs in high-salt condition compared to the frequency of IL-17A + tTregs in standard media (Fig. 7C, F). Moreover, regardless of whether under the high-salt condition or lack of CCAR2, iTregs were fully stable, which few IFNγ + or IL-17A + iTregs were detected (Fig. 7D, G, H). These data are suggestive of an important role for CCAR2 deficiency in Treg cell stable programming that can be explained for an intrinsic resistance to high salt damage. tTregs and splenic naïve CD4 + T cells were isolated either from the WT or from CCAR2 deficient mice. tTregs were subsequently expanded and activated for 72 h. Simultaneously, naive CD4 + T cells were activated using anti-CD3/28 microbeads in the presence of IL-2 and TGF-β for 72 h. All cells were harvested synchronously followed by re-stimulation in either standard media or media containing 40 mM NaCl for another 72 h. At the onset of pre-activation and after a 72-hour pre-activation period, an aliquot of Treg subsets were analyzed. Intranuclear protein levels of Foxp3 were calculated and depicted in tTregs (A) and iTregs (B) by FACS (n = 3). Flow cytometry analysis of IFNγ and IL-17A production by intracellular staining of cytokines in tTregs (C) and iTregs (D), and data were gated on CD4 + Foxp3 + cells (n = 3). Quantitative analysed of the frequency of IFNγ- -expressing tTregs (E), IL-17A-expressing tTregs (F), IFNγ- -expressing iTregs (G), IL-17A-expressing iTregs (H). Statistical analyses were performed using one-way ANOVA, with significance levels represented as follows: * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns. for non-significant results. 3.8 The inhibiting DC maturation by CCAR2-deficient Treg cells was negligibly affected by high salt. To delineate the functional impact of CCAR2-deficient tTregs and iTregs on DCs, a coculture system was established using splenic CD11c + DCs. Four Treg groups (CCAR2-expressing/deficient tTregs and iTregs) were pretreated with or without NaCl (48 h) and cocultured with DCs (1:5 ratio, 48 h). LPS-stimulated DCs served as positive controls, displaying maximal CD80 and CD86 expression. Compared to untreated tTregs, NaCl-pretreated tTregs (Na-tTreg) induced significantly higher CD80 expression in DCs (p < 0.05). However, CCAR2-deficient tTregs resisted salt-induced instability, as DCs cocultured with NaCl-treated CCAR2-KO tTregs (Na-KO-tTreg) showed no significant CD80 elevation versus untreated CCAR2-KO tTregs (KO-tTreg) (Fig. 8A, B). In contrast, iTregs exhibited stable suppressive function regardless of CCAR2 status or NaCl exposure, with no intergroup differences in CD80 inhibition (Fig. 8A, C). Both tTregs and iTregs significantly suppressed CD80 expression compared to LPS controls (p < 0.05). Similarly, Na-tTreg enhanced DC CD86 expression versus untreated tTreg (p < 0.05), while CCAR2 deficiency abrogated this effect, maintaining low CD86 levels irrespective of NaCl exposure. Notably, KO-tTreg exhibited enhanced suppressive capacity, as DCs cocultured with these cells showed lower CD86 expression than those with wild-type tTregs (p < 0.05) (Fig. 8D, E). iTregs uniformly suppressed CD86 expression across all groups, unaffected by CCAR2 status or NaCl. Both tTreg and iTreg significantly inhibited CD86 expression versus LPS controls (p < 0.05) (Fig. 8D, F). To investigate whether CCAR2-deficient tTregs regulate the AKT/mTOR pathway in DCs, we analyzed phosphorylation events following Treg-DC coculture. For p-AKT modulation, DCs cocultured with Na-KO-tTregs exhibited significantly elevated p-AKT levels compared to those cocultured with untreated tTregs (p < 0.05). In contrast, CCAR2-deficient tTregs maintained stable suppression, as DCs cocultured with Na-KO-tTregs showed no significant p-AKT increase versus untreated KO-tTregs (Fig. 8G, H). iTregs also exhibited stable suppressive function regardless of CCAR2 status or NaCl exposure, with no intergroup differences in p-AKT expression in DCs (Fig. 8G, I). For phospho-S6 (pS6, an mTORC1 readout), LPS-stimulated DCs (positive controls) displayed maximal pS6 mean fluorescence intensity (MFI). Na-tTregs induced significantly higher pS6 MFI in DCs compared to untreated tTregs (p < 0.01) (Fig. 8J, K), whereas iTregs showed no NaCl-dependent differences in pS6 suppression regardless of CCAR2 expression or deficiency (Fig. 8J, L). Both wild-type tTregs and iTregs significantly inhibited pS6 expression compared to LPS controls (p < 0.01), with CCAR2-deficient tTregs demonstrating comparable efficacy to wild-type counterparts (Fig. 8J, K). Notably, both CCAR2-expressing and CCAR2-deficient iTregs similarly inhibited pS6 expression in DCs, with no NaCl-dependent effects (Fig. 8J, L). These results indicate that CCAR2 deficiency protects tTregs from high salt-induced functional impairment by preserving AKT/mTOR suppression in DCs, whereas iTregs retain stable immunosuppressive capacity unaffected by CCAR2 knockout or NaCl exposure. The two subsets of Tregs were subjected to stimulation either in the presence or ab-sence of 40 mM NaCl, alongside anti-CD3/CD28 microbeads (at a ratio of 5 cells per bead) and rhIL-2 at 50 U/ml, for a period of 3 days. Following this, cells from each group were thoroughly washed and subsequently co-cultured with splenic CD11c + DCs for an additional 24 hours, maintaining a T cell to DC ratio of 5:1. Representative flow cytometry data depicting CD80 expression levels on CD11c + cells are presented, comparing co-culture conditions derived from the four distinct tTreg groups (A, B) and the four iTreg groups (A, C). Representative FACS plots showing CD86 expression in CD11c + cells co-cultured either from the 4 different tTreg groups (D, E), or from the 4 different iTreg groups (D, F). Total cells were treated with PMA, ionomycin, and monensin to induce cellular activation. Then cells were subjected to immunostaining for the surface marker CD11c, followed by the intracellular staining p-AKT. Flow cytometry analysis of the expression of p-AKT in CD11c + DCs cultured from these tTreg groups (G, up), or from the 4 identical treatment iTreg groups (G, down). Data were summarized in the bar graph, which provides a quantitative analysis of the relative frequency of CD11c + p-AKT + DCs within the total cell population under the systems co-cultured from tTreg groups (H) or from the 4 different iTreg groups (I). Flow cytometry analysis of ps6 expression in each group shows MFI values, representing ps6 expression from five different tTreg groups (J,K), as well as five different iTreg groups(J,L). The statistical comparisons between tTreg and Na-tTreg, KO-tTreg and Na-KO-tTreg, iTreg and Na-iTreg, as well as KO-iTreg and Na-KO-iTreg were performed using paired t-tests, while comparisons among tTreg, Na-tTreg, and control were conducted using one-way ANOVA. Significance levels are indicated as follows: * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns. for non-significant results. 4 Discussion While the initial identification of Tregs occurred within the thymus, subsequent research rapidly revealed that this cell population can be induced and differentiated from non-Treg cells with TGF-β and IL-2.[ 29 , 30 ] TGF-β initiates the phosphorylation and activation of Smad2 and Smad3, pivotal for the induction of Foxp3 during iTreg generation.[ 31 ] Both Treg subsets express canonical Treg markers, such as Foxp3, CD25, GITR, and CTLA4. However, tTregs exhibit higher expression levels of PD-1, neuropilin-1 (Nrp-1), Helios (Ikzf2), and CD73 compared to their iTreg counterparts.[ 32 ] Certain studies have proposed that Foxp3 expression stability in tTregs is maintained via demethylation of CpG islands within the conserved non-coding sequence 2 (CNS2) region of the Foxp3 locus. This region serves as a binding site for various transcription factors such as Stat5 and Runx1/Cbfb, contributing to tTreg stability. Conversely, unstable Foxp3 expression in iTregs is purportedly associated with pronounced demethylation of CNS2.[ 33 ] However, observations from our study and those of others challenge the notion of tTreg stability, particularly under arthritic and inflammatory conditions. Under such circumstances, tTregs exhibit susceptibility to redifferentiation into alternative T effector cell subsets, accompanied by functional changes. In contrast, iTregs demonstrate a lack of this plasticity and display enhanced suppression of osteoclastogenesis and bone erosion compared to tTregs.[ 34 ] High salt levels can promote the differentiation of pathogenic Th17 cells while concurrently dampening the suppressive capacity of tTregs, thereby expediting the onset of EAE.[ 28 ] By using a high-salt diet in a Rag1 −/− colitis model, we provided supplementary evidence indicating that iTregs, but not tTregs, significantly ameliorate intestinal inflammation.[ 8 ] Additionally, we present compelling evidence demonstrating the greater stability of iTregs compared to tTregs, as evidenced by the heightened resistance of iTreg destabilization in the presence of exogenous IL-6 and NaCl combination stimulation in vitro. In the specific EAE model used in our study, tTregs effectively curtailed disease progression. However, their efficacy was compromised in inhibiting EAE advancement in mice subjected to a high-salt diet. Notably, iTregs exhibited superior efficacy in mitigating disease progression, even under high-salt or pro-inflammatory conditions. The therapeutic efficacy of iTregs can be elucidated by several factors. Firstly, the suppressive function of tTreg cells may be compromised by pro-inflammatory cytokines owing to their expression of IL-6R. Numerous studies have reported the abrogation of tTreg suppressive activity in response to IL-6.[ 12 , 35 ] Notably, iTregs demonstrate diminished expression of IL-6 receptor compared to tTregs, rendering them resistant to this cytokine induced transformation and preserving their phenotype and function.[ 20 ] Hence, it is plausible that CD126-negative tTregs may exhibit superior functional activity.[ 36 ] Secondly, iTregs, but not tTregs, exhibit negligible levels of SOCS1 and SOCS3 in response to IL-6 stimulation. SOCS1 has been extensively implicated in mediating Th17 cell differentiation.[ 37 ] Therefore, the differential expression of SOCS proteins may contribute to the activation of STAT-3 in tTreg cells and the potential conversion of tTreg cells into Th17 cells.[ 38 ] Additionally, we demonstrated that Foxp3 exhibits instability following TNF-α treatment in a manner that is dependent on CCAR2/ DBC1, which the deficiency of CCAR2 protects Foxp3 from degradation and preserves Treg cell functionality in response to inflammatory stimulation.[ 12 ] Whether iTregs express CCAR2 at lower levels compared to tTregs, and whether high salt environments are associated with CCAR2 expression, both require further investigation. Thirdly, TGF-β could upregulate Bcl-2 expression and diminish T cell apoptosis in recovered iTregs, indicating that iTregs may exhibit reduced susceptibility to apoptosis compared to tTregs.[ 39 ] Additionally, our preliminary findings indicate that atRA, a crucial metabolite of vitamin A with diverse immunoregulatory functions, has the potential to inhibit the pro-inflammatory response induced by high salt. In this study, we observed high salt only induces barely detectable IFNγ and IL-17A production in splenic Treg cells. In other words, thymus-derived Treg cells and spleen-derived Treg cells may exhibit phenotypic and functional differences under high-salt stimulation. And this suggests, to some extent, that a high salt environment may serve as an effective external medium for identifying the heterogeneity in Treg cell populations.[ 40 ] DCs represent a pivotal cell subset responsible for initiating immune responses, with their pro- or anti-inflammatory polarization influenced by various environmental cues. Prior investigations have indicated that iTreg cells possess the capability to confer immunoregulatory properties upon DCs.[ 41 ] MS constitutes an autoimmune disease characterized by immune dysregulation, culminating in the infiltration of immune cells into the CNS, thereby instigating demyelination, axonal injury, and neurodegeneration.[ 41 ] Our research has unveiled a novel biological role for iTregs in directing the differentiation of profoundly tolerogenic DCs. This significant finding holds promise for the development of new immunotherapeutic strategies targeting a spectrum of autoimmune disorders. Our findings demonstrate that iTreg cell function, in part, by modulating DCs, thereby orchestrating the delicate immuno-balance, consequently contributing to the amelioration of EAE. In MS, DCs exhibit an activated phenotype and initiatively induce pathogenic Th17 cell development. Subsequently, these Th17 cells migrate to the CNS where they instigate attacks on oligodendrocytes, leading to demyelination. In our study, administration of Treg cells not only inhibits the activation of splenic DCs but also confers tolerogenic activities upon them. This tolerogenic capacity relies on DCs establishing a complex network of cell-to-cell interactions.[ 42 ] Various factors contribute to the induction of a tolerogenic DC phenotype, including IL-10, TGF-β, and vitamin D. Studies have revealed the mechanisms by which Treg cells confer tolerogenic attributes to DCs; the most critical signaling pathways identified to date encompass IL-10, CTLA-4, and TGF-β.[ 43 ] Notably, the effects mediated by IL-10 were observed predominantly when immature DCs were exposed to IL-10, while mature DCs remained insensitive to IL-10 stimulation, maintaining a stable, mature phenotype.[ 44 ] In asthmatic mice, Treg cells secrete IL-10, mediating the induction of tol-DCs, and blockade of IL-10 leading to suboptimal generation of tol-DCs.[ 45 ] However, in lupus mice, IL-10 appears dispensable for tol-DC induction.[ 42 ] TGF-β partially exerts suppressive functions by inducing Foxp3 gene and protein expression in T cells.[ 46 ] Protective effects of transferred iTreg cells require both IL-10 and TGF-β. However, the influence of iTregs on the induction of tol-DCs in the EAE model remains unexplored. The AKT/mTOR signaling axis plays a pivotal role in modulating the maturation, activation, and survival of DCs.[ 47 ] mTOR regulates protein synthesis by directly phosphorylating and inactivating the repressor of mRNA translation, and by phosphorylating and activating S6 kinase (p70S6K).[ 48 ] However, mTOR signaling in DC’s function during inflammatory immune responses remains contentious. Mainstream research indicates that inhibition of the Akt/mTOR pathway, particularly through rapamycin, enhances Treg induction and diminishes the immunological effects of DCs.[ 49 ] Additionally, inhibition of AKT and p70S6K phosphorylation facilitates the differentiation of iTreg cells.[ 50 ] Notably, 1,25(OH) 2 D3 has emerged as a significant regulator of the immune system, exerting its effects by inducing immune tolerance in DCs. Treatment with 1,25(OH) 2 D3 suppresses the maturation of bone marrow-derived DCs and promotes a dominant tolerogenic function, achieved by the inhibition of the AKT/mTOR signaling.[ 51 ] However, from a cellular metabolism standpoint, prior research has underscored the critical role of the PI3K/Akt/mTOR pathway in maintaining the tolerogenic phenotype of 1,25(OH) 2 D3-modulated DCs.[ 52 ] Our study corroborate that iTregs suppress the Akt/mTOR pathway, which contribute to the induction of an anti-inflammatory profile in murine DCs. Notably, the expression of the Foxp3 gene, a classical Treg marker, is regulated by the HIF-1α/mTOR pathway, underscoring the significance of this signaling cascade in Treg function. Excessive intake of salt enhances pathogenic Th17 cell differentiation, leading to the progress of a highly pathogenic phenotype that exacerbates EAE. However, contrary to this effect, a prior study has demonstrated that the function of myeloid DCs remains largely unaffected by salt in vitro. Furthermore, high salt intake exacerbates neuroinflammation in EAE mice independently of mature DCs.[ 53 ] This indicates that distinct subsets of immune cells exhibit differential responses to NaCl. Notably, the induction of a pro-inflammatory environment by salt appears to involve specific effects on immune cells rather than non-specific activation of all lymphocytes and APCs.[ 54 ] Consistent with this notion, our experiments demonstrated that high salt intake significantly compromises the therapeutic efficacy of tTreg cells, whereas iTreg cells remain relatively unaffected in EAE. Consequently, iTregs foster tolerogenic DC generation, in contrast to tTregs, thereby contributing to the amelioration of symptoms in EAE mice subjected to a salt-rich diet. In summary, our findings offer further evidence supporting the notion that iTregs exhibit distinct biological properties compared to tTregs. Also, these results underscore the potential clinical relevance of iTregs in patients diagnosed with autoimmune and inflammatory conditions, specifically highlighting the importance of considering the complex influence of environmental factors such as diet. Abbreviations The following abbreviations are used in this manuscript: iTregs induced CD4+Foxp3+ regulatory T cells; tTregs thymus-derived CD4+CD25+ regulatory T cells; Treg subsets (tTregs and iTregs); CCAR2 cell cycle and apoptosis regulator 2; CFSE carboxyfluorescein diacetate succinimidyl ester; EAE experimental autoimmune encephalomyelitis. Declarations Data availability statement All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author. Acknowledgements We would like to acknowledge the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study. Funding The National Natural Science Foundation of China (81960293); the China Postdoctoral Foundation project (2023M731460); the Natural Science Foundation of Gansu Province (20JR5RA3); the Joint Research Fund of Gansu Province (23JRRA1495); the Lanzhou Chengguan District talent innovation and entrepreneurship project (2023RCCX0021), the Student Innovation and Entrepreneurship Program of Lanzhou University (LZU-JZH2634, 202410730187, 20240060201), the Medical Research Improvement Project of Lanzhou University (lzuyxcx-2022-165), the Major scientific and technological innovation project of Health industry in Gansu Province (GSWSQNPY2024-11). Authors’ contributions Yang Luo: Conceptualization, Methodology, Funding acquisition, Figure Supervision. Yating Li, Lingxiao Song and Jun Yang: Conceptualization, Funding acquisition, Investigation, Data curation, writing – original draft. Jiale Tian, Xiaonan Li and Li Zhang: Data curation, Software. Haitao Yu: Resources and Editing. Youquan Gu: Investigation, Resources. All authors read and approved the final draft. Ethics approval and informed consent I confirm that I have read the Editorial Policy pages. All animal experiments in this study were approved by the ethics committee of the first hospital of Lanzhou university (protocol code: LDYYLL-2023-454). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Consent for publication Not Applicable. Conflicts of Interest All authors declare that they have no conflict of interest. References Raffin C, Vo LT, Bluestone JA: T(reg) cell-based therapies: challenges and perspectives . Nature reviews. Immunology 2020, 20 (3):158-172 doi.org/10.1038/s41577-019-0232-6. 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Additional Declarations No competing interests reported. Supplementary Files FigureS1andS2.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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6528083","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451097091,"identity":"16766774-9ce0-472c-a4de-cd6eac330af4","order_by":0,"name":"Yating Li","email":"","orcid":"","institution":"The First Clinical Medical College, Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yating","middleName":"","lastName":"Li","suffix":""},{"id":451097092,"identity":"c45d45b7-6bb6-4b63-a4b6-eb748de6a342","order_by":1,"name":"Linxiao Song","email":"","orcid":"","institution":"The First Clinical Medical College, Lanzhou 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2","display":"","copyAsset":false,"role":"figure","size":240558,"visible":true,"origin":"","legend":"\u003cp\u003eiTregs outperform tTregs in terms of functional activity during EAE-induced brain inflammation\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6528083/v1/c6ed5c9e1d9bb381fa41c3bb.png"},{"id":82086478,"identity":"2e3badb4-390a-4cb7-8da7-4f8d30cb49c9","added_by":"auto","created_at":"2025-05-06 15:14:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":271220,"visible":true,"origin":"","legend":"\u003cp\u003eIn the EAE environment, iTregs suppress the pro-inflammatory DC phe-notype while inducing tolerogenic activities\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6528083/v1/49211e4774ddb9143a103f82.png"},{"id":82086480,"identity":"4c46ce50-e04d-481f-9a0c-c96eec3dd9ae","added_by":"auto","created_at":"2025-05-06 15:14:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":325311,"visible":true,"origin":"","legend":"\u003cp\u003eiTregs give DCs a tolerogenic phenotype and function primarily through TGF-β, not IL-10 signaling\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6528083/v1/1939a5d7b87733b7d889b571.png"},{"id":82086961,"identity":"ddc1eee2-cd57-4b9c-aad8-8c9d2136e021","added_by":"auto","created_at":"2025-05-06 15:22:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":196222,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of the AKT/mTOR pathway causes iTreg cells to gain DC tolerogenic capacity\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6528083/v1/d130dec7d3b9cb9a7eb8b234.png"},{"id":82086962,"identity":"24869466-de48-4a81-b523-42283da1571a","added_by":"auto","created_at":"2025-05-06 15:22:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":241059,"visible":true,"origin":"","legend":"\u003cp\u003eiTregs, but not tTregs, have a therapeutic effect on EAE mice by reducing high-salt diet-induced brain inflammation\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6528083/v1/3289148e9e6404a6e16e85fe.png"},{"id":82086485,"identity":"0aad9c43-e3ee-4c76-ba51-4588e2db3b62","added_by":"auto","created_at":"2025-05-06 15:14:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":205861,"visible":true,"origin":"","legend":"\u003cp\u003eFoxp3 Expression and Treg Conversion Remain Unaffected in CCAR2 deficiency Treg cell subsets Under High-Salt Conditions In Vitro.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6528083/v1/48c6663b85393df7cc3c960d.png"},{"id":82086488,"identity":"1fd437ee-361d-424f-8565-523798a202b9","added_by":"auto","created_at":"2025-05-06 15:14:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":410099,"visible":true,"origin":"","legend":"\u003cp\u003eThe ability of CCAR2-deficient Treg cell subsets to inhibit DC maturation was only minimally impacted by high salt levels.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6528083/v1/47c01a7f2d033c38ce0089d6.png"},{"id":98421864,"identity":"1c094e8a-dbe0-4ed2-aba1-f164dacbbb8a","added_by":"auto","created_at":"2025-12-17 16:29:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5631527,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6528083/v1/33a2ce83-d07b-4ea5-8c31-99bfb0ea6bac.pdf"},{"id":82086477,"identity":"f3af8a47-fbf1-4c68-bae6-439d50f03334","added_by":"auto","created_at":"2025-05-06 15:14:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":322472,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1andS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6528083/v1/33d511f218adc8dfa0705985.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"CCAR2 Dictates tTreg Instability and iTreg-Driven Dendritic Cell Tolerance via Divergent AKT/mTOR Modulation in High-Salt Microenvironments","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e regulatory T cells (Tregs) exert their immunosuppressive actions to limit tissue injury and control hyperinflammatory responses contributing to homeostasis.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Tregs display heterogeneity and are broadly categorized into three subsets: thymus-derived naturally occurring Treg cells (tTregs), peripherally derived Treg cells (pTregs), and TGF-β-induced Treg cells (iTregs). [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Previous studies, including our own, have underscored both the similarities and distinctions among specific Treg subsets.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Despite their notable preventive role in controlling autoimmune diseases in various animal models such as experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis (CIA), dextran sodium sulfate (DSS)-induced colitis, and streptozotocin-induced type 1 diabetes (T1DM), the therapeutic efficacy of tTregs remains suboptimal.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Komatsu et al. reported that adoptively transferred tTregs preferentially lose Foxp3 expression and undergo trans differentiation into pathogenic T helper 17 (Th17) cells, thereby exacerbating the onset and severity of the CIA model.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Furthermore, other scholars, in conjunction with our findings, have reported tTregs are prone to convert into other T effector cell subsets, including IFNγ-producing (Th1), IL-17A-producing (Th17) T cells, concomitant with the Foxp3 degradation and a reduction in their immunosuppressive functionality.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] These changes often occur in the disease-microenvironment with autoimmune abnormalities.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Furthermore, our studies have documented the resistance of iTregs, but not tTregs, to IL-6-driven conversion into Th17 cells. Notably, adoptive transfer of iTregs, but not tTregs, significantly mitigated bone erosion in the CIA model, attributed to the enhanced stability and functionality of iTregs compared to tTregs following cell infusion.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eStudies have demonstrated that high-salt conditions diminish the suppressive capacity of tTreg cells.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] Sodium chloride (NaCl) constitutes a fundamental component of daily dietary intake. Preliminary investigations have unveiled that excessive salt promotes the differentiation of Th17 cells, culminating in a highly pathogenic phenotype that exacerbates EAE.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] High-salt intake was also demonstrated that can activate the complement system, inflammasomes, leading to salt-sensitive hypertension.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] High salt could also induce lipid oxidation in DCs, which in turn, exacerbates high blood pressure.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Furthermore, high salt increases the secretion of IFNγ in tTregs, compromising their suppressive functionality and exacerbating experimental graft-versus-host disease.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] In contrast, the results of our study revealed that, unlike tTregs, iTregs remain relatively stable and function effectively under conditions of elevated sodium chloride concentrations. Notably, high salt does not significantly change the transcriptional profiles of either iTreg-specific markers or inflammatory genes. Moreover, we in-vivo corroborated that iTregs exert substantial control over colitis progression, whereas tTregs predominantly lose their inhibitory potency.\u003csup\u003e5\u003c/sup\u003e We previously demonstrated that cell cycle and apoptosis regulator 2 (CCAR2, also called Deleted breast cancer 1, DBC1) functionally cooperates with Foxp3, resulting in triggering Foxp3 degradation in inflammatory cytokines stimulation. CCAR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Treg cells maintained Foxp3 expression and enhanced suppressive function when compared to WT Treg cells under TNF-α treatment.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eEAE serves as the primary experimental model for multiple sclerosis (MS), a human inflammatory demyelinating disorder characterized by immune dysregulation and infiltration of immune cells into the central nervous system (CNS).[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] Notably, T cells play a pivotal role in the pathogenesis of EAE, wherein peripheral T cell activation by viral or other infectious antigens or superantigens leads to the production of inflammatory cytokines and facilitates their traversal across the blood-brain barrier. The severity of EAE is also correlated with the recruitment of dendritic cells (DCs) into the CNS.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Particularly, conventional DCs, which are highly specialized antigen-presenting cells (APCs), assume a critical role in immune activation by bridging innate and adaptive immune system.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] Under immune homeostatic conditions, DCs patrol the CNS microenvironment, functioning as sentinels. Upon activation, these DCs adopt a pro-inflammatory phenotype and migrate to lymph nodes, thereby fostering the generation of self-reactive T cells and other immune cell subsets. Conversely, DCs also possess tolerogenic properties, contributing to the maintenance of central and peripheral tolerance as well as the resolution of ongoing immune reactions.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] Tolerogenic DCs (tol-DCs) restrain effector T cells and promote Treg differentiation through various mechanisms, including cytokine secretion (e.g., IL-10, IL-27, and TGF-β), expression of indoleamine 2,3-dioxygenase (IDO), and regulation of extracellular levels of adenosine triphosphate (ATP) and adenosine. Treg cells possess the ability to confer tolerogenic functions upon conventional DCs, thereby suppressing Th1 and Th17 responses and ameliorating the autoimmune disease phenotype.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] Consequently, despite numerous studies documenting the preventive roles of both tTregs and iTregs in EAE progression, uncertainties persist regarding whether both Treg subsets exert equivalent immunosuppressive effects in inhibiting EAE exacerbation induced by high dietary salt intake. Additionally, elucidating whether Treg cells modulate EAE progression through DC phenotype modulation remains an important avenue of inquiry.\u003c/p\u003e \u003cp\u003eWe demonstrated that iTregs, unlike tTregs, robustly resist high-salt-induced inflammatory conditions, effectively attenuating high-salt diet-aggravated EAE progression. This is primarily attributed to iTregs significantly reducing conventional DC frequency, downregulating antigen-presenting molecules, and upregulating LAP expression. Moreover, CCAR2 depletion stabilizes tTregs, preventing their Th17-like transformation under high-salt conditions, whereas iTregs enhance DC tolerogenic phenotypes independently of CCAR2.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1Mice\u003c/h2\u003e \u003cp\u003eMale C57BL/6J (B6) mice, 6\u0026ndash;8 weeks old, were purchased from Veterinary Research Lanzhou Institute. B6 CCAR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and B6 Foxp3-GFP mice were kindly provided by Pro. Songguo Zheng (School of Cell and Gene Therapy, Shanghai Jiao Tong University School of Medicine, Shanghai, China.) B6 Rag1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were purchased from Sai ye Biotechnology Co., LTD. All mice were kept under specific pathogen-free conditions of Lanzhou University Medical School. Animals were used in accordance with the regulations stipulated by the Animal Care and Use Committee of Lanzhou University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 EAE, high salt diet\u003c/h2\u003e \u003cp\u003eThe anesthetized mice were subcutaneously injected on the upper and lower back with 250 \u0026micro;g of MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e (Proteimax), fully emulsified in an equivalent volume of Complete Freund's Adjuvant (CFA, Sigma, St Louis, MO, USA) containing 4 mg/ml of heat-killed Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA). At approximately 0- and 48- hour post-immunization, intraperitoneal injections of 500 ng pertussis toxin (Alexis, San Diego, USA) were administered. Clinical scores were daily assessed, with the mean score of each mouse being noted every three days. In certain experiments, mice were provided with a high salt-rich chow containing 6% sodium chloride (ssniff, Germany; Xie-tong, Jiangsu, China) and tap water containing 1% NaCl (NaCl high) ad libitum for 2 weeks prior to EAE induction. Throughout the experimental period, no observable differences of weight change were noted (data not shown), indicating similar consumption of food and water between groups. The high-salt diet was maintained until the mice were sacrificed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Adoptive transfer experiments\u003c/h2\u003e \u003cp\u003eC57BL/6 EAE mice were randomly assigned to receive tail intravenous injections of iTregs, tTregs (both 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mouse), or vehicle saline on day 4 post-immunization. To investigate the in-vivo mechanical suppression of Treg subsets, anti-IL-10 receptor or isotype-matched IgG1 antibody (0.25 mg/kg body weight), or ALK5 inhibitor (LY-364947, 0.5 mg/mouse; Sigma) were treated intraperitoneally weekly for a total of 3 injections, with the first injection occurring the day after cell infusion. In most experiments, each group consisted of 5 mice, with experiments repeated at least twice yielding similar results, and the data presented here represents one of those experiments. On day 30 after MOG injection, all mice (n\u0026thinsp;=\u0026thinsp;5 mice per group) were euthanized, and cells from spleens and brains were harvested. Proportions of Treg cells, Th17 cells, and Th1 cells, or CD11c\u003csup\u003e+\u003c/sup\u003e DCs were examined by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Tissue sampling and immune cell isolation\u003c/h2\u003e \u003cp\u003eAt day 30, animals were euthanized using the perfusion with 4% (w/v) paraformaldehyde under deep anesthesia (10% chloralhydrate, 0.2ml/mouse, i.p.), and then perfused transcardially using cold phosphate buffered saline. Subsequently, brain, spleen and lymph nodes were collected for further processing. Specifically, spleens and lymph nodes were placed in cell strainers (70 \u0026micro;m) and homogenized using syringe plungers, followed by washing with in RPMI-1640 containing 2% NCS. Brain tissues were minced and then subjected to digestion using 2 mg/ml collagenase IV (Sigma Aldrich) in 1640 containing 2% NCS at 37\u0026deg;C with agitation for 60 minutes. Following digestion, brain tissues were filtered and subsequently separated using a 30%/70% Percoll solution (Cytiva) via centrifugation (800 \u0026times; \u003cem\u003eg\u003c/em\u003e, 20 min). Then isolated cell pellets were collected for staining and flow detection procedure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Histology\u003c/h2\u003e \u003cp\u003eMice in each group underwent perfusion with 4% (w/v) paraformaldehyde under deep anesthesia induced by intraperitoneal injection of 10% chloralhydrate (0.2 ml/mouse). Subsequently, brains were meticulously extracted and embedded in paraffin, and the segments were dissected and subjected to staining with hematoxylin and eosin (H\u0026amp;E) following established protocols, which the degree of inflammation was blindly assessed according to the published criteria.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] Scores were in a semiquantitative fashion for inflammation graded: 0, no inflammatory cell infiltration; 1, scattered cell infiltration can be seen throughout the visual field; 2, organization of perivascular infiltrates, or mild cell infiltration into the parenchyma; 3, perivascular cuffing with extension into the adjacent tissue.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 tTreg isolation, iTreg generation, Treg subset conversion in vitro and in vivo\u003c/h2\u003e \u003cp\u003eThymus-derived Tregs (tTregs) from B6 WT, B6 CCAR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e or B6 Foxp3-GFP mice through gating of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003e or CD4\u003csup\u003e+\u003c/sup\u003eGFP\u003csup\u003e+\u003c/sup\u003ecells, achieving a purity of 95\u0026ndash;99%. These cells were subsequently stimulated with anti-CD3/CD28-coated beads (Invitrogen, Carlsbad, CA) at a ratio of 1 bead: 2 cells and rhIL-2 (100 U/ml; R\u0026amp;D Systems, Minneapolis, MN) for 72-hour expansion. All tTregs utilized in the in-vitro experiments of this study underwent prior expansion.\u003c/p\u003e \u003cp\u003eFor iTreg cell generation, na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e\u0026minus;\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e T cells from WT, CCAR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e or Foxp3-GFP B6 mice (purity\u0026thinsp;\u0026gt;\u0026thinsp;95%) were stimulated with anti-CD3/CD28 dynabeads (cells: beads\u0026thinsp;=\u0026thinsp;5:1, Invitrogen) in the presence of rhTGF-β (2 ng/ml) and rh-IL-2 (50 U/ml) (both from R\u0026amp;D System) for 72 hours. Both Treg subsets were cultured in X-VIVO 15 medium (LONZA) in the presence or absence of NaCl (40 mM, Sigma-Aldrich), IL-6 (100 ng/ml, R\u0026amp;D System), or a combination of both.\u003c/p\u003e \u003cp\u003eFor in-vivo Treg subset conversion, primary isolated tTregs or generated iTregs from Foxp3-GFP reporter mice were further sorted into CD4\u003csup\u003e+\u003c/sup\u003eGFP\u003csup\u003e+\u003c/sup\u003e cells with a purity of 99%. Then, a total of 0.5x10\u003csup\u003e6\u003c/sup\u003e purified tTregs or iTregs were intravenously administrated into Rag1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. In some groups, tTregs or iTregs were pre-treated with 40 mM NaCl and 100 ng/ml IL-6 for 48 hours prior to sorting and adoptive transfer. Mice were euthanized on days 5 and 10, and T cells from the mesenteric lymph nodes (MLN) were stained for IFNγ and IL-17A.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Treg suppression assay in vitro\u003c/h2\u003e \u003cp\u003eInitially, both tTregs and iTregs were pre-treated with or without 40 mM NaCl and 100 ng/ml IL-6 in the presence of anti-CD3/CD28 dynabeads (5 cells per bead) for 72 hours. Subsequently, the pretreated cells were co-cultured with 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e\u0026minus;\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e T cells (from WT B6 mice) labeled with 2 \u0026micro;M CFSE (BioLegend) at various ratios as indicated. Cell mixtures were simultaneously stimulated with soluble anti-CD3 (0.5 \u0026micro;g/ml) for 3 days. Division index was calculated by assessing the rates and intensity of CFSE dilution using flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Co-culture of naive T responder cells with DC subsets\u003c/h2\u003e \u003cp\u003eSplenic CD11c\u003csup\u003e+\u003c/sup\u003e DCs were isolated from PBS-, iTreg-, tTreg-treated EAE mice at day 30 using the mouse CD11c- cell MicroBeads isolation kit (Miltenyi). For DC-mediated proliferation assay, the WT B6 na\u0026iuml;ve T cells labeled with CFSE (2 uM) were cocultured with these DC subsets in the presence of anti-CD3 (0.1 ug/ml; BD Pharmingen) stimulation for 3 days. T-cell proliferation was assessed by analyzing the CFSE dilution by FACS. For the DC-induced Treg cell differentiation assay, WT CD4\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e\u0026minus;\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e na\u0026iuml;ve T cells sorted from the B6 Foxp3-GFP mice were cocultured with these DC subsets with the anti-CD3 (0.5 ug/ml) and rh-IL2 (50 U/ml) addition for 3 days. GFP expression was assessed using flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Co-culture of Treg cells with splenic DCs\u003c/h2\u003e \u003cp\u003eSplenic CD11c\u003csup\u003e+\u003c/sup\u003e DCs (WT B6 mice) with the purity\u0026thinsp;\u0026gt;\u0026thinsp;90% were cultured in a medium supplemented with GM-CSF (500 U/ml) and IL-4 (200 U/ml) for 72 h. Then DCs were collected, washed, and re-seeded in 48-well plates containing RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) followed by the co-culture with WT-tTregs, WT-iTregs, NaCl-pretreated WT-tTregs and NaCl-pretreated WT-iTregs at a ratio of 5:1 (Tregs:DCs ) in the presence of anti-CD3 (0.5 \u0026micro;g/ml, BD Pharmingen) stimulation for 24 hours. Subsequently, DCs from each group were collected separately followed by WB examination.\u003c/p\u003e \u003cp\u003eIn additional mechanistic studies, WT or CCAR2-knockout tTregs and iTregs, including those pretreated with NaCl for 48 hours, were co-cultured with WT splenic CD11c\u003csup\u003e+\u003c/sup\u003e DCs at the same 5:1 ratio for 24 hours in the presence of anti-CD3 (0.5 \u0026micro;g/ml) stimulation. Similarly, DCs were then collected separately, but followed by FACS examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Flow-cytometric analyses\u003c/h2\u003e \u003cp\u003eCollected cells were resuspended in cold PBS containing 0.1% BSA and then stained with conjugated-antibodies or their isotype controls for CD3, CD4, and CD25 (eBioscience) at 4\u0026deg;C for 15\u0026ndash;20 min. To assess Foxp3 expression, cells were first subjected to surface staining, followed by treatment with a fixation buffer (Biolegend). Subsequently, the cells were permeabilized using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and maintained at 4\u0026deg;C for 1 h, then were incubated with FITC-conjugated rat anti-mouse Foxp3 antibody (FJK-16s, eBioscience) for FACS detection.\u003c/p\u003e \u003cp\u003eFor intracellular staining of cytokines, cultured cells were stimulated \u003cem\u003eex vivo\u003c/em\u003e with PMA and ionomycin (both 0.25ug/ml; Sigma-Aldrich) for 5 hours at 37\u0026deg;C in the presence of brefeldin A (5 ug/ml; BioLegend) for the last 4 hours. After surface staining, cells were fixed and permeabilized using the Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) prior to intracellular staining: anti-mouse IFNγ-PE and anti-mouse IL-17A-APC (TC11-18H10).\u003c/p\u003e \u003cp\u003eFor DC staining, total splenocytes were pretreated with anti-CD16/CD32 antibodies (BD Biosciences), then stained with the surface antibodies: anti-CD11c, anti-MHCII, anti-CD80, anti-CD86, anti-LAP, anti-B7H4 (all from eBioscience). To assess p-Akt and p-P70S6K expression in DCs, surface CD11c stained cells were fixed, then permeabilized, followed by the incubation with monoclonal rabbit (Ser473, Cell Signaling Technology) against p-Akt or Phospho-p70 S6 Kinase (Thr389) Antibody (649704, BioLegend). IL-10 production in DCs was measured by stimulating total splenocytes with 10 ng/ml PMA and 100 \u0026micro;g/ml ionomycin for 6 hours in the presence of 5 \u0026micro;g/ml Monensin. After surface CD11c staining, cells were fixed, permeabilized, and then anti-mouse IL-10-APC (JES5-16E3, Biolegend) was used for intracellular staining. All flow cytometric analyses were conducted using the following isotype controls: PE Rat IgG2a, Rat IgG2k; APC Rat IgG2a, Rat IgG1; PerCP-Cy5.5 Rat IgG2b. Data were analyzed and acquired with FlowJo 10 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Western blotting\u003c/h2\u003e \u003cp\u003eDCs co-cultured with different Treg subsets were collected and lysed in pre-warmed RPMI 1640. Briefly, total protein was extracted from cultured CD11c\u0026thinsp;+\u0026thinsp;cells and transferred onto PVDF membranes, which were subsequently blocked in 5% BSA or 5% non-fat milk in TBST for 1 hour. Following this, the membranes were incubated overnight at 4\u0026deg;C with primary antibodies targeting phospho-Akt (Ser473), total AKT, phospho-P70S6 kinase (Thr389), total P70S6 kinase, and GAPDH, all sourced from Cell Signaling Technology (Danvers, MA). On the following day, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour. Protein bands were visualized using ECL reagent (Beyotime, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical methods\u003c/h2\u003e \u003cp\u003eStatistics were conducted using GraphPad Prism software (Version 5), if not indicated elsewhere, as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. To assess differences between groups, two-tailed Student\u0026rsquo;s t-test was used. For comparisons across multiple groups, one-way ANOVA analysis was performed with Tukey\u0026rsquo;s post hoc tests. Statistical significance was considered as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ns, not significant).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003e \u003cb\u003e3.1 iTregs and tTregs exhibit distinct functional stability and immunosuppressive capacities under high-salt condition combined with pro-inflammatory cytokine stimulation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have reported that the inflammatory cytokine, like IL-6 drives the conversion of tTregs, rather than iTregs, into pathogenic Th17 cells.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Additionally, sodium chloride converts tTregs, but not iTregs, into IFNγ-producing Th1-like cells.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] To further compare the conversion of the two Treg subsets into the Th17 or/and Th1 lineages, NaCl combined with IL-6 was used as a stimulant. CD4\u003csup\u003e+\u003c/sup\u003eGFP\u003csup\u003e+\u003c/sup\u003e cells were sorted from thymic cells (tTregs) in B6 Foxp3-GFP mice, while iTreg cells (iTregs) were generated from splenic na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e\u0026minus;\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003eGFP\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells using a standard protocol as previously described.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Initially, these Tregs were cultured in the presence of NaCl (40 mM) or IL-6 (100 ng/mL) for 3 days, and subsequently, IFNγ- or IL-17A-producing cells were examined by flow cytometry. Consistent with previous findings, the tTreg subset exhibited higher levels of IFNγ and IL-17A expression compared to the iTreg subset under 40 mM NaCl stimulation (Figure S1A, 1B). Furthermore, when exposed to IL-6, tTregs also expressed significantly more IL-17A than iTregs (Figure S1A, S1C). Interestingly, when each Treg subset was co-stimulated with both IL-6 and NaCl in the presence of anti-IFNγ, IL-17A production was robustly increased in the tTreg subset, whereas the iTreg subset remained entirely resistant to expressing IL-17A (Fig.\u0026nbsp;1A, B).\u003c/p\u003e \u003cp\u003eSubsequently, the in-vivo stability of the two Treg subsets was compared. Both tTregs and iTregs were pretreated with IL-6 and NaCl for 48 hours and were then adoptively transferred into Rag1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e recipients at a dose of 0.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e per mouse to assess their stability under lymphopenic conditions for 10 consecutive days. GFP-expressing cells were quantified in the MLN on day 5 and day 10, respectively. As depicted in Fig.\u0026nbsp;1C and 1D, there was some loss of Foxp3 in both Treg populations, but slightly less in iTregs than in tTregs at day 5 after adoptive infusion. However, by day 10, approximately 75% of Foxp3 expression was retained in iTregs, especially in those pretreated with NaCl and IL-6. Conversely, the pretreated tTregs displayed a notable reduction in Foxp3 expression compared to untreated tTregs. Concurrently with changes in Foxp3 expression, both tTregs and pretreated tTregs exhibited robust expression of IL-17A and IFNγ, with the production of these two cytokines significantly higher in pretreated tTregs than in untreated tTregs (Fig.\u0026nbsp;1E, F). In line with the in vitro findings, no significant expression of IL-17A and IFNγ was observed in either iTregs or pretreated iTregs (Fig.\u0026nbsp;1E, F).\u003c/p\u003e \u003cp\u003eFinally, we sought to ascertain whether the two Treg subsets retained their function faithfully when pretreated with IL-6 and NaCl. Carboxy fluorescein (CFSE)-labeled splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells from WT B6 mice were cultured with Treg subsets at various ratios of T responder and Treg cells, which were primed with or without (NaCl\u0026thinsp;+\u0026thinsp;IL-6) for 3 days. Upon addition of iTregs or tTregs to T responder cells, CFSE labeling revealed a marked inhibition of proliferation, demonstrating a dose-dependent inhibitory effect. Nevertheless, tTregs subjected to pretreatment displayed a marked reduction in their inhibitory function, whereas iTregs that were pretreated preserved their suppressive capacity (Fig.\u0026nbsp;1G, H). Furthermore, the suppression exerted by pretreated iTregs against T cell proliferation far surpassed that of pretreated tTregs at the indicated ratios (Fig.\u0026nbsp;1G, H). In summary, these results unequivocally demonstrate that iTreg cells are more stable and exhibit superior functionality compared to tTregs under conditions of inflammatory cytokines combined with sodium chloride.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eiTregs were generated from na\u0026iuml;ve CD4\u0026thinsp;+\u0026thinsp;T cells of Foxp3-GFP mice following the protocol outlined in the Materials and Methods section. Subsequently, iTregs or thy-mus-sorted tTregs were stimulated with soluble anti-CD3 (2 ug/mL) and anti-CD28 (1 ug/mL) for 3 days in the presence or absence of NaCl (40 mM) (Panel A, B) or rm-IL-6 (100 ug/mL) (A, C). The expression levels of na\u0026iuml;ve IFNγ and IL-17A were determined at the same time by flow cytometry. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of three independent experiments (B, C). Statistical analysis was performed using the paired Student\u0026rsquo;s t-test when comparing only two groups, with significance denoted as * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ns indicating not significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003etTregs sorted from thymus expanded and activated. In parallel, iTregs were induced from naive CD4\u0026thinsp;+\u0026thinsp;T cells. Both Treg cell subsets were from Foxp3-GFP mice and cultured in standard media for 72 h. Following this, cells were harvested, thoroughly washed, and then cultured in media supplemented with NaCl (40 mM) and IL-6 (100 ng/mL) for an additional 48 h, denoted as pretreated-tTregs or pretreated-iTregs. Subsequently, these cells were collected, surface-stained for CD4, and intracellularly stained for IL-17A and IFNγ, with measurements performed on CD4\u0026thinsp;+\u0026thinsp;GFP- and CD4\u0026thinsp;+\u0026thinsp;GFP\u0026thinsp;+\u0026thinsp;cells via FACS. Representative FACS plots from three independent experiments are depicted (A, B), (n\u0026thinsp;=\u0026thinsp;6). In a parallel experiment, tTregs, pretreated-tTregs, iTregs, and pretreated-iTregs were transferred into Rag1-/- mice. The mice were euthanized at day 5 or day 10, and total MLN cells were collected. Assessment of Foxp3-GFP loss (C, D) and determination of the frequency of CD4\u0026thinsp;+\u0026thinsp;IL-17A\u0026thinsp;+\u0026thinsp;and CD4\u0026thinsp;+\u0026thinsp;IFNγ\u0026thinsp;+\u0026thinsp;cells (E, F) in the MLN from each group were compared. Representative results, presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from two distinct experiments, are il-lustrated, with statistical significance analyzed using unpaired t-tests (D, F). Furthermore, iTregs were restimulated under 40 mM NaCl for 3 days and subsequently analyzed by flow cytometry for IL-17A and IFNγ. Representative FACS data (Panel G) and data quantification (Panels G, H) are provided. The bar graph summarizes results from independent experiments (n\u0026thinsp;=\u0026thinsp;3), with statistical analyses conducted using paired t tests. Notably, ns denotes not significant, * indicates p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** indicates p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** indicates p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 iTregs exhibit superior functional activity compared to tTregs in EAE-mediated brain inflammation\u003c/h2\u003e \u003cp\u003eTo investigate the distinct effects of the two Treg subsets in mitigating brain inflammation, we used the murine MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e-induced EAE model. Mice disease manifestation was observed at day 12 post-immunization, characterized initially by tail weakness, followed by hindlimb and/or forelimb paralysis, as previously described.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] Intravenous transfer of Treg subsets occurred 4 days after the expression of EAE. As anticipated, both iTregs and tTregs exhibited a significant functional suppression on the clinical expression of EAE, delaying onset and ameliorating symptoms (Fig.\u0026nbsp;2A). H\u0026amp;E staining revealed a markedly lower level of lymphocyte infiltration in the tTreg- or iTreg-treated groups compared to the disease model group, with iTreg groups revealing a more pronounced alleviation of lymphocyte infiltration compared to tTreg groups (Fig.\u0026nbsp;2B). Notably, we observed that iTregs displayed a superior effect on restraining lymphocytic infiltration compared to tTregs, correlating with the clinical symptoms depicted in Fig.\u0026nbsp;2A. Furthermore, we analyzed the change of Th1 and Th17 cells in each group, as these subsets represent crucial pathogenic immune cells in EAE development. Flow cytometric analyses demonstrated significant suppression of Th17 cells in the spleens (Fig.\u0026nbsp;2C) and brains (Fig.\u0026nbsp;2D) of both treatment groups compared to disease model controls. Particularly, the brain -Th17 population was dramatically decreased in mice administered with iTregs compared to those administered with tTregs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;2E, 2G), indicating a distinct therapeutic difference between iTregs and tTregs. Subsequently, the frequency of Th1 cells in the spleens and brains was significantly reduced in iTreg-treated, but not in tTreg-treated group compared to the model group (Fig.\u0026nbsp;2F, 2H). Specifically, tTreg administration only inhibited brain Th1 cell development, with less obvious effects compared to iTreg administration (Fig.\u0026nbsp;2H).\u003c/p\u003e \u003cp\u003eGiven the critical role of DC priming of T lymphocytes in EAE pathogenesis, we analyzed splenic CD11c\u003csup\u003e+\u003c/sup\u003e DCs in each group. Both tTreg and iTreg subsets decreased the frequency of splenic CD11c\u003csup\u003e+\u003c/sup\u003e DCs, with iTregs displaying superior efficacy compared to tTregs (Fig.\u0026nbsp;2I, 2J). These findings collectively support the notion that both tTregs and iTregs alleviate EAE severity, with iTregs exhibiting more potent suppressive activity, potentially involving DCs in this effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e1\u0026times;106 tTregs or iTregs, generated as described previously, were adoptively trans-ferred into B6 male mice on day 4 following MOG35-55/CFA immunization. Clinical scores of the recipient mice were recorded at various time points post-immunization (A). Histological analyses were conducted, showcasing sections of H\u0026amp;E staining of brain tissues from each experimental group (20x) (B). Experiments were terminated on day 30, following which cells from spleens (C) and brains (D) were harvested and stimulated with PMA, ionomycin, and BFA. Subsequently, IFNγ- and IL-17A-producing CD4\u0026thinsp;+\u0026thinsp;T cells from spleens (E, F) and brains (G, H) were analyzed via flow cytometry. Data presented are representative of at least 5 mice per group. Additionally, staining of indicated CD11c\u0026thinsp;+\u0026thinsp;splenic DCs on day 30 was performed via flow cytometry (I, J). All data are representative of at least 3 independent experiments. Statistical analyses were conducted using one-way ANOVA, with significance denoted as * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ns indicating not significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 iTregs inhibit the pro-inflammatory DC phenotype and promote tolerogenic activities in the EAE environment\u003c/h2\u003e \u003cp\u003eThe immunogenicity of dendritic cells (DCs) reflects a balance of pro- and anti-inflammatory signals in EAE. Pro-inflammatory DCs aggravate EAE progress, while tolerogenic DCs play a modulatory role in restoring immune homeostasis and relieve EAE pathogenesis.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Subsequently, we assessed the immunophenotype and function of splenic CD11c\u003csup\u003e+\u003c/sup\u003e DCs from the three groups: PBS-treated (model-DCs, m-DCs), tTreg-treated (t-DCs), and iTreg-treated (i-DCs) EAE mice. The mean fluorescence intensity (MFI) of the co-stimulatory molecule CD80 was significantly lower only in the i-DCs compared to the m-DCs and t-DCs (Fig.\u0026nbsp;3A, 3B). Likewise, the MFI of another co-stimulatory molecule CD86 in CD11c\u003csup\u003e+\u003c/sup\u003e DCs was significantly decreased in mice treated with the iTreg subset versus the control and tTreg-treated mice. Interestingly, i-DCs expressed much lower levels of CD86 compared to t-DCs (Fig.\u0026nbsp;3A, 3C). Additionally, the MFI of the major histocompatibility class II molecule (I-Ad), as well as activation molecules such as CD40 and CD83, were also decreased in both Treg-treated groups compared to the model group, although not reaching statistical significance (data not shown) (Fig.\u0026nbsp;3A, 3B). Investigation of well-known tolerogenic DC cells also revealed that i-DCs significantly expressed higher levels of latency-associated peptide (LAP) (Fig.\u0026nbsp;3A, 3D) and intracellular IL-10 (Fig.\u0026nbsp;3E) compared to that of m-DCs, as indicated by MFI or frequency respectively. And we did not observe significant expression of the two tolerogenic markers in t-DCs, but only observed an increasing trend (Fig.\u0026nbsp;3A, 3D, 3E). These findings indicate that iTregs are more capable of inducing DC immune tolerance than tTregs, which may help to explain why the iTreg subset is more effective than tTregs in treating EAE.\u003c/p\u003e \u003cp\u003eBased on these findings, we hypothesize that i-DCs would capably suppress T cell proliferation and promote Treg cell differentiation. As expected, when the i-DC subset was cocultured with CFSE-labeled naive T cells (CD4\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e\u0026minus;\u003c/sup\u003e), the CFSE proliferation was significantly restrained compared to that cocultured with m-DCs or t-DCs (Fig.\u0026nbsp;3F, 3G). Similarly, i-DCs also exhibited a remarkable ability to induce Treg cell development. Approximately 10% of the na\u0026iuml;ve T cells became CD25\u003csup\u003e+\u003c/sup\u003eGFP\u003csup\u003e+\u003c/sup\u003e regulatory T cells (Fig.\u0026nbsp;3H, 3I). And this was not observed from the PBS-DCs coculture system. Although t-DCs also obviously generated a significant amount of Foxp3\u003csup\u003e+\u003c/sup\u003e cells, i-DCs had a robustly powerful ability in the induction of naive T cells into Treg cells compared to t-DCs (Fig.\u0026nbsp;3I). Thus, these results indicate that iTreg-modified DCs are rendered hyporesponsive and have anti-inflammatory function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePBS-treated, tTreg-treated, and iTreg-treated EAE mice (n\u0026thinsp;=\u0026thinsp;5 per group) were eu-thanized on day 30. Total splenocytes were collected, and DCs were stained and gated on CD11c. Expression levels of CD80, CD86, and LAP on CD11chi splenic DCs were depicted (A), with corresponding statistical graphs presented (B, C, D). The data revealed are representative of at least 3 independent experiments. Some splenocytes were stimulated with PMA, ionomycin, and monensin, followed by staining for CD11c and IL-10. The bar graph illustrates the frequency of CD11c\u0026thinsp;+\u0026thinsp;IL-10\u0026thinsp;+\u0026thinsp;DCs (E). Additionally, PBS-treated DCs, tTreg-treated DCs, and iTreg-treated DCs were sorted from the spleens of EAE mice, then added to naive B6 T cells labeled with CFSE, and T cell proliferation was assessed by CFSE dilution (F), with statistical data indicated (G). Furthermore, a portion of these three groups of DCs was added to naive T cells (from B6 Foxp3-GFP mice) at a 1:5 ratio in the presence of anti-CD3 and rhIL-2 for 3 days, and Foxp3-GFP expression was measured using flow cytometry (H, I). All data presented are representative of at least 3 independent experiments. Statistical analyses were conducted using one-way ANOVA, with significance denoted as * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ns indicating not significant.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 iTregs endow DCs with a tolerogenic phenotype and function mainly via TGF-β but not IL-10 signaling\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt has been well documented that both Treg subsets secrete soluble TGF-β and IL-10, which are crucial for exerting immunosuppressive function.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] To further determine whether membrane-bound TGF-β and IL-10 receptors are also required for Treg-mediated suppression in EAE, we used an inhibitor of TGF-β receptor I (ALK5) and an anti-IL-10R antibody in the EAE model. EAE induction and iTreg cell infusion were performed, as depicted in Fig.\u0026nbsp;2A. Additionally, some of the mice treated with iTreg cells were injected with a neutralizing anti-IL-10R antibody (isotype-matched IgG1 antibody as its control) or ALK5 inhibitor (ALK5i). Our results revealed that iTreg infusion exerted a significant inhibitory function on the treatment of EAE, as demonstrated by a reduction in clinical scores (Fig.\u0026nbsp;4A) and histological brain inflammation (Fig.\u0026nbsp;4B). Administration of DMSO or IgG1 alone did not influence disease progression in the EAE mice compared to the model group (data not shown). IL-10 signaling blockade resulted in a statistically significant effect on the therapeutic efficacy of iTregs, as determined by clinical scores. Nevertheless, the unchanged neuroinflammation scores following anti-IL-10R blockade suggest that IL-10 signaling may play a limited role in the immunosuppressive capability of iTreg cells in the treatment of EAE (Fig.\u0026nbsp;4B). Conversely, the clinical scores and the pathological inflammation scores were both completely reversed in the iTreg\u0026thinsp;+\u0026thinsp;ALK5i group compared to the iTreg only group, indicating that blockade of TGF-β receptor I activity apparently impaired the protective effect of iTreg cells (Fig.\u0026nbsp;4A, 4B). These results preliminarily indicated that TGF-β and IL-10 signaling both play a role in executing suppressive ability of iTreg cells, whereas TGF-β signaling is more crucial. The presence of Th1 and Th17 cells in the brains was also examined using FACS. As expected, iTreg infusion significantly repressed the brain Th17 cell differentiation. However, co-administration of ALK5i mostly restored the Th17 frequency in the brains to EAE levels compared to the iTreg infusion group. Meanwhile, we only observed the reverse trend in the inhibition of Th17 cell frequency by iTregs treatment in the anti-IL-10R antibody administration group (Fig.\u0026nbsp;4C, 4D). Also, IFNγ-producing Th1 cells were also statistically decreased in iTreg-treated brains. Conversely, neither ALK5i nor anti-IL-10R could significantly reverse the inhibitory effect on Th1 differentiation by iTreg infusion (Fig.\u0026nbsp;4C, 4E). These observations illustrate that TGF-β signaling plays a dominant role in the immunosuppressive function of iTregs on EAE, whereas IL-10R signaling plays a partial role, if any.\u003c/p\u003e \u003cp\u003eIn line with the aforementioned findings, we examined the frequency of splenic DCs from each group of EAE mice at day 30 post-immunization. iTreg-treated mice revealed a remarkable decrease in CD11c\u003csup\u003e+\u003c/sup\u003e DCs in spleens. ALK5i administration completely diminished this phenomenon, but anti-IL-10R did not (Fig.\u0026nbsp;4F). CD11c\u003csup\u003e+\u003c/sup\u003e DCs expressed a baseline level of CD80, CD86, and LAP in splenocytes treated with PBS. DCs from iTreg-treated mice exhibited a downregulation of CD80 and CD86 (Figure S2A, S2B), along with an upregulation of LAP expression (Figure S2A, S2C). In comparison to the iTreg-treated splenocytes, co-administration of ALK5i significantly upregulated the expression of CD80 and CD86 (Figure S2A, S2B), while decreasing the expression of LAP (Figure S2A, S2C). However, statistically significant changes in these molecules in the co-administration of the anti-IL-10R group were not observed (Figure S2A-2C). Next, a series of DC-T cell co-culture experiments were conducted to assess the impact of ALK5i and anti-IL10R on the tolerogenic activity of these in vivo-modified DCs. CD11c\u003csup\u003e+\u003c/sup\u003e DCs were sorted from the spleen (splenocytes) of each EAE group on day 30. These DCs were cocultured with B6 na\u0026iuml;ve T cells using the same Treg-polarizing conditions as described in Fig.\u0026nbsp;3. As revealed in Fig.\u0026nbsp;4G, compared to DCs sorted from PBS-treated EAE mice, iTreg-DCs enabled up to 10% of naive T cells to begin expression of Foxp3. However, splenic DCs from the ALK5i co-administration group dramatically lost their tolerogenic activity, resulting in little formation of CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e cells. However, DCs from the anti-IL10-treated group exhibited some degree of tolerogenic function, although the generation of CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e cells were statistically lower compared to iTreg-DCs (Fig.\u0026nbsp;4G, 4H). In another approach, CFSE dilution was also assessed using the coculture system. WT B6 naive T cells cocultured with iTreg-DCs proliferated much less than those cocultured with PBS-DCs. Similarly, naive T cells with ALK5i-co-administered DCs exhibited an obvious capacity to proliferate at a level similar to that of those cultured with PBS-DCs. Conversely, anti-IL10R-co-administered DCs retained some of their immunosuppressive function, however this was not statistically significant, as revealed by the inhibition of CFSE proliferation compared to the iTreg-DCs (Fig.\u0026nbsp;4I, 4J). Taken together, these data reveal that iTregs primarily induce the formation of tolerogenic DCs via membrane-bound TGF-β signaling, rather than IL-10 signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eiTreg cells were generated and administered, and EAE was induced as described previously. Subsequently, iTregs, ALK5i, or anti-IL-10R were administered in separate groups, with DMSO treated as the model group. The severity of EAE was assessed (A). Histological changes in the brains of mice from each group on day 30 post-immunization were assessed by removing brain sections, fixing them, and conducting H\u0026amp;E staining, with typical photographs displayed (B). On day 30 post-immunization, brains were harvested, and single-cell suspensions were prepared. Populations of IL-17-producing Th17 cells (C, D) and IFNγ-producing Th1 cells (C, E) were analyzed by flow cytometry, with representative flow cytometry data indicated for each group. Additionally, the frequency of splenic CD11c\u0026thinsp;+\u0026thinsp;DCs was examined by flow cytometry (F). Splenic CD11c\u0026thinsp;+\u0026thinsp;DCs were sorted from each group on day 30, and some were co-cultured with naive Foxp3-GFP T cells (G), while others were co-cultured with CFSE-labeled naive B6 T cells (I). Foxp3-GFP expression (H) and CFSE dilution (J) were assessed by flow cytometry after 3 days of culture. In vitro results presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of triplicate wells from three independent experiments, with n\u0026thinsp;=\u0026thinsp;5 mice per group. Statistical analyses were conducted using unpaired t-tests (D, F) and paired t-tests (H, J), with * indicating p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** indicating p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** indicating p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ns. indicating not significant. (PBS-DC vs. iTreg-DC), (iTreg-DC vs. (iTreg\u0026thinsp;+\u0026thinsp;ALK5i)-DC), (iTreg-DC vs. (iTreg\u0026thinsp;+\u0026thinsp;anti-IL-10R)-DC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eiTreg cells were generated and administered, and EAE was induced as described previously. Subsequently, iTregs, ALK5i, or anti-IL-10R were administered in separate groups, with DMSO treated as the model group. All mice were sacrificed on day 30, and total splenocytes were collected for analysis. DCs were stained and gated on CD11c (A). The expression levels of CD80 and CD86 (A, B) as well as LAP (A, C) on CD11c\u0026thinsp;+\u0026thinsp;DCs were detected by FACS. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of three separate experiments, with significance denoted as * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ns indicating not significant. (PBS-DC vs. iTreg-DC), (iTreg-DC vs. (iTreg\u0026thinsp;+\u0026thinsp;ALK5i)-DC), (iTreg-DC vs. (iTreg\u0026thinsp;+\u0026thinsp;anti-IL-10R)-DC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Inhibition of the AKT/mTOR pathway causes iTreg cells to gain DC tolerogenic capacity\u003c/h2\u003e \u003cp\u003eThe participation of AKT/mTOR signaling pathway in the inflammatory and mature process of DCs was well demonstrated.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] AKT Phosphorylation leads to the activation of mTOR signaling along with several downstream targets. P70S6K, one of the most important substrates of mTOR signaling, contributes to the regulation of inflammatory cytokine production in DCs, participating in their antigen presenting and proinflammatory function.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] To investigate whether iTregs modify the immunosuppressive capacity of splenic DCs via AKT/mTOR signaling, intracellular levels of total AKT (t-AKT), phospho-AKT (p-AKT), total P70S6K (t-P70S6K), and phospho-P70S6K (p-P70S6K) in splenic DCs treated with iTregs or tTregs were compared. As measured by WB, the expression of total AKT and P70S6K was similar in DCs treated with iTregs or tTregs (Fig.\u0026nbsp;5A, 5B). However, p-AKT and p-P70S6K decreased considerably only in the iTreg-treated group, and these differences were statistically significant (Fig.\u0026nbsp;5A, 5C). Given that high salt and/or inflammatory cytokines distinctly influence the stability and function of both Treg subsets in vitro and in vivo, we immediately assessed the phenotype of DCs cocultured with the Treg subsets that had been pretreated with a combination of NaCl and IL-6. Pretreated iTregs significantly inhibited p-Akt and p-P70S6K expression on DCs compared to that of pretreated tTregs (Fig.\u0026nbsp;5A, 5E). We did not find statistical differences in t-Akt and t-P70S6K between the two groups (Fig.\u0026nbsp;5A, 5D). In sum, these findings suggest that AKT/mTOR pathway possibly plays an important role in mediating the suppressive effects of iTregs on the immunomodulatory activity of DCs. Additionally, it can be elucidated that the iTreg subset exhibits enhanced suppressive activity in facilitating the tolerogenic function of DCs in comparison to the tTreg subset. This superior suppressive capability may be achieved through the inhibition of the AKT/mTOR signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBoth subsets of Tregs were stimulated with or without NaCl at a concentration of 40 mM, in the presence of anti-CD3/CD28 microbeads (5 cells per bead) and rhIL-2 at a concentration of 50 U/ml, for a duration of 3 days. Subsequently, the cells were washed and cocultured with splenic CD11c\u0026thinsp;+\u0026thinsp;DCs for an additional 48 hours, at a T to DC ratio of 2:1 (A). Representative immunoblots depicting the expression levels of Akt, phosphorylated Akt (p-Akt), P70S6K, and phosphorylated P70S6K proteins are displayed (B-E). The data presented are representative of three independent experiments, with statistical significance determined using a paired t-test. Significance levels are denoted as * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ns indicating not significant.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6 iTregs, but not tTregs, have a therapeutic effect on EAE mice by reducing high-salt diet-induced brain inflammation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHigh-salt diet exacerbates EAE progression.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Previous studies have identified a population of pro-inflammatory tTreg cells that are modified by high salt. These cells are characterized by the secretion of IFNγ and have been found to be dysfunctional both in vitro and in vivo.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] However, we demonstrated that TGF-β-induced iTreg populations are highly stable and functional under high salt conditions.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Therefore, to determine the varying immunosuppressive capabilities of the two Treg subsets in regulating high-salt diet-fed EAE mice, we conducted an adoptive transfer study using a therapeutic regimen. As depicted in Fig.\u0026nbsp;6A, we observed that iTregs exerted a significant inhibitory function on the treatment of high salt fed EAE, leading to an amelioration of the clinical scores. However, tTregs had a significantly reduced inhibitory effect on high salt-fed EAE mice progression (Fig.\u0026nbsp;6A). Similarly, results from the detection of IL-17A\u003csup\u003e+\u003c/sup\u003e and IFNγ\u003csup\u003e+\u003c/sup\u003e of CD4\u003csup\u003e+\u003c/sup\u003eGFP\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells in the brains revealed that iTregs significantly reduced the frequency of Th1 and Th17 cells, whereas tTregs failed to suppress both inflammatory T effector cells (Fig.\u0026nbsp;6B, 6C). Next, as a matter of course, we examined the frequency and phenotype of splenic DCs from each group. CD11c\u003csup\u003e+\u003c/sup\u003e DCs significantly decreased in the group that received iTreg administration compared to the PBS controls. However, the administration of tTreg had only a slight impact on the frequency of splenic DCs (Fig.\u0026nbsp;6D, 6E). Meanwhile, iTregs induced a decreased expression of CD80 and CD86 on splenic DCs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the MFI index of these two DC maturation markers returned to levels similar to those in the PBS control group after treatment with tTregs (Fig.\u0026nbsp;6F, 6G). Furthermore, LAP expression was augmented in splenic DCs in the iTreg-treated group but not in the tTreg-treated group (Fig.\u0026nbsp;6F, 6G). Taken together, based on a face-to-face comparison experiment, we validated that both Treg subsets exhibit distinct biological characteristics in a complex environment, such as a high salt environment and under inflammatory conditions. Thus, TGF-β-induced Treg cells may have some advantages in clinical application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWild-type mice were subjected to a high-salt diet for two weeks prior to active im-munization with MOG35-55 peptide. Subsequently, both subsets of Tregs were gen-erated and administered as described previously. The mean clinical scores of EAE from each group are depicted (A). CD4\u0026thinsp;+\u0026thinsp;T cells from the brains were analyzed on day 30, with flow cytometric analysis conducted to determine the frequencies of IL17A\u0026thinsp;+\u0026thinsp;and IFNγ\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;cells in the respective mouse groups (B, C). The frequency of splenic CD11c\u0026thinsp;+\u0026thinsp;DCs was assessed by FACS on day 30 (D, E). Kinetic analysis of CD80, CD86, and LAP expression in splenic CD11c\u0026thinsp;+\u0026thinsp;cells was performed using flow cytometry (n\u0026thinsp;=\u0026thinsp;5 per group and time point) (Panel F, G). Statistical analyses were conducted using one-way ANOVA, with significance indicated as * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ns indicating not significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.7 CCAR2 deficiency maintains tTreg cell stability in high-salt condition\u003c/h2\u003e \u003cp\u003eSince the high-salt condition weakened the tTreg cell stability endowing these cells Th17-like phenotype, and had little impact on iTreg cells, we decided to evaluate CCAR2-deficient and CCAR2-expressing tTregs in the same environment (NaCl 40 mM). In this regard, thymic Treg cells (CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e T cells) were MACS-sorted, and then were stimulated with anti-CD3/CD28 microbeads and IL-2 in the presence or absence of NaCl \u003cem\u003ein vitro\u003c/em\u003e. The Foxp3 level was examined after 72 h. CCAR2 deficiency slightly promoted Foxp3 expression in tTreg cells, and this trend was also observed by the addition of salt (Fig.\u0026nbsp;7A). iTreg cells were induced from the WT or CCAR2 deficiency na\u0026iuml;ve T cells using the standard protocol either in standard media or in high-salt media for 72 h. We observed that in the absence of CCAR2, differentiation of iTreg cells was markedly enhanced. Identically, there was no significant change of iTreg cell development under high-salt conditions (Fig.\u0026nbsp;7B). To further validate whether CCAR2 deficiency in Treg cells has the potential resistance to NaCl modification, IL-17A and IFNγ expression were then assessed in these groups. High-salt condition significantly induced IFNγ expression in tTregs. However, in the absence of CCAR2, IFNγ expression in tTregs was decrease to some extent compared to WT tTregs. More importantly, high salt failed to induce IFNγ expression in CCAR2 deficient tTregs (Fig.\u0026nbsp;7C, E). Similar to IFNγ, high-salt condition also significantly induced IL-17A expression in tTregs. However, CCAR2 deficiency significantly abolished the effect of high salt, as we found a distinct reduction in the frequency of IL-17A\u003csup\u003e+\u003c/sup\u003e tTregs in high-salt condition compared to the frequency of IL-17A\u003csup\u003e+\u003c/sup\u003e tTregs in standard media (Fig.\u0026nbsp;7C, F). Moreover, regardless of whether under the high-salt condition or lack of CCAR2, iTregs were fully stable, which few IFNγ\u003csup\u003e+\u003c/sup\u003e or IL-17A\u003csup\u003e+\u003c/sup\u003e iTregs were detected (Fig.\u0026nbsp;7D, G, H). These data are suggestive of an important role for CCAR2 deficiency in Treg cell stable programming that can be explained for an intrinsic resistance to high salt damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003etTregs and splenic na\u0026iuml;ve CD4\u0026thinsp;+\u0026thinsp;T cells were isolated either from the WT or from CCAR2 deficient mice. tTregs were subsequently expanded and activated for 72 h. Simultaneously, naive CD4\u0026thinsp;+\u0026thinsp;T cells were activated using anti-CD3/28 microbeads in the presence of IL-2 and TGF-β for 72 h. All cells were harvested synchronously followed by re-stimulation in either standard media or media containing 40 mM NaCl for another 72 h. At the onset of pre-activation and after a 72-hour pre-activation period, an aliquot of Treg subsets were analyzed. Intranuclear protein levels of Foxp3 were calculated and depicted in tTregs (A) and iTregs (B) by FACS (n\u0026thinsp;=\u0026thinsp;3). Flow cytometry analysis of IFNγ and IL-17A production by intracellular staining of cytokines in tTregs (C) and iTregs (D), and data were gated on CD4\u0026thinsp;+\u0026thinsp;Foxp3\u0026thinsp;+\u0026thinsp;cells (n\u0026thinsp;=\u0026thinsp;3). Quantitative analysed of the frequency of IFNγ- -expressing tTregs (E), IL-17A-expressing tTregs (F), IFNγ- -expressing iTregs (G), IL-17A-expressing iTregs (H). Statistical analyses were performed using one-way ANOVA, with significance levels represented as follows: * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ns. for non-significant results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.8 The inhibiting DC maturation by CCAR2-deficient Treg cells was negligibly affected by high salt.\u003c/h2\u003e \u003cp\u003eTo delineate the functional impact of CCAR2-deficient tTregs and iTregs on DCs, a coculture system was established using splenic CD11c\u0026thinsp;+\u0026thinsp;DCs. Four Treg groups (CCAR2-expressing/deficient tTregs and iTregs) were pretreated with or without NaCl (48 h) and cocultured with DCs (1:5 ratio, 48 h). LPS-stimulated DCs served as positive controls, displaying maximal CD80 and CD86 expression. Compared to untreated tTregs, NaCl-pretreated tTregs (Na-tTreg) induced significantly higher CD80 expression in DCs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, CCAR2-deficient tTregs resisted salt-induced instability, as DCs cocultured with NaCl-treated CCAR2-KO tTregs (Na-KO-tTreg) showed no significant CD80 elevation versus untreated CCAR2-KO tTregs (KO-tTreg) (Fig.\u0026nbsp;8A, B). In contrast, iTregs exhibited stable suppressive function regardless of CCAR2 status or NaCl exposure, with no intergroup differences in CD80 inhibition (Fig.\u0026nbsp;8A, C). Both tTregs and iTregs significantly suppressed CD80 expression compared to LPS controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, Na-tTreg enhanced DC CD86 expression versus untreated tTreg (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while CCAR2 deficiency abrogated this effect, maintaining low CD86 levels irrespective of NaCl exposure. Notably, KO-tTreg exhibited enhanced suppressive capacity, as DCs cocultured with these cells showed lower CD86 expression than those with wild-type tTregs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;8D, E). iTregs uniformly suppressed CD86 expression across all groups, unaffected by CCAR2 status or NaCl. Both tTreg and iTreg significantly inhibited CD86 expression versus LPS controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;8D, F).\u003c/p\u003e \u003cp\u003eTo investigate whether CCAR2-deficient tTregs regulate the AKT/mTOR pathway in DCs, we analyzed phosphorylation events following Treg-DC coculture. For p-AKT modulation, DCs cocultured with Na-KO-tTregs exhibited significantly elevated p-AKT levels compared to those cocultured with untreated tTregs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, CCAR2-deficient tTregs maintained stable suppression, as DCs cocultured with Na-KO-tTregs showed no significant p-AKT increase versus untreated KO-tTregs (Fig.\u0026nbsp;8G, H). iTregs also exhibited stable suppressive function regardless of CCAR2 status or NaCl exposure, with no intergroup differences in p-AKT expression in DCs (Fig.\u0026nbsp;8G, I). For phospho-S6 (pS6, an mTORC1 readout), LPS-stimulated DCs (positive controls) displayed maximal pS6 mean fluorescence intensity (MFI). Na-tTregs induced significantly higher pS6 MFI in DCs compared to untreated tTregs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;8J, K), whereas iTregs showed no NaCl-dependent differences in pS6 suppression regardless of CCAR2 expression or deficiency (Fig.\u0026nbsp;8J, L). Both wild-type tTregs and iTregs significantly inhibited pS6 expression compared to LPS controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with CCAR2-deficient tTregs demonstrating comparable efficacy to wild-type counterparts (Fig.\u0026nbsp;8J, K). Notably, both CCAR2-expressing and CCAR2-deficient iTregs similarly inhibited pS6 expression in DCs, with no NaCl-dependent effects (Fig.\u0026nbsp;8J, L). These results indicate that CCAR2 deficiency protects tTregs from high salt-induced functional impairment by preserving AKT/mTOR suppression in DCs, whereas iTregs retain stable immunosuppressive capacity unaffected by CCAR2 knockout or NaCl exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe two subsets of Tregs were subjected to stimulation either in the presence or ab-sence of 40 mM NaCl, alongside anti-CD3/CD28 microbeads (at a ratio of 5 cells per bead) and rhIL-2 at 50 U/ml, for a period of 3 days. Following this, cells from each group were thoroughly washed and subsequently co-cultured with splenic CD11c\u0026thinsp;+\u0026thinsp;DCs for an additional 24 hours, maintaining a T cell to DC ratio of 5:1. Representative flow cytometry data depicting CD80 expression levels on CD11c\u0026thinsp;+\u0026thinsp;cells are presented, comparing co-culture conditions derived from the four distinct tTreg groups (A, B) and the four iTreg groups (A, C). Representative FACS plots showing CD86 expression in CD11c\u0026thinsp;+\u0026thinsp;cells co-cultured either from the 4 different tTreg groups (D, E), or from the 4 different iTreg groups (D, F). Total cells were treated with PMA, ionomycin, and monensin to induce cellular activation. Then cells were subjected to immunostaining for the surface marker CD11c, followed by the intracellular staining p-AKT. Flow cytometry analysis of the expression of p-AKT in CD11c\u0026thinsp;+\u0026thinsp;DCs cultured from these tTreg groups (G, up), or from the 4 identical treatment iTreg groups (G, down). Data were summarized in the bar graph, which provides a quantitative analysis of the relative frequency of CD11c\u0026thinsp;+\u0026thinsp;p-AKT\u0026thinsp;+\u0026thinsp;DCs within the total cell population under the systems co-cultured from tTreg groups (H) or from the 4 different iTreg groups (I). Flow cytometry analysis of ps6 expression in each group shows MFI values, representing ps6 expression from five different tTreg groups (J,K), as well as five different iTreg groups(J,L). The statistical comparisons between tTreg and Na-tTreg, KO-tTreg and Na-KO-tTreg, iTreg and Na-iTreg, as well as KO-iTreg and Na-KO-iTreg were performed using paired t-tests, while comparisons among tTreg, Na-tTreg, and control were conducted using one-way ANOVA. Significance levels are indicated as follows: * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ns. for non-significant results.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eWhile the initial identification of Tregs occurred within the thymus, subsequent research rapidly revealed that this cell population can be induced and differentiated from non-Treg cells with TGF-β and IL-2.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] TGF-β initiates the phosphorylation and activation of Smad2 and Smad3, pivotal for the induction of Foxp3 during iTreg generation.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] Both Treg subsets express canonical Treg markers, such as Foxp3, CD25, GITR, and CTLA4. However, tTregs exhibit higher expression levels of PD-1, neuropilin-1 (Nrp-1), Helios (Ikzf2), and CD73 compared to their iTreg counterparts.[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] Certain studies have proposed that Foxp3 expression stability in tTregs is maintained via demethylation of CpG islands within the conserved non-coding sequence 2 (CNS2) region of the Foxp3 locus. This region serves as a binding site for various transcription factors such as Stat5 and Runx1/Cbfb, contributing to tTreg stability. Conversely, unstable Foxp3 expression in iTregs is purportedly associated with pronounced demethylation of CNS2.[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] However, observations from our study and those of others challenge the notion of tTreg stability, particularly under arthritic and inflammatory conditions. Under such circumstances, tTregs exhibit susceptibility to redifferentiation into alternative T effector cell subsets, accompanied by functional changes. In contrast, iTregs demonstrate a lack of this plasticity and display enhanced suppression of osteoclastogenesis and bone erosion compared to tTregs.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eHigh salt levels can promote the differentiation of pathogenic Th17 cells while concurrently dampening the suppressive capacity of tTregs, thereby expediting the onset of EAE.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] By using a high-salt diet in a Rag1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e colitis model, we provided supplementary evidence indicating that iTregs, but not tTregs, significantly ameliorate intestinal inflammation.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Additionally, we present compelling evidence demonstrating the greater stability of iTregs compared to tTregs, as evidenced by the heightened resistance of iTreg destabilization in the presence of exogenous IL-6 and NaCl combination stimulation in vitro. In the specific EAE model used in our study, tTregs effectively curtailed disease progression. However, their efficacy was compromised in inhibiting EAE advancement in mice subjected to a high-salt diet. Notably, iTregs exhibited superior efficacy in mitigating disease progression, even under high-salt or pro-inflammatory conditions.\u003c/p\u003e \u003cp\u003eThe therapeutic efficacy of iTregs can be elucidated by several factors. Firstly, the suppressive function of tTreg cells may be compromised by pro-inflammatory cytokines owing to their expression of IL-6R. Numerous studies have reported the abrogation of tTreg suppressive activity in response to IL-6.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] Notably, iTregs demonstrate diminished expression of IL-6 receptor compared to tTregs, rendering them resistant to this cytokine induced transformation and preserving their phenotype and function.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Hence, it is plausible that CD126-negative tTregs may exhibit superior functional activity.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] Secondly, iTregs, but not tTregs, exhibit negligible levels of SOCS1 and SOCS3 in response to IL-6 stimulation. SOCS1 has been extensively implicated in mediating Th17 cell differentiation.[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] Therefore, the differential expression of SOCS proteins may contribute to the activation of STAT-3 in tTreg cells and the potential conversion of tTreg cells into Th17 cells.[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] Additionally, we demonstrated that Foxp3 exhibits instability following TNF-α treatment in a manner that is dependent on CCAR2/ DBC1, which the deficiency of CCAR2 protects Foxp3 from degradation and preserves Treg cell functionality in response to inflammatory stimulation.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Whether iTregs express CCAR2 at lower levels compared to tTregs, and whether high salt environments are associated with CCAR2 expression, both require further investigation. Thirdly, TGF-β could upregulate Bcl-2 expression and diminish T cell apoptosis in recovered iTregs, indicating that iTregs may exhibit reduced susceptibility to apoptosis compared to tTregs.[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] Additionally, our preliminary findings indicate that atRA, a crucial metabolite of vitamin A with diverse immunoregulatory functions, has the potential to inhibit the pro-inflammatory response induced by high salt. In this study, we observed high salt only induces barely detectable IFNγ and IL-17A production in splenic Treg cells. In other words, thymus-derived Treg cells and spleen-derived Treg cells may exhibit phenotypic and functional differences under high-salt stimulation. And this suggests, to some extent, that a high salt environment may serve as an effective external medium for identifying the heterogeneity in Treg cell populations.[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eDCs represent a pivotal cell subset responsible for initiating immune responses, with their pro- or anti-inflammatory polarization influenced by various environmental cues. Prior investigations have indicated that iTreg cells possess the capability to confer immunoregulatory properties upon DCs.[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] MS constitutes an autoimmune disease characterized by immune dysregulation, culminating in the infiltration of immune cells into the CNS, thereby instigating demyelination, axonal injury, and neurodegeneration.[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] Our research has unveiled a novel biological role for iTregs in directing the differentiation of profoundly tolerogenic DCs. This significant finding holds promise for the development of new immunotherapeutic strategies targeting a spectrum of autoimmune disorders.\u003c/p\u003e \u003cp\u003eOur findings demonstrate that iTreg cell function, in part, by modulating DCs, thereby orchestrating the delicate immuno-balance, consequently contributing to the amelioration of EAE. In MS, DCs exhibit an activated phenotype and initiatively induce pathogenic Th17 cell development. Subsequently, these Th17 cells migrate to the CNS where they instigate attacks on oligodendrocytes, leading to demyelination. In our study, administration of Treg cells not only inhibits the activation of splenic DCs but also confers tolerogenic activities upon them. This tolerogenic capacity relies on DCs establishing a complex network of cell-to-cell interactions.[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] Various factors contribute to the induction of a tolerogenic DC phenotype, including IL-10, TGF-β, and vitamin D. Studies have revealed the mechanisms by which Treg cells confer tolerogenic attributes to DCs; the most critical signaling pathways identified to date encompass IL-10, CTLA-4, and TGF-β.[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] Notably, the effects mediated by IL-10 were observed predominantly when immature DCs were exposed to IL-10, while mature DCs remained insensitive to IL-10 stimulation, maintaining a stable, mature phenotype.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] In asthmatic mice, Treg cells secrete IL-10, mediating the induction of tol-DCs, and blockade of IL-10 leading to suboptimal generation of tol-DCs.[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] However, in lupus mice, IL-10 appears dispensable for tol-DC induction.[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] TGF-β partially exerts suppressive functions by inducing Foxp3 gene and protein expression in T cells.[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] Protective effects of transferred iTreg cells require both IL-10 and TGF-β. However, the influence of iTregs on the induction of tol-DCs in the EAE model remains unexplored.\u003c/p\u003e \u003cp\u003eThe AKT/mTOR signaling axis plays a pivotal role in modulating the maturation, activation, and survival of DCs.[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] mTOR regulates protein synthesis by directly phosphorylating and inactivating the repressor of mRNA translation, and by phosphorylating and activating S6 kinase (p70S6K).[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] However, mTOR signaling in DC\u0026rsquo;s function during inflammatory immune responses remains contentious. Mainstream research indicates that inhibition of the Akt/mTOR pathway, particularly through rapamycin, enhances Treg induction and diminishes the immunological effects of DCs.[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] Additionally, inhibition of AKT and p70S6K phosphorylation facilitates the differentiation of iTreg cells.[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] Notably, 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD3 has emerged as a significant regulator of the immune system, exerting its effects by inducing immune tolerance in DCs. Treatment with 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD3 suppresses the maturation of bone marrow-derived DCs and promotes a dominant tolerogenic function, achieved by the inhibition of the AKT/mTOR signaling.[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] However, from a cellular metabolism standpoint, prior research has underscored the critical role of the PI3K/Akt/mTOR pathway in maintaining the tolerogenic phenotype of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD3-modulated DCs.[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] Our study corroborate that iTregs suppress the Akt/mTOR pathway, which contribute to the induction of an anti-inflammatory profile in murine DCs. Notably, the expression of the Foxp3 gene, a classical Treg marker, is regulated by the HIF-1α/mTOR pathway, underscoring the significance of this signaling cascade in Treg function.\u003c/p\u003e \u003cp\u003eExcessive intake of salt enhances pathogenic Th17 cell differentiation, leading to the progress of a highly pathogenic phenotype that exacerbates EAE. However, contrary to this effect, a prior study has demonstrated that the function of myeloid DCs remains largely unaffected by salt in vitro. Furthermore, high salt intake exacerbates neuroinflammation in EAE mice independently of mature DCs.[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] This indicates that distinct subsets of immune cells exhibit differential responses to NaCl. Notably, the induction of a pro-inflammatory environment by salt appears to involve specific effects on immune cells rather than non-specific activation of all lymphocytes and APCs.[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] Consistent with this notion, our experiments demonstrated that high salt intake significantly compromises the therapeutic efficacy of tTreg cells, whereas iTreg cells remain relatively unaffected in EAE. Consequently, iTregs foster tolerogenic DC generation, in contrast to tTregs, thereby contributing to the amelioration of symptoms in EAE mice subjected to a salt-rich diet.\u003c/p\u003e \u003cp\u003eIn summary, our findings offer further evidence supporting the notion that iTregs exhibit distinct biological properties compared to tTregs. Also, these results underscore the potential clinical relevance of iTregs in patients diagnosed with autoimmune and inflammatory conditions, specifically highlighting the importance of considering the complex influence of environmental factors such as diet.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eThe following abbreviations are used in this manuscript:\u003c/p\u003e\n\u003cp\u003eiTregs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; induced CD4+Foxp3+ regulatory T cells;\u003c/p\u003e\n\u003cp\u003etTregs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;thymus-derived CD4+CD25+ regulatory T cells;\u003c/p\u003e\n\u003cp\u003eTreg subsets \u0026nbsp; (tTregs and iTregs);\u003c/p\u003e\n\u003cp\u003eCCAR2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;cell cycle and apoptosis regulator 2;\u003c/p\u003e\n\u003cp\u003eCFSE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;carboxyfluorescein diacetate succinimidyl ester;\u003c/p\u003e\n\u003cp\u003eEAE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; experimental autoimmune encephalomyelitis.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.\u003c/p\u003e\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study.\u003c/p\u003e\n\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe National Natural Science Foundation of China (81960293); the China Postdoctoral Foundation project (2023M731460); the Natural Science Foundation of Gansu Province (20JR5RA3); the Joint Research Fund of Gansu Province (23JRRA1495); the Lanzhou Chengguan District talent innovation and entrepreneurship project (2023RCCX0021), the Student Innovation and Entrepreneurship Program of Lanzhou University (LZU-JZH2634, 202410730187, 20240060201), the Medical Research Improvement Project of Lanzhou University (lzuyxcx-2022-165), the Major scientific and technological innovation project of Health industry in Gansu Province (GSWSQNPY2024-11). \u0026nbsp;\u003c/p\u003e\n\n\u003cp\u003eAuthors\u0026rsquo; contributions\u003c/p\u003e\n\u003cp\u003eYang Luo: Conceptualization, Methodology, Funding acquisition, Figure Supervision. Yating Li, Lingxiao Song and Jun Yang: Conceptualization, Funding acquisition, Investigation, Data curation, writing \u0026ndash; original draft. Jiale Tian, Xiaonan Li and Li Zhang: Data curation, Software. Haitao Yu: Resources and Editing. Youquan Gu: Investigation, Resources. All authors read and approved the final draft.\u003c/p\u003e\n\n\u003cp\u003eEthics approval and informed consent\u003c/p\u003e\n\u003cp\u003eI confirm that I have read the Editorial Policy pages. All animal experiments in this study were approved by the ethics committee of the first hospital of Lanzhou university (protocol code: LDYYLL-2023-454). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\n\u003cp\u003eConflicts of Interest\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflict of interest.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRaffin C, Vo LT, Bluestone JA: \u003cstrong\u003eT(reg) cell-based therapies: challenges and perspectives\u003c/strong\u003e. \u003cem\u003eNature reviews. 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doi.org/10.1038/cr.2015.87.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"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":"Experimental autoimmune encephalomyelitis (EAE), High-salt diet, IL-10, TGF-β-induced regulatory T cells (iTregs), TGF-β, Thymus-derived natural regulatory T cells (tTregs), Tolerogenic DCs (tDCs), CCAR2","lastPublishedDoi":"10.21203/rs.3.rs-6528083/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6528083/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eA high-salt environment serves as a pro-inflammatory milieu that induces autoimmune responses by triggering self-reactive immune activation. While thymus-derived regulatory T cells (tTregs) exhibit significantly impaired immunosuppressive function under high-salt diet (HSD) conditions, the TGF-β-induced Treg subset (iTregs) retains full stability and functional integrity in high-salt environments. Despite these findings, endogenous salt-resistant molecular mechanisms that preserve Treg-mediated immunosuppression remain unidentified. Therefore, to address this gap, we propose to investigate the therapeutic potential of Treg cell adoptive transfer in experimental autoimmune encephalomyelitis (EAE) mouse models. By systematically analyzing the differential capacity of tTregs and iTregs to reprogram pro-inflammatory dendritic cells (DCs) into tolerogenic DCs under high-salt conditions, this study aims to identify the mechanistic distinctions that confer resistance to salt-induced inflammatory perturbations in iTregs, while tTregs remain susceptible.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eBoth Treg cell subsets generated from Foxp3-GFP mice were transferred into na\u0026iuml;ve Rag1-/- mice, GFP frequency were dynamically detected and compared within each time point. Subsequently, an EAE mouse model was established, and either iTregs or tTregs were intravenously administrated. Clinical scores were continuously recorded, while brain inflammation was evaluated using hematoxylin and eosin (H\u0026amp;E) staining. Additionally, brain-infiltrating Th1/Th17 cells and the presence of splenic CD11c\u0026thinsp;+\u0026thinsp;dendritic cells (DCs) were analyzed by flow cytometry. A DC-T co-culture assay was then conducted, followed by mechanistic studies using western blotting and FACS. Finally, CCAR2-deficient tTregs and iTregs were generated and co-cultured with DCs with or without NaCl addition. The expression of antigen-presenting molecules and the activation of the AKT/mTOR signaling pathway were then systematically evaluated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eiTregs demonstrate superior efficacy over tTregs in alleviating brain inflammation in both EAE and high-salt diet (HSD)-exacerbated EAE. Unlike tTregs, iTregs suppress pro-inflammatory dendritic cells (DCs) and promote their conversion to an anti-inflammatory phenotype, primarily via membrane-bound TGF-β signaling rather than IL-10R signaling. This functional transformation of DCs is likely mediated by iTreg-induced inhibition of the AKT/mTOR signaling pathway. Notably, under high-salt conditions, this regulatory crosstalk appears specific to iTregs, as tTregs conversely upregulate AKT/mTOR in DCs. Furthermore, CCAR2 contributes to tTreg instability, and its knockdown restores tTreg functionality. In contrast, iTregs enhance DC tolerogenic phenotypes independently of CCAR2.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study delineates a previously unrecognized functional dichotomy between Treg subsets, revealing that iTregs uniquely endow DC tolerance in high-salt environments through membrane-bound TGF-β-dependent suppression of AKT/mTOR signaling, whereas tTregs exacerbate DC immunogenicity via CCAR2-mediated pathway activation. By identifying CCAR2 as a critical destabilizing factor in tTregs and demonstrating the salt-resistant mechanistic signature of iTregs, our findings not only redefine microenvironment-specific regulatory paradigms in autoimmune pathogenesis but also establish iTregs as a superior therapeutic modality for inflammation-dominated disorders, particularly under metabolically stressful conditions such as high-salt exposure.\u003c/p\u003e","manuscriptTitle":"CCAR2 Dictates tTreg Instability and iTreg-Driven Dendritic Cell Tolerance via Divergent AKT/mTOR Modulation in High-Salt Microenvironments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 15:14:34","doi":"10.21203/rs.3.rs-6528083/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":"bc96f9cb-d646-4194-9260-5806f1f1a62a","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-11T01:08:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 15:14:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6528083","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6528083","identity":"rs-6528083","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}