CD28-mediated linear and parallel costimulatory signaling cooperatively regulate CAR-T cell functions via CAR-CD28 microclusters

CD28-mediated linear and parallel costimulatory signaling cooperatively regulate CAR-T cell functions via CAR-CD28 microclusters | 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 CD28-mediated linear and parallel costimulatory signaling cooperatively regulate CAR-T cell functions via CAR-CD28 microclusters Tadashi Yokosuka, Tetsushi Nishikawa, Arata Takeuchi, Hiroaki Machiyama, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5443061/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Oct, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Various improvements have been made to chimeric antigen receptor (CAR)-T cells to enhance their antitumor effects and expand their indications to various cancer types. Although second-generation CARs directly induce second signals in a linear pathway via their own costimulatory domain such as CD28, CAR-T cells themselves express costimulatory receptors, which induce additional second signals in parallel. To clarify the differences between these two types of second signals, we analyzed CD28-mediated signalosomes using high-resolution imaging. CAR constructed using CD3ζ and CD28 (CD28ζ.CAR)-T cells demonstrated intense and persistent accumulation of the CD28 downstream kinase protein kinase C θ (PKCθ) at CAR signalosomes, and endogenous CD28 on CAR-T cells prolonged the CD28-CAR association. This assembling of PKCθ was correlated with IL-2 production and in vivo tumor suppression by CAR-T cells. These results indicate that the development and improvement of CARs requires analysis of intrinsic T-cell responses, which can be effectively assessed from signalosome dynamics evaluated by molecular imaging. Biological sciences/Immunology/Tumour immunology Biological sciences/Immunology/Imaging the immune system Biological sciences/Molecular biology Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Full Text Additional Declarations There is NO Competing Interest. Supplementary Files NishikawaCD28CARSupplement.pdf Dataset 1 Cite Share Download PDF Status: Published Journal Publication published 29 Oct, 2025 Read the published version in Communications Biology → 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. 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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-5443061","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":382217823,"identity":"c866c0e5-06d3-4000-87ba-a1f7b6c0e8f9","order_by":0,"name":"Tadashi 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