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Paradoxically, increasing evidence indicates that superantigens can also induce immunosuppression by promoting regulatory T cell (Treg) development. In this study, we demonstrate that stimulation strength plays a critical role in superantigen-mediated induction of immunosuppressive human CD4+CD25+FOXP3+ T cells. Suboptimal stimulation by a low dose (1 ng\/ml) of staphylococcal enterotoxin C1 (SEC1) led to de novo generation of Treg-like CD4+CD25+FOXP3+ T cells with strong suppressive activity. In contrast, CD4+CD25+ T cells induced by optimal stimulation with high-dose SEC1 (1 \u00b5g\/ml) were not immunosuppressive, despite high FOXP3 expression. Signal transduction pathway analysis revealed differential activation of the PI3K signaling pathway and expression of PTEN in optimal and suboptimal stimulation with SEC1. Additionally, we identified that FOXP3 isoforms in Treg-like cells from the suboptimal condition were located in the nucleus, whereas FOXP3 in nonsuppressive cells from the optimal condition localized in cytoplasm. Sequencing analysis of FOXP3 isoform transcripts identified five isoforms, including a FOXP3 isoform lacking partial exon 3. Overexpression of FOXP3 isoforms confirmed that both an exon 2\u2013lacking isoform and a partial exon 3\u2013lacking isoform confer suppressive activity. Furthermore, blockade of PI3K in optimal stimulation conditions led to induction of suppressive Treg-like cells with nuclear translocation of FOXP3, suggesting that PI3K signaling impairs induction of Tregs in a SEC1 dose-dependent manner. Taken together, these data demonstrate that the strength of activation signals determined by superantigen dose regulates subcellular localization of FOXP3 isoforms, which confers suppressive functionality.<\/jats:p>","DOI":"10.4049\/jimmunol.2300019","type":"journal-article","created":{"date-parts":[[2023,12,18]],"date-time":"2023-12-18T14:12:42Z","timestamp":1702908762000},"page":"421-432","source":"Crossref","is-referenced-by-count":6,"title":["Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4+CD25+FOXP3+ T Cells"],"prefix":"10.1093","volume":"212","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8276-5020","authenticated-orcid":false,"given":"Juyeun","family":"Lee","sequence":"first","affiliation":[{"name":"Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University , Mississippi State, MS"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nogi","family":"Park","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University , Mississippi State, MS"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2295-0763","authenticated-orcid":false,"given":"Michael","family":"Nicosia","sequence":"additional","affiliation":[{"name":"Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic , Cleveland, OH"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8375-0178","authenticated-orcid":false,"given":"Joo Youn","family":"Park","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University , Mississippi State, MS"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4574-5965","authenticated-orcid":false,"given":"Stephen B","family":"Pruett","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University , Mississippi State, MS"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9280-9680","authenticated-orcid":false,"given":"Keun Seok","family":"Seo","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University , Mississippi State, MS"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2023,12,18]]},"reference":[{"key":"2025020405434500700_r1","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1128\/CMR.00134-14","article-title":"Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management","volume":"28","author":"Tong","year":"2015","journal-title":"Clin. 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