NR4A1 limits CD8⁺ T Cell effector responses and protection in tuberculosis

NR4A1 limits CD8⁺ T Cell effector responses and protection in tuberculosis | 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 NR4A1 limits CD8⁺ T Cell effector responses and protection in tuberculosis Amit Singhal, Samreen Fatima, Yao Chen, Lorissa Smulan, Basanthi Satish, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8363336/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract During Mycobacterium tuberculosis (Mtb) infection, CD8⁺ T cells exhibit dysfunction with impaired cytotoxicity and limited localization to granuloma cores. Using knockout mice, adoptive-transfer models and validation in macaque and human datasets, we identified the nuclear receptor NR4A1 as a key restrainer of CD8+ T cell immunity in tuberculosis (TB). Mtb-infected Nr4a1-/- mice displayed reduced bacterial burden, attenuated pathology, higher lung CD8⁺/CD4⁺ T cell ratios, and enhanced CD8⁺ T cell effector functions. Bulk and single-cell RNA sequencing revealed suppression of gene expression program linked with exhaustion, and expansion of Nkg7+ and Granzyme+ cytotoxic CD8⁺ T cell subsets in Nr4a1-/- mice. Spatial analyses demonstrated increased infiltration of Nkg7+ activated CD8⁺ T cells in Nr4a1-/- lesions. ChIP-qPCR showed NR4A1 binding to Nkg7 promoter, and Nkg7 knockdown abrogated the enhanced cytotoxicity of Nr4a1-/- CD8+ T cells. Pharmacologic inhibition of NR4A1 reduced Mtb burden and pathology, and restored Nkg7 expression and CD8+ T cell infiltration in the lung. Together, these findings identify NR4A1 as a negative regulator of CD8⁺ T cell–mediated immunity in TB and suggest the NR4A1-NKG7 axis as a novel host-directed therapeutic target. Biological sciences/Immunology/Infectious diseases/Tuberculosis Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte activation Tuberculosis CD8⁺ T cells NR4A1 granuloma T cell infiltration host-directed therapy Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1 DEGs identified from the time-course RNA-seq analysis of splenic CD8⁺ T cells from WT and Nr4a1 ⁻/⁻ mice. SupplementaryTable2.xlsx Supplementary Table 2 DEGs in splenic CD8⁺ T cells from WT and Nr4a1 ⁻/⁻ mice at 4 and 8 wk after Mtb infection (Fig. 2d). Includes cluster assignments for 4 wk DEGs (clusters i–x; Extended Data Fig. 2b) and 8 wk DEGs (clusters A–J; Fig. 2e). SupplementaryTable3.xlsx Supplementary Table 3 DEGs from scRNA-seq analysis of lung CD8⁺ T cell subsets from WT and Nr4a1 ⁻/⁻ mice at 4 wk. Includes gene lists for Gzmk⁺ effector-memory (cluster g) and Gzma⁺ effector (cluster m) populations (Fig. 4e). SupplementaryTable4.xls Supplementary Table 4 DEGs from GeoMx DSP whole-transcriptome analysis of CD3⁺ T cell ROIs in lung lesions at 4 wk. SupplementaryTable5.xlsx Supplementary Table 5 DEGs stratified by NR4A1 expression state (NR4A1 lo versus NR4A1 hi ) in CD8⁺ T cells from macaques and human TB datasets SupplementaryTable6.xlsx Supplementary Table 6 Sequences of primers NR4APaperextendedfigures19thNov.pdf Extended Data Figures Cite Share Download PDF Status: Under Review 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-8363336","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":572270145,"identity":"96f05d8b-a746-4d37-8178-2acbb2bdc058","order_by":0,"name":"Amit Singhal","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-3913-8798","institution":"A*STAR Infectious Diseases Labs","correspondingAuthor":true,"prefix":"","firstName":"Amit","middleName":"","lastName":"Singhal","suffix":""},{"id":572270146,"identity":"629727ea-ef84-4894-b38d-f5993244cc86","order_by":1,"name":"Samreen Fatima","email":"","orcid":"","institution":"Department of Medicine, University of Massachusetts Chan Medical School","correspondingAuthor":false,"prefix":"","firstName":"Samreen","middleName":"","lastName":"Fatima","suffix":""},{"id":572270147,"identity":"205b2c30-4315-4977-8115-e3a3228fa88b","order_by":2,"name":"Yao Chen","email":"","orcid":"","institution":"A*STAR Infectious Diseases Labs","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Chen","suffix":""},{"id":572270148,"identity":"70abbc3a-cc05-4393-ba91-08b4e8062d95","order_by":3,"name":"Lorissa Smulan","email":"","orcid":"","institution":"Department of Medicine, University of Massachusetts Chan Medical School","correspondingAuthor":false,"prefix":"","firstName":"Lorissa","middleName":"","lastName":"Smulan","suffix":""},{"id":572270149,"identity":"c498b973-aa22-4f30-80e9-10ef847d4e68","order_by":4,"name":"Basanthi Satish","email":"","orcid":"","institution":"Department of Medicine, University of Massachusetts Chan Medical School","correspondingAuthor":false,"prefix":"","firstName":"Basanthi","middleName":"","lastName":"Satish","suffix":""},{"id":572270150,"identity":"ca3cf84f-8601-4e85-96e1-b21f4b8e77e8","order_by":5,"name":"Mike Jameson","email":"","orcid":"","institution":"Department of Medicine, University of Massachusetts Chan Medical School","correspondingAuthor":false,"prefix":"","firstName":"Mike","middleName":"","lastName":"Jameson","suffix":""},{"id":572270151,"identity":"29ba4bb9-f9bf-4a0c-8347-f666bebad2bb","order_by":6,"name":"Sheetal Saini","email":"","orcid":"","institution":"A*STAR Infectious Diseases Labs","correspondingAuthor":false,"prefix":"","firstName":"Sheetal","middleName":"","lastName":"Saini","suffix":""},{"id":572270152,"identity":"4f4e5b16-f03f-4a8d-aa7b-451f692d0fee","order_by":7,"name":"Calvin Johnson","email":"","orcid":"","institution":"Department of Medicine, University of Massachusetts Chan Medical School","correspondingAuthor":false,"prefix":"","firstName":"Calvin","middleName":"","lastName":"Johnson","suffix":""},{"id":572270153,"identity":"e1351a8a-e3fe-43f1-82b2-9ee4d6da0c1c","order_by":8,"name":"Alicia Tay","email":"","orcid":"","institution":"Singapore Immunology Network","correspondingAuthor":false,"prefix":"","firstName":"Alicia","middleName":"","lastName":"Tay","suffix":""},{"id":572270154,"identity":"0e845866-f66a-44bf-bcfd-9fd5f0c309f0","order_by":9,"name":"Shihui Foo","email":"","orcid":"","institution":"Singapore Immunology Network","correspondingAuthor":false,"prefix":"","firstName":"Shihui","middleName":"","lastName":"Foo","suffix":""},{"id":572270155,"identity":"6853c586-40de-495d-9281-ef7ea82523b3","order_by":10,"name":"Hedayathulla Rahmathulla","email":"","orcid":"","institution":"Singapore Immunology Network","correspondingAuthor":false,"prefix":"","firstName":"Hedayathulla","middleName":"","lastName":"Rahmathulla","suffix":""},{"id":572270156,"identity":"3cbafa2d-5884-4219-a763-fca1f0f4aaaf","order_by":11,"name":"Akhila Balachander","email":"","orcid":"","institution":"Singapore Immunology Network","correspondingAuthor":false,"prefix":"","firstName":"Akhila","middleName":"","lastName":"Balachander","suffix":""},{"id":572270157,"identity":"75767f1a-760f-47f0-9ba6-98bfa0a4ba4d","order_by":12,"name":"Shanshan Howland","email":"","orcid":"https://orcid.org/0000-0003-4794-1564","institution":"Singapore Immunology Network","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"Howland","suffix":""},{"id":572270158,"identity":"a192d221-d23b-4548-a00e-602c0e25235c","order_by":13,"name":"Hardy Kornfeld","email":"","orcid":"https://orcid.org/0000-0002-8970-7306","institution":"University of Massachusetts Medical School","correspondingAuthor":false,"prefix":"","firstName":"Hardy","middleName":"","lastName":"Kornfeld","suffix":""}],"badges":[],"createdAt":"2025-12-15 08:07:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8363336/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8363336/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100382230,"identity":"7373d07d-66fc-42dd-a16e-25e79b993d32","added_by":"auto","created_at":"2026-01-16 10:41:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2569090,"visible":true,"origin":"","legend":"","description":"","filename":"NR4A1paperforNatMicro.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8363336/v1_covered_c17c94b4-4ba9-4abc-a70c-93438c59d5ba.pdf"},{"id":100102412,"identity":"4a807d2d-d347-4017-920f-58c1e249664d","added_by":"auto","created_at":"2026-01-13 03:43:41","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e DEGs identified from the time-course RNA-seq analysis of splenic CD8⁺ T cells from WT and \u003cem\u003eNr4a1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice.\u003c/p\u003e","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8363336/v1/9863e7935d9996980b9a2457.xlsx"},{"id":100365010,"identity":"07000945-fa1d-4377-ba24-0611c5e18425","added_by":"auto","created_at":"2026-01-16 07:54:35","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":189158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e\u0026nbsp; DEGs in splenic CD8⁺ T cells from WT and \u003cem\u003eNr4a1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice at 4 and 8 wk after \u003cem\u003eMtb\u003c/em\u003e infection (Fig. 2d). Includes cluster assignments for 4 wk DEGs (clusters i–x; Extended Data Fig. 2b) and 8 wk DEGs (clusters A–J; Fig. 2e).\u003c/p\u003e","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8363336/v1/400e55fe7cadaf05bc8d15ac.xlsx"},{"id":100366472,"identity":"2a35ae10-fd17-4be8-bcc0-c6b76a92a7fd","added_by":"auto","created_at":"2026-01-16 07:56:19","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 3 \u003c/strong\u003eDEGs from scRNA-seq analysis of lung CD8⁺ T cell subsets from WT and \u003cem\u003eNr4a1\u003c/em\u003e\u003csup\u003e\u003cem\u003e⁻/⁻\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice at 4 wk. Includes gene lists for \u003cem\u003eGzmk⁺\u003c/em\u003e effector-memory (cluster g) and \u003cem\u003eGzma⁺\u003c/em\u003e effector (cluster m) populations (Fig. 4e).\u003c/p\u003e","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8363336/v1/7020d917d087c181d2832520.xlsx"},{"id":100366542,"identity":"59fba6bd-02f6-48d7-9dde-a79006166ee0","added_by":"auto","created_at":"2026-01-16 07:56:20","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":69632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 4 \u003c/strong\u003eDEGs from GeoMx DSP whole-transcriptome analysis of CD3⁺ T cell ROIs in lung lesions at 4 wk.\u003c/p\u003e","description":"","filename":"SupplementaryTable4.xls","url":"https://assets-eu.researchsquare.com/files/rs-8363336/v1/4fa6dea630df3a05db15936e.xls"},{"id":100102414,"identity":"b13dbdd9-238d-4d25-a629-2c1f0380f6cb","added_by":"auto","created_at":"2026-01-13 03:43:41","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":56608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 5 \u003c/strong\u003eDEGs stratified by NR4A1 expression state (NR4A1\u003csup\u003elo\u003c/sup\u003e versus NR4A1\u003csup\u003ehi\u003c/sup\u003e) in CD8⁺ T cells from macaques and human TB datasets\u003c/p\u003e","description":"","filename":"SupplementaryTable5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8363336/v1/22f1c98d93fd238c01c697d4.xlsx"},{"id":100365311,"identity":"68b2ad75-1a24-4b7e-8616-d266861e08c9","added_by":"auto","created_at":"2026-01-16 07:55:02","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":10152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 6 \u003c/strong\u003eSequences of primers\u003c/p\u003e","description":"","filename":"SupplementaryTable6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8363336/v1/eecbcf8564fbfdebb97baa49.xlsx"},{"id":100102417,"identity":"f092ab2e-faf0-48ae-b13d-d7328c169086","added_by":"auto","created_at":"2026-01-13 03:43:41","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1173764,"visible":true,"origin":"","legend":"Extended Data Figures","description":"","filename":"NR4APaperextendedfigures19thNov.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8363336/v1/08423177f09bc27e448d0afa.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"NR4A1 limits CD8⁺ T Cell effector responses and protection in tuberculosis","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"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":"Tuberculosis, CD8⁺ T cells, NR4A1, granuloma, T cell infiltration, host-directed therapy","lastPublishedDoi":"10.21203/rs.3.rs-8363336/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8363336/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"During Mycobacterium tuberculosis (Mtb) infection, CD8⁺ T cells exhibit dysfunction with impaired cytotoxicity and limited localization to granuloma cores. Using knockout mice, adoptive-transfer models and validation in macaque and human datasets, we identified the nuclear receptor NR4A1 as a key restrainer of CD8+ T cell immunity in tuberculosis (TB). Mtb-infected Nr4a1-/- mice displayed reduced bacterial burden, attenuated pathology, higher lung CD8⁺/CD4⁺ T cell ratios, and enhanced CD8⁺ T cell effector functions. Bulk and single-cell RNA sequencing revealed suppression of gene expression program linked with exhaustion, and expansion of Nkg7+ and Granzyme+ cytotoxic CD8⁺ T cell subsets in Nr4a1-/- mice. Spatial analyses demonstrated increased infiltration of Nkg7+ activated CD8⁺ T cells in Nr4a1-/- lesions. ChIP-qPCR showed NR4A1 binding to Nkg7 promoter, and Nkg7 knockdown abrogated the enhanced cytotoxicity of Nr4a1-/- CD8+ T cells. Pharmacologic inhibition of NR4A1 reduced Mtb burden and pathology, and restored Nkg7 expression and CD8+ T cell infiltration in the lung. Together, these findings identify NR4A1 as a negative regulator of CD8⁺ T cell–mediated immunity in TB and suggest the NR4A1-NKG7 axis as a novel host-directed therapeutic target.","manuscriptTitle":"NR4A1 limits CD8⁺ T Cell effector responses and protection in tuberculosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 03:43:37","doi":"10.21203/rs.3.rs-8363336/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-microbiology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nmicrobiol","sideBox":"Learn more about [Nature Microbiology](http://www.nature.com/nmicrobiol/)","snPcode":"","submissionUrl":"","title":"Nature Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c021b033-e80e-4280-a914-92931eb389f9","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60901571,"name":"Biological sciences/Immunology/Infectious diseases/Tuberculosis"},{"id":60901572,"name":"Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte activation"}],"tags":[],"updatedAt":"2026-02-13T12:35:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 03:43:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8363336","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8363336","identity":"rs-8363336","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}