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In the present study, we aimed to determine the presence and the frequency of these cells in draining lymph nodes of patients with bladder cancer (BC). Forty-six patients with BC who had undergone radical cystectomy and pelvic lymph node dissection were enrolled. Following routine pathological examination, a portion of the dissected lymph nodes was minced to obtain a single-cell suspension. Mononuclear cells were then separated using Ficoll-Hypaque gradient centrifugation, and the samples with proper viability (&gt;\u200995%) were subjected to further analysis. To phenotype the follicular subsets, cells were stained with appropriate fluorochrome-conjugated antibodies specific for CD4, CXCR5, BCL6, and FOXP3. The cells were then acquired on a four-color flow cytometer. The data were analyzed with the FlowJo software version 10.8.1 package. Our analysis indicated that, on average 37.89\u2009\u00b1\u200916.36% of CD4<jats:sup>+<\/jats:sup> lymphocytes in draining lymph nodes of patients with BC expressed CXCR5. The majority of them were negative for FOXP3, representing helper subsets (28.73\u2009\u00b1\u200913.66). A small percent simultaneously expressed BCL6 transcription factor (1.65%\u2009\u00b1\u20091.35), designated as Tfh (CD4<jats:sup>+<\/jats:sup>BCL6<jats:sup>+<\/jats:sup>CXCR5<jats:sup>+<\/jats:sup>FOXP3<jats:sup>-<\/jats:sup>). While less than 10% of CD4<jats:sup>+<\/jats:sup> lymphocytes expressed CXCR5 and FOXP3, 1.78\u2009\u00b1\u20092.54 were also positive for BCL6, known as TFR. Statistical analysis revealed that the frequency of both Tfh and TFR cells was higher in draining lymph nodes of patients with tumor-infiltrated nodes (P\u2009=\u20090.035 and P\u2009=\u20090.079, respectively) compared to those with negative ones. The percentage of these cells  was also higher in high-grade tumors compared to low-grade ones\u00a0(P\u2009=\u20090.031 for both). Our data collectively indicated that however approximately one third of CD4<jats:sup>+<\/jats:sup> lymphocytes expressed CXCR5 and accordingly had the capacity to enter the follicles, less than 2% of them represented Tfh and TFR phenotypes. The percentage of these cells increased in progressed tumors and showed an association with negative prognostic factors.<\/jats:p>","DOI":"10.1038\/s41598-024-70675-1","type":"journal-article","created":{"date-parts":[[2024,9,2]],"date-time":"2024-09-02T13:05:37Z","timestamp":1725282337000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Clinical and prognostic significance of follicular helper and regulatory T cells in bladder cancer draining lymph nodes"],"prefix":"10.1038","volume":"14","author":[{"given":"Zahra","family":"Mansourabadi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ali","family":"Ariafar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nooshafarin","family":"Chenari","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hossein","family":"Hakimellahi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasmin","family":"Vahidi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zahra","family":"Faghih","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,9,2]]},"reference":[{"issue":"4","key":"70675_CR1","doi-asserted-by":"publisher","first-page":"529","DOI":"10.1016\/j.immuni.2014.10.004","volume":"41","author":"S Crotty","year":"2014","unstructured":"Crotty, S. T follicular helper cell differentiation, function, and roles in disease. Immunity 41(4), 529\u2013542 (2014).","journal-title":"Immunity"},{"issue":"22","key":"70675_CR2","doi-asserted-by":"publisher","first-page":"8524","DOI":"10.3390\/ijms21228524","volume":"21","author":"H Law","year":"2020","unstructured":"Law, H., Venturi, V., Kelleher, A. & Munier, C. M. L. Tfh cells in health and immunity: Potential targets for systems biology approaches to vaccination. Int. J. Mol. Sci. 21(22), 8524 (2020).","journal-title":"Int. J. Mol. Sci."},{"issue":"5943","key":"70675_CR3","doi-asserted-by":"publisher","first-page":"1006","DOI":"10.1126\/science.1175870","volume":"325","author":"RJ Johnston","year":"2009","unstructured":"Johnston, R. J. et al. Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation. 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