{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T10:05:14Z","timestamp":1790244314602,"version":"4.1.0"},"reference-count":26,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:00:00Z","timestamp":1790208000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Rheumatoid arthritis (RA) involves alterations in cytotoxic lymphoid differentiation, yet the precise phenotypic remodeling of circulating helper T cells and natural killer (NK) cells remains incompletely understood. We evaluated circulating CD4+CD28+ and CD4+CD28\u2212 T-cell subsets and NKG2A+CD56+ NK cells, alongside granule-mediated cytotoxic markers (granzyme B and perforin) and the surface-bound death receptor Fas ligand (FasL), in 28 female patients with RA and 12 age-matched healthy female controls using multiparameter flow cytometry. CD4+CD28\u2212 cells were increased in RA compared with controls (16.6% versus 6.5% of CD4+ cells). Within CD4+CD28\u2212 cells, granzyme B positivity was markedly lower in RA (5.0% versus 62.0%), whereas FasL expression was higher (40.3% versus 22.5%). RA patients also exhibited a higher proportion of circulating NKG2A+CD56+ NK cells (6.1% versus 2.7% of PBMCs) with substantially lower granzyme B expression (8.4% versus 55.6%). Higher disease activity (DAS28-CRP) correlated positively with total NKG2A+CD56+ NK cell frequencies and intracellular perforin expression in both CD4+CD28+ T cells and NKG2A+CD56+ NK cells, as well as lower FasL expression in CD4+CD28\u2212 cells. These findings support a disease-associated phenotypic remodeling of circulating cytotoxic markers in RA, characterized by shifted granzyme B and FasL expressions in CD4+CD28\u2212 T cells and NKG2A+CD56+ NK cells, although whether these profiles translate into altered cytotoxic kinetics remains to be functionally determined.<\/jats:p>","DOI":"10.3390\/ijms27198532","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T09:07:54Z","timestamp":1790240874000},"page":"8532","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Differential Expression of Cytotoxic-Associated Markers in Circulating CD4+CD28\u2212 T Cells and NK Cells in Rheumatoid Arthritis"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-3301-1338","authenticated-orcid":false,"given":"Julio","family":"Sesma-Soto","sequence":"first","affiliation":[{"name":"Department of Immunology, Instituto Nacional de Cardiolog\u00eda Ignacio Ch\u00e1vez, Mexico City 14080, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6660-7388","authenticated-orcid":false,"given":"Mariana","family":"Patl\u00e1n","sequence":"additional","affiliation":[{"name":"Basic Research Subdirectorate, Instituto Nacional de Cardiolog\u00eda Ignacio Ch\u00e1vez, Mexico City 14080, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rashidi","family":"Springall","sequence":"additional","affiliation":[{"name":"Department of Immunology, Instituto Nacional de Cardiolog\u00eda Ignacio Ch\u00e1vez, Mexico City 14080, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"}]},{"given":"Araceli","family":"P\u00e1ez","sequence":"additional","affiliation":[{"name":"UNAM\/INC Translational Research Unit, Instituto Nacional de Cardiolog\u00eda Ignacio Ch\u00e1vez, Mexico City 14080, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8671-3367","authenticated-orcid":false,"given":"Felipe","family":"Mass\u00f3","sequence":"additional","affiliation":[{"name":"UNAM\/INC Translational Research Unit, Instituto Nacional de Cardiolog\u00eda Ignacio Ch\u00e1vez, Mexico City 14080, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"}]},{"given":"Luis H.","family":"Silveira","sequence":"additional","affiliation":[{"name":"Department of Rheumatology, Instituto Nacional de Cardiolog\u00eda Ignacio Ch\u00e1vez, Mexico City 14080, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6258-5732","authenticated-orcid":false,"given":"Luis M.","family":"Amezcua-Guerra","sequence":"additional","affiliation":[{"name":"Department of Immunology, Instituto Nacional de Cardiolog\u00eda Ignacio Ch\u00e1vez, Mexico City 14080, Mexico"},{"name":"Health Care Department, Universidad Aut\u00f3noma Metropolitana\u2013Xochimilco, Mexico City 04960, Mexico"},{"name":"School of Medicine, Universidad La Salle, Mexico City 06140, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2019","DOI":"10.1016\/S0140-6736(23)01525-8","article-title":"Rheumatoid arthritis","volume":"402","author":"Bathon","year":"2023","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1038\/ncprheum0249","article-title":"Mechanisms of disease: Genetic susceptibility and environmental triggers in the development of rheumatoid arthritis","volume":"2","author":"Klareskog","year":"2006","journal-title":"Nat. Clin. Pract. Rheumatol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1896","DOI":"10.1093\/rheumatology\/keu171","article-title":"CD70-expressing CD4 T cells produce IFN-\u03b3 and IL-17 in rheumatoid arthritis","volume":"53","author":"Park","year":"2014","journal-title":"Rheumatology"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Broadley, I., Pera, A., Morrow, G., Davies, K.A., and Kern, F. (2017). Expansions of cytotoxic CD4+CD28\u2212 T cells drive excess cardiovascular mortality in rheumatoid arthritis and other chronic inflammatory conditions and are triggered by CMV infection. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.00195"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1111\/imm.12506","article-title":"The life (and death) of CD4+CD28null T cells in inflammatory diseases","volume":"146","author":"Dumitriu","year":"2015","journal-title":"Immunology"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e1250","DOI":"10.1002\/cti2.1250","article-title":"Natural killer cells in inflammatory autoimmune diseases","volume":"10","author":"Yang","year":"2021","journal-title":"Clin. Transl. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5858","DOI":"10.1038\/s41598-020-62654-z","article-title":"Phenotypic and functional characterization of natural killer cells in rheumatoid arthritis-regulation with interleukin-15","volume":"10","author":"Lin","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1063","DOI":"10.1007\/s00011-021-01504-8","article-title":"The role of NK cells in rheumatoid arthritis","volume":"70","author":"Fathollahi","year":"2021","journal-title":"Inflamm. Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1351","DOI":"10.1038\/s41598-018-37448-z","article-title":"High percentages and activity of synovial fluid NK cells present in patients with advanced stage active Rheumatoid Arthritis","volume":"9","author":"Yamin","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Zheng, Y., Zhao, J., Shan, Y., Guo, S., Schrodi, S.J., and He, D. (2023). Role of the granzyme family in rheumatoid arthritis: Current Insights and future perspectives. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1137918"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2569","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":"ref_12","doi-asserted-by":"crossref","first-page":"111807","DOI":"10.1016\/j.intimp.2024.111807","article-title":"CD28null T cells in aging and diseases: From biology to assessment and intervention","volume":"131","author":"Guan","year":"2024","journal-title":"Int. Immunopharmacol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"109924","DOI":"10.1016\/j.clim.2024.109927","article-title":"Involvement of expanded cytotoxic and proinflammatory CD28null T cells in primary Sj\u00f6gren\u2019s syndrome","volume":"261","author":"Deng","year":"2024","journal-title":"Clin. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"R210","DOI":"10.1186\/ar766","article-title":"The expansion of CD4+CD28\u2212 T cells in patients with rheumatoid arthritis","volume":"5","author":"Pawlik","year":"2003","journal-title":"Arthritis Res. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4509","DOI":"10.7150\/thno.27428","article-title":"CD28null pro-atherogenic CD4 T cells explain the link between CMV infection and an increased risk of cardiovascular death","volume":"8","author":"Pera","year":"2018","journal-title":"Theranostics"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"R87","DOI":"10.1186\/ar2286","article-title":"Skewed distribution of proinflammatory CD4+CD28null T cells in rheumatoid arthritis","volume":"9","author":"Fasth","year":"2007","journal-title":"Arthritis Res. Ther."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1111\/sji.12139","article-title":"Peripheral and site-specific CD4+CD28null T cells from rheumatoid arthritis patients show distinct characteristics","volume":"79","author":"Pieper","year":"2014","journal-title":"Scand. J. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1016\/j.molmed.2012.06.003","article-title":"Pathogenic features of CD4+CD28\u2212 T cells in immune disorders","volume":"18","author":"Broux","year":"2012","journal-title":"Trends Mol. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1002\/eji.200939399","article-title":"Activating NK-cell receptors co-stimulate CD4+CD28\u2212 T cells in patients with rheumatoid arthritis","volume":"40","author":"Fasth","year":"2010","journal-title":"Eur. J. Immunol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"6514","DOI":"10.4049\/jimmunol.179.10.6514","article-title":"CD4+CD28null T cells in autoimmune disease: Pathogenic features and decreased susceptibility to immunoregulation","volume":"179","author":"Thewissen","year":"2007","journal-title":"J. Immunol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"S197","DOI":"10.1186\/ar557","article-title":"Studies of T-cell activation in chronic inflammation","volume":"4","author":"Cope","year":"2002","journal-title":"Arthritis Res. Ther."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Kucuksezer, U.C., Aktas Cetin, E., Esen, F., Tahrali, I., Akdeniz, N., Gelmez, M.Y., and Deniz, G. (2021). The Role of Natural Killer Cells in Autoimmune Diseases. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.622306"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Yang, M., and Zhu, L. (2024). Osteoimmunology: The crosstalk between T cells, B cells, and osteoclasts in rheumatoid arthritis. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25052688"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1150","DOI":"10.14336\/AD.2021.0110","article-title":"Premature senescence of T cells favors bone loss during osteolytic diseases: A new concern in the osteoimmunology arena","volume":"12","author":"Rojas","year":"2021","journal-title":"Aging Dis."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1186\/s12967-014-0363-2","article-title":"Reduced T-cell repertoire restrictions in abatacept-treated rheumatoid arthritis patients","volume":"13","author":"Imberti","year":"2015","journal-title":"J. Transl. Med."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1111\/1756-185X.12056","article-title":"Reduced numbers of circulating CD28-negative CD4+ cells in patients with rheumatoid arthritis chronically treated with abatacept","volume":"16","author":"Vargas","year":"2013","journal-title":"Int. J. Rheum. Dis."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/19\/8532\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T09:15:28Z","timestamp":1790241328000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/19\/8532"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,24]]},"references-count":26,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["ijms27198532"],"URL":"https:\/\/doi.org\/10.3390\/ijms27198532","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,24]]}}}