{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T05:13:39Z","timestamp":1783574019666,"version":"3.55.0"},"reference-count":39,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,6,25]],"date-time":"2026-06-25T00:00:00Z","timestamp":1782345600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>FOXP3 is the master transcriptional regulator of regulatory T cells (Tregs) and is expressed in humans as two main alternatively spliced isoforms: full-length FOXP3 (FOXP3-FL) and the exon 2-deficient variant (FOXP3-\u03942). While the role of these isoforms has been mainly studied in CD4+ T cells, their distribution across peripheral blood leukocyte populations and their relationship with immune checkpoint expression remain incompletely defined. In this study, we used a multiparametric flow cytometry panel allowing isoform-specific detection of FOXP3-FL and FOXP3-\u03942, together with PD-1 and CTLA-4, to analyze peripheral blood samples from six healthy donors under basal conditions. Major leukocyte populations, including CD4+CD25+ and CD4+CD25\u2212 T cells, CD8+ T cells, monocytes, and neutrophils, were evaluated. FOXP3-FL predominated in CD4+CD25+ T cells, whereas FOXP3-\u03942 was more frequently detected in CD8+ T cells, monocytes, and neutrophils. However, the absolute frequencies of these FOXP3-\u03942-positive populations were low, consistent with the overall low levels of FOXP3 expression observed in these cell types. In CD4+ T-cell subsets, PD-1 expression was generally higher than CTLA-4, regardless of FOXP3 isoform, and FOXP3-\u03942+ cells showed relatively higher PD-1 expression compared to FOXP3-FL+ cells. In contrast, checkpoint expression in non-CD4+ populations was limited. The observed FOXP3-FL+\/FOXP3-\u03942+ ratios across immune cell populations were consistent with a predominant role of FOXP3-FL in maintaining immune tolerance under basal conditions; whether these patterns are preserved or altered in pathological settings warrants further investigation. These results provide a descriptive overview of FOXP3 isoform distribution and checkpoint expression across peripheral blood immune cell subsets in healthy individuals, which may serve as a reference for future studies in immune-mediated diseases.<\/jats:p>","DOI":"10.3390\/biom16070948","type":"journal-article","created":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T03:06:34Z","timestamp":1782875194000},"page":"948","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["First Comprehensive Analysis of Full-Length and \u03942 Foxp3 Isoforms Distribution in PBMCs from Healthy Volunteers"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3684-2712","authenticated-orcid":false,"given":"Manuel","family":"Fern\u00e1ndez-Delgado","sequence":"first","affiliation":[{"name":"Service of Hematology and Hemotherapy, Hospital General Universitario de Castell\u00f3n, 12004 Castell\u00f3 de la Plana, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3176-9763","authenticated-orcid":false,"given":"Luis","family":"Sendra","sequence":"additional","affiliation":[{"name":"Gene Therapy and Pharmacogenomics, Department of Pharmacology, Universitat de Val\u00e8ncia, 46010 Valencia, Spain"},{"name":"Unit of Pharmacogenetics and Gene Therapy, La Fe Health Research Institute, 46026 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6042-4185","authenticated-orcid":false,"given":"Mar\u00eda Jos\u00e9","family":"Herrero","sequence":"additional","affiliation":[{"name":"Gene Therapy and Pharmacogenomics, Department of Pharmacology, Universitat de Val\u00e8ncia, 46010 Valencia, Spain"},{"name":"Unit of Pharmacogenetics and Gene Therapy, La Fe Health Research Institute, 46026 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7698-2122","authenticated-orcid":false,"given":"Gladys G.","family":"Olivera-Pasquini","sequence":"additional","affiliation":[{"name":"Unit of Pharmacogenetics and Gene Therapy, La Fe Health Research Institute, 46026 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2542-1776","authenticated-orcid":false,"given":"Enrique G.","family":"Zucchet","sequence":"additional","affiliation":[{"name":"Unit of Pharmacogenetics and Gene Therapy, La Fe Health Research Institute, 46026 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raimundo","family":"Garc\u00eda-Boyero","sequence":"additional","affiliation":[{"name":"Service of Hematology and Hemotherapy, Hospital General Universitario de Castell\u00f3n, 12004 Castell\u00f3 de la Plana, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7830-8713","authenticated-orcid":false,"given":"Salvador F.","family":"Ali\u00f1o","sequence":"additional","affiliation":[{"name":"Gene Therapy and Pharmacogenomics, Department of Pharmacology, Universitat de Val\u00e8ncia, 46010 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1038\/nri2785","article-title":"FOXP3+ Regulatory T Cells in the Human Immune System","volume":"10","author":"Sakaguchi","year":"2010","journal-title":"Nat. Rev. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Blinova, V.G., Gladilina, Y.A., Abramova, A.A., Eliseeva, D.D., Vtorushina, V.V., Shishparenok, A.N., and Zhdanov, D.D. (2023). Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA. Cells, 13.","DOI":"10.3390\/cells13010077"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e2023739118","DOI":"10.1073\/pnas.2023739118","article-title":"Treg-Expressed CTLA-4 Depletes CD80\/CD86 by Trogocytosis, Releasing Free PD-L1 on Antigen-Presenting Cells","volume":"118","author":"Tekguc","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3276","DOI":"10.1172\/JCI24685","article-title":"The Role of 2 FOXP3 Isoforms in the Generation of Human CD4+ Tregs","volume":"115","author":"Allan","year":"2005","journal-title":"J. Clin. Investig."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6121","DOI":"10.4049\/jimmunol.0803646","article-title":"Regulatory T Cell Suppression Is Potentiated by Target T Cells in a Cell Contact, IL-35- and IL-10-Dependent Manner","volume":"182","author":"Collison","year":"2009","journal-title":"J. Immunol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1322","DOI":"10.1038\/ni.3540","article-title":"An Essential Role for the IL-2 Receptor in Treg Cell Function","volume":"17","author":"Chinen","year":"2016","journal-title":"Nat. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1038\/nri.2017.108","article-title":"The Diverse Functions of the PD1 Inhibitory Pathway","volume":"18","author":"Sharpe","year":"2018","journal-title":"Nat. Rev. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1038\/ni904","article-title":"Foxp3 Programs the Development and Function of CD4+CD25+ Regulatory T Cells","volume":"4","author":"Fontenot","year":"2003","journal-title":"Nat. Immunol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Chang, H., Wang, Y., Gao, L., and Zheng, S.J. (2026). Beyond Conventional Transcriptional Regulation Function: FOXP3 as an Integrative Hub for Chromatin Interactions and Protein Complexes in Immune Regulation. Biology, 15.","DOI":"10.3390\/biology15030254"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"112877","DOI":"10.1016\/j.celrep.2023.112877","article-title":"The Splicing Isoform Foxp3\u03942 Differentially Regulates tTreg and pTreg Homeostasis","volume":"42","author":"Gu","year":"2023","journal-title":"Cell Rep."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Luo, Z., Zhang, Y., Saleh, Q.W., Zhang, J., Zhu, Z., and Tepel, M. (2023). Metabolic Regulation of Forkhead Box P3 Alternative Splicing Isoforms and Their Impact on Health and Disease. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1278560"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Mailer, R.K. (2020). IPEX as a Consequence of Alternatively Spliced FOXP3. Front. Pediatr., 8.","DOI":"10.3389\/fped.2020.594375"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1093\/intimm\/dxae049","article-title":"A Splice of Life: The Discovery, Function, and Clinical Implications of FOXP3 Isoforms in Autoimmune Disease","volume":"37","author":"Weinstein","year":"2024","journal-title":"Int. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Mailer, R.K.W., Falk, K., and R\u00f6tzschke, O. (2009). Absence of Leucine Zipper in the Natural FOXP3\u03942\u03947 Isoform Does Not Affect Dimerization but Abrogates Suppressive Capacity. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0006104"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"eabo5407","DOI":"10.1126\/sciimmunol.abo5407","article-title":"FOXP3 Exon 2 Controls Treg Stability and Autoimmunity","volume":"7","author":"Du","year":"2022","journal-title":"Sci. Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"eadr9933","DOI":"10.1126\/sciimmunol.adr9933","article-title":"Reprogramming Intratumoral Treg Cells by Morpholino-Mediated Splicing of FOXP3 for Cancer Immunotherapy","volume":"10","author":"Li","year":"2025","journal-title":"Sci. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"989","DOI":"10.1016\/j.immuni.2016.05.001","article-title":"Lag-3, Tim-3, and TIGIT: Co-Inhibitory Receptors with Specialized Functions in Immune Regulation","volume":"44","author":"Anderson","year":"2016","journal-title":"Immunity"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1038\/s41392-025-02326-4","article-title":"Regulatory T Cells in Homeostasis and Disease: Molecular Mechanisms and Therapeutic Potential","volume":"10","author":"Wang","year":"2025","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"36828","DOI":"10.1074\/jbc.M608848200","article-title":"Transcriptional Regulation by Foxp3 Is Associated with Direct Promoter Occupancy and Modulation of Histone Acetylation","volume":"281","author":"Chen","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5155","DOI":"10.1182\/blood-2011-11-388918","article-title":"Basis of CTLA-4 Function in Regulatory and Conventional CD4+ T Cells","volume":"119","author":"Tai","year":"2012","journal-title":"Blood"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2517","DOI":"10.1056\/NEJMe1205943","article-title":"Tumor Immunotherapy Directed at PD-1","volume":"366","author":"Ribas","year":"2012","journal-title":"N. Engl. J. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1381","DOI":"10.1002\/eji.200737590","article-title":"Forced Overexpression of Either of the Two Common Human Foxp3 Isoforms Can Induce Regulatory T Cells from CD4+ CD25\u2212 Cells","volume":"38","author":"Emmelot","year":"2008","journal-title":"Eur. J. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"651","DOI":"10.1038\/s41577-020-0306-5","article-title":"A Guide to Cancer Immunotherapy: From T Cell Basic Science to Clinical Practice","volume":"20","author":"Waldman","year":"2020","journal-title":"Nat. Rev. Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"9543","DOI":"10.1128\/MCB.25.21.9543-9553.2005","article-title":"CTLA-4 and PD-1 Receptors Inhibit T-Cell Activation by Distinct Mechanisms","volume":"25","author":"Parry","year":"2005","journal-title":"Mol. Cell. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e31307","DOI":"10.1097\/MD.0000000000031307","article-title":"T Lymphocyte Subsets and PD-1 Expression on Lymphocytes in Peripheral Blood of Patients with Non-Small Cell Lung Cancer","volume":"101","author":"Chen","year":"2022","journal-title":"Medicine"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1046\/j.1365-3083.2001.00985.x","article-title":"Regulation of Surface and Intracellular Expression of CTLA-4 on Human Peripheral T Cells","volume":"54","author":"Wang","year":"2001","journal-title":"Scand. J. Immunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1133","DOI":"10.1189\/jlb.0511244","article-title":"Higher Proportions of Circulating FOXP3+ and CTLA-4+ Regulatory T Cells Are Associated with Lower Fractions of Memory CD4+ T Cells in Infants","volume":"90","author":"Rabe","year":"2011","journal-title":"J. Leukoc. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1002\/eji.200940022","article-title":"Co-expression of TNFR2 and CD25 Identifies More of the Functional CD4+ FOXP3+ Regulatory T Cells in Human Peripheral Blood","volume":"40","author":"Chen","year":"2010","journal-title":"Eur. J. Immunol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1701","DOI":"10.1084\/jem.20060772","article-title":"CD127 Expression Inversely Correlates with FoxP3 and Suppressive Function of Human CD4+ T Reg Cells","volume":"203","author":"Liu","year":"2006","journal-title":"J. Exp. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1111\/cei.12557","article-title":"Altered Balance between Self-Reactive T Helper (Th)17 Cells and Th10 Cells and between Full-Length Forkhead Box Protein 3 (FoxP3) and FoxP3 Splice Variants in Hashimoto\u2019s Thyroiditis","volume":"180","author":"Kristensen","year":"2015","journal-title":"Clin. Exp. Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Zhdanov, D.D., Gladilina, Y.A., Blinova, V.G., Abramova, A.A., Shishparenok, A.N., and Eliseeva, D.D. (2024). Induction of FoxP3 Pre-mRNA Alternative Splicing to Enhance the Suppressive Activity of Regulatory T Cells from Amyotrophic Lateral Sclerosis Patients. Biomedicines, 12.","DOI":"10.3390\/biomedicines12051022"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1922","DOI":"10.1002\/art.37959","article-title":"Patients With Antineutrophil Cytoplasmic Antibody\u2013Associated Vasculitis Have Defective Treg Cell Function Exacerbated by the Presence of a Suppression-Resistant Effector Cell Population","volume":"65","author":"Free","year":"2013","journal-title":"Arthritis Rheum."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"869","DOI":"10.1007\/s00262-014-1581-4","article-title":"Foxp3 Expression in T Regulatory Cells and Other Cell Lineages","volume":"63","author":"Devaud","year":"2014","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.dci.2019.02.019","article-title":"Regulation of FOXP3 Expression in Myeloid Cells in Response to All-Trans-Retinoic Acid, Interleukin 2 and Transforming Growth Factor \u03b2","volume":"96","author":"Ilnicka","year":"2019","journal-title":"Dev. Comp. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3674","DOI":"10.1038\/s41598-018-21861-5","article-title":"FoxP3 Isoforms and PD-1 Expression by T Regulatory Cells in Multiple Sclerosis","volume":"8","author":"Sambucci","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"108957","DOI":"10.1016\/j.clim.2022.108957","article-title":"The FOXP3 Full-Length Isoform Controls the Lineage-Stability of CD4+FOXP3+ Regulatory T Cells","volume":"237","author":"Seitz","year":"2022","journal-title":"Clin. Immunol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Sato, Y., Liu, J., Lee, E., Perriman, R., Roncarolo, M.G., and Bacchetta, R. (2021). Co-Expression of FOXP3FL and FOXP3\u03942 Isoforms Is Required for Optimal Treg-Like Cell Phenotypes and Suppressive Function. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.752394"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"12112","DOI":"10.1038\/s41598-024-62149-1","article-title":"FOXP3 Splice Variant Expression in Males and Females in Healthy Populations and in Kidney Transplant Recipients","volume":"14","author":"Saleh","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Nie, J., Li, Y.Y., Zheng, S.G., Tsun, A., and Li, B. (2015). FOXP3+ Treg Cells and Gender Bias in Autoimmune Diseases. Front. Immunol., 6.","DOI":"10.3389\/fimmu.2015.00493"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/7\/948\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T04:17:09Z","timestamp":1783570629000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/7\/948"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,25]]},"references-count":39,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["biom16070948"],"URL":"https:\/\/doi.org\/10.3390\/biom16070948","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,25]]}}}