Chronic TCR Signaling-Driven Suppression of the FOXO-KLHL6 Axis Promotes T Cell Exhaustion

Chronic TCR Signaling-Driven Suppression of the FOXO-KLHL6 Axis Promotes T Cell Exhaustion | 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 Short Report Chronic TCR Signaling-Driven Suppression of the FOXO-KLHL6 Axis Promotes T Cell Exhaustion Xiaoli Pan, Yujia Pan, Yapeng Su, Yue Xu, Jing Du, Hongcheng Cheng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7913669/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract No abstract is needed for a brief report. Figures Figure 1 Full Text Although chronic antigenic stimulation is known to drive T cell exhaustion, the molecular mechanisms by which persistent TCR signaling promotes this process remain incompletely understood. In our previous work, we identified the E3 ubiquitin ligase KLHL6 as a master regulator that restrains T cell exhaustion by controlling TOX stability and mitochondrial function [1]. Chronic antigen exposure, however, markedly downregulates KLHL6 expression, thereby compromising its ability to maintain effective antitumor T cell responses. The mechanism underlying this transcriptional repression has yet to be elucidated. To address this critical gap, we reanalyzed a publicly available single-cell RNA sequencing (scRNA-seq) dataset of antigen-specific T cells from LCMV-Clone13 infected mice [2]. UMAP analysis revealed nine distinct T cell subsets defined by their differentiation states (Supplementary Fig. 1a). KLHL6 was highly expressed in cluster 7 (progenitor exhausted T cells), cluster 3 (memory-like T cells), and cluster 1 (effector T cells), but was markedly downregulated in cluster 0 (terminally exhausted T cells), consistent with Tcf7 expression (Supplementary Fig. 1a), supporting the notion that chronic TCR signaling transcriptionally regulates KLHL6 expression. Indeed, we observed dynamic changes in Klhl6 mRNA following T cell activation and re-stimulation in vitro (Supplementary Fig. 1b), and repeated TCR stimulation substantially reduced both KLHL6 mRNA and protein levels in T cells (Fig. 1a, b and Supplementary Fig. 1c, d). To further explore the mechanism by which chronic TCR stimulation transcriptionally suppresses KLHL6 expression, we performed computational analysis using the JASPAR database, and identified FOXO1 as a predicted transcription factor binding to the Klhl6 promoter (Fig. 1c). To experimentally validate this, we performed a chromatin immunoprecipitation assay, which revealed high FOXO1 occupancy in the conserved region of the Klhl6 promoter in both mouse and human T cells (Fig. 1c). Notably, this binding was markedly diminished following TCR stimulation, indicating that FOXO1 directly regulates Klhl6 transcription in CD8⁺ T cells in response to TCR engagement. Consistent with previous reports [3, 4, 5, 6], we found activated T cells displayed increased AKT phosphorylation , reduced FOXO1 expression , and enhanced FOXO1 phosphorylation (Fig. 1d), preventing its nuclear translocation and attenuating its transcriptional activity. Gene set enrichment analysis (GSEA) further revealed a marked activation of the PI3K-AKT pathway accompanied by a decline in FOXO1-dependent transcriptional activity upon either acute or chronic TCR stimulation (Supplementary Fig. 1e, f), consistent with the observed downregulation of KLHL6 expression. (Fig. 1a, b and Supplementary Fig. 1b, d). To further confirm FOXO1's role in KLHL6 transcriptional regulation, we overexpressed wild-type FOXO1 (FOXO1 WT ) and a phosphorylation-resistant mutant of FOXO1 (FOXO1 AAA ) in OT-I T cells. Compared with FOXO1 WT , the constitutively active mutant FOXO1 AAA markedly enhanced KLHL6 expression, particularly following TCR stimulation (Fig. 1e and Supplementary Fig. 1g). In contrast, FOXO1 knockdown led to a pronounced reduction in KLHL6 expression at both the mRNA and protein levels (Fig. 1f and Supplementary Fig. 1h). To further validate the role of PI3K-AKT-FOXO1 axis in regulating KLHL6, we treated activated T cells with the highly specific AKT inhibitor, AKTi-1/2, which partially rescued the expression of KLHL6 following TCR engagement (Supplementary Fig. 1i). This result is aligned well with our observation that disrupted phosphorylation of FOXO1 in T cells reinforced KLHL6 expression (Fig. 1e, f). Together, these findings strongly support that PI3K-AKT-mediated suppression of FOXO1 transcriptional activity leads to the downregulation of KLHL6 following TCR stimulation. FOXO1 has been shown to promote memory formation and restrain exhaustion in human CAR T cells. Loss of FOXO1 downregulates memory-associated genes, induces an exhaustion-like phenotype, and impairs the antitumor efficacy of CAR T cells [7, 8, 9]. Given that KLHL6 is a direct transcriptional target of FOXO1, we next investigated whether KLHL6 overexpression could rescue the impaired antitumor function of FOXO1-deficient T cells using an in vivo T cell transfer model. Consistent with previous reports, FOXO1 knockdown alone ( shFoxo1 + vec ) markedly compromised T cell-mediated tumor control, as evidenced by reduced tumor control, increased terminal exhaustion (PD-1⁺TIM-3⁺), decreased cytokine production (IFN-γ⁺TNF-α⁺), and reduced Tcm (central memory T cell; CD62L⁺CD44⁺) formation in draining lymph nodes (dLNs) compared to control ( shCtrl + vec ) group (Fig. 1g-j and Supplementary Fig. 1j, k). KLHL6 overexpression alone markedly inhibits T cell exhaustion and enhances anti-tumor efficacy, and KLHL6 overexpression significantly rescued the numerical and functional defects of FOXO1-knockdown T cells, reduced T cell exhaustion, and restored effector function (Fig. 1g-j and Supplementary Fig. 1j, k). These results indicate that FOXO1-mediated T cell anti-tumor immunity is at least partly dependent on KLHL6. Collectively, our findings reveal that chronic TCR stimulation suppresses KLHL6 expression at the transcriptional level through the PI3K-AKT-FOXO1 signaling axis , thereby driving T cell exhaustion and compromising antitumor immunity. Persistent TCR antigen stimulation is a key driver of CD8 + T cell exhaustion in both tumors and chronic infections, accompanied by the remodeling of transcriptional, epigenetic and metabolism programs [10, 11]. During this process, multiple transcription factors such as NFAT, TOX, T-BET, NR4A, and EOMES play central roles in the establishment of T cell exhaustion. For instance, chronic antigen exposure leads to sustained TCR activation and continuous engagement of the Ca²⁺/NFAT signaling pathway . This, in turn, disrupts the NFAT: AP-1 balance, resulting in partnerless NFAT (NFAT acting without AP-1), which transcriptionally upregulates exhaustion-associated genes such as TOX , NR4A1 , and PDCD1 , thereby accelerating the onset and progression of T cell exhaustion [12, 13, 14, 15] . However, the central hub linking chronic TCR stimulation to T cell exhaustion remains unclear. In this and our recent work, we demonstrated that persistent TCR signaling suppresses FOXO1 activity, leading to transcriptional downregulation of KLHL6, an E3 ubiquitin ligase that restrains T cell exhaustion by regulating TOX stability and PGAM5-mediated mitochondrial function [1] . These findings identify KLHL6 as a master regulator of T cell exhaustion, targeting both TOX and PGAM5, and extend our mechanistic understanding of how sustained TCR engagement drives exhaustion (Fig. 1k). Given the multi-protein targeting nature of KLHL6, it remains to be determined whether KLHL6 directly regulates other exhaustion-associated proteins. FOXO1 is known to promote memory T cell differentiation by transactivating Tcf7. Whether chronic TCR signaling may also modulate TCF-1 levels through direct or indirect regulation of the ubiquitination machinery warrants further investigation. Collectively, our findings uncover a previously unrecognized FOXO–KLHL6–TOX/PGAM5 regulatory axis that functions during chronic antigen stimulation in both cancer and infection. This work provides important mechanistic insight into the molecular basis of T cell exhaustion and helps to answer a long-standing question of how chronic TCR signaling drives T cell exhaustion. Considering the multifaceted role of KLHL6 in controlling T cell exhaustion, the development of small-molecule agonists that enhance KLHL6 activity could represent a promising therapeutic strategy to reinvigorate T cell function and improve the efficacy of T cell –based cancer immunotherapy. Declarations Acknowledgements We thank the Suzhou Municipal Key Laboratory (SZS2023005) and the NCTIB Fund for R&D Platform for Cell and Gene Therapy. The schematic diagrams in this study were created using BioRender (https://biorender.com). Authors’ contributions H.H.C. and G.D.L. conceived the project and designed experiments; X.L.P., Y.X., J.D., and Y.J.P. performed most experiments and interpreted data; Y.P.S., and H.H.C. performed all bioinformatics analyses; H.H.C., G.D.L., X.L.P., and Y.J.P. wrote and edited the manuscript; G.D.L. and H.H.C supervised the project and provided overall direction. All authors reviewed the manuscript. Funding This work was supported by the Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0520600), the National Natural Science Foundation of China (32525028 and 32270994 to G. L., 32571074 and 32300764 to H.C.), the Basic Research Program of Jiangsu (BK20250003 to G. L., BK20230280 to H.C.), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2021-RC310-014 and 2024-JKCS-15 to G. L.), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-047, 2021-I2M-1-061, 2022-I2M-2-004, 2023-I2M-2-010, 2023-I2M-QJ-019, and 2024-I2M-ZD-009 to G.L., 2025-I2M-TS-15 to H.C.). Data availability The datasets supporting the findings of this study are included in the manuscript and Supplementary Information. Source data, full uncropped images of gels and blots are available in the supplementary files. Ethics approval and consent to participate This study involving all the mice were kept in a specific-pathogen-free facility, and all animal experiments were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of Suzhou Institute of Systems Medicine (ISM-IACUC-0151-R). All tumor burdens did not exceed the permission of the Institutional Animal Care and Use Committee of Suzhou Institute of Systems Medicine. Age-and sex-matched mice were assigned randomly to experimental and control groups. Human PBMCs from healthy donors were purchased from Sailybio (Shanghai, China). Consent for publication Not applicable. Competing of interests The corresponding author Guideng Li is a member of the Editorial Board of the journal Immunity & Inflammation. However, he was not involved in the peer-review or decision-making process for this manuscript. The authors declare no other competing interests. References Cheng H, Su Y, Pan X, et al. The ubiquitin ligase KLHL6 drives resistance to CD8+ T cell dysfunction. Nature. (in press) Miller BC, Sen DR, Al Abosy R, et al. Subsets of exhausted CD8 + T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol. 2019;20(3):326-336. Hasegawa J, Wada Y, Sageshima M, et al. Structure and pulmonary toxicity relationship on O,O-dimethyl S-alkyl phosphorothioate esters. Pharmacol Toxicol. 1990;66(5):367-372. Zhang L, Tschumi BO, Lopez-Mejia IC, et al. Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner. Cell Rep. 2016;14(5):1206-1217. Rao RR, Li Q, Gubbels Bupp MR, Shrikant PA. Transcription factor Foxo1 represses T-bet-mediated effector functions and promotes memory CD8(+) T cell differentiation. Immunity. 2012;36(3):374-387. Luo CT, Li MO. Foxo transcription factors in T cell biology and tumor immunity. Semin Cancer Biol. 2018;50:13-20. Doan AE, Mueller KP, Chen AY, et al. FOXO1 is a master regulator of memory programming in CAR T cells. Nature. 2024;629(8010):211-218. Chan JD, Scheffler CM, Munoz I, et al. FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy. Nature. 2024;629(8010):201-210. Marchal I. FOXO1 enhances CAR T cell fitness and function. Nat Biotechnol. 2024;42(5):699. Cheng H, Qiu Y, Xu Y, et al. Extracellular acidosis restricts one-carbon metabolism and preserves T cell stemness. Nat Metab. 2023;5(2):314-330. Qiu Y, Su Y, Xie E, et al. Mannose metabolism reshapes T cell differentiation to enhance anti-tumor immunity. Cancer Cell. 2025;43(1):103-121.e8. Martinez GJ, Pereira RM, Äijö T, et al. The transcription factor NFAT promotes exhaustion of activated CD8⁺ T cells. Immunity. 2015;42(2):265-278. Tillé L, Cropp D, Charmoy M, et al. Activation of the transcription factor NFAT5 in the tumor microenvironment enforces CD8+ T cell exhaustion. Nat Immunol. 2023;24(10):1645-1653. Seo H, Chen J, González-Avalos E, et al. TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8 + T cell exhaustion. Proc Natl Acad Sci U S A. 2019;116(25):12410-12415. Cheng H, Ma K, Zhang L, Li G. The tumor microenvironment shapes the molecular characteristics of exhausted CD8 + T cells. Cancer Lett. 2021;506:55-66. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial2.docx Supplementarymaterial1.pdf SupplementaryFig.1.jpg Supplementary Fig. 1 FOXO1 transcriptionally regulates KLHL6 expression in CD8 + T cells, related to Figure 1. (a) UMAP plot of a published scRNA-seq dataset of gp33 tetramer + CD8 + T cells from chronic LCMV-infected mice at day 28 post-infection. Each dot corresponds to an individual cell. The cells are categorized into 9 clusters. Cluster signatures are defined as follows: 0, exhaustion; 1, effector; 2, intermediate; 3, memory-like; 4, post-progenitor; 5, pre-exhaustion; 6, pre-proliferation; 7, progenitor; 8, proliferation. UMAP plot shows gene expression as indicated. (b) Relative Klhl6 mRNA levels in secondary stimulation of OT-I T cells in vitro. OT-I T cells were isolated from the spleen of OT-I mice, activated with anti-CD3/CD28, and cultured in media containing mIL-2 for 4 days. Subsequently, the cells were re-activated on day 4 and cultured until day 7 for analysis. (c) Schematic representation of the chronic stimulation of OT-I T cells in vitro . (d) Quantitative mRNA expression of Klhl6 in chronically stimulated OT-I T cells in vitro . Cell treated as indicated in (c), and the expression of Klhl6 was analyzed by qPCR at day 10 (n = 3 independent samples). (e) GSEA plots of gene sets associated with the PI3K-AKT pathway and FOXO1 targets in naive versus activated (day 2) OT-I T cells. NES, normalized enrichment score. (f) GSEA plots of gene sets associated with the PI3K-AKT pathway and FOXO1 targets in PD-1 - TIM-3 - versus PD-1 + TIM-3 + CD8 + TILs isolated from tumors in B16-OVA bearing mice after adoptive transfer at day 14. NES, normalized enrichment score. (g) Relative Klhl6 mRNA levels in OT-I T cells under the conditions indicated in Fig.1e (n = 3 independent samples). (h) Relative Klhl6 mRNA levels in Jurkat cells under the conditions indicated in Fig.1f (n = 3 independent samples). (i) Assessment of the protein levels of KLHL6 in Resting (without anti-CD3) and activated (with anti-CD3) OT-I T cells treated with or without the AKT inhibitor (AKTi-1/2, 1 μM). The activated OT-I T cells were cultured for 4 days in vitro and then re-stimulated by anti-CD3 antibody for 2 days with or without the AKT inhibitor. n = 3 independent samples. (j) The levels of PD-1 and TIM-3 in transferred CD8 + TILs at day 14 after ACT (n = 5 mice), related to Fig. 1g. (k) The percentage of CD44 + CD62L + subsets of transferred CD8 + T cells from dLN and spleen in B16-OVA tumor-bearing mice at day 14 after ACT (n=5 mice), related to Fig. 1g. Data are presented as mean ± SEM. Statistical analyses were determined by unpaired two­tailed Student’s t ­test (d) or two­way ANOVA with Tukey’s multiple-comparisons test (g,h,i-k). * p <0.05, ** p <0.01, *** p <0.001, and **** p <0.0001; ns, no significance. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 31 Oct, 2025 Reviews received at journal 31 Oct, 2025 Reviews received at journal 30 Oct, 2025 Reviews received at journal 28 Oct, 2025 Reviewers agreed at journal 28 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers invited by journal 27 Oct, 2025 Editor assigned by journal 27 Oct, 2025 Submission checks completed at journal 26 Oct, 2025 First submitted to journal 21 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":1026775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChronic TCR stimulation suppresses KLHL6 expression via FOXO1 inhibition to promote T cell exhaustion and impair anti-tumor immunity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eb\u003c/strong\u003e) Immunoblots and quantification of KLHL6 expression in OT-I T cells (\u003cstrong\u003ea\u003c/strong\u003e) or human T cells (\u003cstrong\u003eb\u003c/strong\u003e) after acute or chronic TCR stimulation (n = 3 independent samples). (\u003cstrong\u003ec\u003c/strong\u003e) Binding sites of FOXO1 to the promoter of KLHL6 analyzed using JASPAR (left). FOXO1 occupancy on the conserved region of \u003cem\u003eKlhl6\u003c/em\u003e promoter in both mouse and human T cells was analyzed (right). The activated mouse and human T cells were cultured for 4 days and then re-stimulated with anti-CD3 (2 μg/mL) antibody for 2 days. The cells with (anti-CD3) or without (Resting) re-stimulation were collected and analyzed using CUT\u0026amp;RUN-qPCR. IgG: negative control. n = 3 independent samples. (\u003cstrong\u003ed\u003c/strong\u003e) Assessment of AKT-FOXO axis by western blot in resting or stimulated OT-I T cells. The activated OT-I T cells were cultured for 4 days \u003cem\u003ein vitro \u003c/em\u003eand then re-stimulated by anti-CD3 (2 μg/mL) antibody for 2 days. The cells stimulated (with anti-CD3) or not re-stimulated (Resting) were collected and analyzed. n = 3 independent samples. (\u003cstrong\u003ee\u003c/strong\u003e) Immunoblot analysis and quantification of KLHL6 in OT-I T cells transduced with FOXO1\u003csup\u003eWT\u003c/sup\u003e, FOXO1\u003csup\u003eAAA\u003c/sup\u003e, or empty plasmid (Ctrl). The transduced cells were cultured for 4 days \u003cem\u003ein vitro\u003c/em\u003e, and then re-stimulated with anti-CD3 (2 μg/mL) for 48 h for analysis. n = 3 independent samples. (\u003cstrong\u003ef\u003c/strong\u003e) Immunoblots and quantification of KLHL6 expression in \u003cem\u003eshFOXO1\u003c/em\u003e or \u003cem\u003eshCtrl \u003c/em\u003eJurkat cells after with anti-CD3 (2 μg/mL) for 24 h (n = 3 independent samples). (\u003cstrong\u003eg-j\u003c/strong\u003e) CD45.1\u003csup\u003e+\u003c/sup\u003e OT-I CD8\u003csup\u003e+\u003c/sup\u003e T cells were transduced with\u003cem\u003e \u003c/em\u003eindicated plasmids, and adoptively transferred into CD45.2⁺ mice bearing B16-OVA tumor that had been implanted 9 days earlier. Tumor weight of tumor-bearing mice were measured on day 14 after ACT (\u003cstrong\u003eg\u003c/strong\u003e, n = 9 mice); TOX expression in transferred CD8⁺ TILs at day 14 after ACT (\u003cstrong\u003eh\u003c/strong\u003e, n = 5 mice). Representative plots (left) and percentages (right) of TIM-3\u003csup\u003e+\u003c/sup\u003ePD-1\u003csup\u003e+\u003c/sup\u003e populations in transferred CD8\u003csup\u003e+\u003c/sup\u003e TILs at day 14 after ACT (\u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003en = 5 mice); TNF-α and IFN-γ production in transferred CD8⁺ TILs after 4.5 h PMA+BFA stimulation at day 14 after ACT (\u003cstrong\u003ej\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003en = 5 mice). (\u003cstrong\u003ek\u003c/strong\u003e) Schematic diagram illustrates chronic TCR engagement promoting CD8⁺ T cell exhaustion. Data are presented as mean ± SEM. Statistical analyses were determined by unpaired two­tailed Student’s \u003cem\u003et\u003c/em\u003e­test (\u003cstrong\u003ea-d\u003c/strong\u003e) or two­way ANOVA with Tukey’s multiple-comparisons test (\u003cstrong\u003ee-j\u003c/strong\u003e). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, and ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001; ns, no significance.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7913669/v1/201d5025f419d3a931a70f99.jpg"},{"id":95531524,"identity":"d2f1dca0-75f9-4b6b-a05e-3207019d0caa","added_by":"auto","created_at":"2025-11-10 10:23:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1554722,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7913669/v1/3457186a-95a3-4999-ab32-a4b9e531f035.pdf"},{"id":95391486,"identity":"c951c526-2290-4885-a467-a4d34609257d","added_by":"auto","created_at":"2025-11-07 14:07:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28185,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7913669/v1/7d7123a17487fe0acfd893ce.docx"},{"id":95525648,"identity":"38521952-a84c-432a-b3ce-e1112755ee0a","added_by":"auto","created_at":"2025-11-10 10:05:31","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":629151,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7913669/v1/8f606a4a75c561a412fcfcf9.pdf"},{"id":95391489,"identity":"121802c6-40b8-4458-a8f3-e7f257954627","added_by":"auto","created_at":"2025-11-07 14:07:34","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":867484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1 FOXO1 transcriptionally regulates KLHL6 expression in CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells, related to Figure 1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) UMAP plot of a published scRNA-seq dataset of gp33 tetramer\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells from chronic LCMV-infected mice at day 28 post-infection. Each dot corresponds to an individual cell. The cells are categorized into 9 clusters. Cluster signatures are defined as follows: 0, exhaustion; 1, effector; 2, intermediate; 3, memory-like; 4, post-progenitor; 5, pre-exhaustion; 6, pre-proliferation; 7, progenitor; 8, proliferation. UMAP plot shows gene expression as indicated. (\u003cstrong\u003eb\u003c/strong\u003e) Relative\u003cem\u003e Klhl6\u003c/em\u003e mRNA levels in secondary stimulation of OT-I T cells \u003cem\u003ein vitro.\u003c/em\u003e OT-I T cells were isolated from the spleen of OT-I mice, activated with anti-CD3/CD28, and cultured in media containing mIL-2 for 4 days. Subsequently, the cells were re-activated on day 4 and cultured until day 7 for analysis. (\u003cstrong\u003ec\u003c/strong\u003e) Schematic representation of the chronic stimulation of OT-I T cells \u003cem\u003ein vitro\u003c/em\u003e. (\u003cstrong\u003ed\u003c/strong\u003e) Quantitative mRNA expression of \u003cem\u003eKlhl6\u003c/em\u003e in chronically stimulated OT-I T cells \u003cem\u003ein vitro\u003c/em\u003e. Cell treated as indicated in (\u003cstrong\u003ec\u003c/strong\u003e), and the expression of \u003cem\u003eKlhl6 \u003c/em\u003ewas analyzed by qPCR at day 10 (n = 3 independent samples). (\u003cstrong\u003ee\u003c/strong\u003e) GSEA plots of gene sets associated with the PI3K-AKT pathway and FOXO1 targets in naive versus activated (day 2) OT-I T cells. NES, normalized enrichment score. (\u003cstrong\u003ef\u003c/strong\u003e) GSEA plots of gene sets associated with the PI3K-AKT pathway and FOXO1 targets in PD-1\u003csup\u003e-\u003c/sup\u003eTIM-3\u003csup\u003e-\u003c/sup\u003e versus PD-1\u003csup\u003e+\u003c/sup\u003eTIM-3\u003csup\u003e+ \u003c/sup\u003eCD8\u003csup\u003e+ \u003c/sup\u003eTILs isolated from tumors in B16-OVA bearing mice after adoptive transfer at day 14. NES, normalized enrichment score. (\u003cstrong\u003eg\u003c/strong\u003e) Relative \u003cem\u003eKlhl6\u003c/em\u003e mRNA levels in OT-I T cells under the conditions indicated in Fig.1e (n = 3 independent samples). (\u003cstrong\u003eh\u003c/strong\u003e) Relative \u003cem\u003eKlhl6\u003c/em\u003e mRNA levels in Jurkat cells under the conditions indicated in Fig.1f (n = 3 independent samples). (\u003cstrong\u003ei\u003c/strong\u003e) Assessment of the protein levels of KLHL6 in Resting (without anti-CD3) and activated (with anti-CD3) OT-I T cells treated with or without the AKT inhibitor (AKTi-1/2, 1 μM). The activated OT-I T cells were cultured for 4 days \u003cem\u003ein vitro \u003c/em\u003eand then re-stimulated by anti-CD3 antibody for 2 days with or without the AKT inhibitor. n = 3 independent samples. (\u003cstrong\u003ej\u003c/strong\u003e) The levels of PD-1 and TIM-3 in transferred CD8\u003csup\u003e+\u003c/sup\u003e TILs at day 14 after ACT (n = 5 mice), related to Fig. 1g. (\u003cstrong\u003ek\u003c/strong\u003e) The percentage of CD44\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e subsets of transferred CD8\u003csup\u003e+\u003c/sup\u003e T cells from dLN and spleen in B16-OVA tumor-bearing mice at day 14 after ACT (n=5 mice), related to Fig. 1g. Data are presented as mean ± SEM. Statistical analyses were determined by unpaired two­tailed Student’s \u003cem\u003et\u003c/em\u003e­test (\u003cstrong\u003ed\u003c/strong\u003e) or two­way ANOVA with Tukey’s multiple-comparisons test (\u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003ei-k\u003c/strong\u003e). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, and ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001; ns, no significance.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"SupplementaryFig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7913669/v1/42154466c9a48321ab69be91.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chronic TCR Signaling-Driven Suppression of the FOXO-KLHL6 Axis Promotes T Cell Exhaustion","fulltext":[{"header":"Full Text","content":"\u003cp\u003eAlthough chronic antigenic stimulation is known to drive T cell exhaustion, the molecular mechanisms by which persistent TCR signaling promotes this process remain incompletely understood.\u0026nbsp;In our previous work, we identified the E3 ubiquitin ligase \u003cstrong\u003eKLHL6\u003c/strong\u003e as a \u003cstrong\u003emaster regulator\u003c/strong\u003e that restrains T cell exhaustion by controlling \u003cstrong\u003eTOX stability\u003c/strong\u003e and \u003cstrong\u003emitochondrial function\u0026nbsp;\u003c/strong\u003e[1]. Chronic antigen exposure, however, markedly downregulates \u003cstrong\u003eKLHL6\u003c/strong\u003e expression, thereby compromising its ability to maintain effective antitumor T cell responses. The mechanism underlying this transcriptional repression has yet to be elucidated.\u003c/p\u003e\n\u003cp\u003eTo address this critical gap, we reanalyzed a publicly available single-cell RNA sequencing (scRNA-seq) dataset of antigen-specific T cells from LCMV-Clone13 infected mice [2]. UMAP analysis revealed nine distinct T cell subsets defined by their differentiation states (Supplementary Fig. 1a). KLHL6 was highly expressed in cluster 7 (progenitor exhausted T cells), cluster 3 (memory-like T cells), and cluster 1 (effector T cells), but was markedly downregulated in cluster 0 (terminally exhausted T cells), consistent with \u003cem\u003eTcf7\u003c/em\u003e expression (Supplementary Fig. 1a), supporting the notion that chronic TCR signaling transcriptionally regulates KLHL6 expression. Indeed, we observed dynamic changes in \u003cem\u003eKlhl6\u003c/em\u003e mRNA following T cell activation and re-stimulation \u003cem\u003ein vitro\u003c/em\u003e (Supplementary Fig. 1b), and repeated TCR stimulation substantially reduced both KLHL6 mRNA and protein levels in T cells (Fig. 1a, b and Supplementary Fig. 1c, d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further explore the mechanism by which chronic TCR stimulation transcriptionally suppresses \u003cstrong\u003eKLHL6\u003c/strong\u003e expression,\u0026nbsp;we performed computational analysis using the JASPAR database, and\u0026nbsp;identified \u003cstrong\u003eFOXO1\u003c/strong\u003e as a predicted transcription factor binding to the \u003cem\u003eKlhl6\u003c/em\u003e promoter\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Fig. 1c).\u0026nbsp;To experimentally validate this, we performed a chromatin immunoprecipitation assay, which revealed high FOXO1 occupancy in the conserved region of the \u003cem\u003eKlhl6\u003c/em\u003e promoter in both mouse and human T cells (Fig. 1c).\u0026nbsp;Notably,\u0026nbsp;this binding\u0026nbsp;was markedly diminished following TCR stimulation, indicating that \u003cstrong\u003eFOXO1 directly regulates\u0026nbsp;\u003c/strong\u003e\u003cem\u003eKlhl6\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;transcription\u003c/strong\u003e in CD8⁺ T cells in response to TCR engagement.\u003c/p\u003e\n\u003cp\u003eConsistent with previous reports [3, 4, 5, 6], we found activated T cells\u0026nbsp;displayed increased \u003cstrong\u003eAKT phosphorylation\u003c/strong\u003e, reduced \u003cstrong\u003eFOXO1 expression\u003c/strong\u003e, and enhanced \u003cstrong\u003eFOXO1 phosphorylation\u003c/strong\u003e (Fig. 1d), preventing its nuclear translocation and\u0026nbsp;attenuating\u0026nbsp;its transcriptional activity. Gene set enrichment analysis (GSEA) further revealed\u0026nbsp;a marked activation of the \u003cstrong\u003ePI3K-AKT pathway\u003c/strong\u003e accompanied by a decline in \u003cstrong\u003eFOXO1-dependent transcriptional activity\u003c/strong\u003e upon either acute or chronic TCR stimulation\u0026nbsp;(Supplementary Fig. 1e, f),\u0026nbsp;consistent with the observed downregulation of \u003cstrong\u003eKLHL6\u003c/strong\u003e expression.\u0026nbsp;(Fig. 1a, b and Supplementary Fig. 1b, d). To further confirm FOXO1\u0026apos;s role in KLHL6 transcriptional regulation, we overexpressed wild-type FOXO1 (FOXO1\u003csup\u003eWT\u003c/sup\u003e) and a phosphorylation-resistant mutant of FOXO1 (FOXO1\u003csup\u003eAAA\u003c/sup\u003e) in OT-I T cells. Compared with FOXO1\u003csup\u003eWT\u003c/sup\u003e, the constitutively active mutant FOXO1\u003csup\u003eAAA\u003c/sup\u003e markedly enhanced KLHL6 expression, particularly following TCR stimulation (Fig. 1e and Supplementary Fig. 1g). In contrast, FOXO1 knockdown\u0026nbsp;led to a pronounced reduction in \u003cstrong\u003eKLHL6\u003c/strong\u003e expression at both the mRNA and protein levels\u0026nbsp;(Fig. 1f and Supplementary Fig. 1h).\u0026nbsp;To further validate the role of PI3K-AKT-FOXO1 axis in regulating KLHL6, we treated activated T cells with the highly\u0026nbsp;specific\u0026nbsp;AKT inhibitor, AKTi-1/2, which\u0026nbsp;partially rescued the expression of KLHL6 following TCR engagement\u0026nbsp;(Supplementary Fig. 1i). This result is aligned well with our observation that\u0026nbsp;disrupted phosphorylation of FOXO1 in T cells reinforced KLHL6 expression\u0026nbsp;(Fig. 1e, f). Together, these findings strongly support that PI3K-AKT-mediated suppression of FOXO1 transcriptional activity leads to the downregulation of KLHL6 following TCR stimulation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFOXO1 has been shown to promote memory formation and restrain exhaustion in human CAR T cells. Loss of FOXO1 downregulates memory-associated genes, induces an exhaustion-like phenotype, and impairs the antitumor efficacy of CAR T cells [7, 8, 9]. Given that KLHL6 is a direct transcriptional target of FOXO1, we next investigated whether KLHL6 overexpression could rescue the impaired antitumor function of FOXO1-deficient T cells using an \u003cstrong\u003e\u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e T cell transfer model. Consistent with previous reports,\u0026nbsp;FOXO1 knockdown alone (\u003cem\u003eshFoxo1\u003c/em\u003e+\u003cem\u003evec\u003c/em\u003e)\u0026nbsp;markedly compromised T cell-mediated tumor control, as evidenced by reduced tumor control, increased terminal exhaustion (PD-1⁺TIM-3⁺), decreased cytokine production (IFN-\u0026gamma;⁺TNF-\u0026alpha;⁺), and reduced Tcm (central memory T cell; CD62L⁺CD44⁺) formation in draining lymph nodes (dLNs) compared to control (\u003cem\u003eshCtrl\u003c/em\u003e+\u003cem\u003evec\u003c/em\u003e) group (Fig. 1g-j and Supplementary Fig. 1j, k). KLHL6 overexpression alone markedly inhibits T cell exhaustion and enhances anti-tumor efficacy, and KLHL6 overexpression significantly rescued the numerical and functional defects of FOXO1-knockdown T cells, reduced T cell exhaustion, and restored effector function (Fig. 1g-j and Supplementary Fig. 1j, k). These results indicate that FOXO1-mediated T cell anti-tumor immunity is at least partly dependent on KLHL6.\u0026nbsp;Collectively, our findings reveal that chronic TCR stimulation suppresses \u003cstrong\u003eKLHL6\u003c/strong\u003e expression at the transcriptional level through the \u003cstrong\u003ePI3K-AKT-FOXO1 signaling axis\u003c/strong\u003e, thereby driving T cell exhaustion and compromising antitumor immunity.\u003c/p\u003e\n\u003cp\u003ePersistent TCR antigen stimulation is a key driver of CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cell exhaustion in both tumors and chronic infections, accompanied by the remodeling of transcriptional, epigenetic and metabolism programs\u0026nbsp;[10, 11]. During this process, multiple transcription factors such as NFAT, TOX, T-BET, NR4A, and EOMES play central roles in the establishment of T cell exhaustion. For instance, chronic antigen exposure leads to sustained TCR activation and continuous engagement of the \u003cstrong\u003eCa\u0026sup2;⁺/NFAT signaling pathway\u003c/strong\u003e. This, in turn, disrupts the NFAT: AP-1 balance, resulting in \u003cstrong\u003epartnerless NFAT\u003c/strong\u003e (NFAT acting without AP-1), which transcriptionally upregulates exhaustion-associated genes such as \u003cstrong\u003e\u003cem\u003eTOX\u003c/em\u003e, \u003cem\u003eNR4A1\u003c/em\u003e,\u003c/strong\u003e and \u003cstrong\u003e\u003cem\u003ePDCD1\u003c/em\u003e\u003c/strong\u003e, thereby accelerating the onset and progression of T cell exhaustion\u0026nbsp;[12, 13, 14, 15]\u003cem\u003e. However, the central hub linking chronic TCR stimulation to T cell exhaustion remains unclear. In this and our recent work, we demonstrated that persistent TCR signaling suppresses FOXO1 activity, leading to transcriptional downregulation of KLHL6, an E3 ubiquitin ligase that restrains T cell exhaustion by regulating TOX stability and PGAM5-mediated mitochondrial function\u0026nbsp;\u003c/em\u003e[1]\u003cem\u003e. These findings identify KLHL6 as a master regulator of T cell exhaustion, targeting both TOX and PGAM5, and extend our mechanistic understanding of how sustained TCR engagement drives exhaustion (Fig. 1k). Given the multi-protein targeting nature of KLHL6, it remains to be determined whether KLHL6 directly regulates other exhaustion-associated proteins. FOXO1 is known to promote memory T cell differentiation by transactivating Tcf7. Whether chronic TCR signaling may also modulate TCF-1 levels through direct or indirect regulation of the ubiquitination machinery warrants further investigation.\u003c/em\u003e \u003cem\u003eCollectively, our findings uncover a previously unrecognized FOXO\u0026ndash;KLHL6\u0026ndash;TOX/PGAM5 regulatory axis that functions during chronic antigen stimulation in both cancer and infection. This work provides important mechanistic insight into the molecular basis of T cell exhaustion and helps to answer a long-standing question of how chronic TCR signaling drives T cell exhaustion.\u003c/em\u003e\u003cem\u003e\u0026nbsp;Considering the multifaceted role of KLHL6 in controlling T cell exhaustion, the development of small-molecule agonists that enhance KLHL6 activity could represent a promising therapeutic strategy to reinvigorate T cell function and improve the efficacy of T cell\u003c/em\u003e\u003cem\u003e\u0026ndash;based cancer immunotherapy.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Suzhou Municipal Key Laboratory (SZS2023005) and the NCTIB Fund for R\u0026amp;D Platform for Cell and Gene Therapy. The schematic diagrams in this study were created using BioRender (https://biorender.com).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.H.C. and G.D.L. conceived the project and designed experiments; X.L.P., Y.X., J.D., and Y.J.P. performed most experiments and interpreted data; Y.P.S., and H.H.C. performed all bioinformatics analyses; H.H.C., G.D.L., X.L.P., and Y.J.P. wrote and edited the manuscript; G.D.L. and H.H.C supervised the project and provided overall direction. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0520600), the National Natural Science Foundation of China (32525028 and 32270994 to G. L., 32571074 and 32300764 to H.C.), the Basic Research Program of Jiangsu (BK20250003 to G. L., BK20230280 to H.C.), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2021-RC310-014 and 2024-JKCS-15 to G. L.), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-047, 2021-I2M-1-061, 2022-I2M-2-004, 2023-I2M-2-010, 2023-I2M-QJ-019, and 2024-I2M-ZD-009 to G.L., 2025-I2M-TS-15 to H.C.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the findings of this study are included in the manuscript and Supplementary Information. Source data, full uncropped images of gels and blots are available in the supplementary files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study involving all the mice were kept in a specific-pathogen-free facility, and all animal experiments were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of Suzhou Institute of Systems Medicine (ISM-IACUC-0151-R). All tumor burdens did not exceed the permission of the Institutional Animal Care and Use Committee of Suzhou Institute of Systems Medicine. Age-and sex-matched mice were assigned randomly to experimental and control groups. Human PBMCs from healthy donors were purchased from Sailybio (Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding author Guideng Li is a member of the Editorial Board of the journal Immunity \u0026amp; Inflammation. However, he was not involved in the peer-review or decision-making process for this manuscript. The authors declare no other competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCheng H, Su Y, Pan X, et al. The ubiquitin ligase KLHL6 drives resistance to CD8+ T cell dysfunction. Nature. (in press)\u003c/li\u003e\n\u003cli\u003eMiller BC, Sen DR, Al Abosy R, et al. 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The transcription factor NFAT promotes exhaustion of activated CD8⁺ T cells. Immunity. 2015;42(2):265-278. \u003c/li\u003e\n\u003cli\u003eTill\u0026eacute; L, Cropp D, Charmoy M, et al. Activation of the transcription factor NFAT5 in the tumor microenvironment enforces CD8+ T cell exhaustion. Nat Immunol. 2023;24(10):1645-1653. \u003c/li\u003e\n\u003cli\u003eSeo H, Chen J, Gonz\u0026aacute;lez-Avalos E, et al. TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8\u003csup\u003e+\u003c/sup\u003e T cell exhaustion. Proc Natl Acad Sci U S A. 2019;116(25):12410-12415.\u003c/li\u003e\n\u003cli\u003eCheng H, Ma K, Zhang L, Li G. The tumor microenvironment shapes the molecular characteristics of exhausted CD8\u003csup\u003e+\u003c/sup\u003e T cells. Cancer Lett. 2021;506:55-66. \u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"immunity-and-inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Immunity \u0026 Inflammation](https://link.springer.com/journal/44466)","snPcode":"44466","submissionUrl":"https://submission.springernature.com/new-submission/44466/3","title":"Immunity \u0026 Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7913669/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7913669/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"No abstract is needed for a brief report.","manuscriptTitle":"Chronic TCR Signaling-Driven Suppression of the FOXO-KLHL6 Axis Promotes T Cell Exhaustion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 14:07:29","doi":"10.21203/rs.3.rs-7913669/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-31T09:19:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-31T07:15:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-31T00:12:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T07:05:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69053138712044458963636223135212532533","date":"2025-10-28T05:53:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122328305570944890108502616677209837774","date":"2025-10-28T03:17:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282640161050830189945751150003819158594","date":"2025-10-28T03:14:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-28T03:04:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-28T02:51:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-27T02:52:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Immunity \u0026 Inflammation","date":"2025-10-21T10:41:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"immunity-and-inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Immunity \u0026 Inflammation](https://link.springer.com/journal/44466)","snPcode":"44466","submissionUrl":"https://submission.springernature.com/new-submission/44466/3","title":"Immunity \u0026 Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"585c2f2a-fe23-4e46-8631-0d5e79bbec38","owner":[],"postedDate":"November 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-21T02:53:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-07 14:07:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7913669","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7913669","identity":"rs-7913669","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}