Effects of testosterone and estrogen on human CD4+ T-cell subsets during gender-affirming hormone therapy

Effects of testosterone and estrogen on human CD4+ T-cell subsets during gender-affirming hormone therapy | 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 Brief Communication Effects of testosterone and estrogen on human CD4+ T-cell subsets during gender-affirming hormone therapy Sebastian Riemann, Jeroen Vervalcke, Carolien Bonroy, Yaël Sertons, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9281100/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 Women have a higher burden of Type 2-driven diseases as well as Type 1-/Type 17-driven autoimmune diseases, suggesting insufficient counterbalancing by regulatory T cells (Tregs) as a possible shared mechanism. We investigated early immunological effects of gender-affirming hormone therapy (GAHT) in 19 transmen and 15 transwomen by analyzing CD4⁺ T-cell subsets before and three months after treatment initiation. Using 14-color flow cytometry and unsupervised FlowSOM clustering, we quantified 20 CD4⁺ T-cell metaclusters in peripheral blood. Feminizing GAHT in transwomen significantly reduced Treg frequencies, without affecting T-helper (Th)-1, Th2, or Th17 subsets. In contrast, masculinizing GAHT induced no major shifts in helper or regulatory subsets in transmen. These findings reveal early, hormone-specific immune remodeling during GAHT, characterized by reduced Tregs following feminizing therapy. Our results highlight Tregs as a key target of sex hormone-driven immune modulation and provide a cellular correlate to recent proteomic and transcriptomic studies. Biological sciences/Immunology/Adaptive immunity/Cellular immunity Biological sciences/Immunology/Lymphocytes/T cells/CD4-positive T cells/Regulatory T cells Biological sciences/Computational biology and bioinformatics/Functional clustering Full Text Additional Declarations Yes there is potential Competing Interest. The Department of Respiratory Medicine is funded by FWO Flanders (G0H1222N, G025123N, funding paid to the institution) and Ghent University (bof/baf/2y/2024/01/023; bof/baf/1y/2026/01/008, funding paid to the institution); S.R. is funded by a predoctorate mandate provided by Ghent University Special Research Fund (UGent- BOF23/DOC/013). Outside this work, S.R. has received non-financial support from AstraZeneca, GlaxoSmithKline, and Sanofi Regeneron; and lecture fees from GlaxoSmithKline; T.M. reports grants or contracts from Chiesi (Chiesi Chair on the role of environmental factors in asthma development) and GlaxoSmithKline (Chair on eosinophilic airway diseases, ended 31/12/2024) and stocks/stock options from Oryzon Genomics (shareholder, outside the submitted work) and Mendelion Lifesciences SL (shareholder, outside the submitted work); G.B. has received speaker honoraria from AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Merck Sharp & Dohme, Novartis, and Sanofi-Regeneron; he is past President of the Belgian Respiratory Society. J.V., C.B., Y.S., P.B., M.B., S.V.G., G.T., M.H. and B.L. report no conflicts of interest. Supplementary Files ImmunoTSupplementsFinal.docx Online supplement Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9281100","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Brief Communication","associatedPublications":[],"authors":[{"id":615460084,"identity":"348f9ea8-1980-4835-b072-851d964191c0","order_by":0,"name":"Sebastian Riemann","email":"data:image/png;base64,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","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Riemann","suffix":""},{"id":615460085,"identity":"2261dada-4079-49aa-aecb-c18ffe79ebb6","order_by":1,"name":"Jeroen Vervalcke","email":"","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jeroen","middleName":"","lastName":"Vervalcke","suffix":""},{"id":615460086,"identity":"08d93fad-46f7-4136-b23a-48b545aa0033","order_by":2,"name":"Carolien Bonroy","email":"","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Carolien","middleName":"","lastName":"Bonroy","suffix":""},{"id":615460087,"identity":"fd3f312c-e535-40b6-b209-dcf6a7a96fff","order_by":3,"name":"Yaël Sertons","email":"","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yaël","middleName":"","lastName":"Sertons","suffix":""},{"id":615460088,"identity":"0b742e7c-fa03-47d9-aaee-35c6a05c5d45","order_by":4,"name":"Pauline Breughe","email":"","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pauline","middleName":"","lastName":"Breughe","suffix":""},{"id":615460089,"identity":"a26a223f-2db5-4a73-acf0-4d6f006476c8","order_by":5,"name":"Malicorne Buysse","email":"","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Malicorne","middleName":"","lastName":"Buysse","suffix":""},{"id":615460090,"identity":"b4baf71c-7811-4328-a07a-8f6c0168ead3","order_by":6,"name":"Sofie Van Gassen","email":"","orcid":"https://orcid.org/0000-0002-7119-5330","institution":"Data Mining and Modelling for Biomedicine, VIB-UGent, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Sofie","middleName":"Van","lastName":"Gassen","suffix":""},{"id":615460091,"identity":"be732d9d-6e93-4661-888e-5c01b3e8bd51","order_by":7,"name":"Tania Maes","email":"","orcid":"https://orcid.org/0000-0002-3037-6928","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Tania","middleName":"","lastName":"Maes","suffix":""},{"id":615460092,"identity":"290f52cb-6597-402f-8035-95de969e00f1","order_by":8,"name":"Guy T'Sjoen","email":"","orcid":"https://orcid.org/0000-0003-0457-9673","institution":"Ghent University Hospital; 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J.V., C.B., Y.S., P.B., M.B., S.V.G., G.T., M.H. and B.L. report no conflicts of interest.","formattedTitle":"Effects of testosterone and estrogen on human CD4+ T-cell subsets during gender-affirming hormone therapy","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-9281100/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9281100/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Women have a higher burden of Type 2-driven diseases as well as Type 1-/Type 17-driven autoimmune diseases, suggesting insufficient counterbalancing by regulatory T cells (Tregs) as a possible shared mechanism. We investigated early immunological effects of gender-affirming hormone therapy (GAHT) in 19 transmen and 15 transwomen by analyzing CD4⁺ T-cell subsets before and three months after treatment initiation. Using 14-color flow cytometry and unsupervised FlowSOM clustering, we quantified 20 CD4⁺ T-cell metaclusters in peripheral blood. Feminizing GAHT in transwomen significantly reduced Treg frequencies, without affecting T-helper (Th)-1, Th2, or Th17 subsets. In contrast, masculinizing GAHT induced no major shifts in helper or regulatory subsets in transmen. These findings reveal early, hormone-specific immune remodeling during GAHT, characterized by reduced Tregs following feminizing therapy. 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