Impaired CD4+ T cell help through the CD40L-CD40 pathway contributes to reduced tumor control in males

Impaired CD4+ T cell help through the CD40L-CD40 pathway contributes to reduced tumor control in males | 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 Impaired CD4+ T cell help through the CD40L-CD40 pathway contributes to reduced tumor control in males Katey S. Hunt, Samantha Cooke, Emily Ebert, Lindsey M. Kuehm, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6568498/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Sex-based differences in cancer incidence are incompletely understood, but potential roles for the immune system are beginning to emerge. CD4 + T cells play a central role in organizing antitumor immunity. In addition to cytokine production, CD40L expression on CD4 + T cell provides necessary helper signaling to dendritic cells that is required for the priming of cytotoxic tumor-specific CD8 + T cells. Despite these critical functions, the impact of biological sex on the CD4 + T cell response to cancer remains unknown. Here, we demonstrate that impaired immune-mediated tumor control in male compared to female mice is driven by disparate helper CD4 + T cell responses between the sexes. We find that CD40L expression is reduced on CD4 + T cells isolated from males via a mechanism predominantly driven by sex hormones, resulting in decreased CD40 signaling in dendritic cells within tumor-draining lymph nodes. These deficits resulted in decreased helper CD4 + T cell frequencies and impaired CD8 + T cell function within the male tumor microenvironment which could be rescued by targeting the CD40L-CD40 axis. Our findings identify a novel mechanism of CD4 + T cell-based sex differences in the immune response to cancer that impairs tumor control. *Stephen T. Ferris & Elise Alspach are co-senior authors. Biological sciences/Cancer/Tumour immunology Biological sciences/Immunology/Adaptive immunity/Cellular immunity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files SFig1.jpg Sup Fig 1: MB49 tumor cells do not elicit immune responses against the Y chromosome SFig2.jpg Sup Fig 2: MB49 tumors display sex differences in T cell phenotypes SFig3.jpg Sup Fig 3: CD4 + T cells from MB49 tumors show sex-based phenotypic differences SFig4.jpg Sup Fig 4: Flow cytometry analysis of CD4 + T cells SFig5.jpg Sup Fig 5: Sex-based differences in CD4 + T cells infiltrating human bladder tumors SFig6.jpg Sup Fig 6: cDC1s from MB49 tumor-bearing male and female mice display equivalent expression levels of CD40 SFig7.jpg Sup Fig 7: Modulation of CD40L-CD40 signaling impacts both CD4 + and CD8 + T cell function SFig8.jpg Sup Fig 8: Mixed male-female bone marrow chimeras SupFiglegends.pdf Legends for supplemental figures Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6568498","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":457235023,"identity":"4f934ab7-b239-428e-bd7b-6eb1ba134f76","order_by":0,"name":"Katey S. Hunt","email":"","orcid":"https://orcid.org/0000-0001-7720-3365","institution":"Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO","correspondingAuthor":false,"prefix":"","firstName":"Katey","middleName":"S.","lastName":"Hunt","suffix":""},{"id":457235024,"identity":"e1a9680e-3aaa-453f-a4f4-12ad1d155d72","order_by":1,"name":"Samantha Cooke","email":"","orcid":"","institution":"Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO","correspondingAuthor":false,"prefix":"","firstName":"Samantha","middleName":"","lastName":"Cooke","suffix":""},{"id":457235025,"identity":"9b3b5a24-39fc-44e6-89a4-8812d0abb07d","order_by":2,"name":"Emily Ebert","email":"","orcid":"https://orcid.org/0009-0000-1293-3180","institution":"Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"","lastName":"Ebert","suffix":""},{"id":457235026,"identity":"3b8a2bc3-57ba-441c-8801-c0e7beb065f1","order_by":3,"name":"Lindsey M. Kuehm","email":"","orcid":"","institution":"Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO","correspondingAuthor":false,"prefix":"","firstName":"Lindsey","middleName":"M.","lastName":"Kuehm","suffix":""},{"id":457235027,"identity":"ac97e8b0-711e-44cd-a65e-04b0e1330f15","order_by":4,"name":"Smrithi Beeram","email":"","orcid":"","institution":"Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO","correspondingAuthor":false,"prefix":"","firstName":"Smrithi","middleName":"","lastName":"Beeram","suffix":""},{"id":457235028,"identity":"dabaa20e-9fa9-4435-8219-fd47c4387fb5","order_by":5,"name":"Ryan M. Teague","email":"","orcid":"https://orcid.org/0000-0002-8426-2870","institution":"Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO","correspondingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"M.","lastName":"Teague","suffix":""},{"id":457235029,"identity":"20a07085-275c-4a37-bbae-4b323896ed93","order_by":6,"name":"Michelle Brennan","email":"","orcid":"https://orcid.org/0000-0002-3460-5599","institution":"Department of Biochemistry, Saint Louis University School of Medicine, St. Louis, MO","correspondingAuthor":false,"prefix":"","firstName":"Michelle","middleName":"","lastName":"Brennan","suffix":""},{"id":499566550,"identity":"55e6784b-62cd-4208-8fc2-bd2202bc580a","order_by":7,"name":"Melissa M. Berrien-Elliott","email":"","orcid":"","institution":"Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO","correspondingAuthor":false,"prefix":"","firstName":"Melissa","middleName":"M.","lastName":"Berrien-Elliott","suffix":""},{"id":457235030,"identity":"cc0e4efc-e207-4b4d-9418-a6aa1abb3f12","order_by":8,"name":"Stephen T. Ferris*","email":"","orcid":"","institution":"Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"T.","lastName":"Ferris*","suffix":""},{"id":457235022,"identity":"1f786c6c-856d-425f-b1c5-39e183696ecb","order_by":9,"name":"Elise Alspach*","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-2740-9768","institution":"Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO","correspondingAuthor":true,"prefix":"","firstName":"Elise","middleName":"","lastName":"Alspach*","suffix":""}],"badges":[],"createdAt":"2025-05-01 01:50:07","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6568498/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-6568498/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89105112,"identity":"41e00e74-a882-4fa7-b08b-ea1e8b62943e","added_by":"auto","created_at":"2025-08-14 17:14:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":616050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSex differences in tumor control are mediated by the adaptive immune system. A)\u003c/strong\u003e Survival of \u003cem\u003eRAG2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e and wildtype (WT) male and female mice treated subcutaneously with 100mg 3-methylcholanthrene (MCA). n of each cohort is indicated in panel legend. p values calculated using Kaplan-Meier analysis. \u003cstrong\u003eB)\u003c/strong\u003e Tumor incidence of animals described in (A). p values calculated using Fischer’s exact test. \u003cstrong\u003eC)\u003c/strong\u003e Tumor growth curves of control IgG- or aCD4+aCD8-treated male and female mice implanted with MB49 cells. Data is shown as mean tumor volume + SEM of n=9-14 animals per cohort from 2-3 independent experiments. p values calculated using 2-way ANOVA with Bonferroni correction for multiple comparisons. \u003cstrong\u003eD) \u003c/strong\u003eMB49 tumor infiltrating CD8\u003csup\u003e+ \u003c/sup\u003eand CD4\u003csup\u003e+ \u003c/sup\u003eT cells were isolated from male and female mice 14 days post tumor inoculation and analyzed by scRNAseq. Uniform manifold approximation and projection (UMAP) plots from 3,363 male- and 3,276 female-derived cells are shown. \u003cstrong\u003eE)\u003c/strong\u003e Pie charts show the proportion of male- and female-derived T cells within clusters identified from scRNAseq analysis described in (D). ns, non-significant, p\u0026gt;0.05; ***, p£0.0001; ****, p£0.0001.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/c0ed8befec0d5b8f8b986245.jpg"},{"id":89105111,"identity":"741ada89-01d2-4ccc-9e42-c65593fd411b","added_by":"auto","created_at":"2025-08-14 17:14:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":663767,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTumors from males are deficient in CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cell help. A)\u003c/strong\u003e UMAP plots from 895 male- and 720 female-derived CD4\u003csup\u003e+ \u003c/sup\u003eT cells isolated from MB49 tumors 14 days post transplantation. \u003cstrong\u003eB)\u003c/strong\u003e Pie charts show the proportion of male- and female-derived CD4\u003csup\u003e+\u003c/sup\u003e T cells within clusters identified by scRNAseq in (A). \u003cstrong\u003eC)\u003c/strong\u003e Dot plot comparing the expression level of the indicated transcripts in CD4\u003csup\u003e+ \u003c/sup\u003eT cell C0 and C1 from (A). Dot size indicates proportion of cells expressing the transcript within the indicated cluster while dot color indicates transcript expression level. \u003cstrong\u003eD)\u003c/strong\u003e Representative flow cytometry analysis of CD4\u003csup\u003e+ \u003c/sup\u003eT cells infiltrating MB49 tumors 14 days post-transplant. Cells were previously gated on viable, CD90.2\u003csup\u003e+ \u003c/sup\u003eCD4\u003csup\u003e+ \u003c/sup\u003epopulation. \u003cstrong\u003eE)\u003c/strong\u003e Quantification of flow cytometry analysis of CD4\u003csup\u003e+ \u003c/sup\u003eT cells as described in (D). Data is shown as mean percent TBET\u003csup\u003e+ \u003c/sup\u003eFOXP3\u003csup\u003e- \u003c/sup\u003e(Th1) cells within the CD4\u003csup\u003e+ \u003c/sup\u003eT cell population ± SEM (left), and mean FOXP3\u003csup\u003e+ \u003c/sup\u003e(T\u003csub\u003ereg\u003c/sub\u003e):Th1 CD4\u003csup\u003e+ \u003c/sup\u003eT cell ratio ± SEM (right) from 10 male- and 14 female-derived MB49 tumors from 3 independent experiments. p values calculated using unpaired, two-tailed t test with Welch’s correction (left) and unpaired, two-tailed Mann-Whitney test (right). One outlier in the male cohort was excluded. **, p£0.01; ***, p£0.0001. \u003cstrong\u003eF)\u003c/strong\u003e UMAP of CD4\u003csup\u003e+ \u003c/sup\u003eT cells isolated from patients with bladder cancer (n=3 males and 2 females). Plot is overlaid with expression level of \u003cem\u003eTBX21\u003c/em\u003e and \u003cem\u003eFOXP3\u003c/em\u003e transcripts to identify Th1 and T\u003csub\u003ereg\u003c/sub\u003e clusters, respectively. Dashed lines indicate cluster borders. Pie charts on right show the total proportion of male- and female-derived T cells within T\u003csub\u003ereg\u003c/sub\u003e and Th1 clusters.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/e6fe9e095aeb6ca4e63ed55b.jpg"},{"id":89105119,"identity":"4190b918-1377-415b-b441-7a06bf3c1f1d","added_by":"auto","created_at":"2025-08-14 17:14:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":345037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSex differences in CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cell function contribute to disparate tumor control between males and females. \u003c/strong\u003eTumor growth curves of \u003cstrong\u003eA)\u003c/strong\u003e vehicle control- or diphtheria toxin (DTX)-treated male and female \u003cem\u003eFoxp3\u003c/em\u003e\u003csup\u003e\u003cem\u003eDTR\u003c/em\u003e\u003c/sup\u003e mice (n=9-15 mice per cohort from 3 independent experiments); \u003cstrong\u003eB)\u003c/strong\u003e male and female WT or \u003cem\u003eCd40lg\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice (n=5-9 mice per cohort from 2 independent experiments); and \u003cstrong\u003eC)\u003c/strong\u003e control IgG- or agonistic aCD40-treated male and female mice (n=12-15 mice per cohort from 3 independent experiments) implanted with MB49 cells. Data is shown as mean tumor volume + SEM. p values calculated using 2-way ANOVA with Bonferroni correction for multiple comparisons. ns, non-significant, p\u0026gt;0.05; ***, p£0.001; ****, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/cdf90de13ac197053e0ec14c.jpg"},{"id":89105546,"identity":"8e935d05-1827-4e6b-b731-573be5069e5c","added_by":"auto","created_at":"2025-08-14 17:22:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":362832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecDC1s from males display a deficit in CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cell-mediated CD40L-CD40 signaling. A, B)\u003c/strong\u003e Flow cytometry analysis of CD70 expression in migratory cDC1 from male- and female-derived tdLNs 7 days post MB49 implantation. Quantification in (A) is shown as mean percent CD70\u003csup\u003e+ \u003c/sup\u003ecells within the migratory cDC1 population ± SEM of 8- 12 animals per group from 2 independent experiments. Representative flow plots are shown in (B). p value calculated using unpaired, two-tailed t test. \u003cstrong\u003eC, D)\u003c/strong\u003e Flow cytometry analysis of CD40L expression in CD4\u003csup\u003e+ \u003c/sup\u003eT cells from day 7 MB49 tdLNs. Quantification in (C) is shown as mean percent CD40L\u003csup\u003e+ \u003c/sup\u003ecells within the CD4\u003csup\u003e+ \u003c/sup\u003eT cell population ± SEM of 9 animals per group from 3 independent experiments. Representative flow plots are shown in (D). Cells were previously gated on viable, CD90.2\u003csup\u003e+ \u003c/sup\u003eCD4\u003csup\u003e+ \u003c/sup\u003ecells. p value calculated using unpaired, two-tailed t test. \u003cstrong\u003eE)\u003c/strong\u003e Tumor growth curves of \u003cem\u003eXcr1\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003exCd40\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003e(cDC1\u003csup\u003eCD40KO\u003c/sup\u003e) or \u003cem\u003eCd40\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e (cDC1\u003csup\u003eCD40WT\u003c/sup\u003e) male and female mice implanted with MB49 cells. Data is shown as mean tumor volume + SEM of 10-15 animals per cohort from 3 independent experiments. p values calculated using 2-way ANOVA with Bonferroni correction for multiple comparisons. ns, non-significant, p\u0026gt;0.05; *, p\u0026lt;0.05; **, p£0.01; ****, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/ab4cb2460895dc5d0d5d8763.jpg"},{"id":89105121,"identity":"3b75d79f-5ca4-409d-8d5c-b6e493f60d90","added_by":"auto","created_at":"2025-08-14 17:14:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":594133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD40L-CD40 signaling contributes to sex-based differences in CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eand CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cell function. A)\u003c/strong\u003e Flow cytometry analysis of CD4\u003csup\u003e+\u003c/sup\u003e T cells isolated from MB49 tumors 14 days following implantation in WT or \u003cem\u003eCd40lg\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. Data is shown as the mean percent Th1 (TBET\u003csup\u003e+ \u003c/sup\u003eFOXP3\u003csup\u003e-\u003c/sup\u003e) cells within the CD4\u003csup\u003e+ \u003c/sup\u003eT cell population ± SEM in 4-8 tumors per cohort from 3 independent experiments. p value calculated using 2-way ANOVA with Bonferroni correction for multiple comparisons. \u003cstrong\u003eB)\u003c/strong\u003e Flow cytometry analysis of CD4\u003csup\u003e+ \u003c/sup\u003eT cells isolated from day 14 MB49 tumors implanted in cDC1\u003csup\u003eCD40WT\u003c/sup\u003e or cDC1\u003csup\u003eCD40KO\u003c/sup\u003e mice. Data is shown as the mean percent Th1 cells within the CD4\u003csup\u003e+ \u003c/sup\u003eT cell population ± SEM in 7-12 tumors per cohort from 3 independent experiments. p value calculated using 2-way ANOVA with Bonferroni correction for multiple comparisons. One and 2 outliers were removed from the WT male and female cohorts, respectively. \u003cstrong\u003eC-F)\u003c/strong\u003e Flow cytometry analysis of CD4\u003csup\u003e+ \u003c/sup\u003eand CD8\u003csup\u003e+ \u003c/sup\u003eT cells from day 14 MB49 tumors implanted in mice treated with IgG control or agonistic aCD40 antibody. Data is shown as mean percent Th1 cells within the CD4\u003csup\u003e+ \u003c/sup\u003eT cell population (C) and mean percent PD-1\u003csup\u003e+\u003c/sup\u003eTIM-3\u003csup\u003e+ \u003c/sup\u003ecells within the CD8\u003csup\u003e+ \u003c/sup\u003eT cell population (D) ± SEM in 10-18 tumors per cohort from 4 independent experiments. Representative flow plots are shown in (E) and (F). p values calculated using 2-way ANOVA with Bonferroni correction for multiple comparisons. ns, non-significant, p\u0026gt;0.05; *, p\u0026lt;0.05; **, p£0.01; ***, p£0.001; ****, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/d2c451c63eedd10ac13a454b.jpg"},{"id":89106044,"identity":"09676aef-f4f1-4b31-a010-faa172791d7f","added_by":"auto","created_at":"2025-08-14 17:30:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":510991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSex differences in CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cell expression of CD40L are hormonally driven. A)\u003c/strong\u003e Experimental design of mixed male-female bone marrow chimeras. \u003cstrong\u003eB, C)\u003c/strong\u003e Flow cytometry analysis of CD40L expression in male donor, female donor and host-derived CD4\u003csup\u003e+ \u003c/sup\u003eT cells from day 7 MB49 tdLNs. Quantification in (B) is shown as mean percent CD40L\u003csup\u003e+ \u003c/sup\u003ecells within the CD4\u003csup\u003e+ \u003c/sup\u003eT cell population ± SEM of 8-10 mice from 2 independent experiments. Representative flow plots are shown in (C). p values were calculated using 2-way ANOVA with Bonferroni correction for multiple comparisons. ns, non-significant, p\u0026gt;0.05; *, p\u0026lt;0.05; **, p£0.01; ***, p£0.001. \u003cstrong\u003eD)\u003c/strong\u003e Graphical summary of findings.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/08b1b7a988d5ea2c6256d561.jpg"},{"id":89155478,"identity":"fa4e44cc-b516-45e6-abf2-acf8570e86cd","added_by":"auto","created_at":"2025-08-15 14:08:30","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3828191,"visible":true,"origin":"","legend":"","description":"","filename":"sexdiffCD4v4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2_covered_762d0263-50d7-4d8c-9bf8-470f633b9e6f.pdf"},{"id":89105544,"identity":"ae227331-fed1-4f02-a811-c16da4f9fea6","added_by":"auto","created_at":"2025-08-14 17:22:24","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":561181,"visible":true,"origin":"","legend":"\u003cp\u003eSup Fig 1: MB49 tumor cells do not elicit immune responses against the Y chromosome\u003c/p\u003e","description":"","filename":"SFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/1795def3ac5bc32abd9a1440.jpg"},{"id":89105545,"identity":"e41b70af-4c2b-4c8a-b21f-6440561d3a45","added_by":"auto","created_at":"2025-08-14 17:22:24","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1205595,"visible":true,"origin":"","legend":"\u003cp\u003eSup Fig 2: MB49 tumors display sex differences in T cell phenotypes\u003c/p\u003e","description":"","filename":"SFig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/7d3f0552bc24efcd3c4a1cc8.jpg"},{"id":89105120,"identity":"d6f9d89f-92c4-44c1-b9c0-0b18425ddea6","added_by":"auto","created_at":"2025-08-14 17:14:24","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":903982,"visible":true,"origin":"","legend":"\u003cp\u003eSup Fig 3: CD4\u003csup\u003e+ \u003c/sup\u003eT cells from MB49 tumors show sex-based phenotypic differences\u003c/p\u003e","description":"","filename":"SFig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/a9788935c6df5cbe4430488d.jpg"},{"id":89105116,"identity":"4d546bd8-ea69-4461-847c-af2bc52f7eb8","added_by":"auto","created_at":"2025-08-14 17:14:24","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":503187,"visible":true,"origin":"","legend":"\u003cp\u003eSup Fig 4: Flow cytometry analysis of CD4\u003csup\u003e+ \u003c/sup\u003eT cells\u003c/p\u003e","description":"","filename":"SFig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/9d191ac3181c98a9480ace17.jpg"},{"id":89105122,"identity":"25645fc6-cf38-4d60-9747-e928aade6eee","added_by":"auto","created_at":"2025-08-14 17:14:24","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1659093,"visible":true,"origin":"","legend":"\u003cp\u003eSup Fig 5: Sex-based differences in CD4\u003csup\u003e+ \u003c/sup\u003eT cells infiltrating human bladder tumors\u003c/p\u003e","description":"","filename":"SFig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/b8c665abfdc241d92c5b8578.jpg"},{"id":89105126,"identity":"dcc9ba03-b93d-4c4e-87c2-c01db95f5db3","added_by":"auto","created_at":"2025-08-14 17:14:24","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":599834,"visible":true,"origin":"","legend":"\u003cp\u003eSup Fig 6: cDC1s from MB49 tumor-bearing male and female mice display equivalent expression levels of CD40\u003c/p\u003e","description":"","filename":"SFig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/7f034c30d72c43e850706e75.jpg"},{"id":89105548,"identity":"62803fc3-7dfc-4b5c-a1c3-563a93e1b6c9","added_by":"auto","created_at":"2025-08-14 17:22:24","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":847335,"visible":true,"origin":"","legend":"\u003cp\u003eSup Fig 7: Modulation of CD40L-CD40 signaling impacts both CD4\u003csup\u003e+ \u003c/sup\u003eand CD8\u003csup\u003e+ \u003c/sup\u003eT cell function\u003c/p\u003e","description":"","filename":"SFig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/47a5246b976002a40585ee06.jpg"},{"id":89105129,"identity":"3f11dfc0-04e7-4e5b-b268-d30d9123bcd1","added_by":"auto","created_at":"2025-08-14 17:14:24","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":364087,"visible":true,"origin":"","legend":"\u003cp\u003eSup Fig 8: Mixed male-female bone marrow chimeras\u003c/p\u003e","description":"","filename":"SFig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/2c57d9a50b82c03868d72551.jpg"},{"id":89105550,"identity":"a5e76b27-838f-4584-bc9c-a6ffa405f143","added_by":"auto","created_at":"2025-08-14 17:22:24","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":57084,"visible":true,"origin":"","legend":"\u003cp\u003eLegends for supplemental figures\u003c/p\u003e","description":"","filename":"SupFiglegends.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6568498/v2/e258fd60b8c8eb4694e23761.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"Impaired CD4+ T cell help through the CD40L-CD40 pathway contributes to reduced tumor control in males","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-6568498/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6568498/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSex-based differences in cancer incidence are incompletely understood, but potential roles for the immune system are beginning to emerge. CD4\u003csup\u003e+\u003c/sup\u003e T cells play a central role in organizing antitumor immunity. In addition to cytokine production, CD40L expression on CD4\u003csup\u003e+\u003c/sup\u003e T cell provides necessary helper signaling to dendritic cells that is required for the priming of cytotoxic tumor-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells. Despite these critical functions, the impact of biological sex on the CD4\u003csup\u003e+\u003c/sup\u003e T cell response to cancer remains unknown. Here, we demonstrate that impaired immune-mediated tumor control in male compared to female mice is driven by disparate helper CD4\u003csup\u003e+\u003c/sup\u003e T cell responses between the sexes. We find that CD40L expression is reduced on CD4\u003csup\u003e+\u003c/sup\u003e T cells isolated from males via a mechanism predominantly driven by sex hormones, resulting in decreased CD40 signaling in dendritic cells within tumor-draining lymph nodes. These deficits resulted in decreased helper CD4\u003csup\u003e+\u003c/sup\u003e T cell frequencies and impaired CD8\u003csup\u003e+\u003c/sup\u003e T cell function within the male tumor microenvironment which could be rescued by targeting the CD40L-CD40 axis. Our findings identify a novel mechanism of CD4\u003csup\u003e+\u003c/sup\u003e T cell-based sex differences in the immune response to cancer that impairs tumor control.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e*Stephen T. Ferris \u0026amp; Elise Alspach are co-senior authors.\u003c/strong\u003e\u003c/p\u003e","manuscriptTitle":"Impaired CD4+ T cell help through the CD40L-CD40 pathway contributes to reduced tumor control in males","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-08-14 17:14:19","doi":"10.21203/rs.3.rs-6568498/v2","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}},{"code":1,"date":"2025-05-16 09:50:23","doi":"10.21203/rs.3.rs-6568498/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":"0b8f0b0f-9105-4994-ac9b-ffde31232f5d","owner":[],"postedDate":"August 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48597260,"name":"Biological sciences/Cancer/Tumour immunology"},{"id":48597261,"name":"Biological sciences/Immunology/Adaptive immunity/Cellular immunity"}],"tags":[],"updatedAt":"2025-08-07T18:26:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-14 17:14:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-6568498","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6568498","identity":"rs-6568498","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}