Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response

Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response | 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 Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response Terry Fry, Kole DeGolier, Etienne Danis, Marc D’Antonio, Jennifer Cimons, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3712137/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Nature Immunology → Version 1 posted You are reading this latest preprint version Abstract Chimeric antigen receptor T cells are an effective therapy for B-lineage malignancies. However, many patients relapse and this therapeutic has yet to show strong efficacy in other hematologic or solid tumors. One opportunity for improvement lies in the ability to generate T cells with desirable functional characteristics. Here, we dissect the biology of CD8+ CAR T cells (CAR8) by controlling whether the T cell has encountered cognate TCR antigen prior to CAR generation. We find that prior antigen experience influences multiple aspects of in vitro and in vivo CAR8 functionality, resulting in superior effector function and leukemia clearance in the setting of limiting target antigen density compared to antigen-inexperienced T cells. However, this comes at the expense of inferior proliferative capacity, susceptibility to phenotypic exhaustion and dysfunction, and inability to clear wildtype leukemia in the setting of limiting CAR+ cell dose. Epigenomic and transcriptomic comparisons of these cell populations identified overexpression of the Runx2 transcription factor as a novel strategy to enhance CAR8 function, with a differential impact depending on prior cell state. Collectively, our data demonstrate that prior antigen experience determines functional attributes of a CAR T cell, as well as amenability to functional enhancement by transcription factor modulation. Biological sciences/Immunology/Gene regulation in immune cells/Epigenetics in immune cells Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Biological sciences/Immunology/Lymphocytes/T cells/CD8-positive T cells Full Text Additional Declarations Yes there is potential Competing Interest. Patent applicant (whether author or institution): University of Colorado Anschutz Medical Campus Name of inventor(s): Kole R DeGolier, James P Scott-Browne, Terry J Fry Application number: U.S. Provisional Application No. 63/595,612 Status of application: Pending Aspect covered: Methods of enhancing potency of engineered immune cells via Runx2 modulation Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Nature Immunology → 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-3712137","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":260813500,"identity":"84459ee0-a0e1-4590-a2e8-b57d554ff3ed","order_by":0,"name":"Terry Fry","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIie3Rv2oCMRzA8RxCutzRTXIE9BUiB7f6KgmFmwTXDkUjwrnpqkufwck5chCXg65uRQq6dDhx0CFif1edCpdbO+Q7hZAP+YeQy/UP635+HE4+ug3ZZrwnIh202bZ/LjxjhKwgmPgx9ZESy1zH5KizznIrJPOktpJwDmQx7zXDxV7xB8mshBRAZuE3pgEQBoRbSXt2KDgyQ0x7QFYD3gWivIttl+eJ4ki9PUij3GUkaw6mOZAEhzmm1/dfMkY1JGFAXmDQpMG0UV4/rSMx4UyJ1E+iKJjCI+frFAmpowoB18+BvAJ5ynZfQfmVm9GkOBrTqiL32N8Jbl/vcrlcrpp+AFLAcJd3N0I8AAAAAElFTkSuQmCC","orcid":"","institution":"University of Colorado Anschutz Medical Campus","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Terry","middleName":"","lastName":"Fry","suffix":""},{"id":260813501,"identity":"f17d3fd9-2a26-4089-9877-d1bd9e700efb","order_by":1,"name":"Kole DeGolier","email":"","orcid":"https://orcid.org/0000-0002-4144-7277","institution":"University of Colorado Anschutz Medical Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kole","middleName":"","lastName":"DeGolier","suffix":""},{"id":260813502,"identity":"32fd269c-27ce-4925-8fe4-1adb708720cf","order_by":2,"name":"Etienne Danis","email":"","orcid":"","institution":"University of Colorado Anschutz Medical Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Etienne","middleName":"","lastName":"Danis","suffix":""},{"id":260813503,"identity":"82c3eea4-1133-4dd1-89dc-c17edfcbb3af","order_by":3,"name":"Marc D’Antonio","email":"","orcid":"","institution":"University of Colorado School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marc","middleName":"","lastName":"D’Antonio","suffix":""},{"id":260813504,"identity":"7e1dac91-c727-40a4-826a-8ef66c16f290","order_by":4,"name":"Jennifer Cimons","email":"","orcid":"https://orcid.org/0000-0002-8111-8697","institution":"University of Colorado Anschutz Medical Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Cimons","suffix":""},{"id":260813505,"identity":"dcaa44a0-4f64-49b7-8867-61652756c48f","order_by":5,"name":"Michael Yarnell","email":"","orcid":"https://orcid.org/0000-0002-4557-7255","institution":"University of Colorado Anschutz Medical Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Yarnell","suffix":""},{"id":260813506,"identity":"c3830881-f290-499a-a044-c67dfa109f75","order_by":6,"name":"Ross Kedl","email":"","orcid":"https://orcid.org/0000-0002-9065-7895","institution":"University of Colorado Anschutz Medical Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ross","middleName":"","lastName":"Kedl","suffix":""},{"id":260813507,"identity":"34cb125d-5d30-4526-b250-25f692e738a3","order_by":7,"name":"Mark kohler","email":"","orcid":"","institution":"University of Colorado School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"kohler","suffix":""},{"id":260813508,"identity":"660da788-aeb1-4913-b5a6-f54435d6a9a8","order_by":8,"name":"James Scott-Browne","email":"","orcid":"","institution":"National Jewish Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"James","middleName":"","lastName":"Scott-Browne","suffix":""}],"badges":[],"createdAt":"2023-12-05 23:15:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3712137/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3712137/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41590-024-02034-1","type":"published","date":"2025-01-02T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72874812,"identity":"7f21a656-af69-453d-9a7c-bfb754a695ab","added_by":"auto","created_at":"2025-01-03 08:05:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8206420,"visible":true,"origin":"","legend":"","description":"","filename":"2023125FINALDeGolieretalNatureImmunologyManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3712137/v1_covered_79c4a686-ec46-4422-b752-962b289b7659.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nPatent applicant (whether author or institution): University of Colorado Anschutz Medical Campus\r\nName of inventor(s): Kole R DeGolier, James P Scott-Browne, Terry J Fry\r\nApplication number: U.S. Provisional Application No. 63/595,612 \r\nStatus of application: Pending\r\nAspect covered: Methods of enhancing potency of engineered immune cells via Runx2 modulation","formattedTitle":"Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3712137/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3712137/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChimeric antigen receptor T cells are an effective therapy for B-lineage malignancies. However, many patients relapse and this therapeutic has yet to show strong efficacy in other hematologic or solid tumors. One opportunity for improvement lies in the ability to generate T cells with desirable functional characteristics. Here, we dissect the biology of CD8+ CAR T cells (CAR8) by controlling whether the T cell has encountered cognate TCR antigen prior to CAR generation. We find that prior antigen experience influences multiple aspects of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e CAR8 functionality, resulting in superior effector function and leukemia clearance in the setting of limiting target antigen density compared to antigen-inexperienced T cells. However, this comes at the expense of inferior proliferative capacity, susceptibility to phenotypic exhaustion and dysfunction, and inability to clear wildtype leukemia in the setting of limiting CAR+ cell dose. Epigenomic and transcriptomic comparisons of these cell populations identified overexpression of the Runx2 transcription factor as a novel strategy to enhance CAR8 function, with a differential impact depending on prior cell state. Collectively, our data demonstrate that prior antigen experience determines functional attributes of a CAR T cell, as well as amenability to functional enhancement by transcription factor modulation.\u003c/p\u003e","manuscriptTitle":"Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-21 21:03:30","doi":"10.21203/rs.3.rs-3712137/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-immunology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ni","sideBox":"Learn more about [Nature Immunology](http://www.nature.com/ni/)","snPcode":"","submissionUrl":"","title":"Nature Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0ad2f43e-6c2c-41d2-a07e-54c9fb4f71f1","owner":[],"postedDate":"December 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":27528805,"name":"Biological sciences/Immunology/Gene regulation in immune cells/Epigenetics in immune cells"},{"id":27528806,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":27528807,"name":"Biological sciences/Immunology/Lymphocytes/T cells/CD8-positive T cells"}],"tags":[],"updatedAt":"2025-01-03T08:05:25+00:00","versionOfRecord":{"articleIdentity":"rs-3712137","link":"https://doi.org/10.1038/s41590-024-02034-1","journal":{"identity":"nature-immunology","isVorOnly":false,"title":"Nature Immunology"},"publishedOn":"2025-01-02 05:00:00","publishedOnDateReadable":"January 2nd, 2025"},"versionCreatedAt":"2023-12-21 21:03:30","video":"","vorDoi":"10.1038/s41590-024-02034-1","vorDoiUrl":"https://doi.org/10.1038/s41590-024-02034-1","workflowStages":[]},"version":"v1","identity":"rs-3712137","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3712137","identity":"rs-3712137","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}