Endotype dependent loss of tolerance in cow milk allergies is driven by antigen-specific CD4+ T cell outgrowth and gut microbial dysbiosis

Endotype dependent loss of tolerance in cow milk allergies is driven by antigen-specific CD4+ T cell outgrowth and gut microbial dysbiosis | 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 Endotype dependent loss of tolerance in cow milk allergies is driven by antigen-specific CD4+ T cell outgrowth and gut microbial dysbiosis Nicholas Panhuys, Tracy Augustine, Asmma Doudin, Selvasankar Murugesan, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7103135/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 Cow milk allergy (CMA) is one of the most common infant food allergies, presenting in IgE- and non-IgE-mediated forms. Currently, the allergen-reactive CD4+ T cell inflammatory responses that distinguish CMA endotypes are not well characterized. In this study, we isolated allergen-reactive conventional (Tcon) and regulatory (Treg) CD4+ T cells from IgE and non-IgE mediated pediatric CMA patients and performed an in-depth cellular and molecular characterization of the composition and function of both T helper (TH) and TH-like subsets. Bacteria present in the gut microbiota were also analysed to assess potential differences between CMA endotypes, and associations between TH and TH-like subsets were mapped against the composition of gut bacterial species. Our findings show that alterations in the balance between allergen-reactive Tcon and Treg cells underlies the development of food allergen sensitization in both IgE- and non-IgE-mediated forms of CMA. Additionally, differences in the composition of TH and TH-like subsets, as well as in the bacterial species present, showed significant associations with the development of specific CMA endotypes. Together, these findings provide a rational explanation for the distinct immunopathologies observed in endotype-specific immune responses against a common allergen and offer insights into the development of therapeutic strategies for distinct CMA endotypes. Biological sciences/Immunology/Immunological disorders/Inflammatory diseases/Allergy Biological sciences/Immunology/Lymphocytes/T cells/CD4-positive T cells/Regulatory T cells Biological sciences/Immunology/Translational immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations (Not answered) Supplementary Files TableS1GenelistofRNAseqDEGs.xlsx Table S1-Gene list of RNAseq DEGs TableS2GSEAgenelists.xlsx Table S2-GSEA gene lists TableS4LEfSeanalysisofgutbacteriaatspecieslevel.xlsx Table S4-LEfSe analysis of gut bacteria at species level SupplementaryMaterialsFile.pdf Supplemental Materials File SupplementaryFiguresFile.pdf Supplementary Figures File TableS3Genuslevelanalysisofgutbacteria.xlsx Table S3-Genus level analysis of gut bacteria 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. 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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-7103135","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":497186444,"identity":"c9e53042-cb85-4d6f-b91a-38c94882b2a5","order_by":0,"name":"Nicholas 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15:41:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7103135/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7103135/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91560648,"identity":"e61bb52e-b0af-471c-b14a-49941ab6738e","added_by":"auto","created_at":"2025-09-17 18:43:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":722761,"visible":true,"origin":"","legend":"\u003cp\u003eAntigen-specific inflammatory CD4+ T cells dominate the CM-specific response in cow milk allergic pediatric subjects: (A) Schematic overview of the study design, summarizing subject groups, analysis of Cow’s Milk (CM) reactive CD4+ T cells and assessment of microbial components. (B) Total IgE levels in the study subjects. (C) Cow milk specific IgE levels. (D) CM-specific T cell response in CM allergic subjects after in vitro stimulation of PBMCs for 6hrs. (E) Comparison of Treg/Tcon markers in activated cells (CD137+ cells in blue, CD154+ cells in red). (F) Analysis of genes that define Treg and Tcon subsets in sorted CD137+ TregCM+ and CD154+ TconCM+ cells. (G) Comparative frequencies of TregCM+ and TconCM+ cells present in each group. (H) Subject-wise paired analysis of TregCM+ and TconCM+. Percentages indicate the proportion of subjects with TregCM+ \u0026lt; TconCM+ in each group. (I) TregCM+, TconCM+ ratios across CMA subject groups. Quantitative variables were compared using Kruskal Wallis with Dunn’s test for multiple comparison (B \u0026amp; C) and One-way ANOVA with Tukey’s test for multiple comparison (G \u0026amp; I). Statistical significance is defined as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. Data are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"MainFiguresFile1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/55845fbe9d50ab4e9c9fedb7.jpg"},{"id":91560650,"identity":"abcc1123-8f3a-4feb-b2ab-9866b5fce136","added_by":"auto","created_at":"2025-09-17 18:43:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":602836,"visible":true,"origin":"","legend":"\u003cp\u003eAntigen-specific inflammatory CD4+ TH cell subsets are elevated in allergic subjects: Antigen-specific T helper cell subsets in CMA patients analyzed by multiparametric flow cytometry analysis using a combination of surface and chemokine markers specific for (A) antigen-specific TH subsets, as % of total CD154+ cells. (B) Antigen-specific TH-like subsets in CMA patients, as % of total CD137+ cells. (C) Comparative analysis of the distribution of TH subsets present within CD154+ T cells and CD137+ T cells. (D) Antigen- specific TH Lin- subsets in CMA patients, present in CD154+ and CD137+ subsets. (E) Comparative ratio analysis of total TH/TH-like antigen-specific subsets in total CD4+ T cells. Quantitative variables were compared using One-way ANOVA with Tukey’s test for multiple comparison. Statistical significance is defined as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. Data are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"MainFiguresFile2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/adfe6c18c8ccdcb5e1b2f869.jpg"},{"id":91560662,"identity":"1a05953b-6e55-484b-96c8-7c50bd4acf44","added_by":"auto","created_at":"2025-09-17 18:43:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":705435,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression mapping of antigen-reactive Tcons and Tregs in CMA patients: (A) Heat-map of z-score-normalized expression levels of Treg associated markers, transcription factors, homing associated genes and cytokines from bulk RNA-seq analysis of sorted TregCM+ and TconCM+ 974 subsets. (B) Venn diagrams indicating the number of up- and down-regulated differentially expressed genes from TregCM+ (left) and TconCM+ (right) in comparison with CON subjects. (C) Scatter plot of log2 fold change between IgE+ 976 (ALL and AD) groups and IgE- (CON and ENP) groups (y-axis) compared to CM allergic subjects (ALL, AD, ENP) and CON subjects (x-axis) in TconCM+ subset (left) and TregCM+ subset (right). Colored circles indicate genes with ≥2-fold increase in expression, and an adjusted p-value (p.adj) of \u0026lt;0.05: red for IgE comparison, blue for CMA comparison and purple for both. Normalized enrichment scores (NES) from gene set enrichment analysis (GSEA) of (D) T- helper subsets and (E) non-canonical hyporesponsive T-helper subsets. q, false discovery rate (FDR) for NES, *\u0026lt;0.25, **\u0026lt;0.05, ***\u0026lt;0.05. (F) Heatmap of z-score-normalized expression using leading-edge genes from ALL, AD and ENP Treg pathway GSEAs contributing to Treg pathway enrichment in TregCM+ cells. (G) Median-centered violin plots display the RNA levels of significantly altered leading-edge genes from GSEA across the full min-max range. Statistical differences was assessed using One-way ANOVA with Dunnett’s post-hoc test, *p ≤ 0.05.\u003c/p\u003e","description":"","filename":"MainFiguresFile3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/247d7285e3b5c872936ae1cb.jpg"},{"id":91562810,"identity":"a1689d1f-1a5b-482f-bbba-aa96dfd21f18","added_by":"auto","created_at":"2025-09-17 18:59:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":617033,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential expression and association mapping of transcription factors and cytokines in CMAs: (A) Partial least squares discriminant analysis (PLS-DA) of TregCM+ and TconCM+ subsets, with 95% confidence interval ellipses (shaded), arrows indicate major genes contributing to group separation. Heat map of TregCM+ and TconCM+ subsets (B) transcription factors and (C) cytokines organized by hierarchical clustering of z-score-normalized RNAseq expression data.\u003c/p\u003e","description":"","filename":"MainFiguresFile4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/98e5a2f2faa0772c988bc225.jpg"},{"id":91560666,"identity":"4cd8158e-0b47-4460-bebf-2b21693387d9","added_by":"auto","created_at":"2025-09-17 18:43:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":534259,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial signatures associated with CM specific response in allergic patients: (A) Phyla distribution among the different study groups. (B) Alpha diversity measures using Observed, Chao, Shannon and Simpson Indexes. (C) Upset plot of microbial occurrence across sample groups at the genus level. (D) Relative abundance of microbes at the genus level based on z-score. (E) Microbial dysbiosis analysis using Centroid distance score. (F) Louvain-based clustering of microbes at the genus level and the variations of each cluster among the study groups. Quantitative variables were compared using Kruskal Wallis with Dunn’s test for multiple comparison. Statistical significance is defined as *p ≤ 0.05, and **p ≤ 0.01. Data are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"MainFiguresFile5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/bbefb7f762d5495be728eb0d.jpg"},{"id":91560654,"identity":"f8adc07e-b7eb-4d39-89ec-30b85d42ff03","added_by":"auto","created_at":"2025-09-17 18:43:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":486530,"visible":true,"origin":"","legend":"\u003cp\u003eGut microbial interactions modulate inflammatory and regulatory CM-reactive CD4+ T cell endotypes: Z-score analysis of relative abundance amongst butyrate producers (A) and non-butyrate producers (B) identified from LEfSe analysis of the gut microbiome in the study subjects. (C) Network map identifying significant positive and negative associations of the butyrate producers and non-butyrate producers with CM-reactive CD4+ T cell subsets in the study groups.\u003c/p\u003e","description":"","filename":"MainFiguresFile6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/a0f2ad496737738da2368019.jpg"},{"id":95312751,"identity":"3129bbce-8bd8-4395-b79d-993d6d3ae602","added_by":"auto","created_at":"2025-11-06 15:50:12","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4352254,"visible":true,"origin":"","legend":"Article File","description":"","filename":"MainManuscriptFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1_covered_e87da8ae-aaad-4b6e-bdec-79f02d5d5332.pdf"},{"id":91560647,"identity":"b2bda4c5-e5da-413b-868a-20832ce93fd5","added_by":"auto","created_at":"2025-09-17 18:43:42","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":49129,"visible":true,"origin":"","legend":"Table S1-Gene list of RNAseq DEGs","description":"","filename":"TableS1GenelistofRNAseqDEGs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/c9b15b3a3a4e95cac8934a6b.xlsx"},{"id":91560646,"identity":"a92a9ee3-dfc4-4165-a45b-0e1fa8595d89","added_by":"auto","created_at":"2025-09-17 18:43:42","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":44896,"visible":true,"origin":"","legend":"Table S2-GSEA gene lists","description":"","filename":"TableS2GSEAgenelists.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/524831f3f3b2aba1868734ae.xlsx"},{"id":91560665,"identity":"4485d85c-fa5d-4a81-82ed-62ae7c3178b9","added_by":"auto","created_at":"2025-09-17 18:43:43","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20461,"visible":true,"origin":"","legend":"Table S4-LEfSe analysis of gut bacteria at species level","description":"","filename":"TableS4LEfSeanalysisofgutbacteriaatspecieslevel.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/df61b6626a48e0309ba18fcd.xlsx"},{"id":91560651,"identity":"92bd618c-6b90-496f-aaae-8e2c1390c72c","added_by":"auto","created_at":"2025-09-17 18:43:42","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":169358,"visible":true,"origin":"","legend":"Supplemental Materials File","description":"","filename":"SupplementaryMaterialsFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/24b982ea2d2bad124d046821.pdf"},{"id":91560664,"identity":"94e27a49-b526-4fb8-968f-17bb745e9266","added_by":"auto","created_at":"2025-09-17 18:43:43","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":7178882,"visible":true,"origin":"","legend":"Supplementary Figures File","description":"","filename":"SupplementaryFiguresFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/e3b7e0f9fd9dd03e456a6810.pdf"},{"id":91560657,"identity":"51cbc1e2-8057-4fe6-a772-98c644dd4675","added_by":"auto","created_at":"2025-09-17 18:43:43","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":24794,"visible":true,"origin":"","legend":"Table S3-Genus level analysis of gut bacteria","description":"","filename":"TableS3Genuslevelanalysisofgutbacteria.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7103135/v1/b60d138e7cc5abc1c21bdcc3.xlsx"}],"financialInterests":"(Not answered)","formattedTitle":"Endotype dependent loss of tolerance in cow milk allergies is driven by antigen-specific CD4+ T cell outgrowth and gut microbial dysbiosis","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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-7103135/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7103135/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cow milk allergy (CMA) is one of the most common infant food allergies, presenting in IgE- and non-IgE-mediated forms. Currently, the allergen-reactive CD4+ T cell inflammatory responses that distinguish CMA endotypes are not well characterized. In this study, we isolated allergen-reactive conventional (Tcon) and regulatory (Treg) CD4+ T cells from IgE and non-IgE mediated pediatric CMA patients and performed an in-depth cellular and molecular characterization of the composition and function of both T helper (TH) and TH-like subsets. Bacteria present in the gut microbiota were also analysed to assess potential differences between CMA endotypes, and associations between TH and TH-like subsets were mapped against the composition of gut bacterial species. Our findings show that alterations in the balance between allergen-reactive Tcon and Treg cells underlies the development of food allergen sensitization in both IgE- and non-IgE-mediated forms of CMA. Additionally, differences in the composition of TH and TH-like subsets, as well as in the bacterial species present, showed significant associations with the development of specific CMA endotypes. Together, these findings provide a rational explanation for the distinct immunopathologies observed in endotype-specific immune responses against a common allergen and offer insights into the development of therapeutic strategies for distinct CMA endotypes.","manuscriptTitle":"Endotype dependent loss of tolerance in cow milk allergies is driven by antigen-specific CD4+ T cell outgrowth and gut microbial dysbiosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 18:43:38","doi":"10.21203/rs.3.rs-7103135/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":"341760b6-fc0c-4916-bcdf-cd4ebf65df69","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52816205,"name":"Biological sciences/Immunology/Immunological disorders/Inflammatory diseases/Allergy"},{"id":52816206,"name":"Biological sciences/Immunology/Lymphocytes/T cells/CD4-positive T cells/Regulatory T cells"},{"id":52816207,"name":"Biological sciences/Immunology/Translational immunology"}],"tags":[],"updatedAt":"2025-11-05T18:10:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 18:43:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7103135","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7103135","identity":"rs-7103135","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}