Adipocyte OX40L Promotes Adipose T Cell Activation and Insulin Resistance in Obesity

Adipocyte OX40L Promotes Adipose T Cell Activation and Insulin Resistance in Obesity | 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 Article Adipocyte OX40L Promotes Adipose T Cell Activation and Insulin Resistance in Obesity Tuo Deng, Jianfeng Song, Qin Zeng, Yayi Jiao, Xiaoxiao Sun, Limin Xie, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7361491/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 T cells contribute critically to obesity-induced adipose inflammation and insulin resistance, yet the co-stimulatory signals that govern their activation in adipose tissue remain unclear. Here, we systematically profile co-stimulatory molecules in adipocytes and adipose tissue macrophages and identify OX40 ligand (OX40L) as the most robustly upregulated in obesity. OX40L is also elevated in adipocytes from obese, insulin-resistant humans. While macrophage-specific OX40L deletion has no metabolic impact, global OX40 deficiency or adipocyte-specific OX40L deletion reduces Th1 cell accumulation in visceral adipose tissue, attenuates inflammation, and improves insulin sensitivity without affecting adiposity. These benefits are reversed by Th1 cell transfer. Therapeutic blockade of OX40L with a neutralizing antibody mimics the protective effects of genetic deletion. Our findings identify adipocyte-derived OX40L as a critical mediator of obesity-associated immune dysfunction and establish it as a targetable checkpoint for tissue-specific immunotherapy in metabolic disease. Biological sciences/Immunology/Immunotherapy/Immunosuppression Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte activation obesity adipose inflammation adipocyte T cell OX40 OX40L Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The rising prevalence of obesity poses a significant global challenge [1], heightening the risk of associated comorbidities such as type 2 diabetes mellitus (T2DM), cardiovascular disease, non-alcoholic steatohepatitis (NASH), and cancer [2]. Chronic low-grade inflammation associated with obesity is a critical mechanistic link between excess adiposity and these serious disorders [3, 4]. Among various tissues, visceral adipose tissue (VAT) inflammation plays a pivotal role in the development of insulin resistance and other obesity-related complications, primarily through alterations in immune cell composition [5-7]. Thus, targeting VAT inflammation represents a promising therapeutic strategy for combating obesity-associated diseases. A variety of immune cells are involved in obesity-induced VAT inflammation [7, 8]. Adipose tissue macrophages (ATMs) are substantially increased in obesity [9] and serve as key regulators of inflammatory processes [10, 11]. However, adipose tissue T cells (ARTs) also play a central role by modulating the phenotypes of pro-inflammatory ATMs and adipocyte. In lean individuals, CD4⁺ T helper 2 (Th2) cells and regulatory T (Treg) cells dominate the ARTs population and help maintain an anti-inflammatory microenvironment through the secretion of cytokines such as interleukin-10 (IL-10) [12]. High-fat diet (HFD) feeding induces the expansion of Th1 and CD8 + T cells, leading to enhanced interferon gamma (IFNγ) production, which promotes pro-inflammatory macrophage polarization and reduces Th2 and Treg cell abundance [13, 14]. Depletion of Th1 or CD8 + T cells ameliorates HFD-induced adipose inflammation and insulin resistance [15, 16]. While Th1 and CD8 + T cells are now recognized as key components of obesity-induced adipose inflammation, the specific signals that initiate their activation are remain elusive. Uncovering these signals would guide new strategies to control obesity-induced inflammation. T cell activation requires both major histocompatibility complex (MHC)-mediated antigen presentation and co-stimulation. CD4 + and CD8 + T cells, respectively, recognize antigens presented by MHCII and MHCI, and usually share co-stimulation signals [17]. Our previous work demonstrated that MHCII expression in adipocytes increases during obesity and plays a crucial role in promoting adipose inflammation and insulin resistance [18, 19]. HFD-fed MHCII -/- mice show reduced Th1 activation and improved insulin sensitivity despite comparable adiposity to wild-type (WT) controls [18, 20]. Moreover, adipocyte-specific deletion of MHCII recapitulates these effects, further implicating adipocytes as functional antigen-presenting cells (APCs) in obesity [21]. In parallel, Dr. Lumeng’s group identified ATMs as dominant APCs within the stromal vascular fraction of VAT and showed that macrophage-specific MHCII deletion also improved adipose inflammation and insulin sensitivity under HFD feeding [20]. These studies suggest that both adipocytes and ATMs contribute to adipose CD4⁺ T cell activation during obesity through MHCII-dependent antigen presentation. However, antigen recognition alone is insufficient, and co-stimulatory signals are also required for full T cell activation. Several T cell co-stimulators have been investigated in the context of obesity. CD40, CD80 (B7-1), and CD86 (B7-2) are induced in adipose tissue from obese mice [22, 23]. Nevertheless, they are unlikely to co-stimulate ARTs activation in obesity, because both CD40 and CD80/CD86 show protective effects on HFD-induced adipose inflammation and insulin resistance [23-25]. Both CD40 knockout mice and CD80/CD86 double knockout mice fed a HFD have aggravated inflammation in adipose tissue because of increased CD8 + ARTs and ATMs [23, 24] or decreased adipose Tregs [25], respectively. The 4-1BB ligand is modestly elevated in obese adipose tissue, and while 4-1BB deficiency improves metabolic outcomes, this effect has been attributed to enhanced brown adipose activity and reduced weight gain [26]. The co-stimulatory molecule ICOS is highly expressed on Tregs and limits their VAT accumulation by suppressing CCR3 expression [27]. Although ICOS-deficient mice show improved insulin sensitivity, the regulation of ICOS or ICOSL in adipose tissue during obesity remains unclear [27]. Collectively, these findings underscore the need to identify specific co-stimulatory molecules that directly drive ARTs activation in obese VAT. In this study, we identify OX40 ligand (OX40L) as a key co-stimulatory molecule that is markedly upregulated in adipocytes and ATMs during obesity. We demonstrate that adipocyte-derived, but not macrophage-derived, OX40L is essential for Th1 activation in VAT and contributes to obesity-induced insulin resistance. Importantly, treatment with an OX40L-blocking antibody alleviates VAT inflammation and improves metabolic parameters in obese mice. These findings uncover a critical role for adipocyte-T cell OX40L-OX40 signaling in driving obesity-induced immune activation and metabolic dysfunction, and highlight this pathway as a potential therapeutic target. 2. Results 2.1 Obesity induces OX40L expression in adipocytes and ATMs Given that ATMs [20] and adipocytes [18, 19] function as APCs in obese VAT, we assessed the expression of known T cell co-stimulatory molecules [17] in these cell types isolated from the VAT of lean and diet-induced obesity (DIO) mice. Among the candidates, OX40L was most strongly upregulated in both adipocytes and ATMs from obese mice (Figure S1 A and S1B). Similarly, in transgenic ob/ob mice, a genetic model of obesity, OX40L expression was markedly elevated in both adipocytes and ATMs (Figure S1 C and S1D). Notably, HFD feeding specifically enhanced OX40L gene expression in VAT, without similar upregulation in other examined tissues (Figure S1 E), suggesting a VAT-specific regulation in obesity. Flow cytometry analysis revealed a significant increase in OX40L + adipocytes and ATMs in both DIO and ob/ob mice (Fig. 1 A and 1 B). Given the technical challenges of performing flow cytometry on buoyant and fragile adipocytes, we present the complete gating strategy (Figure S2A), accompanied by bright-field images that depict adipocyte morphology of ND, HFD, and ob/ob mice (Figure S2B), as well as fluorescence images validating OX40L staining in adipocytes (Figure S2C). Consistently, western blotting confirmed elevated OX40L protein levels in these cells under obese conditions (Fig. 1 C-F). To examine the translational relevance of these findings, we analyzed VAT samples from lean and obese human subjects. Adipocytes from obese individuals exhibited a 2.5- to 4-fold increase in OX40L mRNA levels compared to lean controls (Figure S1 F). Due to limited access to human ATMs, RT-qPCR analysis of these cells was not performed. Collectively, these murine and human data demonstrate that OX40L is upregulated in adipocytes and ATMs within obese VAT, implicating the OX40L-OX40 costimulatory axis as a key mediator of obesity-driven ARTs activation and insulin resistance. 2.2 OX40 deficiency attenuates HFD-induced adipose inflammation and insulin resistance To clarify the role of OX40L-OX40 costimulatory signaling in obesity-induced adipose inflammation and insulin resistance, we examined OX40-deficient ( OX40 ⁻ / ⁻) mice under HFD conditions. After 12 weeks of HFD feeding, KO mice and their WT littermates exhibited comparable body weight, fat mass, and fasting blood glucose levels (Fig. 2 A, 2 B and 2 E). However, OX40 ⁻ / ⁻ mice displayed significantly improved insulin sensitivity, enhanced glucose tolerance, and lower fasting insulin levels (Fig. 2 C, 2 D and 2 F), indicating that OX40 deficiency mitigates HFD-induced insulin resistance. Histological analysis revealed a reduced abundance of crown-like structures (CLSs) in the eWAT of obese OX40 ⁻ / ⁻ mice relative to controls (Fig. 2 G). The size of adipocytes was comparable between the two groups (Fig. 2 H), suggesting that the reduction in CLS abundance was not attributable to differences in adipocyte hypertrophy-induced cell death. Flow cytometric analysis further revealed a marked reduction in ARTs in the eWAT of OX40 ⁻ / ⁻ mice, whereas ATMs numbers remained unchanged compared to WT controls (Fig. 2 I, Flow cytometry gating strategy is shown in Figure S3). Given that T cells are a key component of CLS [12, 16, 28], the decline in CLS abundance is likely associated with the reduction in ARTs. Notably, HFD-fed OX40 ⁻ / ⁻ mice exhibited a significant decrease in the proportion and number of CD8⁺ T and Th1 cells in eWAT, with no corresponding changes observed in the spleen or liver (Fig. 2 J-M). This observation aligns with the upregulation of OX40L expression in the eWAT of obese mice, supporting a specific role for OX40 signaling in facilitating CD8⁺ T and Th1 cell activation within adipose tissue during obesity. Collectively, these findings demonstrate that OX40 deficiency alleviates adipose tissue inflammation and enhances insulin sensitivity under HFD-induced metabolic stress, highlighting the crucial role of OX40L-OX40 signaling in obesity-associated metabolic dysfunction. 2.3 Macrophage OX40L deficiency does not affect HFD-induced insulin resistance To investigate the role of macrophage-derived OX40L in obesity-associated metabolic dysfunction, we generated macrophage-specific OX40L knockout mice (MKO) mice by crossing Tnfsf4 flox/flox mice to Lysm -Cre mice. MKO mice were viable and born at expected Mendelian ratios. In HFD-fed MKO mice, OX40L expression was markedly reduced in ATMs and bone marrow-derived macrophages (BMDMs), but remained unchanged in adipocytes (Figure S4A). Flow cytometry further revealed that the proportion of OX40L + ATMs in MKO mice was significantly lower than in their WT littermates (MWT) (Figure S4B), confirming successful macrophage-specific deletion of OX40L. Following 12 weeks of HFD feeding, MKO and MWT mice exhibited comparable body weight and adipose tissue mass (Figure S4C-F). There were no significant differences between the two groups in insulin sensitivity, glucose tolerance, fasting blood glucose, or fasting insulin levels (Figure S4G-J), indicating that macrophage-specific OX40L deficiency does not affect HFD-induced insulin resistance. These findings suggest that macrophage-derived OX40L is dispensable for obesity-induced metabolic dysfunction. 2.4 Adipocyte OX40L deficiency attenuates HFD-induced adipose inflammation and insulin resistance To determine the role of adipocyte-derived OX40L in obesity-induced adipose inflammation and insulin resistance, we generated adipocyte-specific OX40L knockout mice (AKO) by crossing Tnfsf4 flox/flox mice with Adipoq -Cre mice. The AKO mice were viable, born at expected Mendelian ratios, and exhibited no discernible morphological differences from their AWT ( Tnfsf4 flox/flox ) littermates. As anticipated, OX40L expression was reduced in all examined adipose depots of HFD-fed AKO mice compared with AWT controls, with no changes detected in non-adipose tissues (Figure S5A). OX40L reduction was specific to adipocytes, with no changes in the SVF or BMDMs (Figure S5B). The protein levels of OX40L in adipocytes were consistently and substantially lower in AKO mice than in AWT mice (Figures S5C and S5D), while OX40L levels in SVF remained unchanged between groups (Figure S5C). These results confirm the successful generation of a mouse model with adipocyte-specific OX40L deletion. AKO and AWT littermate mice were fed either ND or HFD for 12 weeks and analyzed for differences in metabolic phenotypes. Under ND conditions, AKO mice exhibited similar body weight, insulin sensitivity, and glucose tolerance to AWT controls (Figures S6A-E). Additionally, the frequency of CD4 + T, CD8 + T, Th1, Th2, Th17 and Treg cells remained unchanged in both eWAT and the spleen of the AKO group compared with the AWT group (Figures S6F-J). Under HFD conditions, AKO mice showed comparable adiposity and glucose tolerance to AWT controls (Fig. 3 A-D, F-G), but exhibited lower fasting insulin levels and improved insulin sensitivity (Fig. 3 E and 3 H). Western blotting of eWAT, skeletal muscle and liver demonstrated enhanced post-insulin AKT phosphorylation in AKO mice (Fig. 3 I-K), indicating improved insulin responsiveness. Consistent with their improved insulin sensitivity, AKO mice exhibited reduced expression of pro-inflammatory genes and increased expression of anti-inflammatory genes in adipocytes (Fig. 4 A). Specifically, they showed reduced expression of MHCⅡ-related genes ( H2Ab1 , H2Eb1 , and Ciita ), T-cell co-stimulatory genes ( OX40L , Cd40 and Cd80 ), and inflammatory factors ( Il1b , Tnfα , and MCP-1 ), along with increased expression of the anti-inflammatory adipokine adiponectin. Additionally, the Th1 transcription factor Tbet was significantly lower in the eWAT of AKO mice compared with AWT mice, whereas macrophage-related gene expression remained unchanged between the two groups (Figs. 4 B). The frequency of CD4⁺ and CD8⁺ T cells in eWAT and spleen was similar between groups (Fig. 4 C), though their numbers in eWAT trended downward in AKO mice (Fig. 4 D). Notably, Th1 cells were significantly reduced in eWAT, with no changes in spleen (Fig. 4 E and 4 F). In contrast, Treg cell frequency increased in eWAT, though total Treg numbers were unchanged (Fig. 4 G and 4 H). Th2 and Th17 cell infiltration was not affected (Fig. 4 E-H). Furthermore, the proportion of M1 macrophages was reduced (Fig. 4 I), and fewer CLSs were observed in eWAT of AKO mice (Fig. 4 J). Together, these findings indicate that adipocyte-specific OX40L deletion mitigates HFD-induced adipose inflammation and insulin resistance. 2.5 Adipocyte OX40L-OX40 signaling promotes insulin resistance by activating Th1 cells in adipose tissue To determine whether the reduction in adipose Th1 cells observed in HFD-fed AKO mice was responsible for their improved insulin resistance, obese AWT and AKO mice were intraperitoneally injected with inflammatory Th1 cells, and their insulin responsiveness was assessed via glucose tolerance test (GTT) and insulin tolerance test (ITT) (Figs. 5 A). No significant differences in weight gain were observed between AWT and AKO mice before or after Th1 cell transfer (Fig. 5 B). Following adoptive transfer, AKO mice exhibited a marked increase in the proportion of Th1 cells within VAT, restoring their levels to those observed in AWT cotrols (Fig. 5 C), thus confirming successful Th1 cell infiltration. Prior to Th1 cells transfer, AKO mice displayed significantly enhanced insulin sensitive compared to AWT mice, although glucose tolerance was comparable (Figs. 5 D and 5 F). However, following Th1 cells transfer insulin sensitivity and glucose tolerance were indistinguishable between AWT and AKO mice, indicating that restoration of Th1 cells reversed the metabolic improvement in AKO mice (Figs. 5 E and 5 G). To further investigate the mechanism underlying the reduction of adipose Th1 cells in AKO mice, we examined the proliferation and activation of these cells. For proliferation analysis, we used two markers, 5-ethynyl-2'-deoxyuridine (EdU) incorporation and Ki67 staining. Flow cytometry revealed that the proportions of EdU + and Ki67 + Th1 cells in eWAT were significantly lower in AKO mice than in AWT mice (Figs. 6 A and 6 B), indicating that reduced proliferation contributes to the decline of Th1 cells in eWAT. Next, to assess adipocyte-mediated CD4 + T cell activation, we cocultured CD45-depleted adipocytes from AKO and AWT mice with naïve CD4 + T cells from OT-II mice, with or without OVA (Fig. 6 C). After three days, IFNγ levels in the culture supernatant were measured by ELISA, and Th1 differentiation was analyzed by flow cytometry. In the absence of OVA, both AKO and AWT adipocytes exhibited minimal Th1 cell activation. However, in the presence of OVA, AKO adipocytes showed a significantly reduced capacity to activate Th1 cells (Fig. 6 D), which was associated with a marked reduction in IFNγ production (Fig. 6 E). These findings suggest that the reduction of Th1 cells in AKO eWAT results from both impaired proliferation and diminished activation. 2.6 OX40L-blocking antibody ameliorates obesity-induced ARTs activation and insulin resistance To evaluate the therapeutic potential of OX40L inhibition, we treated HFD-induced obese mice with either an OX40L blocking antibody (α-OX40L) or an isotype control antibody (IgG2b) (Fig. 7 A). Body weight gain and composition did not differ significantly between α-OX40L- and IgG2b-treated mice during HFD feeding (Figs. 7 B, S7A and S7B). Similarly, glucose and insulin tolerance were comparable between the two groups before treatment (Figures S7C and S7D). However, following treatment, α-OX40L mice exhibited significantly improved insulin sensitivity and glucose tolerance compared with IgG2b controls (Figs. 7 C and 7 D). Fasting insulin levels were markedly reduced in α-OX40L mice, whereas fasting glucose levels remained unchanged (Figs. 7 E and 7 F). In eWAT, α-OX40L treatment increased the proportion of CD4 + T cells while decreasing the proportion of CD8 + T cells compared with IgG2b treatment (Fig. 7 G), a phenomenon not observed in the spleen. The absolute number of CD4 + T cells in eWAT was significantly higher in α-OX40L-treated mice than in IgG2b-treated mice, whereas no significant difference was detected in absolute CD8 + T counts (Fig. 7 H). Additionally, α-OX40L treatment significantly reduced the proportion of Th1 cells in eWAT compared with IgG2b treatment, although absolute Th1 cell counts remained unchanged (Fig. 7 I and 7 J). Moreover, after α-OX40L treatment, both the proportion and number of Treg cells in eWAT were significantly increased (Fig. 7 K and 7 L). These findings indicated that OX40L blockade effectively ameliorates obesity-induced insulin resistance and ARTs activation. 3. Discussion Th1 cells play a pivotal role in driving obesity-induced adipose tissue inflammation and insulin resistance [15, 29, 30]. Understanding the mechanisms underlying Th1 cell accumulation in adipose tissue during obesity is critical for developing therapies against obesity-associated metabolic disorders. The pathogenic expansion of Th1 cells in VAT during obesity can result from multiple mechanisms. For instance, leptin is a key initiator of the adipose inflammatory cascade, with its receptor expression significantly upregulated on ARTs in the obese state [31]. Hyperleptinemia, driven by adipose tissue expansion, promotes IFNγ secretion by T cells, skewing differentiation toward Th1 while suppressing Th2 responses [32, 33]. Our previous work demonstrated that leptin-stimulated IFNγ production in ARTs induces adipocyte MHCII expression, enabling adipocytes to act as APCs that further amplify Th1 activation [18]. These finding establish that obese adipocytes provide MHCII-mediated antigen presentation to promote Th1 activation. More recently, we identified that adipose stem cells (ASCs) orchestrate T cell infiltration into VAT during the early phase of HFD-induced obesity through TNFα/NF-κB-dependent secretion of CCL5, a potent chemokine that attracts T cells [34]. However, targeting these mechanisms lacks specificity for Th1 suppression and may inadvertently disrupt systemic immune homeostasis. Therefore, more selective molecular strategies that specifically inhibit Th1 cells within obese adipose tissue are urgently needed. Accumulating preclinical and clinical evidence identifies co-stimulatory molecules as promising therapeutic targets due to their spatiotemporally regulated expression [35–37]. Unlike broadly active immune mediators such as MHC molecules, co-stimulatory signals are selectively upregulated following T cell receptor engagement, allowing targeted immunomodulation with reduced off-target effects [38, 39]. These molecules act as critical secondary signals that determine the magnitude and polarization of immune responses, offering unique flexibility to modulate immunity as needed. In this study, we systematically examined the expression profiles of all known T cell co-stimulators in adipocytes and ATMs and found that OX40L is the most prominently upregulated in both cell types under obese conditions. Genetic ablation of OX40 signaling, either through global OX40-knockout ( OX40 ⁻ / ⁻) (Fig. 2 ) or adipocyte-specific OX40L deletion (OX40L-AKO) (Fig. 3 and Fig. 4 ), conferred substantial protection against obesity-induced adipose inflammation and systemic insulin resistance. This phenotype was therapeutically recapitulated by treatment with an OX40L-neutralizing antibody, which alleviated obesity-associated metabolic dysfunction (Fig. 7 ). These results indicate that adipocytes provide essential co-stimulatory signals through the OX40L-OX40 axis to sustain Th1 effector function and drive adipose inflammation in obesity. Notably, although OX40L expression is more strongly induced in macrophages than in adipocytes under obese conditions (Fig. 1 and Figure S1 ), macrophage-specific OX40L knockout (OX40L-MKO) mice failed to exhibit metabolic improvements (Figure S4). This observation suggests that increased macrophage-derived OX40L may be a secondary effect of adipocyte-initiated inflammation rather than a primary pathogenic driver. Further mechanistic studies are warranted to elucidate the spatiotemporal and cell type-specific roles of OX40L in the pathogenesis of adipose inflammation. Nevertheless, our data establish adipocyte-derived OX40L as a key regulator of obesity-induced adipose inflammation and insulin resistance, highlighting its potential as a therapeutic target for metabolic disease. The OX40-OX40L axis has emerged as a promising therapeutic target in T cell-mediated diseases [40]. Antibody-mediated blockade of OX40 or OX40L has shown efficacy in multiple autoimmune models, including experimental autoimmune encephalomyelitis (EAE) [41, 42], systemic lupus erythematosus (SLE) [43], rheumatoid arthritis (RA) [44, 45], and inflammatory bowel disease (IBD) [46–48]. In addition, clinical trials targeting the OX40/OX40L costimulatory pathway have demonstrated therapeutic efficacy in atopic dermatitis [49, 50]. Notably, Amgen’s rocatinlimab (an anti-OX40 monoclonal antibody) and Sanofi’s amlitelimab (an anti-OX40L monoclonal antibody), two novel biologics disrupting T cell activation via the OX40/OX40L axis, have shown durable clinical responses in patients with atopic dermatitis [51–53]. Our findings extend this paradigm to metabolic disease, demonstrating that treatment with an OX40L-blocking antibody reduces Th1 cell accumulation in VAT and improves insulin sensitivity in obese mice (Fig. 7 ). Importantly, systemic inhibition of the OX40 pathway has not been associated with serious side effects in clinical trials [40]. However, given the pleiotropic functions of OX40 in T cell biology, selectively targeting adipocyte-derived OX40L represents a more precise therapeutic strategy to mitigate adipose inflammation without compromising systemic immune function. To this end, we propose the development of adipocyte-targeted delivery systems. Innovative platforms with adipocyte specificity could enable adipose-restricted OX40L blockade, thereby conferring anti-inflammatory effects that are localized to adipose depots while preserving systemic immune competence. Such spatially targeted approaches may address a critical unmet need in the development of tissue-specific immunotherapies for obesity-related metabolic dysfunction. Previous studies have examined the response of global OX40-knockout ( OX40 ⁻ / ⁻) mice to HFD challenge [54]. However, the present study is the first to provide an in-depth investigation of adipose OX40L’s role in obesity-induced adipose inflammation and insulin resistance. Bing Liu et al. reported that OX40 ⁻ / ⁻ mice fed an HFD exhibited reduced weight gain along with improved adipose inflammation and insulin resistance compared to WT mice [54]. Since weight loss is inherently linked to improvements in adipose inflammation and insulin resistance, their study could not rule out the possibility that these metabolic benefits were secondary to reduced weight gain. In contrast, we observed no significant differences in HFD-induced weight gain between OX40 ⁻ / ⁻, AKO, or MKO mice and their respective WT controls. Notably, Bing Liu et al. purchased age-matched male WT and OX40 ⁻ / ⁻ mice from The Jackon Laboratory, and mice of different genotypes were likely housed separately. In our study, by contrast, WT and OX40 ⁻ / ⁻ mice were littermates generated through heterozygous breeding and were cohoused. The discrepancy in HFD-induced weight gain between our findings and those of Bing Liu et al. may be attributed to differences in breeding and housing conditions, as well as potential variations in microbiome composition. Thus, the current study provides a comprehensive analysis using multiple independent measures to establish a direct role of adipocyte-derived OX40L in obesity-induced Th1 activation in adipose tissue and insulin resistance. The results presented here clearly demonstrate that adipocyte OX40L plays a pivotal role in promoting CD4 + ARTs activation and IFNγ production in HFD-fed mice, thereby driving adipose inflammation and systemic insulin resistance. Disruption of this signaling, either through reduced adipocyte OX40L expression or administration of an OX40L-blocking antibody, ameliorates adipose inflammation and preserves systemic insulin sensitivity. These findings position adipose OX40L-OX40 signaling as a potential therapeutic target for mitigating obesity-associated metabolic dysfunction (Fig. 8 ). 4. Methods Human samples This study was conducted in compliance with the Declaration of Helsinki and was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University (No. LYF2022207). Written informed consent was obtained from all human donors prior to their enrollment in the study. Human VAT (omental WAT) samples were obtained from two distinct groups: obese donors (BMI ≥ 30 kg/m 2 ) eligible for bariatric surgery, and nonobese donors (BMI < 30 kg/m 2 ) undergoing non-acute cholecystectomy surgery. Freshly collected VAT specimens (200 mg tissue blocks) were immediately processed (≤ 2 h post-excision) for mechanical dissociation, followed by collagenase digestion to isolate mature adipocytes. Animals All animal studies were performed in accordance with procedures approved by the Central South University Animal Care and Use Committee. Mice were housed in specific pathogen-free facilities under controlled environmental conditions (22°C, 50–60% humidity) with a 12 h light/dark cycle. Animals were provided ad libitum access to water and either a ND (1010001, Jiangsu Xietong Pharmaceutical Bio-engineering Co.LTD) or a 60% HFD (D12492, Wuhan BIOPIKE Bioscience Co.LTD). 6-week-old C57BL/6J mice were obtained from Slac Laboratory Animal Inc, while 12-week-old C57BL/6J ob/ob ( Lep ob /Lep ob ) mice were purchased from the Model Animal Research Center of Nanjing University. We acquired Tnfsf4 tm1a embryonic stem cells (clone number: HEPD0717_6_A11) from the European Conditional Mouse Mutagenesis Program (EUCOMM) and generated Tnfsf4 tm1a mice in the Charles River laboratories. We then crossed the Tnfsf4 tm1a mice with FLP transgenic mice (Jackson Laboratory, Stock No.009086) to breed and remove the lacZ-neo cassette, thereby obtaining Tnfsf4 flox/flox mice. Tnfrsf4 KO mice (Stock No. 012838), Adipoq -Cre mice (Stock No.028020), Lysm -Cre mice (Stock No.004781) and OT-II (Stock No.004194) mice were obtained from the Jackson Laboratory. AKO mice were generated by breeding Adipoq -Cre mice with Tnfsf4 flox/flox mice. Similarly, MKO mice were produced by breeding Lyz2 -Cre mice with Tnfsf4 flox/flox mice. 6-week-old male mice were fed with HFD for 12 weeks to establish the DIO model. These male mice were humanely euthanized via carbon dioxide (CO₂) inhalation for primary euthanasia, followed by cervical dislocation as a secondary confirmatory method, prior to tissue collection. Adipose tissue fractionation Adipose tissues (eWAT and iWAT) were minced and digested with 1 mg/mL type II collagenase (Worthington, Cat#LS004177) and 1% bovine serum albumin at 37°C for 30 min with gentle shaking at 120 rpm. The mixture was filtered to remove undigested fragments and centrifuged at 300 g for 5 min. The upper layer, containing mature adipocyte fraction, was aspirated and washed with PBS containing 2 mM EDTA. The pellet (SVF) was washed with PBS and incubated with red blood cell lysis buffer on ice for 5 min. After lysis, the SVF was washed with PBS and collected by centrifugation at 500 g for 5 min. The purified adipocytes and SVF were then prepared for further analysis. RNA isolation and qRT-PCR According to the manufacturer's instructions, Trizol (Invitrogen, Cat#15596018CN) was used to extract total RNA from cells and tissues. Accurate Biology's cDNA Synthesis kit (Cat#AG11728) facilitated the reverse transcription of RNA. The SYBR Green Master Mix (Vazyme, Cat#Q511-02) was utilized for Real-Time PCR on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The calculation of normalized mRNA expression in mouse samples utilized β-actin or 36b4 as reference genes, whereas 36B4 was used for human samples. Relative mRNA expression was assessed using the ΔΔCt method. Primer sequences are listed in Table S1 . Flow cytometry Following standardized adipose tissue fractionation, 60 µL of isolated adipocytes were collected and incubated with anti-CD16/32 antibody (1:100, Biolegend, Cat#101302) for FcR blockade (7 min, room temperature, dark). To identify OX40L + adipocyte population, cells were stained with anti-CD45 (1:200, Biolegend, Cat#103132) and anti-OX40L (1:100, Biolegend, Cat#108810) in PBS containing 1% FBS and 2 mM EDTA (7 min, room temperature, dark). The reaction was quenched with 1 mL PBS, followed by 5 min incubation for phase separation at room temperature. The lower aqueous phase was carefully aspirated using a 1 mL syringe, and the adipocytes were resuspended in 100 µL PBS, transferred to flow cytometry tubes, and stained with 0.6 µL propidium iodide (PI, Biolegend, Cat#421301) for viability assessment prior to flow cytometric analysis. The instrument used for flow cytometry analysis of adipocytes was the NovoCyte Quanteon Flow Cytometer. Before loading, the adipocytes were thoroughly mixed and introduced into the machine at a low flow rate. The SVF was isolated by centrifugation at 300 g for 5 min at room temperature, separating it from floating adipocytes. For surface marker detection, cells were first incubated with FcR blocking antibody (1:100, Biolegend, Cat#101302) for 7 min at room temperature, followed by incubation with antibodies against surface markers for 30 min at 4°C in the dark. After washing with PBS, cell viability was assessed using PI for 5 min at room temperature. For intracellular staining, cells were incubated with Zombie NIR™ (1:200, Biolegend, Cat#423106) for 7 min at room temperature, and then blocked with anti-CD16/32 antibody (1:100, Biolegend, Cat#101302) and stained with fluorochrome-labeled mAbs against cell-surface antigens for 30 min at 4°C. Cells subsequently were fixed and permeabilized using the Foxp3/transcription factor staining buffer set (eBioscience, Cat#00-5523-00) according to manufacturer's protocol, followed by intracellular staining with fluorochrome-conjugated antibodies for an additional 45 min at 4°C in the dark. The following mAbs were used: anti-CD45 (1:200, Biolegend, Cat#103132), anti-CD45.1 (1:200, Biolegend, Cat#110728), anti-CD3 (1:200, Biolegend, Cat#100306 and Cat#100236), anti-CD8 (1:200, Biolegend, Cat#100730), anti-CD4 (1:200, Biolegend, Cat#100428), anti-CD11b (1:200, Biolegend, Cat#101222), anti-F4/80 (1:200, Biolegend, Cat#123114), anti-CD11c (1:200, Biolegend, Cat#117308), anti-CD206 (1:200, Biolegend, Cat#141707), anti-Foxp3 (1:200, eBiosicence, Cat#17-5773-82), anti-IFNγ (1:200, Biolegend, Cat#505808; 1:200, BD Bioscience, Cat#566097), anti-IL17A (1:200, Biolegend, Cat#506938), anti-IL4 (1:200, Biolegend, Cat#144806), anti-Ki67 (1:200, Biolegend, Cat#652410). Immunofluorescence Staining Adipocytes were isolated from mouse epididymal white adipose tissue (eWAT). The cells were washed 3–5 times with PBS containing 2 mM EDTA to remove debris. For staining, 50 µL of adipocyte suspension was transferred to a fresh tube and mixed with 100 µL of PBS. To block Fc receptors, anti-CD16/32 antibody (1:100) was added and incubated at room temperature for 7 minutes. Subsequently, BODIPY (100 µg/mL, Invitrogen, D3922), Hoechst (10 µg/mL, Beyotime, Cat#C1022), and PE anti-mouse OX40L antibody (1:100, Biolegend, Cat#108806) were added to the mixture and incubated at room temperature for 10 minutes. After staining, the adipocytes were washed with 1 mL of PBS. The bottom aqueous phase was removed, and the cells were resuspended in 50 µL of PBS. The stained adipocyte suspension was transferred to a confocal dish, and images were acquired using a confocal fluorescence microscope (Zeiss). Glucose tolerance test (GTT) and insulin tolerance test (ITT) To conduct glucose tolerance tests, mice were fasted for 16 h before receiving an intraperitoneal injection of glucose at a dose of 1 g/kg. At 0, 15, 30, 60, 90 and 120 min, blood samples from the tail vein were measured with a Roche glucometer. For insulin tolerance tests, mice were fasted for 6 h and injected with insulin at a dose of 0.75 U/kg. Blood samples taken from tail vein were measured at 0, 15, 30, 45, 60 and 90 min using Roche glucometer. Fasting blood glucose (FPG) and insulin (FPI) measurements Mice were fasted for 6 hours prior to sample collection. Fasting FPG levels were measured using a Roche glucometer. For insulin measurement, blood samples were collected from the retro-orbital sinus into EDTA-coated tubes. Plasma was obtained by centrifugation at 3000 rpm for 15 minutes, and insulin levels were quantified using a commercial ELISA kit (AiFang biological company, Cat#AF2579-A)) according to the manufacturer’s instructions. Western blot To examine the OX40L protein expression, adipocytes, macrophages and SVFs were isolated from eWAT of mice fed either a HFD or ND. To check the insulin signalling, mice were fasted for 6 h prior to intraperitoneal injection with either insulin (4 U/kg) or PBS as control. Skeletal muscle and eWAT were collected 15 min after injection. Cells and tissues were lysed in RIPA lysis buffer (Beyotime, Cat#P0013B) supplemented with cOmplete™ Mini Protease Inhibitor Cocktail Tables (Roche, Cat#11836153001) and cOmplete™ EDTA-free (Sigma-Aldrich, Cat#4693132001). Protein quantification was performed using BCA assays optimized for different sample types: BCA Protein Assay Kit (Beijing Dingguo Changsheng Biotech, Cat# P0398M) for non-adipose tissues and Pierce Microplate BCA Protein Assay kit (Thermo scientific, Cat#23252) for adipocyte samples, following manufacturers' protocols. For immunoblotting, 30 µg of total protein were loaded onto 10% SDS-polyacrylamide gels and transferred to PVDF membranes (Merck Millipore, Burlington, MA). After blocking with 5% BSA in PBS for 1 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies against: OX40L (1:1000, AiFang biological, Cat#AF07062), β-actin (1:40000, Sigma-Aldrich, Cat#A5316), p-AKT (1:1000, Cell Signaling Technology, Cat#13038S) and AKT (1:1000, Cell Signaling Technology, Cat#4691S). Following phosphotyrosine detection, membranes were stripped with buffer containing 100 mM β-mercaptoethanol, 2% SDS, and 62.5 mMTris-HCl (pH 7.6) at 50°C for 30 min, and reincubated with total AKT antibody at 4°C overnight. Membranes were washed three times (15 min each) with TBST, followed by incubation with anti-HRP secondary antibody (1:20000, Cell Signaling Technology, Cat#7074P2) in 2% BSA for 1 h at room temperature with gentle agitation. Wash the membrane as previously. Apply ClarityTM Western ECL Substrate (Bio-rad, Cat#1705061) and expose to film. Bio-Rad Image Lab and Image-Pro Plus software were used for analysis. Differentiation and adoptive transfer of Th1 cells Naive CD4 + T cells were purified from splenocytes of 6–8 weeks old male CD45.1 mice using magnetic bead cell sorting (Miltenyi Biotec, Cat#130-104-453). Cell purity was confirmed by flow cytometry analysis of CD4 + CD44 low CD62L high T cell subset. These naïve CD4 + T cells were stimulated with 2 µg/mL pre-coated anti-CD3ε antibody (Selleck, Cat#A2104) and 2 µg/mL soluble anti-CD28 antibody (Selleck, Cat#A2108) in culture medium containing 20 ng/mL IL12 (Peprotech, Cat#210 − 12), 10 ng/mL IL2 (Peprotech, Cat#200-02) and 10 µg/mL anti–IL4 antibody (BD Biosciences, Cat#554432) for 3 days. The culture medium was RPMI 1640 medium (plus 50 mM β-mercaptoethanol) supplemented with 10% FBS, 1% GlutaMax, and 1% Pen/Strep (Gibco, Shanghai, China). The purity of Th1 cells after differentiation was validated by flow cytometry. Male 8-week-old AKO and AWT mice were maintained on a HFD for 11 weeks prior to receiving 1×10 7 in vitro-differentiated Th1 cells via intraperitoneal injection. 3 days after the injection, ITT were performed. 5 days after the injection, GTT were performed. 7 days after the injection, the mice were sacrificed. In addition, to verify Th1 cell recruitment, cells from eWAT and spleen were analyzed by flow cytometry. Adipocyte-T cell coculture and antigen-specific T cell activation assays Adipocytes of AKO and AWT mice fed 12 weeks of HFD were incubated with medium alone or 10 µg/mL OVA peptide (OVA323-339, Sigma, Cat#O1641) at 37°C on a shaker. Naïve CD4 + T cells were purified from the splenocytes of OT-II mice using the MojoSort™ Mouse CD4 Naïve T Cell Isolation Kit (Miltenyi Biotec, Cat#130-104-453). The pulsed adipocytes were then cocultured with isolated T cells in RPMI-1640 complete medium supplemented with 10 ng/mL IL2 (Peprotech, Cat#200-02) on a shaker. Three days later, T cells were stained for flow cytometry, and the Th1-cell cytokine IFNγ in the media was measured by ELISA (R&D Systems, Cat#DY485). Cell proliferation assay DNA synthesis was quantified using the Click-iT® EdU Flow Cytometry Assay Kits (Invitrogen, Cat#C10424) according to the manufacturer's protocol. Briefly, mice received intraperitoneal injections of EdU at 20 µg/g. Six hours post-injection, SVF were isolated from mice as described previously. Cells were first stained with appropriate surface markers, washed with PBS, then fixed and permeabilized with 50 µL Component D (provided in the kit) for 15 min at room temperature protected from light. Following additional washes with PBS containing 1% BSA, cells were incubated with 100 µL 1×Click-iT saponin-based permeabilization/wash reagent and 125 µL Click-iT reaction cocktail for 30 min at room temperature in the dark. For intracellular cytokine staining, cells were subsequently treated with 1×permeabilization/wash reagent at 4°C for 30 min containing anti-IFNγ antibody (1:200, Biolegend, Cat#505808). OX40L antibody blockade assay C57BL/6J mice fed a HFD for 12 weeks were randomly assigned to receive intraperitoneal injections of either an OX40L-blocking monoclonal antibody (BioXcell, Cat#BE0033-1) or an isotype-matched control antibody IgG2b (BioXcell, Cat#BE0090), administered at a dose of 300 µg per mouse, twice weekly. Metabolic assessments, including insulin tolerance tests (ITT) and glucose tolerance tests (GTT), were performed at week 16 of HFD feeding. At week 18, mice were euthanized, and T cell subsets in VAT and spleen were profiled by flow cytometry. Statistical analysis All data are expressed as the mean ± SEM. Normal distribution of populations at the 0.05 level was calculated. Unless stated otherwise, significance was assessed by Student’s t-test for two-groups comparisons, while ANOVA for multiple-group comparisons. Non-parametric data were evaluated using the Mann-Whitney U test. For longitudinal metabolic data (ITT and GTT), two-way repeated-measures ANOVA was performed. All statistical analyses were conducted using SPSS version 25.0, with statistical significance defined as P < 0.05. The sample size (n) for each experiment is specified in the corresponding figure legend. Declarations Conflict of Interest The authors declare no conflicts of interest. Author contributions J.F.S., Q.Z., and Y.Y.J. contributed equally. J.F.S., Q.Z., and Y.Y.J. designed the study, conducted the majority of experiments, analyzed data, and drafted the manuscript. X.X.S., L.M.X., and J.P.Q. performed animal studies. W.Y.H., F.Q.W., B.L.H., and W.Q.M. conducted cellular experiments. Y.J.D. and W.L. provided human adipose tissue samples and performed related analyses. Y.M. and D.D.W. and L.X. contributed to data interpretation and manuscript revision. X.X., W.A.H. and X.C.L. provided expert knowledge and experimental guidance. T.D. conceived and supervised the project. All authors participated in result discussions and manuscript review. Acknowledgments We thank Dr. Xian Chang Li (Immunobiology & Transplant Science Center, Houston Methodist Hospital, Texas Medical Center) and Dr. Xiang Xiao (Immunobiology & Transplant Science Center, Houston Methodist Hospital, Texas Medical Center) for their scientific advice in the research. This work was supported by the National Key R&D Program of China (2020YFA0803604 and 2023YFC3603404), the Key Program of the National Natural Science Foundation of China (82130024, T2341005), the Fund for International Cooperation and Exchange of the National Natural Science Foundation of China (82361168636), the Natural Science Foundation of Hunan Province (2023JJ40809), and the Science and Technology Innovation Program of Hunan Province (2024RC3052). References Collaboration NCDRF. 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China.","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Mei","suffix":""},{"id":504800190,"identity":"b744a3a9-3fec-4524-ada3-b99921229a52","order_by":13,"name":"Dandan Wang","email":"","orcid":"","institution":"National Clinical Research Center for Metabolic Diseases, and Department of Metabolism and Endocrinology, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, China.","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Wang","suffix":""},{"id":504800191,"identity":"db52f850-aa23-4517-8adb-d2aef4e69539","order_by":14,"name":"Lan Xie","email":"","orcid":"","institution":"Tsinghua University, Intelligent Chinese Medicine Engineering Research Center, Beijing 100084, China","correspondingAuthor":false,"prefix":"","firstName":"Lan","middleName":"","lastName":"Xie","suffix":""},{"id":504800192,"identity":"53f676d7-ab0b-448b-8acd-daa659487f56","order_by":15,"name":"Xiang Xiao","email":"","orcid":"","institution":"Houston Methodist Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Xiao","suffix":""},{"id":504800193,"identity":"e2b6b2c6-72e2-4341-ac81-76ac52434497","order_by":16,"name":"Wei Liu","email":"","orcid":"","institution":"Department of General Surgery, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, China.","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Liu","suffix":""},{"id":504800194,"identity":"f463cd4e-8b06-4519-b415-abb8df258a07","order_by":17,"name":"Willa Hsueh","email":"","orcid":"","institution":"The Diabetes and Metabolism Research Center, Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, The Ohio State University, Columbus, OH 43210, 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08:03:39","extension":"pptx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5018404,"visible":true,"origin":"","legend":"unprocessed WB images","description":"","filename":"unprocessedWBimages.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7361491/v1/8aecec9eb94a0a2721d07e16.pptx"},{"id":90293548,"identity":"22346bca-9520-4431-9c3f-2a3e48297180","added_by":"auto","created_at":"2025-09-01 07:47:40","extension":"pptx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4483206,"visible":true,"origin":"","legend":"Unprocessed images","description":"","filename":"unprocessedimages.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7361491/v1/0ebb18aac1b713ec841ea5a0.pptx"}],"financialInterests":"(Not answered)","formattedTitle":"Adipocyte OX40L Promotes Adipose T Cell Activation and Insulin Resistance in Obesity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe rising prevalence of obesity poses a significant global challenge\u0026nbsp;[1], heightening the risk of associated comorbidities such as type 2 diabetes mellitus (T2DM), cardiovascular disease, non-alcoholic steatohepatitis (NASH), and cancer [2]. Chronic low-grade inflammation associated with obesity is a critical mechanistic link between excess adiposity and these serious disorders [3, 4]. Among various tissues, visceral adipose tissue (VAT) inflammation plays a pivotal role in the development of insulin resistance and other obesity-related complications, primarily through alterations in immune cell composition [5-7]. Thus, targeting VAT inflammation represents a promising therapeutic strategy for combating obesity-associated diseases.\u003c/p\u003e\n\u003cp\u003eA variety of immune cells are involved in obesity-induced VAT inflammation [7, 8]. Adipose tissue macrophages (ATMs) are substantially increased in obesity [9] and serve as key regulators of inflammatory processes [10, 11]. However, adipose tissue T cells (ARTs) also play a central role by modulating the phenotypes of pro-inflammatory ATMs and adipocyte. In lean individuals, CD4⁺ T helper 2 (Th2) cells and regulatory T (Treg) cells dominate the ARTs population and help maintain an anti-inflammatory microenvironment through the secretion of cytokines such as interleukin-10 (IL-10) [12]. High-fat diet (HFD) feeding induces the expansion of Th1 and CD8\u003csup\u003e+\u003c/sup\u003e T cells, leading to enhanced interferon gamma (IFN\u0026gamma;) production, which promotes pro-inflammatory macrophage polarization and reduces Th2 and Treg cell abundance [13, 14]. Depletion of Th1 or CD8\u003csup\u003e+\u003c/sup\u003e T cells ameliorates HFD-induced adipose inflammation and insulin resistance [15, 16]. While Th1 and CD8\u003csup\u003e+\u003c/sup\u003e T cells are now recognized as key components of obesity-induced adipose inflammation, the specific signals that initiate their activation are remain elusive. Uncovering these signals would guide new strategies to control obesity-induced inflammation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eT cell activation requires both major histocompatibility complex (MHC)-mediated antigen presentation and co-stimulation. CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells, respectively, recognize antigens presented by MHCII and MHCI, and usually share co-stimulation signals [17]. Our previous work demonstrated that MHCII expression in adipocytes increases during obesity and plays a crucial role in promoting adipose inflammation and insulin resistance [18, 19]. HFD-fed \u003cem\u003eMHCII\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice show reduced Th1 activation and improved insulin sensitivity despite comparable adiposity to wild-type (WT) controls [18, 20]. Moreover, adipocyte-specific deletion of MHCII recapitulates these effects, further implicating adipocytes as functional antigen-presenting cells (APCs) in obesity [21]. In parallel, Dr. Lumeng\u0026rsquo;s group identified ATMs as dominant APCs within the stromal vascular fraction of VAT and showed that macrophage-specific MHCII deletion also improved adipose inflammation and insulin sensitivity under HFD feeding [20]. These studies suggest that both adipocytes and ATMs contribute to adipose CD4⁺ T cell activation during obesity through MHCII-dependent antigen presentation. However, antigen recognition alone is insufficient, and co-stimulatory signals are also required for full T cell activation.\u003c/p\u003e\n\u003cp\u003eSeveral T cell co-stimulators have been investigated in the context of obesity. CD40, CD80 (B7-1), and CD86 (B7-2) are induced in adipose tissue from obese mice [22, 23]. Nevertheless, they are unlikely to co-stimulate ARTs activation in obesity, because both CD40 and CD80/CD86 show protective effects on HFD-induced adipose inflammation and insulin resistance [23-25]. Both CD40 knockout mice and CD80/CD86 double knockout mice fed a HFD have aggravated inflammation in adipose tissue because of increased CD8\u003csup\u003e+\u003c/sup\u003e ARTs and ATMs [23, 24] or decreased adipose Tregs [25], respectively. The 4-1BB ligand is modestly elevated in obese adipose tissue, and while 4-1BB deficiency improves metabolic outcomes, this effect has been attributed to enhanced brown adipose activity and reduced weight gain [26]. The co-stimulatory molecule ICOS is highly expressed on Tregs and limits their VAT accumulation by suppressing CCR3 expression [27]. Although ICOS-deficient mice show improved insulin sensitivity, the regulation of ICOS or ICOSL in adipose tissue during obesity remains unclear [27]. Collectively, these findings underscore the need to identify specific co-stimulatory molecules that directly drive ARTs activation in obese VAT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we identify OX40 ligand (OX40L) as a key co-stimulatory molecule that is markedly upregulated in adipocytes and ATMs during obesity. We demonstrate that adipocyte-derived, but not macrophage-derived, OX40L is essential for Th1 activation in VAT and contributes to obesity-induced insulin resistance. Importantly, treatment with an OX40L-blocking antibody alleviates VAT inflammation and improves metabolic parameters in obese mice. These findings uncover a critical role for adipocyte-T cell OX40L-OX40 signaling in driving obesity-induced immune activation and metabolic dysfunction, and highlight this pathway as a potential therapeutic target.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Obesity induces OX40L expression in adipocytes and ATMs\u003c/h2\u003e\u003cp\u003eGiven that ATMs [20] and adipocytes [18, 19] function as APCs in obese VAT, we assessed the expression of known T cell co-stimulatory molecules [17] in these cell types isolated from the VAT of lean and diet-induced obesity (DIO) mice. Among the candidates, OX40L was most strongly upregulated in both adipocytes and ATMs from obese mice (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B). Similarly, in transgenic \u003cem\u003eob/ob\u003c/em\u003e mice, a genetic model of obesity, OX40L expression was markedly elevated in both adipocytes and ATMs (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC and S1D). Notably, HFD feeding specifically enhanced OX40L gene expression in VAT, without similar upregulation in other examined tissues (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE), suggesting a VAT-specific regulation in obesity. Flow cytometry analysis revealed a significant increase in OX40L\u003csup\u003e+\u003c/sup\u003e adipocytes and ATMs in both DIO and \u003cem\u003eob/ob\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Given the technical challenges of performing flow cytometry on buoyant and fragile adipocytes, we present the complete gating strategy (Figure S2A), accompanied by bright-field images that depict adipocyte morphology of ND, HFD, and \u003cem\u003eob/ob\u003c/em\u003e mice (Figure S2B), as well as fluorescence images validating OX40L staining in adipocytes (Figure S2C). Consistently, western blotting confirmed elevated OX40L protein levels in these cells under obese conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo examine the translational relevance of these findings, we analyzed VAT samples from lean and obese human subjects. Adipocytes from obese individuals exhibited a 2.5- to 4-fold increase in OX40L mRNA levels compared to lean controls (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF). Due to limited access to human ATMs, RT-qPCR analysis of these cells was not performed. Collectively, these murine and human data demonstrate that OX40L is upregulated in adipocytes and ATMs within obese VAT, implicating the OX40L-OX40 costimulatory axis as a key mediator of obesity-driven ARTs activation and insulin resistance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.2 OX40 deficiency attenuates HFD-induced adipose inflammation and insulin resistance\u003c/h2\u003e\u003cp\u003eTo clarify the role of OX40L-OX40 costimulatory signaling in obesity-induced adipose inflammation and insulin resistance, we examined OX40-deficient (\u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻) mice under HFD conditions. After 12 weeks of HFD feeding, KO mice and their WT littermates exhibited comparable body weight, fat mass, and fasting blood glucose levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). However, \u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ mice displayed significantly improved insulin sensitivity, enhanced glucose tolerance, and lower fasting insulin levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), indicating that OX40 deficiency mitigates HFD-induced insulin resistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHistological analysis revealed a reduced abundance of crown-like structures (CLSs) in the eWAT of obese \u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ mice relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The size of adipocytes was comparable between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), suggesting that the reduction in CLS abundance was not attributable to differences in adipocyte hypertrophy-induced cell death. Flow cytometric analysis further revealed a marked reduction in ARTs in the eWAT of \u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ mice, whereas ATMs numbers remained unchanged compared to WT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, Flow cytometry gating strategy is shown in Figure S3). Given that T cells are a key component of CLS [12, 16, 28], the decline in CLS abundance is likely associated with the reduction in ARTs. Notably, HFD-fed \u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ mice exhibited a significant decrease in the proportion and number of CD8⁺ T and Th1 cells in eWAT, with no corresponding changes observed in the spleen or liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-M). This observation aligns with the upregulation of OX40L expression in the eWAT of obese mice, supporting a specific role for OX40 signaling in facilitating CD8⁺ T and Th1 cell activation within adipose tissue during obesity. Collectively, these findings demonstrate that OX40 deficiency alleviates adipose tissue inflammation and enhances insulin sensitivity under HFD-induced metabolic stress, highlighting the crucial role of OX40L-OX40 signaling in obesity-associated metabolic dysfunction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Macrophage OX40L deficiency does not affect HFD-induced insulin resistance\u003c/h2\u003e\u003cp\u003eTo investigate the role of macrophage-derived OX40L in obesity-associated metabolic dysfunction, we generated macrophage-specific OX40L knockout mice (MKO) mice by crossing \u003cem\u003eTnfsf4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice to \u003cem\u003eLysm\u003c/em\u003e-Cre mice. MKO mice were viable and born at expected Mendelian ratios. In HFD-fed MKO mice, OX40L expression was markedly reduced in ATMs and bone marrow-derived macrophages (BMDMs), but remained unchanged in adipocytes (Figure S4A). Flow cytometry further revealed that the proportion of OX40L\u003csup\u003e+\u003c/sup\u003e ATMs in MKO mice was significantly lower than in their WT littermates (MWT) (Figure S4B), confirming successful macrophage-specific deletion of OX40L.\u003c/p\u003e\u003cp\u003eFollowing 12 weeks of HFD feeding, MKO and MWT mice exhibited comparable body weight and adipose tissue mass (Figure S4C-F). There were no significant differences between the two groups in insulin sensitivity, glucose tolerance, fasting blood glucose, or fasting insulin levels (Figure S4G-J), indicating that macrophage-specific OX40L deficiency does not affect HFD-induced insulin resistance. These findings suggest that macrophage-derived OX40L is dispensable for obesity-induced metabolic dysfunction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Adipocyte OX40L deficiency attenuates HFD-induced adipose inflammation and insulin resistance\u003c/h2\u003e\u003cp\u003eTo determine the role of adipocyte-derived OX40L in obesity-induced adipose inflammation and insulin resistance, we generated adipocyte-specific OX40L knockout mice (AKO) by crossing \u003cem\u003eTnfsf4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice with \u003cem\u003eAdipoq\u003c/em\u003e-Cre mice. The AKO mice were viable, born at expected Mendelian ratios, and exhibited no discernible morphological differences from their AWT (\u003cem\u003eTnfsf4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e) littermates. As anticipated, OX40L expression was reduced in all examined adipose depots of HFD-fed AKO mice compared with AWT controls, with no changes detected in non-adipose tissues (Figure S5A). OX40L reduction was specific to adipocytes, with no changes in the SVF or BMDMs (Figure S5B). The protein levels of OX40L in adipocytes were consistently and substantially lower in AKO mice than in AWT mice (Figures S5C and S5D), while OX40L levels in SVF remained unchanged between groups (Figure S5C). These results confirm the successful generation of a mouse model with adipocyte-specific OX40L deletion.\u003c/p\u003e\u003cp\u003eAKO and AWT littermate mice were fed either ND or HFD for 12 weeks and analyzed for differences in metabolic phenotypes. Under ND conditions, AKO mice exhibited similar body weight, insulin sensitivity, and glucose tolerance to AWT controls (Figures S6A-E). Additionally, the frequency of CD4\u003csup\u003e+\u003c/sup\u003e T, CD8\u003csup\u003e+\u003c/sup\u003e T, Th1, Th2, Th17 and Treg cells remained unchanged in both eWAT and the spleen of the AKO group compared with the AWT group (Figures S6F-J).\u003c/p\u003e\u003cp\u003eUnder HFD conditions, AKO mice showed comparable adiposity and glucose tolerance to AWT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D, F-G), but exhibited lower fasting insulin levels and improved insulin sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Western blotting of eWAT, skeletal muscle and liver demonstrated enhanced post-insulin AKT phosphorylation in AKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-K), indicating improved insulin responsiveness.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConsistent with their improved insulin sensitivity, AKO mice exhibited reduced expression of pro-inflammatory genes and increased expression of anti-inflammatory genes in adipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Specifically, they showed reduced expression of MHCⅡ-related genes (\u003cem\u003eH2Ab1\u003c/em\u003e, \u003cem\u003eH2Eb1\u003c/em\u003e, and \u003cem\u003eCiita\u003c/em\u003e), T-cell co-stimulatory genes (\u003cem\u003eOX40L\u003c/em\u003e, \u003cem\u003eCd40\u003c/em\u003e and \u003cem\u003eCd80\u003c/em\u003e), and inflammatory factors (\u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eTnfα\u003c/em\u003e, and \u003cem\u003eMCP-1\u003c/em\u003e), along with increased expression of the anti-inflammatory adipokine adiponectin. Additionally, the Th1 transcription factor \u003cem\u003eTbet\u003c/em\u003e was significantly lower in the eWAT of AKO mice compared with AWT mice, whereas macrophage-related gene expression remained unchanged between the two groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The frequency of CD4⁺ and CD8⁺ T cells in eWAT and spleen was similar between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), though their numbers in eWAT trended downward in AKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Notably, Th1 cells were significantly reduced in eWAT, with no changes in spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). In contrast, Treg cell frequency increased in eWAT, though total Treg numbers were unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Th2 and Th17 cell infiltration was not affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H). Furthermore, the proportion of M1 macrophages was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), and fewer CLSs were observed in eWAT of AKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Together, these findings indicate that adipocyte-specific OX40L deletion mitigates HFD-induced adipose inflammation and insulin resistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Adipocyte OX40L-OX40 signaling promotes insulin resistance by activating Th1 cells in adipose tissue\u003c/h2\u003e\u003cp\u003eTo determine whether the reduction in adipose Th1 cells observed in HFD-fed AKO mice was responsible for their improved insulin resistance, obese AWT and AKO mice were intraperitoneally injected with inflammatory Th1 cells, and their insulin responsiveness was assessed via glucose tolerance test (GTT) and insulin tolerance test (ITT) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). No significant differences in weight gain were observed between AWT and AKO mice before or after Th1 cell transfer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Following adoptive transfer, AKO mice exhibited a marked increase in the proportion of Th1 cells within VAT, restoring their levels to those observed in AWT cotrols (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), thus confirming successful Th1 cell infiltration. Prior to Th1 cells transfer, AKO mice displayed significantly enhanced insulin sensitive compared to AWT mice, although glucose tolerance was comparable (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). However, following Th1 cells transfer insulin sensitivity and glucose tolerance were indistinguishable between AWT and AKO mice, indicating that restoration of Th1 cells reversed the metabolic improvement in AKO mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the mechanism underlying the reduction of adipose Th1 cells in AKO mice, we examined the proliferation and activation of these cells. For proliferation analysis, we used two markers, 5-ethynyl-2'-deoxyuridine (EdU) incorporation and Ki67 staining. Flow cytometry revealed that the proportions of EdU\u003csup\u003e+\u003c/sup\u003e and Ki67\u003csup\u003e+\u003c/sup\u003e Th1 cells in eWAT were significantly lower in AKO mice than in AWT mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), indicating that reduced proliferation contributes to the decline of Th1 cells in eWAT. Next, to assess adipocyte-mediated CD4\u003csup\u003e+\u003c/sup\u003e T cell activation, we cocultured CD45-depleted adipocytes from AKO and AWT mice with na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells from OT-II mice, with or without OVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). After three days, IFNγ levels in the culture supernatant were measured by ELISA, and Th1 differentiation was analyzed by flow cytometry. In the absence of OVA, both AKO and AWT adipocytes exhibited minimal Th1 cell activation. However, in the presence of OVA, AKO adipocytes showed a significantly reduced capacity to activate Th1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), which was associated with a marked reduction in IFNγ production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These findings suggest that the reduction of Th1 cells in AKO eWAT results from both impaired proliferation and diminished activation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.6 OX40L-blocking antibody ameliorates obesity-induced ARTs activation and insulin resistance\u003c/h2\u003e\u003cp\u003eTo evaluate the therapeutic potential of OX40L inhibition, we treated HFD-induced obese mice with either an OX40L blocking antibody (α-OX40L) or an isotype control antibody (IgG2b) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Body weight gain and composition did not differ significantly between α-OX40L- and IgG2b-treated mice during HFD feeding (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, S7A and S7B). Similarly, glucose and insulin tolerance were comparable between the two groups before treatment (Figures S7C and S7D). However, following treatment, α-OX40L mice exhibited significantly improved insulin sensitivity and glucose tolerance compared with IgG2b controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Fasting insulin levels were markedly reduced in α-OX40L mice, whereas fasting glucose levels remained unchanged (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn eWAT, α-OX40L treatment increased the proportion of CD4\u003csup\u003e+\u003c/sup\u003e T cells while decreasing the proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells compared with IgG2b treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG), a phenomenon not observed in the spleen. The absolute number of CD4\u003csup\u003e+\u003c/sup\u003e T cells in eWAT was significantly higher in α-OX40L-treated mice than in IgG2b-treated mice, whereas no significant difference was detected in absolute CD8\u003csup\u003e+\u003c/sup\u003e T counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Additionally, α-OX40L treatment significantly reduced the proportion of Th1 cells in eWAT compared with IgG2b treatment, although absolute Th1 cell counts remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). Moreover, after α-OX40L treatment, both the proportion and number of Treg cells in eWAT were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL). These findings indicated that OX40L blockade effectively ameliorates obesity-induced insulin resistance and ARTs activation.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eTh1 cells play a pivotal role in driving obesity-induced adipose tissue inflammation and insulin resistance [15, 29, 30]. Understanding the mechanisms underlying Th1 cell accumulation in adipose tissue during obesity is critical for developing therapies against obesity-associated metabolic disorders. The pathogenic expansion of Th1 cells in VAT during obesity can result from multiple mechanisms. For instance, leptin is a key initiator of the adipose inflammatory cascade, with its receptor expression significantly upregulated on ARTs in the obese state [31]. Hyperleptinemia, driven by adipose tissue expansion, promotes IFNγ secretion by T cells, skewing differentiation toward Th1 while suppressing Th2 responses [32, 33]. Our previous work demonstrated that leptin-stimulated IFNγ production in ARTs induces adipocyte MHCII expression, enabling adipocytes to act as APCs that further amplify Th1 activation [18]. These finding establish that obese adipocytes provide MHCII-mediated antigen presentation to promote Th1 activation. More recently, we identified that adipose stem cells (ASCs) orchestrate T cell infiltration into VAT during the early phase of HFD-induced obesity through TNFα/NF-κB-dependent secretion of CCL5, a potent chemokine that attracts T cells [34]. However, targeting these mechanisms lacks specificity for Th1 suppression and may inadvertently disrupt systemic immune homeostasis. Therefore, more selective molecular strategies that specifically inhibit Th1 cells within obese adipose tissue are urgently needed.\u003c/p\u003e\u003cp\u003eAccumulating preclinical and clinical evidence identifies co-stimulatory molecules as promising therapeutic targets due to their spatiotemporally regulated expression [35\u0026ndash;37]. Unlike broadly active immune mediators such as MHC molecules, co-stimulatory signals are selectively upregulated following T cell receptor engagement, allowing targeted immunomodulation with reduced off-target effects [38, 39]. These molecules act as critical secondary signals that determine the magnitude and polarization of immune responses, offering unique flexibility to modulate immunity as needed.\u003c/p\u003e\u003cp\u003eIn this study, we systematically examined the expression profiles of all known T cell co-stimulators in adipocytes and ATMs and found that OX40L is the most prominently upregulated in both cell types under obese conditions. Genetic ablation of OX40 signaling, either through global OX40-knockout (\u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) or adipocyte-specific OX40L deletion (OX40L-AKO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), conferred substantial protection against obesity-induced adipose inflammation and systemic insulin resistance. This phenotype was therapeutically recapitulated by treatment with an OX40L-neutralizing antibody, which alleviated obesity-associated metabolic dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results indicate that adipocytes provide essential co-stimulatory signals through the OX40L-OX40 axis to sustain Th1 effector function and drive adipose inflammation in obesity. Notably, although OX40L expression is more strongly induced in macrophages than in adipocytes under obese conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), macrophage-specific OX40L knockout (OX40L-MKO) mice failed to exhibit metabolic improvements (Figure S4). This observation suggests that increased macrophage-derived OX40L may be a secondary effect of adipocyte-initiated inflammation rather than a primary pathogenic driver. Further mechanistic studies are warranted to elucidate the spatiotemporal and cell type-specific roles of OX40L in the pathogenesis of adipose inflammation. Nevertheless, our data establish adipocyte-derived OX40L as a key regulator of obesity-induced adipose inflammation and insulin resistance, highlighting its potential as a therapeutic target for metabolic disease.\u003c/p\u003e\u003cp\u003eThe OX40-OX40L axis has emerged as a promising therapeutic target in T cell-mediated diseases [40]. Antibody-mediated blockade of OX40 or OX40L has shown efficacy in multiple autoimmune models, including experimental autoimmune encephalomyelitis (EAE) [41, 42], systemic lupus erythematosus (SLE) [43], rheumatoid arthritis (RA) [44, 45], and inflammatory bowel disease (IBD) [46\u0026ndash;48]. In addition, clinical trials targeting the OX40/OX40L costimulatory pathway have demonstrated therapeutic efficacy in atopic dermatitis [49, 50]. Notably, Amgen\u0026rsquo;s rocatinlimab (an anti-OX40 monoclonal antibody) and Sanofi\u0026rsquo;s amlitelimab (an anti-OX40L monoclonal antibody), two novel biologics disrupting T cell activation via the OX40/OX40L axis, have shown durable clinical responses in patients with atopic dermatitis [51\u0026ndash;53]. Our findings extend this paradigm to metabolic disease, demonstrating that treatment with an OX40L-blocking antibody reduces Th1 cell accumulation in VAT and improves insulin sensitivity in obese mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Importantly, systemic inhibition of the OX40 pathway has not been associated with serious side effects in clinical trials [40]. However, given the pleiotropic functions of OX40 in T cell biology, selectively targeting adipocyte-derived OX40L represents a more precise therapeutic strategy to mitigate adipose inflammation without compromising systemic immune function. To this end, we propose the development of adipocyte-targeted delivery systems. Innovative platforms with adipocyte specificity could enable adipose-restricted OX40L blockade, thereby conferring anti-inflammatory effects that are localized to adipose depots while preserving systemic immune competence. Such spatially targeted approaches may address a critical unmet need in the development of tissue-specific immunotherapies for obesity-related metabolic dysfunction.\u003c/p\u003e\u003cp\u003ePrevious studies have examined the response of global OX40-knockout (\u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻) mice to HFD challenge [54]. However, the present study is the first to provide an in-depth investigation of adipose OX40L\u0026rsquo;s role in obesity-induced adipose inflammation and insulin resistance. Bing Liu et al. reported that \u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ mice fed an HFD exhibited reduced weight gain along with improved adipose inflammation and insulin resistance compared to WT mice [54]. Since weight loss is inherently linked to improvements in adipose inflammation and insulin resistance, their study could not rule out the possibility that these metabolic benefits were secondary to reduced weight gain. In contrast, we observed no significant differences in HFD-induced weight gain between \u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻, AKO, or MKO mice and their respective WT controls. Notably, Bing Liu et al. purchased age-matched male WT and \u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ mice from The Jackon Laboratory, and mice of different genotypes were likely housed separately. In our study, by contrast, WT and \u003cem\u003eOX40\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ mice were littermates generated through heterozygous breeding and were cohoused. The discrepancy in HFD-induced weight gain between our findings and those of Bing Liu et al. may be attributed to differences in breeding and housing conditions, as well as potential variations in microbiome composition. Thus, the current study provides a comprehensive analysis using multiple independent measures to establish a direct role of adipocyte-derived OX40L in obesity-induced Th1 activation in adipose tissue and insulin resistance.\u003c/p\u003e\u003cp\u003eThe results presented here clearly demonstrate that adipocyte OX40L plays a pivotal role in promoting CD4\u003csup\u003e+\u003c/sup\u003e ARTs activation and IFNγ production in HFD-fed mice, thereby driving adipose inflammation and systemic insulin resistance. Disruption of this signaling, either through reduced adipocyte OX40L expression or administration of an OX40L-blocking antibody, ameliorates adipose inflammation and preserves systemic insulin sensitivity. These findings position adipose OX40L-OX40 signaling as a potential therapeutic target for mitigating obesity-associated metabolic dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cp\u003e\u003cb\u003eHuman samples\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study was conducted in compliance with the Declaration of Helsinki and was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University (No. LYF2022207). Written informed consent was obtained from all human donors prior to their enrollment in the study. Human VAT (omental WAT) samples were obtained from two distinct groups: obese donors (BMI\u0026thinsp;\u0026ge;\u0026thinsp;30 kg/m\u003csup\u003e2\u003c/sup\u003e) eligible for bariatric surgery, and nonobese donors (BMI\u0026thinsp;\u0026lt;\u0026thinsp;30 kg/m\u003csup\u003e2\u003c/sup\u003e) undergoing non-acute cholecystectomy surgery. Freshly collected VAT specimens (200 mg tissue blocks) were immediately processed (\u0026le;\u0026thinsp;2 h post-excision) for mechanical dissociation, followed by collagenase digestion to isolate mature adipocytes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll animal studies were performed in accordance with procedures approved by the Central South University Animal Care and Use Committee. Mice were housed in specific pathogen-free facilities under controlled environmental conditions (22\u0026deg;C, 50\u0026ndash;60% humidity) with a 12 h light/dark cycle. Animals were provided \u003cem\u003ead libitum\u003c/em\u003e access to water and either a ND (1010001, Jiangsu Xietong Pharmaceutical Bio-engineering Co.LTD) or a 60% HFD (D12492, Wuhan BIOPIKE Bioscience Co.LTD). 6-week-old C57BL/6J mice were obtained from Slac Laboratory Animal Inc, while 12-week-old C57BL/6J \u003cem\u003eob/ob\u003c/em\u003e (\u003cem\u003eLep\u003c/em\u003e\u003csup\u003e\u003cem\u003eob\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/Lep\u003c/em\u003e\u003csup\u003e\u003cem\u003eob\u003c/em\u003e\u003c/sup\u003e) mice were purchased from the Model Animal Research Center of Nanjing University. We acquired \u003cem\u003eTnfsf4\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1a\u003c/em\u003e\u003c/sup\u003e embryonic stem cells (clone number: HEPD0717_6_A11) from the European Conditional Mouse Mutagenesis Program (EUCOMM) and generated \u003cem\u003eTnfsf4\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1a\u003c/em\u003e\u003c/sup\u003e mice in the Charles River laboratories. We then crossed the \u003cem\u003eTnfsf4\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1a\u003c/em\u003e\u003c/sup\u003e mice with FLP transgenic mice (Jackson Laboratory, Stock No.009086) to breed and remove the lacZ-neo cassette, thereby obtaining \u003cem\u003eTnfsf4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice. \u003cem\u003eTnfrsf4\u003c/em\u003e KO mice (Stock No. 012838), \u003cem\u003eAdipoq\u003c/em\u003e-Cre mice (Stock No.028020), \u003cem\u003eLysm\u003c/em\u003e-Cre mice (Stock No.004781) and OT-II (Stock No.004194) mice were obtained from the Jackson Laboratory. AKO mice were generated by breeding \u003cem\u003eAdipoq\u003c/em\u003e-Cre mice with \u003cem\u003eTnfsf4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice. Similarly, MKO mice were produced by breeding \u003cem\u003eLyz2\u003c/em\u003e-Cre mice with \u003cem\u003eTnfsf4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice. 6-week-old male mice were fed with HFD for 12 weeks to establish the DIO model. These male mice were humanely euthanized via carbon dioxide (CO₂) inhalation for primary euthanasia, followed by cervical dislocation as a secondary confirmatory method, prior to tissue collection.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdipose tissue fractionation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAdipose tissues (eWAT and iWAT) were minced and digested with 1 mg/mL type II collagenase (Worthington, Cat#LS004177) and 1% bovine serum albumin at 37\u0026deg;C for 30 min with gentle shaking at 120 rpm. The mixture was filtered to remove undigested fragments and centrifuged at 300 g for 5 min. The upper layer, containing mature adipocyte fraction, was aspirated and washed with PBS containing 2 mM EDTA. The pellet (SVF) was washed with PBS and incubated with red blood cell lysis buffer on ice for 5 min. After lysis, the SVF was washed with PBS and collected by centrifugation at 500 g for 5 min. The purified adipocytes and SVF were then prepared for further analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA isolation and qRT-PCR\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAccording to the manufacturer's instructions, Trizol (Invitrogen, Cat#15596018CN) was used to extract total RNA from cells and tissues. Accurate Biology's cDNA Synthesis kit (Cat#AG11728) facilitated the reverse transcription of RNA. The SYBR Green Master Mix (Vazyme, Cat#Q511-02) was utilized for Real-Time PCR on the Applied Biosystems ViiA\u0026trade; 7 Real-Time PCR System. The calculation of normalized mRNA expression in mouse samples utilized β-actin or 36b4 as reference genes, whereas 36B4 was used for human samples. Relative mRNA expression was assessed using the ΔΔCt method. Primer sequences are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFlow cytometry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollowing standardized adipose tissue fractionation, 60 \u0026micro;L of isolated adipocytes were collected and incubated with anti-CD16/32 antibody (1:100, Biolegend, Cat#101302) for FcR blockade (7 min, room temperature, dark). To identify OX40L\u003csup\u003e+\u003c/sup\u003e adipocyte population, cells were stained with anti-CD45 (1:200, Biolegend, Cat#103132) and anti-OX40L (1:100, Biolegend, Cat#108810) in PBS containing 1% FBS and 2 mM EDTA (7 min, room temperature, dark). The reaction was quenched with 1 mL PBS, followed by 5 min incubation for phase separation at room temperature. The lower aqueous phase was carefully aspirated using a 1 mL syringe, and the adipocytes were resuspended in 100 \u0026micro;L PBS, transferred to flow cytometry tubes, and stained with 0.6 \u0026micro;L propidium iodide (PI, Biolegend, Cat#421301) for viability assessment prior to flow cytometric analysis. The instrument used for flow cytometry analysis of adipocytes was the NovoCyte Quanteon Flow Cytometer. Before loading, the adipocytes were thoroughly mixed and introduced into the machine at a low flow rate.\u003c/p\u003e\u003cp\u003eThe SVF was isolated by centrifugation at 300 g for 5 min at room temperature, separating it from floating adipocytes. For surface marker detection, cells were first incubated with FcR blocking antibody (1:100, Biolegend, Cat#101302) for 7 min at room temperature, followed by incubation with antibodies against surface markers for 30 min at 4\u0026deg;C in the dark. After washing with PBS, cell viability was assessed using PI for 5 min at room temperature. For intracellular staining, cells were incubated with Zombie NIR\u0026trade; (1:200, Biolegend, Cat#423106) for 7 min at room temperature, and then blocked with anti-CD16/32 antibody (1:100, Biolegend, Cat#101302) and stained with fluorochrome-labeled mAbs against cell-surface antigens for 30 min at 4\u0026deg;C. Cells subsequently were fixed and permeabilized using the Foxp3/transcription factor staining buffer set (eBioscience, Cat#00-5523-00) according to manufacturer's protocol, followed by intracellular staining with fluorochrome-conjugated antibodies for an additional 45 min at 4\u0026deg;C in the dark. The following mAbs were used: anti-CD45 (1:200, Biolegend, Cat#103132), anti-CD45.1 (1:200, Biolegend, Cat#110728), anti-CD3 (1:200, Biolegend, Cat#100306 and Cat#100236), anti-CD8 (1:200, Biolegend, Cat#100730), anti-CD4 (1:200, Biolegend, Cat#100428), anti-CD11b (1:200, Biolegend, Cat#101222), anti-F4/80 (1:200, Biolegend, Cat#123114), anti-CD11c (1:200, Biolegend, Cat#117308), anti-CD206 (1:200, Biolegend, Cat#141707), anti-Foxp3 (1:200, eBiosicence, Cat#17-5773-82), anti-IFNγ (1:200, Biolegend, Cat#505808; 1:200, BD Bioscience, Cat#566097), anti-IL17A (1:200, Biolegend, Cat#506938), anti-IL4 (1:200, Biolegend, Cat#144806), anti-Ki67 (1:200, Biolegend, Cat#652410).\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence Staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAdipocytes were isolated from mouse epididymal white adipose tissue (eWAT). The cells were washed 3\u0026ndash;5 times with PBS containing 2 mM EDTA to remove debris. For staining, 50 \u0026micro;L of adipocyte suspension was transferred to a fresh tube and mixed with 100 \u0026micro;L of PBS. To block Fc receptors, anti-CD16/32 antibody (1:100) was added and incubated at room temperature for 7 minutes. Subsequently, BODIPY (100 \u0026micro;g/mL, Invitrogen, D3922), Hoechst (10 \u0026micro;g/mL, Beyotime, Cat#C1022), and PE anti-mouse OX40L antibody (1:100, Biolegend, Cat#108806) were added to the mixture and incubated at room temperature for 10 minutes. After staining, the adipocytes were washed with 1 mL of PBS. The bottom aqueous phase was removed, and the cells were resuspended in 50 \u0026micro;L of PBS. The stained adipocyte suspension was transferred to a confocal dish, and images were acquired using a confocal fluorescence microscope (Zeiss).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGlucose tolerance test (GTT) and insulin tolerance test (ITT)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo conduct glucose tolerance tests, mice were fasted for 16 h before receiving an intraperitoneal injection of glucose at a dose of 1 g/kg. At 0, 15, 30, 60, 90 and 120 min, blood samples from the tail vein were measured with a Roche glucometer. For insulin tolerance tests, mice were fasted for 6 h and injected with insulin at a dose of 0.75 U/kg. Blood samples taken from tail vein were measured at 0, 15, 30, 45, 60 and 90 min using Roche glucometer.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFasting blood glucose (FPG) and insulin (FPI) measurements\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMice were fasted for 6 hours prior to sample collection. Fasting FPG levels were measured using a Roche glucometer. For insulin measurement, blood samples were collected from the retro-orbital sinus into EDTA-coated tubes. Plasma was obtained by centrifugation at 3000 rpm for 15 minutes, and insulin levels were quantified using a commercial ELISA kit (AiFang biological company, Cat#AF2579-A)) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo examine the OX40L protein expression, adipocytes, macrophages and SVFs were isolated from eWAT of mice fed either a HFD or ND. To check the insulin signalling, mice were fasted for 6 h prior to intraperitoneal injection with either insulin (4 U/kg) or PBS as control. Skeletal muscle and eWAT were collected 15 min after injection. Cells and tissues were lysed in RIPA lysis buffer (Beyotime, Cat#P0013B) supplemented with cOmplete\u0026trade; Mini Protease Inhibitor Cocktail Tables (Roche, Cat#11836153001) and cOmplete\u0026trade; EDTA-free (Sigma-Aldrich, Cat#4693132001). Protein quantification was performed using BCA assays optimized for different sample types: BCA Protein Assay Kit (Beijing Dingguo Changsheng Biotech, Cat# P0398M) for non-adipose tissues and Pierce Microplate BCA Protein Assay kit (Thermo scientific, Cat#23252) for adipocyte samples, following manufacturers' protocols. For immunoblotting, 30 \u0026micro;g of total protein were loaded onto 10% SDS-polyacrylamide gels and transferred to PVDF membranes (Merck Millipore, Burlington, MA). After blocking with 5% BSA in PBS for 1 h at room temperature, membranes were incubated overnight at 4\u0026deg;C with the following primary antibodies against: OX40L (1:1000, AiFang biological, Cat#AF07062), β-actin (1:40000, Sigma-Aldrich, Cat#A5316), p-AKT (1:1000, Cell Signaling Technology, Cat#13038S) and AKT (1:1000, Cell Signaling Technology, Cat#4691S). Following phosphotyrosine detection, membranes were stripped with buffer containing 100 mM β-mercaptoethanol, 2% SDS, and 62.5 mMTris-HCl (pH 7.6) at 50\u0026deg;C for 30 min, and reincubated with total AKT antibody at 4\u0026deg;C overnight. Membranes were washed three times (15 min each) with TBST, followed by incubation with anti-HRP secondary antibody (1:20000, Cell Signaling Technology, Cat#7074P2) in 2% BSA for 1 h at room temperature with gentle agitation. Wash the membrane as previously. Apply ClarityTM Western ECL Substrate (Bio-rad, Cat#1705061) and expose to film. Bio-Rad Image Lab and Image-Pro Plus software were used for analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDifferentiation and adoptive transfer of Th1 cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNaive CD4\u003csup\u003e+\u003c/sup\u003e T cells were purified from splenocytes of 6\u0026ndash;8 weeks old male CD45.1 mice using magnetic bead cell sorting (Miltenyi Biotec, Cat#130-104-453). Cell purity was confirmed by flow cytometry analysis of CD4\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003elow\u003c/sup\u003eCD62L\u003csup\u003ehigh\u003c/sup\u003e T cell subset. These na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells were stimulated with 2 \u0026micro;g/mL pre-coated anti-CD3ε antibody (Selleck, Cat#A2104) and 2 \u0026micro;g/mL soluble anti-CD28 antibody (Selleck, Cat#A2108) in culture medium containing 20 ng/mL IL12 (Peprotech, Cat#210\u0026thinsp;\u0026minus;\u0026thinsp;12), 10 ng/mL IL2 (Peprotech, Cat#200-02) and 10 \u0026micro;g/mL anti\u0026ndash;IL4 antibody (BD Biosciences, Cat#554432) for 3 days. The culture medium was RPMI 1640 medium (plus 50 mM β-mercaptoethanol) supplemented with 10% FBS, 1% GlutaMax, and 1% Pen/Strep (Gibco, Shanghai, China). The purity of Th1 cells after differentiation was validated by flow cytometry.\u003c/p\u003e\u003cp\u003eMale 8-week-old AKO and AWT mice were maintained on a HFD for 11 weeks prior to receiving 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e in vitro-differentiated Th1 cells via intraperitoneal injection. 3 days after the injection, ITT were performed. 5 days after the injection, GTT were performed. 7 days after the injection, the mice were sacrificed. In addition, to verify Th1 cell recruitment, cells from eWAT and spleen were analyzed by flow cytometry.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdipocyte-T cell coculture and antigen-specific T cell activation assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAdipocytes of AKO and AWT mice fed 12 weeks of HFD were incubated with medium alone or 10 \u0026micro;g/mL OVA peptide (OVA323-339, Sigma, Cat#O1641) at 37\u0026deg;C on a shaker. Na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells were purified from the splenocytes of OT-II mice using the MojoSort\u0026trade; Mouse CD4 Na\u0026iuml;ve T Cell Isolation Kit (Miltenyi Biotec, Cat#130-104-453). The pulsed adipocytes were then cocultured with isolated T cells in RPMI-1640 complete medium supplemented with 10 ng/mL IL2 (Peprotech, Cat#200-02) on a shaker. Three days later, T cells were stained for flow cytometry, and the Th1-cell cytokine IFNγ in the media was measured by ELISA (R\u0026amp;D Systems, Cat#DY485).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell proliferation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDNA synthesis was quantified using the Click-iT\u0026reg; EdU Flow Cytometry Assay Kits (Invitrogen, Cat#C10424) according to the manufacturer's protocol. Briefly, mice received intraperitoneal injections of EdU at 20 \u0026micro;g/g. Six hours post-injection, SVF were isolated from mice as described previously. Cells were first stained with appropriate surface markers, washed with PBS, then fixed and permeabilized with 50 \u0026micro;L Component D (provided in the kit) for 15 min at room temperature protected from light. Following additional washes with PBS containing 1% BSA, cells were incubated with 100 \u0026micro;L 1\u0026times;Click-iT saponin-based permeabilization/wash reagent and 125 \u0026micro;L Click-iT reaction cocktail for 30 min at room temperature in the dark. For intracellular cytokine staining, cells were subsequently treated with 1\u0026times;permeabilization/wash reagent at 4\u0026deg;C for 30 min containing anti-IFNγ antibody (1:200, Biolegend, Cat#505808).\u003c/p\u003e\u003cp\u003e\u003cb\u003eOX40L antibody blockade assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eC57BL/6J mice fed a HFD for 12 weeks were randomly assigned to receive intraperitoneal injections of either an OX40L-blocking monoclonal antibody (BioXcell, Cat#BE0033-1) or an isotype-matched control antibody IgG2b (BioXcell, Cat#BE0090), administered at a dose of 300 \u0026micro;g per mouse, twice weekly. Metabolic assessments, including insulin tolerance tests (ITT) and glucose tolerance tests (GTT), were performed at week 16 of HFD feeding. At week 18, mice were euthanized, and T cell subsets in VAT and spleen were profiled by flow cytometry.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Normal distribution of populations at the 0.05 level was calculated. Unless stated otherwise, significance was assessed by Student\u0026rsquo;s t-test for two-groups comparisons, while ANOVA for multiple-group comparisons. Non-parametric data were evaluated using the Mann-Whitney U test. For longitudinal metabolic data (ITT and GTT), two-way repeated-measures ANOVA was performed. All statistical analyses were conducted using SPSS version 25.0, with statistical significance defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The sample size (n) for each experiment is specified in the corresponding figure legend.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eJ.F.S., Q.Z., and Y.Y.J. contributed equally. J.F.S., Q.Z., and Y.Y.J. designed the study, conducted the majority of experiments, analyzed data, and drafted the manuscript. X.X.S., L.M.X., and J.P.Q. performed animal studies. W.Y.H., F.Q.W., B.L.H., and W.Q.M. conducted cellular experiments. Y.J.D. and W.L. provided human adipose tissue samples and performed related analyses. Y.M. and D.D.W. and L.X. contributed to data interpretation and manuscript revision. X.X., W.A.H. and X.C.L. provided expert knowledge and experimental guidance. T.D. conceived and supervised the project. All authors participated in result discussions and manuscript review.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eWe thank Dr. Xian Chang Li (Immunobiology \u0026amp; Transplant Science Center, Houston Methodist Hospital, Texas Medical Center) and Dr. Xiang Xiao (Immunobiology \u0026amp; Transplant Science Center, Houston Methodist Hospital, Texas Medical Center) for their scientific advice in the research. This work was supported by the National Key R\u0026amp;D Program of China (2020YFA0803604 and 2023YFC3603404), the Key Program of the National Natural Science Foundation of China (82130024, T2341005), the Fund for International Cooperation and Exchange of the National Natural Science Foundation of China (82361168636), the Natural Science Foundation of Hunan Province (2023JJ40809), and the Science and Technology Innovation Program of Hunan Province (2024RC3052).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCollaboration NCDRF. Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222\u0026nbsp;million children, adolescents, and adults. Lancet. 2024; 403: 1027-50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBluher M. Obesity: global epidemiology and pathogenesis. 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Cell Mol Life Sci. 2017; 74: 3827-40.\u003c/span\u003e\u003c/li\u003e\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":"obesity, adipose inflammation, adipocyte, T cell, OX40, OX40L","lastPublishedDoi":"10.21203/rs.3.rs-7361491/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7361491/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eT cells contribute critically to obesity-induced adipose inflammation and insulin resistance, yet the co-stimulatory signals that govern their activation in adipose tissue remain unclear. Here, we systematically profile co-stimulatory molecules in adipocytes and adipose tissue macrophages and identify OX40 ligand (OX40L) as the most robustly upregulated in obesity. OX40L is also elevated in adipocytes from obese, insulin-resistant humans. While macrophage-specific OX40L deletion has no metabolic impact, global OX40 deficiency or adipocyte-specific OX40L deletion reduces Th1 cell accumulation in visceral adipose tissue, attenuates inflammation, and improves insulin sensitivity without affecting adiposity. These benefits are reversed by Th1 cell transfer. Therapeutic blockade of OX40L with a neutralizing antibody mimics the protective effects of genetic deletion. Our findings identify adipocyte-derived OX40L as a critical mediator of obesity-associated immune dysfunction and establish it as a targetable checkpoint for tissue-specific immunotherapy in metabolic disease.\u003c/p\u003e","manuscriptTitle":"Adipocyte OX40L Promotes Adipose T Cell Activation and Insulin Resistance in Obesity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 07:47:33","doi":"10.21203/rs.3.rs-7361491/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":"c8d1f144-1262-4d02-9f11-100c90558508","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53623595,"name":"Biological sciences/Immunology/Immunotherapy/Immunosuppression"},{"id":53623596,"name":"Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte activation"}],"tags":[],"updatedAt":"2025-09-07T14:50:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-01 07:47:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7361491","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7361491","identity":"rs-7361491","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}