{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T05:58:53Z","timestamp":1776405533578,"version":"3.51.2"},"reference-count":44,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T00:00:00Z","timestamp":1776384000000},"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:p>\n                    Regulatory T cells (Tregs) are essential for immune tolerance and are under active development as cell therapy in transplantation. However, the widespread use of the calcineurin inhibitor tacrolimus may inadvertently suppress Treg proliferation and activation, undermining their therapeutic potential. Tacrolimus binds to the FKBP12 protein in T cells, forming a complex that blocks calcineurin\u2013NFAT signaling and suppresses IL-2 gene transcription, thereby inhibiting T cell activation. In this study, we investigated whether deleting FKBP12 in human Tregs could prevent tacrolimus-mediated suppression. Using CRISPR-Cas9 gene editing, FKBP12 was knocked out in\n                    <jats:italic>ex vivo<\/jats:italic>\n                    expanded human Tregs, which were then cultured for seven days with tacrolimus (10 ng\/mL) or control, under varying IL-2 concentrations (100\u2013500 IU\/mL). We observed that tacrolimus significantly reduced the proliferation of control Tregs, even in conditions with 500 IU\/mL IL-2, whereas FKBP12-knockout Tregs maintained robust proliferation comparable to untreated cells. We found no discernible changes in Treg phenotype or stability following FKBP12 deletion or tacrolimus exposure: edited Tregs retained normal expression of the lineage-defining marker FOXP3, displayed a global transcriptomic profile nearly indistinguishable from controls, and were similarly suppressive, indicating that they remained\n                    <jats:italic>bona fide<\/jats:italic>\n                    Tregs. These findings demonstrate that the antiproliferative effect of tacrolimus on Tregs is critically dependent on FKBP12, mirroring its mechanism in conventional T cells. By genetically uncoupling tacrolimus from its target in Tregs, this approach suggests a strategy to preserve Treg numbers during tacrolimus-based immunosuppression in transplant recipients, potentially enhancing Treg-based therapies for transplantation tolerance.\n                  <\/jats:p>","DOI":"10.3389\/fimmu.2026.1756624","type":"journal-article","created":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T05:22:40Z","timestamp":1776403360000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering human Tregs to resist tacrolimus via FKBP12 gene editing"],"prefix":"10.3389","volume":"17","author":[{"given":"Christopher J.","family":"Requejo Cier","sequence":"first","affiliation":[{"name":"Centre de recherche de l\u2019H\u00f4pital Maisonneuve-Rosemont, Department of Microbiology, Infectiology and Immunology, Universit\u00e9 de Montr\u00e9al","place":["Montreal, QC, Canada"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicolas","family":"Valentini","sequence":"additional","affiliation":[{"name":"Centre de recherche de l\u2019H\u00f4pital Maisonneuve-Rosemont, Department of Microbiology, Infectiology and Immunology, Universit\u00e9 de Montr\u00e9al","place":["Montreal, QC, Canada"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gabrielle","family":"Boudreau","sequence":"additional","affiliation":[{"name":"Centre de recherche de l\u2019H\u00f4pital Maisonneuve-Rosemont","place":["Montreal, QC, Canada"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jean-S\u00e9bastien","family":"Delisle","sequence":"additional","affiliation":[{"name":"Centre de recherche de l\u2019H\u00f4pital Maisonneuve-Rosemont, Department of Medicine, Universit\u00e9 de Montr\u00e9al","place":["Montreal, QC, Canada"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Caroline","family":"Lamarche","sequence":"additional","affiliation":[{"name":"Centre de recherche de l\u2019H\u00f4pital Maisonneuve-Rosemont, Department of Medicine, Universit\u00e9 de Montr\u00e9al","place":["Montreal, QC, Canada"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2026,4,17]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1038\/aps.2017.182","article-title":"Organ preservation: from the past to the future","volume":"39","author":"Jing","year":"2018","journal-title":"Acta Pharmacol Sin"},{"key":"B2","doi-asserted-by":"publisher","first-page":"91","DOI":"10.3324\/haematol.2015.128934","article-title":"Impact of immunosuppressive drugs on the therapeutic efficacy of ex vivo expanded human regulatory T cells","volume":"101","author":"Scott\u00e0","year":"2016","journal-title":"Haematologica"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpg.2021.101762","article-title":"Adverse effects of immunosuppression after liver transplantation","author":"Noble","year":"2021","journal-title":"Best Pract Res Clin Gastroenterol"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1097\/00007890-200206150-00023","article-title":"FKBP12 is the only FK506 binding protein mediating T-cell inhibition by the immunosuppressant FK506","volume":"73","author":"Xu","year":"2002","journal-title":"Transplantation"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.3389\/fphar.2024.1331800","article-title":"Mechanism of tacrolimus in the treatment of lupus nephritis","volume":"15","author":"Wang","year":"2024","journal-title":"Front Pharmacol"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2008.05.009","article-title":"Regulatory T cells and immune tolerance","volume":"133","author":"Sakaguchi","year":"2008","journal-title":"Cell"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2010-07-293795","article-title":"Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics","volume":"117","author":"Brunstein","year":"2011","journal-title":"Blood"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2023.1267762","article-title":"Unlocking the potential of Tregs: innovations in CAR technology","volume":"10","author":"Requejo Cier","year":"2023","journal-title":"Front Mol Biosci"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1097\/mnh.0000000000000737","article-title":"Cellular therapies in kidney transplantation","volume":"30","author":"Leclerc","year":"2021","journal-title":"Curr Opin Nephrol Hypertens"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1016\/j.ekir.2022.04.003","article-title":"Chimeric antigen receptor regulatory T cell in transplantation: the future of cell therapy","volume":"7","author":"Lamarche","year":"2022","journal-title":"Kidney Int Rep"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1097\/tp.0b013e318166910b","article-title":"Impact of immunosuppressive drugs on CD4+CD25+FOXP3+ regulatory T cells: does in vitro evidence translate to the clinical setting","volume":"85","author":"Demirkiran","year":"2008","journal-title":"Transplantation"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1097\/tp.0000000000001379","article-title":"Impact of immune-modulatory drugs on regulatory T cell","volume":"100","author":"Furukawa","year":"2016","journal-title":"Transplantation"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1620835114","article-title":"IL-2 therapy restores regulatory T-cell dysfunction induced by calcineurin inhibitors","volume":"114","author":"Whitehouse","year":"2017","journal-title":"Proc Natl Acad Sci USA"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1102376","article-title":"Constitutive nuclear localization of NFAT in Foxp3+ regulatory T cells independent of calcineurin activity","volume":"188","author":"Li","year":"2012","journal-title":"J Immunol"},{"key":"B15","doi-asserted-by":"publisher","first-page":"1397","DOI":"10.3390\/cells11091397","article-title":"NFAT factors are dispensable for the development but are critical for the maintenance of Foxp3(+) regulatory T cells","volume":"11","author":"Barahona de Brito","year":"2022","journal-title":"Cells"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1007\/s10059-009-0114-z","article-title":"Tacrolimus differentially regulates the proliferation of conventional and regulatory CD4(+) T cells","volume":"28","author":"Kogina","year":"2009","journal-title":"Mol Cells"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2006-01-0329","article-title":"Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent interleukin-2 production","volume":"108","author":"Zeiser","year":"2006","journal-title":"Blood"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1111\/j.1600-6143.2012.04269.x","article-title":"Differing effects of rapamycin or calcineurin inhibitor on T-regulatory cells in pediatric liver and kidney transplant recipients","volume":"12","author":"Akimova","year":"2012","journal-title":"Am J Transplant"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1111\/ajt.12934","article-title":"Superiority of rapamycin over tacrolimus in preserving nonhuman primate Treg half-life and phenotype after adoptive transfer","volume":"14","author":"Singh","year":"2014","journal-title":"Am J Transplant"},{"key":"B20","doi-asserted-by":"publisher","first-page":"32","DOI":"10.21417\/la2020mt","article-title":"CRISPR-Cas9-edited tacrolimus-resistant antiviral T cells for advanced adoptive immunotherapy in transplant recipients","volume":"29","author":"Amini","year":"2021","journal-title":"Mol Ther"},{"key":"B21","doi-asserted-by":"publisher","first-page":"52","DOI":"10.1016\/j.omtm.2022.02.012","article-title":"Tacrolimus-resistant SARS-CoV-2-specific T cell products to prevent and treat severe COVID-19 in immunosuppressed patients","volume":"25","author":"Peter","year":"2022","journal-title":"Mol Ther Methods Clin Dev"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1097\/hs9.0000000000000809","article-title":"Pathogen-specific T cells: targeting old enemies and new invaders in transplantation and beyond","volume":"7","author":"Papadopoulou","year":"2023","journal-title":"Hemasphere"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2009-07-230482","article-title":"Generation of Epstein-Barr virus-specific cytotoxic T lymphocytes resistant to the immunosuppressive drug tacrolimus (FK506)","volume":"114","author":"De Angelis","year":"2009","journal-title":"Blood"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.4049\/immunohorizons.1800032","article-title":"Peptide antigen concentration modulates digital NFAT1 activation in primary mouse naive CD8(+) T cells as measured by flow cytometry of isolated cell nuclei","volume":"2","author":"Gallagher","year":"2018","journal-title":"Immunohorizons"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1097\/mot.0000000000000483","article-title":"Guiding regulatory T cells to the allograft","volume":"23","author":"Lamarche","year":"2018","journal-title":"Curr Opin Organ Transplant"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btp616","article-title":"edgeR: a Bioconductor package for differential expression analysis of digital gene expression data","volume":"26","author":"Robinson","year":"2010","journal-title":"Bioinformatics"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1097\/tp.0b013e3182590d8f","article-title":"In vitro effects of cyclosporine A and tacrolimus on regulatory T-cell proliferation and function","volume":"94","author":"Miroux","year":"2012","journal-title":"Transplantation"},{"key":"B28","doi-asserted-by":"publisher","first-page":"731","DOI":"10.1186\/s40001-025-03000-9","article-title":"From bench to bed: deep insights the tacrolimus-induced diabetes and gut microbiota dysbiosis","volume":"30","author":"Han","year":"2025","journal-title":"Eur J Med Res"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22401","article-title":"Nuclear translocation of nuclear factor of activated T cells (NFAT) as a quantitative pharmacodynamic parameter for tacrolimus","volume":"83","author":"Maguire","year":"2013","journal-title":"Cytometry A"},{"key":"B30","doi-asserted-by":"publisher","first-page":"e2025825118","DOI":"10.1073\/pnas.2025825118","article-title":"Hierarchy of signaling thresholds downstream of the T cell receptor and the Tec kinase ITK","volume":"118","author":"Gallagher","year":"2021","journal-title":"Proc Natl Acad Sci USA"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcyt.2020.01.011","article-title":"Expansion and CD2\/CD3\/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity","volume":"22","author":"Andrews","year":"2020","journal-title":"Cytotherapy"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1111\/ajt.14087","article-title":"Age-dependent metabolic and immunosuppressive effects of tacrolimus","volume":"17","author":"Krenzien","year":"2017","journal-title":"Am J Transplant"},{"key":"B33","doi-asserted-by":"publisher","first-page":"999","DOI":"10.1016\/j.molimm.2008.09.029","article-title":"IL-2 signaling prevents T cell anergy by inhibiting the expression of anergy-inducing genes","volume":"46","author":"Dur\u00e9","year":"2009","journal-title":"Mol Immunol"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.1159\/000236201","article-title":"The effects of FK-506 and cyclosporin A on the proliferation of PHA-stimulated T cells in response to IL-2, IL-4 or IL-6","volume":"98","author":"Liu","year":"1992","journal-title":"Int Arch Allergy Immunol"},{"key":"B35","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1111\/j.1600-065x.2011.01004.x","article-title":"T-cell tolerance and the multi-functional role of IL-2R signaling in T-regulatory cells","volume":"241","author":"Cheng","year":"2011","journal-title":"Immunol Rev"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-017-0002-5","article-title":"Targeting IL-2: an unexpected effect in treating immunological diseases","volume":"3","author":"Ye","year":"2018","journal-title":"Signal Transduct Target Ther"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2024.06.022","article-title":"Immunosuppressant therapy averts rejection of allogeneic FKBP1A-disrupted CAR-T cells","volume":"32","author":"Maldini","year":"2024","journal-title":"Mol Ther"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1016\/s0140-6736(20)30167-7","article-title":"Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1\/2A trials","volume":"395","author":"Sawitzki","year":"2020","journal-title":"Lancet"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1097\/tp.0000000000005065","article-title":"Late treatment with autologous expanded regulatory T-cell therapy after alemtuzumab induction is safe and facilitates immunosuppression minimization in living donor renal transplantation","volume":"108","author":"Brook","year":"2024","journal-title":"Transplantation"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.1136\/bmjopen-2022-061864","article-title":"Transplantation without overimmunosuppression (TWO) study protocol: a phase 2b randomised controlled single-centre trial of regulatory T cell therapy to facilitate immunosuppression reduction in living donor kidney transplant recipients","volume":"12","author":"Brook","year":"2022","journal-title":"BMJ Open"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1016\/j.ekir.2022.03.030","article-title":"Study design: Human leukocyte antigen class I molecule A(\u2217)02-chimeric antigen receptor regulatory T cells in renal transplantation","volume":"7","author":"Schreeb","year":"2022","journal-title":"Kidney Int Rep"},{"key":"B42","doi-asserted-by":"publisher","DOI":"10.1126\/science.7518616","article-title":"Specific interaction of type I receptors of the TGF-beta family with the immunophilin FKBP-12","volume":"265","author":"Wang","year":"1994","journal-title":"Science"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0031956","article-title":"FKBP12 activates the cardiac ryanodine receptor Ca2+-release channel and is antagonised by FKBP12.6","volume":"7","author":"Galfr\u00e9","year":"2012","journal-title":"PloS One"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1111\/trf.15121","article-title":"Leukoreduction system chambers are a reliable source for the manufacturing of T-cell therapeutics","volume":"59","author":"Boudreau","year":"2019","journal-title":"Transfusion"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1756624\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T05:22:42Z","timestamp":1776403362000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1756624\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,17]]},"references-count":44,"alternative-id":["10.3389\/fimmu.2026.1756624"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2026.1756624","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,17]]},"article-number":"1756624"}}