{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T11:39:02Z","timestamp":1783424342961,"version":"3.54.6"},"reference-count":51,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,7,24]],"date-time":"2025-07-24T00:00:00Z","timestamp":1753315200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Objective<\/jats:title><jats:p>Rheumatoid arthritis (RA) is classified into seropositive (SP-RA) and seronegative (SN-RA) types, reflecting distinct immunological profiles. This study aimed to identify the T cell phenotypes associated with each type, thereby enhancing our understanding of their unique pathophysiological mechanisms.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We analyzed peripheral blood T cells from 50 participants, including 16 patients with untreated SP-RA, 17 patients with SN-RA, and 17 healthy controls, utilizing 25 T cell markers. For initial analysis, a dataset was established through manual T cell subset gating analysis. For advanced analysis, two distinct datasets derived from a self-organizing map algorithm, FlowSOM, were used: one encompassing all CD3+ T cells and another focusing on activated T cell subsets. Subsequently, these datasets were rigorously analyzed using adaptive least absolute shrinkage and selection operator in conjunction with leave-one-out cross-validation. This approach enhanced analysis robustness, identifying T cell clusters consistently discriminative between SP-RA and SN-RA.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Our analysis revealed significant differences in T cell subsets between RA patients and healthy controls, including elevated levels of activated T cells (CD3+, CD4+, CD8+) and helper subsets (Th1, Th17, Th17.1, and Tph cells). The Tph\/Treg ratio was markedly higher in SP-RA, underscoring an effector-dominant immune imbalance. FlowSOM-based clustering identified 44 unique T cell clusters, six of which were selected as discriminative T cell clusters (D-TCLs) for distinguishing SP-RA from SN-RA. TCL21, an activated Th1-type Tph-like cell, was strongly associated with SP-RA\u2019s aggressive profile, while TCL02, a central memory CD4+ T cell subset, displayed ICOS+, CTLA-4low+, PD-1low+, and CXCR3+, providing insights into immune memory mechanisms. Additionally, TCL31 and TCL35, both CD4\u2212CD8\u2212 T cells, exhibited unique phenotypes: CD161+ for TCL31 and HLA-DR+CD38+TIM-3+ for TCL35, suggesting distinct pro-inflammatory roles. Support vector machine analysis (bootstrap n = 1000) validated the D-TCLs\u2019 discriminative power, achieving an accuracy of 86.2%, sensitivity of 85.7%, and specificity of 80.9%.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>This study advances our understanding of immunological distinctions between SP-RA and SN-RA, identifying key T cell phenotypes as potential targets for SP-RA disease progression. These findings provide a basis for studies on targeted therapeutic strategies tailored to modulate the markers and improve treatment for SP-RA.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2025.1491041","type":"journal-article","created":{"date-parts":[[2025,7,24]],"date-time":"2025-07-24T05:33:10Z","timestamp":1753335190000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Comprehensive and advanced T cell cluster analysis for discriminating seropositive and seronegative rheumatoid arthritis"],"prefix":"10.3389","volume":"16","author":[{"given":"Shinji","family":"Maeda","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroya","family":"Hashimoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoyo","family":"Maeda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shin-ya","family":"Tamechika","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taio","family":"Naniwa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akio","family":"Niimi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,7,24]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/nrdp.2018.1","article-title":"Rheumatoid arthritis","volume":"4","author":"Smolen","year":"2018","journal-title":"Nat Rev Dis Primers"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1001\/jama.2018.13103","article-title":"Diagnosis and management of rheumatoid arthritis: a review","volume":"320","author":"Aletaha","year":"2018","journal-title":"JAMA"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1136\/ard.2010.138461","article-title":"2010 rheumatoid arthritis classification criteria: an American College of Rheumatology\/European League Against Rheumatism collaborative initiative","volume":"69","author":"Aletaha","year":"2010","journal-title":"Ann Rheum Dis"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1002\/art.27584","article-title":"2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology\/European League Against Rheumatism collaborative initiative","volume":"62","author":"Aletaha","year":"2010","journal-title":"Arthritis Rheum"},{"key":"B5","doi-asserted-by":"publisher","first-page":"60","DOI":"10.1038\/nri.2016.124","article-title":"The immunopathogenesis of seropositive rheumatoid arthritis: from triggering to targeting","volume":"17","author":"Malmstr\u00f6m","year":"2017","journal-title":"Nat Rev Immunol"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1002\/art.23284","article-title":"Induction of macrophage secretion of tumor necrosis factor \u03b1 through Fc\u03b3 receptor IIa engagement by rheumatoid arthritis\u2013specific autoantibodies to citrullinated proteins complexed with fibrinogen","volume":"58","author":"Clavel","year":"2008","journal-title":"Arthritis Rheum"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1172\/JCI60975","article-title":"Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin","volume":"122","author":"Harre","year":"2012","journal-title":"J Clin Invest"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2017.02.006","article-title":"Immunopathogenesis of rheumatoid arthritis","volume":"46","author":"Firestein","year":"2017","journal-title":"Immunity"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1038\/s41590-020-00816-x","article-title":"The immunology of rheumatoid arthritis","volume":"22","author":"Weyand","year":"2021","journal-title":"Nat Immunol"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1136\/annrheumdis-2018-214885","article-title":"Multi-dimensional analysis identified rheumatoid arthritis-driving pathway in human T cell","volume":"78","author":"Takeshita","year":"2019","journal-title":"Ann Rheum Dis"},{"key":"B11","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1016\/j.jaut.2017.12.006","article-title":"Molecular mechanisms underpinning T helper 17 cell heterogeneity and functions in rheumatoid arthritis","volume":"87","author":"van Hamburg","year":"2018","journal-title":"J Autoimmun"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.192.Supp.185.26","article-title":"Multi-drug resistant Th17 cells: new players in autoimmune and steroid-resistant inflammation (HUM8P.351)","volume":"192","author":"Sundrud","year":"2014","journal-title":"J Immunol"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1600737","article-title":"Ex-Th17 (nonclassical Th1) cells are functionally distinct from classical Th1 and Th17 cells and are not constrained by regulatory T cells","volume":"198","author":"Basdeo","year":"2017","journal-title":"J Immunol"},{"key":"B14","doi-asserted-by":"publisher","first-page":"e0215192","DOI":"10.1371\/journal.pone.0215192","article-title":"Circulating Th17.1 cells as candidate for the prediction of therapeutic response to abatacept in patients with rheumatoid arthritis: an exploratory research","volume":"14","author":"Maeda","year":"2019","journal-title":"PloS One"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1038\/nm.3432","article-title":"Pathogenic conversion of Foxp3 + T cells into T H 17 cells in autoimmune arthritis","volume":"20","author":"Komatsu","year":"2013","journal-title":"Nat Med"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1038\/nature20810","article-title":"Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis","volume":"542","author":"Rao","year":"2017","journal-title":"Nature"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1038\/s41423-020-00529-z","article-title":"Shared and distinct roles of T peripheral helper and T follicular helper cells in human diseases","volume":"18","author":"Yoshitomi","year":"2021","journal-title":"Cell Mol Immunol"},{"key":"B18","doi-asserted-by":"publisher","first-page":"319","DOI":"10.1038\/s41467-022-35264-8","article-title":"Cytotoxic CD8+ T cells target citrullinated antigens in rheumatoid arthritis","volume":"14","author":"Moon","year":"2023","journal-title":"Nat Commun"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2021.109902","article-title":"Arthritis flares mediated by tissue-resident memory T cells in the joint","volume":"37","author":"Chang","year":"2021","journal-title":"Cell Rep"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1007\/s100670050089","article-title":"Distribution of double-negative (CD4\u2013 CD8\u2013, DN) T subsets in blood and synovial fluid from patients with rheumatoid arthritis","volume":"18","author":"Liu","year":"1999","journal-title":"Clin Rheumatol"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1111\/imm.13441","article-title":"Double-negative T cells: setting the stage for disease control or progression","volume":"165","author":"Velikkakam","year":"2022","journal-title":"Immunology"},{"key":"B22","doi-asserted-by":"publisher","first-page":"1446","DOI":"10.7150\/thno.47860","article-title":"Tofacitinib restores the balance of \u03b3\u03b4Treg\/\u03b3\u03b4T17 cells in rheumatoid arthritis by inhibiting the NLRP3 inflammasome","volume":"11","author":"Yang","year":"2021","journal-title":"Theranostics"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.3390\/cells9020462","article-title":"The role of gamma delta T cells in autoimmune rheumatic diseases","volume":"9","author":"Bank","year":"2020","journal-title":"Cells"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2018.03055","article-title":"DNA damage response signals transduce stress from rheumatoid arthritis risk factors into T cell dysfunction","volume":"9","author":"Shao","year":"2018","journal-title":"Front Immunol"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2021.673916","article-title":"Mitochondria as key players in the pathogenesis and treatment of rheumatoid arthritis","volume":"12","author":"Clayton","year":"2021","journal-title":"Front Immunol"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1038\/s41590-019-0322-4","article-title":"N-myristoylation of AMPK controls T cell inflammatory function","volume":"20","author":"Finlay","year":"2019","journal-title":"Nat Immunol"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-06708-y","article-title":"Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes","volume":"623","author":"Zhang","year":"2023","journal-title":"Nature"},{"key":"B28","doi-asserted-by":"publisher","first-page":"344","DOI":"10.1186\/s13075-015-0800-5","article-title":"CCR6(+) Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis","volume":"17","author":"Paulissen","year":"2015","journal-title":"Arthritis Res Ther"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1016\/j.coi.2018.03.023","article-title":"Mass cytometry: a powerful tool for dissecting the immune landscape","volume":"51","author":"Simoni","year":"2018","journal-title":"Curr Opin Immunol"},{"key":"B30","doi-asserted-by":"publisher","first-page":"191","DOI":"10.1038\/nri3158","article-title":"Standardizing immunophenotyping for the human immunology project","volume":"12","author":"Maecker","year":"2012","journal-title":"Nat Rev Immunol"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-021-00550-0","article-title":"Analyzing high-dimensional cytometry data using FlowSOM","volume":"16","author":"Quintelier","year":"2021","journal-title":"Nat Protoc"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22625","article-title":"FlowSOM: using self-organizing maps for visualization and interpretation of cytometry data","volume":"87","author":"van Gassen","year":"2015","journal-title":"Cytomet A"},{"key":"B33","doi-asserted-by":"publisher","first-page":"289","DOI":"10.1111\/j.2517-6161.1995.tb02031.x","article-title":"Controlling the false discovery rate: A practical and powerful approach to multiple testing","volume":"57","author":"Benjamini","year":"1995","journal-title":"J R Stat Soc B"},{"key":"B34","doi-asserted-by":"publisher","first-page":"899","DOI":"10.1016\/j.immuni.2009.03.019","article-title":"Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor","volume":"30","author":"Miyara","year":"2009","journal-title":"Immunity"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1038\/s41588-024-02019-8","article-title":"Single-cell RNA sequencing of peripheral blood links cell-type-specific regulation of splicing to autoimmune and inflammatory diseases. Title","volume":"56","author":"Tian","year":"2024","journal-title":"Nat Genet"},{"key":"B36","doi-asserted-by":"publisher","first-page":"4046","DOI":"10.1038\/s41467-022-31519-6","article-title":"Single-cell sequencing identifies clonally expanded synovial CD4+ TPH cells expressing GPR56 in rheumatoid arthritis","volume":"13","author":"Argyriou","year":"2022","journal-title":"Nat Commun"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1097\/CM9.0000000000002678","article-title":"Osteoimmunology research in rheumatoid arthritis: From single-cell omics approach","volume":"136","author":"Hu","year":"2023","journal-title":"Chin Med J"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2024.1491318","article-title":"Applications of single-cell RNA sequencing in rheumatoid arthritis","volume":"15","author":"Figueiredo","year":"2024","journal-title":"Front Immunol"},{"key":"B39","doi-asserted-by":"publisher","first-page":"4977","DOI":"10.1038\/s41467-021-25246-7","article-title":"Single-cell sequencing of immune cells from anticitrullinated peptide antibody positive and negative rheumatoid arthritis","volume":"12","author":"Wu","year":"2021","journal-title":"Nat Commun"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2023.1161148","article-title":"The role of dendritic cells and their immunometabolism in rheumatoid arthritis","volume":"14","author":"Suwa","year":"2023","journal-title":"Front Immunol"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1093\/rheumatology\/kez077","article-title":"Expanded circulating peripheral helper T cells in systemic lupus erythematosus: association with disease activity and B cell differentiation","volume":"58","author":"Makiyama","year":"2019","journal-title":"Rheumatology"},{"key":"B42","doi-asserted-by":"publisher","DOI":"10.1038\/s41590-023-01510-4","article-title":"CD4+ T cell memory","volume":"24","author":"K\u00fcnzli","year":"2023","journal-title":"Nat Immunol"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.1016\/s0896-8411(03)00119-7","article-title":"Regulation of immune and autoimmune responses by ICOS","volume":"21","author":"Dong","year":"2003","journal-title":"J Autoimmun"},{"key":"B44","doi-asserted-by":"publisher","first-page":"S3","DOI":"10.1186\/ar2414","article-title":"T-cell co-stimulatory pathways in autoimmunity","volume":"10","author":"Goronzy","year":"2008","journal-title":"Arthritis Res Ther"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.1186\/s12979-022-00269-w","article-title":"CD161 expression defines new human \u03b3\u03b4 T cell subsets","volume":"19","author":"Karunathilaka","year":"2022","journal-title":"Immun Ageing"},{"key":"B46","doi-asserted-by":"publisher","first-page":"76","DOI":"10.1097\/BOR.0000000000000238","article-title":"The lung in rheumatoid arthritis, cause or consequence","volume":"28","author":"Chatzidionisyou","year":"2016","journal-title":"Curr Opin Rheumatol"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.1186\/s13075-018-1799-1","article-title":"Altered composition and phenotype of mucosal-associated invariant T cells in early untreated rheumatoid arthritis","volume":"21","author":"Koppejan","year":"2019","journal-title":"Arthritis Res Ther"},{"key":"B48","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2020.01014","article-title":"MAIT cell activation and functions","volume":"11","author":"Hinks","year":"2020","journal-title":"Front Immunol"},{"key":"B49","doi-asserted-by":"publisher","DOI":"10.1093\/rheumatology\/keaa417","article-title":"Distinct features between HLA-DR+ and HLA-DR\u2013 PD-1hi CXCR5\u2013 T peripheral helper cells in seropositive rheumatoid arthritis","volume":"60","author":"Yamada","year":"2021","journal-title":"Rheumatology"},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.1016\/j.molimm.2018.11.015","article-title":"A role for Th1-like Th17 cells in the pathogenesis of inflammatory and autoimmune disorders","volume":"105","author":"Kamali","year":"2019","journal-title":"Mol Immunol"},{"key":"B51","doi-asserted-by":"publisher","DOI":"10.1007\/s10067-022-06202-2","article-title":"Th17.1 lymphocytes: emerging players in the orchestra of immune-mediated inflammatory diseases","volume":"41","author":"Misra","year":"2022","journal-title":"Clin Rheumatol"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1491041\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,7,24]],"date-time":"2025-07-24T05:33:11Z","timestamp":1753335191000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1491041\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,24]]},"references-count":51,"alternative-id":["10.3389\/fimmu.2025.1491041"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1491041","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,24]]},"article-number":"1491041"}}