Immune Cell-Based Clustering Reveals Clinically Distinct Endotypes in Type 2 Diabetes

Immune Cell-Based Clustering Reveals Clinically Distinct Endotypes in Type 2 Diabetes | 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 Immune Cell-Based Clustering Reveals Clinically Distinct Endotypes in Type 2 Diabetes Nicolas Venteclef, Bao-Tran Vuong, chloe delepine, jacqueline ratter-rieck, and 43 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7752906/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 Type 2 diabetes (T2D) is a heterogeneous disease, yet current classifications fail to capture its complexity or enable precision care. Here, we introduce a clinically scalable, immune-based endotyping strategy using routine blood immune cell counts - neutrophils, lymphocytes, and monocytes - from over 1,500 individuals with newly diagnosed T2D across three independent longitudinal European cohorts. Unsupervised clustering identified four consistent and clinically meaningful immune endotypes: Severe Inflammatory Diabetes (SIND), Mild Inflammatory Diabetes (MIND), Lymphocyte-Rich Diabetes (LYRD), and Lymphocyte-Deficient Diabetes (LYDD), that named immunotypes. These endotypes were associated with divergent long-term outcomes, with SIND and LYDD showing increased cardiovascular, renal, and mortality risks, and MIND and LYRD linked to more favorable trajectories. Multi-omic profiling revealed endotype-specific inflammatory signatures, including selective expansion of CCR2hi and CD39hi classical monocytes in the SIND endotype. Integration with single-cell RNA-seq uncovered distinct monocyte subsets in SIND, enriched for chemotaxis and myeloid activation transcriptional programs, alongside skewed adaptive immunity. Strikingly, this pro-inflammatory immune profile was attenuated by IL-1β antagonism and bariatric surgery-induced diabetes remission, underscoring its therapeutic relevance. These findings position immune-based endotyping as a robust and accessible tool to stratify risk, uncover disease mechanisms, and guide personalized intervention strategies in T2D. Health sciences/Diseases/Endocrine system and metabolic diseases/Diabetes/Type 2 diabetes Biological sciences/Physiology/Metabolism/Metabolic diseases/Diabetes/Type 2 diabetes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations There is NO Competing Interest. Tables 1 to 3 are available in the Supplementary Files section. Supplementary Files TablessupVuongetal.pdf Supplemental TABLES SupmaterialsVuongetal.pdf Suplemental Material Description TablesmainVuongetal.pdf Clinical Tables FiguresextendedVuongetal.pdf Suplemental Figures 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-7752906","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":524420868,"identity":"8dad3f71-7ee8-441a-acc6-a9eacd821c54","order_by":0,"name":"Nicolas Venteclef","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-6313-1214","institution":"Necker Insitute (INEM)","correspondingAuthor":true,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Venteclef","suffix":""},{"id":524420869,"identity":"895cbfdd-f696-4f54-a207-6cd94c514f5f","order_by":1,"name":"Bao-Tran Vuong","email":"","orcid":"","institution":"Necker Insitute (INEM)","correspondingAuthor":false,"prefix":"","firstName":"Bao-Tran","middleName":"","lastName":"Vuong","suffix":""},{"id":524420870,"identity":"9d2496ec-3409-455d-a894-bd126e5c1fae","order_by":2,"name":"chloe delepine","email":"","orcid":"","institution":"Necker Insitute (INEM)","correspondingAuthor":false,"prefix":"","firstName":"chloe","middleName":"","lastName":"delepine","suffix":""},{"id":524420871,"identity":"a9c77608-8d85-46d6-ba99-ce04ec3f362a","order_by":3,"name":"jacqueline ratter-rieck","email":"","orcid":"","institution":"DDZ","correspondingAuthor":false,"prefix":"","firstName":"jacqueline","middleName":"","lastName":"ratter-rieck","suffix":""},{"id":524420872,"identity":"da88c0b3-1e74-4197-b610-a6b6478ebe11","order_by":4,"name":"elisabetta rinaldi","email":"","orcid":"","institution":"University of Verona and Hospital Trust of Verona","correspondingAuthor":false,"prefix":"","firstName":"elisabetta","middleName":"","lastName":"rinaldi","suffix":""},{"id":524420873,"identity":"fd42e6db-662d-4cc9-9cc7-e15b2de69362","order_by":5,"name":"jeanbaptiste julla","email":"","orcid":"https://orcid.org/0000-0002-4497-5710","institution":"Aphp","correspondingAuthor":false,"prefix":"","firstName":"jeanbaptiste","middleName":"","lastName":"julla","suffix":""},{"id":524420874,"identity":"c0399c0e-7216-46d8-8082-b24f96d6f66f","order_by":6,"name":"elise iu","email":"","orcid":"","institution":"APHP","correspondingAuthor":false,"prefix":"","firstName":"elise","middleName":"","lastName":"iu","suffix":""},{"id":524420875,"identity":"76553a0c-1579-4b54-b3cb-f5ffdfd75d71","order_by":7,"name":"Marc Diedisheim","email":"","orcid":"https://orcid.org/0000-0002-0418-1107","institution":"Institut Cochin, Université de Paris, INSERM, CNRS, APHP","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"","lastName":"Diedisheim","suffix":""},{"id":524420876,"identity":"a653290e-d9f9-4b7a-94ae-8d586db8bba1","order_by":8,"name":"Violeta Raverdy","email":"","orcid":"https://orcid.org/0000-0001-5754-2028","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Violeta","middleName":"","lastName":"Raverdy","suffix":""},{"id":524420877,"identity":"5e9458aa-d73d-457a-8252-966157a48815","order_by":9,"name":"justus fischer","email":"","orcid":"","institution":"unibas","correspondingAuthor":false,"prefix":"","firstName":"justus","middleName":"","lastName":"fischer","suffix":""},{"id":524420878,"identity":"73a4e5c6-4ac5-4ca4-88de-8a702acde302","order_by":10,"name":"Matthias Hepprich","email":"","orcid":"https://orcid.org/0000-0002-6905-6235","institution":"University Hospital Basel","correspondingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"","lastName":"Hepprich","suffix":""},{"id":524420879,"identity":"17c6a031-98e1-4256-a764-704330daf4d9","order_by":11,"name":"Fawaz Alzaid","email":"","orcid":"","institution":"Université de Paris, INSERM, Immunity and Metabolism in Diabetes Laboratory, Centre de Recherche des Cordelier, Sorbonne Paris Cité, F-75006, Paris","correspondingAuthor":false,"prefix":"","firstName":"Fawaz","middleName":"","lastName":"Alzaid","suffix":""},{"id":524420880,"identity":"0e037b62-d42a-4e41-848f-4ff117b1f339","order_by":12,"name":"camille bleriot","email":"","orcid":"","institution":"INEM","correspondingAuthor":false,"prefix":"","firstName":"camille","middleName":"","lastName":"bleriot","suffix":""},{"id":524420881,"identity":"7f6a381c-1865-42b5-ad58-9722520cb581","order_by":13,"name":"Clémence Cornuot Cornuot","email":"","orcid":"","institution":"IGR","correspondingAuthor":false,"prefix":"","firstName":"Clémence","middleName":"Cornuot","lastName":"Cornuot","suffix":""},{"id":524420882,"identity":"8997efc4-ad0a-4bb1-bebb-a9789c34b76d","order_by":14,"name":"joe de.keize","email":"","orcid":"","institution":"univ-poitiers","correspondingAuthor":false,"prefix":"","firstName":"joe","middleName":"","lastName":"de.keize","suffix":""},{"id":524420883,"identity":"14e6a696-4c54-41d5-a539-80ada19a64d1","order_by":15,"name":"macine-bachir abdelouahab","email":"","orcid":"","institution":"univ-lille","correspondingAuthor":false,"prefix":"","firstName":"macine-bachir","middleName":"","lastName":"abdelouahab","suffix":""},{"id":524420884,"identity":"080e312e-38ea-4845-9ca7-ed7df33ea9b9","order_by":16,"name":"magali chiral","email":"","orcid":"","institution":"INEM","correspondingAuthor":false,"prefix":"","firstName":"magali","middleName":"","lastName":"chiral","suffix":""},{"id":524420885,"identity":"a7c54dff-a9c8-465e-b306-662e8463c762","order_by":17,"name":"Rebecca .Pfeil","email":"","orcid":"","institution":"DDZ","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":".Pfeil","suffix":""},{"id":524420886,"identity":"bb7fcd11-ba83-4a7f-9bd6-cf2611de2d45","order_by":18,"name":"Polina Lipaeva","email":"","orcid":"","institution":"DDZ","correspondingAuthor":false,"prefix":"","firstName":"Polina","middleName":"","lastName":"Lipaeva","suffix":""},{"id":524420887,"identity":"2d9eef19-a7d4-4215-8358-cfd17a702524","order_by":19,"name":"daniel doerr","email":"","orcid":"","institution":"DDZ","correspondingAuthor":false,"prefix":"","firstName":"daniel","middleName":"","lastName":"doerr","suffix":""},{"id":524420888,"identity":"7b92d680-e01d-4711-a783-8adfc471a0db","order_by":20,"name":"Oana-Patricia Zaharia","email":"","orcid":"","institution":"Institute for Clinical Diabetology, German Diabetes Center, Leibniz Center for Diabetes Research","correspondingAuthor":false,"prefix":"","firstName":"Oana-Patricia","middleName":"","lastName":"Zaharia","suffix":""},{"id":524420889,"identity":"a66a9e6d-b571-48d0-8f32-40b2bbd9f9e4","order_by":21,"name":"theo jolivet","email":"","orcid":"","institution":"Aphp","correspondingAuthor":false,"prefix":"","firstName":"theo","middleName":"","lastName":"jolivet","suffix":""},{"id":524420890,"identity":"926aba7c-ef69-4863-b734-c2584cdc8a91","order_by":22,"name":"Julie Alberge Alberge","email":"","orcid":"","institution":"INRIA","correspondingAuthor":false,"prefix":"","firstName":"Julie","middleName":"Alberge","lastName":"Alberge","suffix":""},{"id":524420891,"identity":"47a4230e-c298-401c-8206-49bb5e1ba551","order_by":23,"name":"charline. potier","email":"","orcid":"","institution":"APHP","correspondingAuthor":false,"prefix":"","firstName":"charline.","middleName":"","lastName":"potier","suffix":""},{"id":524420892,"identity":"6a61a069-e5a0-4db2-bb6c-d197a314f6e3","order_by":24,"name":"kennan .khider","email":"","orcid":"","institution":"INEM","correspondingAuthor":false,"prefix":"","firstName":"kennan","middleName":"","lastName":".khider","suffix":""},{"id":524420893,"identity":"72475dc7-cbfa-40ec-8343-e2e87bd60645","order_by":25,"name":"frederik oger","email":"","orcid":"","institution":"univ-lille","correspondingAuthor":false,"prefix":"","firstName":"frederik","middleName":"","lastName":"oger","suffix":""},{"id":524420894,"identity":"0aaed36a-4a52-446b-beb8-3302cee8b0fe","order_by":26,"name":"jeanpierre riveline","email":"","orcid":"","institution":"APHP","correspondingAuthor":false,"prefix":"","firstName":"jeanpierre","middleName":"","lastName":"riveline","suffix":""},{"id":524420895,"identity":"0d6a8fd7-bcff-431f-a48b-5979ec03ef7e","order_by":27,"name":"tiphaine vidal-trecan","email":"","orcid":"","institution":"APHP","correspondingAuthor":false,"prefix":"","firstName":"tiphaine","middleName":"","lastName":"vidal-trecan","suffix":""},{"id":524420896,"identity":"61e1b06e-686d-425f-bde8-069784967c13","order_by":28,"name":"Florence Tubach","email":"","orcid":"https://orcid.org/0000-0002-7802-944X","institution":"French Institute of Health and Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Florence","middleName":"","lastName":"Tubach","suffix":""},{"id":524420897,"identity":"71d93363-4900-4bf2-86b3-915f6db4c4a7","order_by":29,"name":"candice estellat","email":"","orcid":"","institution":"Inria","correspondingAuthor":false,"prefix":"","firstName":"candice","middleName":"","lastName":"estellat","suffix":""},{"id":524420898,"identity":"5941d6b9-7c3f-4e43-9f57-594399f69f28","order_by":30,"name":"Judith Abécassis","email":"","orcid":"https://orcid.org/0000-0002-5818-8304","institution":"INRIA","correspondingAuthor":false,"prefix":"","firstName":"Judith","middleName":"","lastName":"Abécassis","suffix":""},{"id":524420899,"identity":"611054ad-cf99-43be-a670-4c193a91e01d","order_by":31,"name":"Aymeric Silvin","email":"","orcid":"https://orcid.org/0000-0003-2748-8555","institution":"Institut National de la Sante et de la Recherche Medicale (INSERM) U1015, Equipe Labellisee - Ligue Nationale contre le Cancer","correspondingAuthor":false,"prefix":"","firstName":"Aymeric","middleName":"","lastName":"Silvin","suffix":""},{"id":524420900,"identity":"2075cc29-0049-4082-bcb2-6ecd4581fd9c","order_by":32,"name":"Samy Hadjadj","email":"","orcid":"","institution":"L'Institut du Thorax, Université de Nantes, CHU Nantes, CNRS, Nantes, INSERM, Nantes Université","correspondingAuthor":false,"prefix":"","firstName":"Samy","middleName":"","lastName":"Hadjadj","suffix":""},{"id":524420901,"identity":"4ef94283-ddcb-4e97-b94b-a1dc8324f346","order_by":33,"name":"Gael Varoquaux","email":"","orcid":"","institution":"Inria Saclay - Île-de-France Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Gael","middleName":"","lastName":"Varoquaux","suffix":""},{"id":524420902,"identity":"659b169b-2dee-4a4a-b4f5-709226c74ca7","order_by":34,"name":"Robert Wagner","email":"","orcid":"","institution":"German Diabetes Center (DDZ), Leibniz Center for Diabetes Research at Heinrich-Heine University","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Wagner","suffix":""},{"id":524420903,"identity":"01e95d2b-da90-42ca-a27e-3cec0da5c0d5","order_by":35,"name":"philippe froguel","email":"","orcid":"","institution":"Univ.Lille","correspondingAuthor":false,"prefix":"","firstName":"philippe","middleName":"","lastName":"froguel","suffix":""},{"id":524420904,"identity":"bf3ec023-980e-4b07-a412-6689fd154f5f","order_by":36,"name":"Amélie Bonnefond","email":"","orcid":"https://orcid.org/0000-0001-9976-3005","institution":"Inserm/CNRS","correspondingAuthor":false,"prefix":"","firstName":"Amélie","middleName":"","lastName":"Bonnefond","suffix":""},{"id":524420905,"identity":"3eb26c21-9e3f-40f9-9505-c8425b0b69d6","order_by":37,"name":"pierre.jean saulnier","email":"","orcid":"https://orcid.org/0000-0003-1862-4252","institution":"Univ Poitiers","correspondingAuthor":false,"prefix":"","firstName":"pierre.jean","middleName":"","lastName":"saulnier","suffix":""},{"id":524420906,"identity":"a6e45ea3-b935-49b3-812d-feb45d248dc7","order_by":38,"name":"François Pattou","email":"","orcid":"https://orcid.org/0000-0001-8388-3766","institution":"Univ Lille, CHU LIlle, Inserm U1190, Lille Pasteur Institute","correspondingAuthor":false,"prefix":"","firstName":"François","middleName":"","lastName":"Pattou","suffix":""},{"id":524420907,"identity":"7497ffdf-40ff-46f2-8ca8-4fcf87a10a6c","order_by":39,"name":"Florent Ginhoux","email":"","orcid":"","institution":"Institut National de la Sante et de la Recherche Medicale (INSERM) U1015, Equipe Labellisee - Ligue Nationale contre le Cancer","correspondingAuthor":false,"prefix":"","firstName":"Florent","middleName":"","lastName":"Ginhoux","suffix":""},{"id":524420908,"identity":"237ef7c3-beab-42fd-b70b-fec27a810d1c","order_by":40,"name":"Michael Roden","email":"","orcid":"https://orcid.org/0000-0001-8200-6382","institution":"[email protected]","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Roden","suffix":""},{"id":524420909,"identity":"2c2975f6-6c92-4f6d-acdf-a2eaac6334f3","order_by":41,"name":"maddalena trombetta","email":"","orcid":"","institution":"Univ. Verona","correspondingAuthor":false,"prefix":"","firstName":"maddalena","middleName":"","lastName":"trombetta","suffix":""},{"id":524420910,"identity":"e7770e7b-c24b-43d9-9d0e-36bb8cca6aac","order_by":42,"name":"louis potier","email":"","orcid":"","institution":"APHP","correspondingAuthor":false,"prefix":"","firstName":"louis","middleName":"","lastName":"potier","suffix":""},{"id":524420911,"identity":"6b4901f1-e692-43cb-8009-37228cffedab","order_by":43,"name":"Christian Herder","email":"","orcid":"https://orcid.org/0000-0002-2050-093X","institution":"German Diabetes Center","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Herder","suffix":""},{"id":524420912,"identity":"6cc03511-1060-496d-91eb-b007309e04d4","order_by":44,"name":"Jean-Francois Gautier","email":"","orcid":"","institution":"Assistance Publique-Hôpitaux de Paris, DHU FIRE, Lariboisière Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jean-Francois","middleName":"","lastName":"Gautier","suffix":""},{"id":524420913,"identity":"9ddc64e4-db7f-4f0a-954b-c75362318dfc","order_by":45,"name":"Marc Donath","email":"","orcid":"","institution":"Centre de Recherche, Centre Hospitalier de l'Universite de Montreal","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"","lastName":"Donath","suffix":""},{"id":524420914,"identity":"5438a021-b7b6-4fca-82c9-0f9d9163699f","order_by":46,"name":"Riccardo Bonadonna","email":"","orcid":"https://orcid.org/0000-0002-9809-1005","institution":"University of Verona","correspondingAuthor":false,"prefix":"","firstName":"Riccardo","middleName":"","lastName":"Bonadonna","suffix":""}],"badges":[],"createdAt":"2025-09-30 15:15:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7752906/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7752906/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93023905,"identity":"917a8c9a-5909-48c4-aace-93a4de52a813","added_by":"auto","created_at":"2025-10-08 09:13:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":314210,"visible":true,"origin":"","legend":"\u003cp\u003eStudy flowchart\u003c/p\u003e\n\u003cp\u003eUnsupervised clustering (k-means) of circulating immune cell counts (neutrophils, lymphocytes,\u003c/p\u003e\n\u003cp\u003emonocytes) identified four T2D endotypes (immunotypes) in the PNDS discovery cohort (n = 496, Paris,\u003c/p\u003e\n\u003cp\u003eFrance). The clustering model was validated by projecting two independent cohorts onto the k-means\u003c/p\u003e\n\u003cp\u003emodel: VNDS (n = 661, Verona, Italy) and GDS (n = 406, Düsseldorf, Germany). Endotype stability and\u003c/p\u003e\n\u003cp\u003etrajectories toward complications were evaluated across the VNDS, GDS, ANGIOSAFE, CODIA,\u003c/p\u003e\n\u003cp\u003eSURDIAGENE, and DESIR cohorts. Multi-omic profiling, including proteomics, immunophenotyping, and\u003c/p\u003e\n\u003cp\u003etranscriptomics (bulk RNA-seq and single-cell RNA-seq), was employed to characterize endotype-specific\u003c/p\u003e\n\u003cp\u003ebiomarkers.\u003c/p\u003e","description":"","filename":"FiguresmainsVuongetal1.png","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1/8d3b4fb5cac62b0684eeb7f1.png"},{"id":93025021,"identity":"1d15d181-5a6a-49bf-80c2-2ad1c997ddde","added_by":"auto","created_at":"2025-10-08 09:21:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1564799,"visible":true,"origin":"","legend":"\u003cp\u003eCharacteristics of four endotypes in newly-diagnosed T2D cohorts\u003c/p\u003e\n\u003cp\u003ea-d, Distribution of immune-based endotypes and their immune profiles across newly-diagnosed T2D\u003c/p\u003e\n\u003cp\u003ecohorts: PNDS (n = 496) (a), VNDS (n = 661) (b), GDS (n = 406) (c), and merged cohort (n = 1,563) (d). e,\u003c/p\u003e\n\u003cp\u003eClinical and biological characteristics of endotypes across newly-dignosed T2D cohorts. f, Insulin sensitivity\u003c/p\u003e\n\u003cp\u003eand total-phase insulin secretion of endotypes in the VNDS and GDS cohort. In the radar plots, dots indicate\u003c/p\u003e\n\u003cp\u003emedian values of immune cell counts. In the line plots, dots represent median values and error bars indicate\u003c/p\u003e\n\u003cp\u003ethe interquartile range (Q1, Q3). Pairwise comparisons between endotypes were performed using Dunn’s\u003c/p\u003e\n\u003cp\u003etest.\u003c/p\u003e","description":"","filename":"FiguresmainsVuongetal2.png","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1/217603bfc6aa214d9ba1fa0f.png"},{"id":93022880,"identity":"593303ef-5ab1-4bbe-bf8a-993338dce0ce","added_by":"auto","created_at":"2025-10-08 09:05:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3990466,"visible":true,"origin":"","legend":"\u003cp\u003eLongitudinal stability of circulating immune cell counts across endotypes and their\u003c/p\u003e\n\u003cp\u003etrajectories toward T2D-related complications\u003c/p\u003e\n\u003cp\u003ea,b, Stability over time of circulating immune cell counts across the four endotypes in the combined cohort\u003c/p\u003e\n\u003cp\u003eof ANGIOSAFE (n = 3,307 T2D participants), GDS (n = 406 participants with T2D and 205 individuals\u003c/p\u003e\n\u003cp\u003ewithout T2D), and DESIR (n = 99 individuals without T2D) (a), and in the CODIA cohort (n = 48,971\u003c/p\u003e\n\u003cp\u003eparticipants with T2D) (b). Colored lines in a-b represents the median values of circulating immune cell\u003c/p\u003e\n\u003cp\u003ecounts at each time point. Shaded ranges in a-b reflect 95% confidence intervals around the median. c,d,\u003c/p\u003e\n\u003cp\u003eIncidence of cardiovascular events (c) and renal events (d, left) across endotypes at diagnosis, 5-year, and\u003c/p\u003e\n\u003cp\u003e10-year follow-up in the VNDS cohort. d, right, Renal functions (eGFR) across endotypes at diagnosis and\u003c/p\u003e\n\u003cp\u003e5-year follow-up in the GDS cohort. e-g, left, Forest plots illustrating hazard ratios (HRs) for cardiovascular\u003c/p\u003e\n\u003cp\u003eevents (e), all-cause mortality (f), and renal events (g) across endotypes, derived from multivariable Cox\u003c/p\u003e\n\u003cp\u003eproportional hazards models in the CODIA (n = 67,375), SURDIAGENE (n = 920), and ANGIOSAFE (n =\u003c/p\u003e\n\u003cp\u003e3,307) cohorts. Renal events were defined as a composite of eGFR ≤ 60 mL/min/1.73 m² or ACR ≥ 30\u003c/p\u003e\n\u003cp\u003emg/mmol in CODIA and ANGIOSAFE, and as a ≥40% decline in eGFR in SURDIAGENE. Models in CODIA\u003c/p\u003e\n\u003cp\u003ewere adjusted for age, gender, smoking status and SBP. Models in SURDIAGENE of CV events and\u003c/p\u003e\n\u003cp\u003emortality were adjusted for age, gender, smoking, SBP, eGFR, BMI, diabetes duration, lipid profile,\u003c/p\u003e\n\u003cp\u003ealbuminuria, statin, ACEI/ARB, diuretics; models in SURDIAGENE of renal events were adjusted for age,\u003c/p\u003e\n\u003cp\u003egender, baseline eGFR and ACR. Models in ANGIOSAFE were adjusted for age, gender. Dots represent\u003c/p\u003e\n\u003cp\u003eHRs, error bars represent the 95% confidence intervals. e-g, right, Cumulative incidence of cardiovascular\u003c/p\u003e\n\u003cp\u003eevents (e), all-cause mortality (f), and renal events (g) across endotypes in the CODIA cohort. In the line\u003c/p\u003e\n\u003cp\u003eplots, dots represent median values and error bars indicate the interquartile range (Q1, Q3). Pairwise\u003c/p\u003e\n\u003cp\u003ecomparisons between endotypes were performed using Dunn’s test.\u003c/p\u003e","description":"","filename":"FiguresmainsVuongetal4.png","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1/9bc699ceb28702dbbe73045a.png"},{"id":93022883,"identity":"2e725ab5-0840-4248-8695-80c2e276afdc","added_by":"auto","created_at":"2025-10-08 09:05:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":773713,"visible":true,"origin":"","legend":"\u003cp\u003eInflammation-related proteomic signatures of T2D endotypes across cohorts\u003c/p\u003e\n\u003cp\u003ea, Schematic overview of the immunoproteomic analysis performed on serum samples. b, Endotypeenriched\u003c/p\u003e\n\u003cp\u003eproteins across cohorts. c, Common proteins enriched in SIND across the cohorts (left). Endotypeenriched\u003c/p\u003e\n\u003cp\u003eproteins in the PNDS (n = 384) , the VNDS (n = 660) and the GDS cohort (n = 354) (right). d,\u003c/p\u003e\n\u003cp\u003eComparison of endotype-enriched proteins between individuals without T2D and participants with newly\u003c/p\u003e\n\u003cp\u003ediagnosed T2D in the DESIR (N = 96 without T2D, N = 156 with T2D) and GDS cohort (N = 369 without\u003c/p\u003e\n\u003cp\u003eT2D, N = 354 with T2D). e, Longitudinal stability of endotype-enriched proteins in the ANGIOSAFE cohort\u003c/p\u003e\n\u003cp\u003eat Visit 1 (N = 1,003) and Visit 2 (N = 477; ~5 years later). Visit 2 data were scaled using the mean and\u003c/p\u003e\n\u003cp\u003estandard deviation of Visit 1. Heatmaps display median normalized NPX values.\u003c/p\u003e","description":"","filename":"FiguresmainsVuongetal6.png","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1/448b4af9711153ef40e5319a.png"},{"id":93023907,"identity":"00fd3562-a7fe-4752-b741-ea35c4dad9e3","added_by":"auto","created_at":"2025-10-08 09:13:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":13460138,"visible":true,"origin":"","legend":"\u003cp\u003eMonocyte-driven inflammation and dysregulated lymphocyte states in the SIND endotype\u003c/p\u003e\n\u003cp\u003ea, Immunophenotyping of PBMCs across the newly-diagnosed T2D endotypes (n = 191) in the PNDS\u003c/p\u003e\n\u003cp\u003ecohort; heatmap of median normalized absolute counts of PBMC subpopulations (left); line plots of PBMC\u003c/p\u003e\n\u003cp\u003esubpopulation counts (right). Asterisks indicate statistically significant differences compared with MIND\u003c/p\u003e\n\u003cp\u003eusing Dunn’s test. b, Nichenet analysis indentifying predicted PBMC subpopulations targets of proteins\u003c/p\u003e\n\u003cp\u003eenriched in SIND. c, UMAP visualization of monocyte subsets derived from scRNA-seq, with heatmap of\u003c/p\u003e\n\u003cp\u003etop marker genes. d, Monocyte subset counts from scRNA-seq in non-T2D group (n = 4), MIND (n = 8) and\u003c/p\u003e\n\u003cp\u003eSIND (n = 10) (left); log₂ fold-change of SIND versus MIND across monocyte subsets (right). e, UMAP plot\u003c/p\u003e\n\u003cp\u003eof monocyte subsets identified by flow cytometry of MIND and SIND (left); absolute counts of monocyte\u003c/p\u003e\n\u003cp\u003eand lymphocyte subsets in PNDS cohort across MIND (n = 13) and SIND (n = 10), in the PNDS +\u003c/p\u003e\n\u003cp\u003eANGIOSAFE cohorts across MIND (n = 24) and SIND (n = 31) (right). f, Enriched pathways of DEGs SIND\u003c/p\u003e\n\u003cp\u003eversus MIND in Mono CCR2hi and Mono CD39hi (scRNA-seq). g, Schematic overview of intergrating\u003c/p\u003e\n\u003cp\u003eCD14+ monocyte bulk RNA-seq with monocyte scRNA-seq; gene set enrichment analysis (GSEA) of\u003c/p\u003e\n\u003cp\u003eCD14⁺ monocytes bulk RNA-seq comparing SIND (n = 24) versus MIND (n = 18) (left); projection of DEGs\u003c/p\u003e\n\u003cp\u003eonto monocyte scRNA-seq dataset (right). In the line plots, dots represent median values and error bars\u003c/p\u003e\n\u003cp\u003eindicate the interquartile range (Q1, Q3). Pairwise comparisons between endotypes were performed using\u003c/p\u003e\n\u003cp\u003eDunn’s test. Comparisons between SIND and MIND were performed using Mann–Whitney U test.\u003c/p\u003e","description":"","filename":"FiguresmainsVuongetal7.png","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1/991d635626eba063f729da0b.png"},{"id":93022884,"identity":"21589536-0de3-4a1b-9e6f-29c641d6de96","added_by":"auto","created_at":"2025-10-08 09:05:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3993547,"visible":true,"origin":"","legend":"\u003cp\u003eIL-1β antagonism and bariatric surgery effects on monocyte-driven inflammation and\u003c/p\u003e\n\u003cp\u003eimmune-based endotype shifts\u003c/p\u003e\n\u003cp\u003ea, Study design of the Hyper-preDIL trial and distribution of immune-based endotypes among participants\u003c/p\u003e\n\u003cp\u003ewith prediabetes at the screening visit (n = 20). At both Visit 1 and Visit 2, blood samples were collected\u003c/p\u003e\n\u003cp\u003efollowing treatment administration the day before and again 60 minutes prior (total dose: anakinra 100 mg\u003c/p\u003e\n\u003cp\u003eor placebo). b, Changes in immune cell counts following placebo and anakinra treatment (left); proportion\u003c/p\u003e\n\u003cp\u003eof changes in immune cell counts and CRP across endotypes between anakinra versus placebo (right). c,\u003c/p\u003e\n\u003cp\u003eShifts in immune-based endotypes from baseline to post-anakinra treatment. d, UMAP visualization of\u003c/p\u003e\n\u003cp\u003emonocyte subsets derived from flow-cytometry in placebo and anakinra treatment (left). e, Changes in\u003c/p\u003e\n\u003cp\u003emonocyte and lymphocyte subset counts following placebo and anakinra treatment; proportion of changes\u003c/p\u003e\n\u003cp\u003ein cell subsets between anakinra versus placebo. f, Study design of the ABOS study and distribution of\u003c/p\u003e\n\u003cp\u003eimmune-based endotypes among individuals with T2D and obesity before bariatric surgery (n = 529). g-h,\u003c/p\u003e\n\u003cp\u003eEvolutions of HbA1c and BMI (g), immune cell counts (h) across endotypes before and during the 5-year\u003c/p\u003e\n\u003cp\u003efollow-up after bariatric surgery. i, Proportions of changes in immune cell counts across the endotypes\u003c/p\u003e\n\u003cp\u003ebefore versus 1 year after bariatric surgery. j, Shifts in immune-based endotypes from before to 1 year after\u003c/p\u003e\n\u003cp\u003ebariatric surgery. In the line graphs, each line represents a paired value of one individual. Paired\u003c/p\u003e\n\u003cp\u003ecomparisons were assessed using the Wilcoxon signed-rank test. In the bar graphs, bar graphs represent\u003c/p\u003e\n\u003cp\u003ethe mean percentage change in immune cell counts following placebo and anakinra treatment; error bars\u003c/p\u003e\n\u003cp\u003eindicate the standard error of the mean (SEM).\u003c/p\u003e","description":"","filename":"FiguresmainsVuongetal9.png","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1/5fafadd6616254355d4e953b.png"},{"id":108804520,"identity":"01efa98e-6b58-4cec-a628-23d4f908fe9e","added_by":"auto","created_at":"2026-05-08 15:21:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12112629,"visible":true,"origin":"","legend":"Article File","description":"","filename":"VuongandDelepineetal20253009.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1_covered_4937afc8-37c5-4846-8bb9-9fc6c37eaa09.pdf"},{"id":93022878,"identity":"5b95eb02-4922-4004-ac99-c291ea7195b8","added_by":"auto","created_at":"2025-10-08 09:05:15","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":228797,"visible":true,"origin":"","legend":"Supplemental TABLES","description":"","filename":"TablessupVuongetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1/6958c66d7705f0654fac523f.pdf"},{"id":93022882,"identity":"5a4bdb7e-4cd9-413e-9bd2-815369db848a","added_by":"auto","created_at":"2025-10-08 09:05:15","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":148423,"visible":true,"origin":"","legend":"Suplemental Material Description","description":"","filename":"SupmaterialsVuongetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1/501487257d9efecbf1f83c98.pdf"},{"id":93022877,"identity":"2db5d7bc-ec27-4fa2-bc00-f59afd86fa13","added_by":"auto","created_at":"2025-10-08 09:05:14","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":32720,"visible":true,"origin":"","legend":"Clinical Tables","description":"","filename":"TablesmainVuongetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1/a7a761858c1c9c08f05bd971.pdf"},{"id":93022885,"identity":"bfd66c62-4b45-4037-bbc2-f7201272699f","added_by":"auto","created_at":"2025-10-08 09:05:15","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10129600,"visible":true,"origin":"","legend":"Suplemental Figures","description":"","filename":"FiguresextendedVuongetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7752906/v1/46332500459dcba0ac4d4140.pdf"}],"financialInterests":"\u003cp\u003eThere is \u003cstrong\u003eNO\u003c/strong\u003e Competing Interest.\u003c/p\u003e\n\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e","formattedTitle":"Immune Cell-Based Clustering Reveals Clinically Distinct Endotypes in Type 2 Diabetes","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-7752906/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7752906/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Type 2 diabetes (T2D) is a heterogeneous disease, yet current classifications fail to capture its complexity or enable precision care. Here, we introduce a clinically scalable, immune-based endotyping strategy using routine blood immune cell counts - neutrophils, lymphocytes, and monocytes - from over 1,500 individuals with newly diagnosed T2D across three independent longitudinal European cohorts. Unsupervised clustering identified four consistent and clinically meaningful immune endotypes: Severe Inflammatory Diabetes (SIND), Mild Inflammatory Diabetes (MIND), Lymphocyte-Rich Diabetes (LYRD), and Lymphocyte-Deficient Diabetes (LYDD), that named immunotypes. These endotypes were associated with divergent long-term outcomes, with SIND and LYDD showing increased cardiovascular, renal, and mortality risks, and MIND and LYRD linked to more favorable trajectories. Multi-omic profiling revealed endotype-specific inflammatory signatures, including selective expansion of CCR2hi and CD39hi classical monocytes in the SIND endotype. Integration with single-cell RNA-seq uncovered distinct monocyte subsets in SIND, enriched for chemotaxis and myeloid activation transcriptional programs, alongside skewed adaptive immunity. Strikingly, this pro-inflammatory immune profile was attenuated by IL-1β antagonism and bariatric surgery-induced diabetes remission, underscoring its therapeutic relevance. These findings position immune-based endotyping as a robust and accessible tool to stratify risk, uncover disease mechanisms, and guide personalized intervention strategies in T2D.","manuscriptTitle":"Immune Cell-Based Clustering Reveals Clinically Distinct Endotypes in Type 2 Diabetes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 09:05:10","doi":"10.21203/rs.3.rs-7752906/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":"cd7e8336-0940-43c7-b21d-a3bef20e0a53","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55741698,"name":"Health sciences/Diseases/Endocrine system and metabolic diseases/Diabetes/Type 2 diabetes"},{"id":55741699,"name":"Biological sciences/Physiology/Metabolism/Metabolic diseases/Diabetes/Type 2 diabetes"}],"tags":[],"updatedAt":"2026-05-05T12:21:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 09:05:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7752906","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7752906","identity":"rs-7752906","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}