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We have previously shown that allogeneic umbilical cord blood-derived (UCB) regulatory T cells (Tregs) can resolve inflammation and treat acute and immune-mediated lung injuries. Allogeneic, cryopreserved UCB Tregs have shown a clinical benefit in patients suffering from COVID-19 acute respiratory distress syndrome. The unique properties of UCB Treg cells include a lack of plasticity under inflammatory micro-environments, no requirement for HLA matching, a long shelf life of cryopreserved cells, and immediate product availability, which makes them attractive for treating acute inflammatory syndromes. Therefore, we hypothesized that adjunct therapy with UCB Tregs may resolve the undesirable inflammation responsible for CAR T cell therapy-associated toxicity. In in vitro analysis, no interference from the addition of UCB Tregs was observed on CD19 CAR T cells\u2019 ability to kill CD19 Raji cells at different CAR T: Raji cell ratios of 8:1 (80.4% vs. 81.5%); 4:1 (62.0% vs. 66.2%); 2:1 (50.1% vs. 54.7%); and 1:1 (35.4% vs. 44.1%). In the xenogeneic B-cell lymphoma model, multiple injections of UCB Tregs were administered 3 days after CD19 CAR T cell injection, and no detrimental effect of add-on Tregs was noted on the circulating CD8+ T effector cells. The distribution of CAR T cells in multiple organs remained unaffected by the addition of the UCB Tregs. Specifically, no difference in the overall tumor burden was detected between the UCB Treg + CAR T vs. CAR T alone recipients. No tumor was detected in the liver or bone marrow in CAR T cells + UCB Tregs recipients, with a notable corresponding decrease in multiple circulating inflammatory cytokines when compared to CART alone recipients. Here we show the proof of concept for adjunct therapy with UCB Tregs to mitigate the hyper-inflammatory state induced by CAR T cells without any interference in their on-target anti-tumor activity. Administration of UCB Tregs after CAR T cells allows sufficient time for their synapse formation with tumor cells and exerts cytotoxicity, such that the UCB Tregs are diverted to interact with the antigen-presenting cells at the site of inflammation. Such a differential distribution of cells would allow for a two-pronged strategy of a UCB Treg \u201ccooling blanket\u201d effect and lay the groundwork for clinical study.<\/jats:p>","DOI":"10.3390\/cells12141880","type":"journal-article","created":{"date-parts":[[2023,7,18]],"date-time":"2023-07-18T01:46:02Z","timestamp":1689644762000},"page":"1880","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Adjunct Therapy with T Regulatory Cells Decreases Inflammation and Preserves the Anti-Tumor Activity of CAR T Cells"],"prefix":"10.3390","volume":"12","author":[{"given":"Ke","family":"Zeng","sequence":"first","affiliation":[{"name":"Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meixian","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mi-Ae","family":"Lyu","sequence":"additional","affiliation":[{"name":"Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2621-3584","authenticated-orcid":false,"given":"Joseph D.","family":"Khoury","sequence":"additional","affiliation":[{"name":"Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7302-8299","authenticated-orcid":false,"given":"Sairah","family":"Ahmed","sequence":"additional","affiliation":[{"name":"Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Krina K.","family":"Patel","sequence":"additional","affiliation":[{"name":"Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Boro","family":"Dropuli\u0107","sequence":"additional","affiliation":[{"name":"Caring Cross, Gaithersburg, MD 20878, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jane","family":"Reese-Koc","sequence":"additional","affiliation":[{"name":"Department of Cellular Therapy, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paolo F.","family":"Caimi","sequence":"additional","affiliation":[{"name":"Department of Hematology and Medical Oncology, Cleveland Clinic, Cleveland, OH 44195, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tara","family":"Sadeghi","sequence":"additional","affiliation":[{"name":"Cellenkos Inc., Houston, TX 77005, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marcos de","family":"Lima","sequence":"additional","affiliation":[{"name":"Division of Hematology, The Ohio State University College of Medicine, Columbus, OH 43210, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher R.","family":"Flowers","sequence":"additional","affiliation":[{"name":"Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simrit","family":"Parmar","sequence":"additional","affiliation":[{"name":"Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,7,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1056\/NEJMra1706169","article-title":"Chimeric Antigen Receptor Therapy","volume":"379","author":"June","year":"2018","journal-title":"N. Engl. J. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e123","DOI":"10.1016\/j.bbmt.2018.12.756","article-title":"Cytokine Release Syndrome with Chimeric Antigen Receptor T Cell Therapy","volume":"25","author":"Frey","year":"2019","journal-title":"Biol. Blood Marrow Transplant."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"S42","DOI":"10.1002\/ajh.25445","article-title":"Toxicities of CD19 CAR-T cell immunotherapy","volume":"94","author":"Hirayama","year":"2019","journal-title":"Am. J. Hematol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"775","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":"ref_5","doi-asserted-by":"crossref","first-page":"260","DOI":"10.1111\/j.1600-065X.2011.01018.x","article-title":"Regulatory T cells and Foxp3","volume":"241","author":"Rudensky","year":"2011","journal-title":"Immunol. Rev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1016\/j.cell.2017.01.017","article-title":"Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy","volume":"168","author":"Sharma","year":"2017","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1158\/1078-0432.CCR-05-1886","article-title":"Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers","volume":"12","author":"Badoual","year":"2006","journal-title":"Clin. Cancer Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/j.jcyt.2022.10.009","article-title":"Allogeneic cord blood regulatory T cells can resolve lung inflammation","volume":"25","author":"Lyu","year":"2023","journal-title":"Cytotherapy"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"852","DOI":"10.7326\/L20-0681","article-title":"Regulatory T Cells for Treating Patients With COVID-19 and Acute Respiratory Distress Syndrome: Two Case Reports","volume":"173","author":"Gladstone","year":"2020","journal-title":"Ann. Intern. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3075","DOI":"10.1182\/bloodadvances.2022009619","article-title":"Randomized, double-blinded, placebo-controlled trial of allogeneic cord blood T-regulatory cells for treatment of COVID-19 ARDS","volume":"7","author":"Gladstone","year":"2023","journal-title":"Blood Adv."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.jcyt.2013.07.009","article-title":"Third-party umbilical cord blood-derived regulatory T cells prevent xenogenic graft-versus-host disease","volume":"16","author":"Parmar","year":"2014","journal-title":"Cytotherapy"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"35611","DOI":"10.18632\/oncotarget.26242","article-title":"Third party, umbilical cord blood derived regulatory T-cells for prevention of graft versus host disease in allogeneic hematopoietic stem cell transplantation: Feasibility, safety and immune reconstitution","volume":"9","author":"Kellner","year":"2018","journal-title":"Oncotarget"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1061","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":"ref_14","doi-asserted-by":"crossref","first-page":"1044","DOI":"10.1182\/blood-2015-06-653667","article-title":"Umbilical cord blood-derived T regulatory cells to prevent GVHD: Kinetics, toxicity profile, and clinical effect","volume":"127","author":"Brunstein","year":"2016","journal-title":"Blood"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1331","DOI":"10.1038\/s41596-020-00467-0","article-title":"Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity","volume":"16","author":"Kiesgen","year":"2021","journal-title":"Nat. Protoc."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.bbrc.2013.07.030","article-title":"CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models","volume":"438","author":"Tsukahara","year":"2013","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"101476","DOI":"10.1016\/j.smim.2021.101476","article-title":"Tumor resident regulatory T cells","volume":"52","author":"Glasner","year":"2021","journal-title":"Semin. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1111\/j.1600-065X.2007.00487.x","article-title":"When three is not a crowd: A Crossregulation model of the dynamics and repertoire selection of regulatory CD4+ T cells","volume":"216","author":"Carneiro","year":"2007","journal-title":"Immunol. Rev."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1973","DOI":"10.3389\/fimmu.2020.01973","article-title":"Neurotoxicity and Cytokine Release Syndrome After Chimeric Antigen Receptor T Cell Therapy: Insights Into Mechanisms and Novel Therapies","volume":"11","author":"Siegler","year":"2020","journal-title":"Front. Immunol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Benmebarek, M.-R., Karches, C.H., Cadilha, B.L., Lesch, S., Endres, S., and Kobold, S. (2019). Killing Mechanisms of Chimeric Antigen Receptor (CAR) T Cells. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20061283"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1038","DOI":"10.1084\/jem.20182375","article-title":"Single-cell imaging of CAR T cell activity in vivo reveals extensive functional and anatomical heterogeneity","volume":"216","author":"Cazaux","year":"2019","journal-title":"J. Exp. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/j.ebiom.2018.12.013","article-title":"Modeling anti-CD19 CAR T cell therapy in humanized mice with human immunity and autologous leukemia","volume":"39","author":"Jin","year":"2019","journal-title":"EBioMedicine"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2040772","DOI":"10.1080\/2162402X.2022.2040772","article-title":"Single-cell analysis by mass cytometry reveals CD19 CAR T cell spatiotemporal plasticity in patients","volume":"11","author":"Goldberg","year":"2022","journal-title":"Oncoimmunology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1111\/j.1750-3639.1991.tb00646.x","article-title":"Migration of hematogenous cells through the blood-brain barrier and the initiation of CNS inflammation","volume":"1","author":"Hickey","year":"1991","journal-title":"Brain Pathol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3598","DOI":"10.1002\/1521-4141(200212)32:12<3598::AID-IMMU3598>3.0.CO;2-6","article-title":"Encephalitogenic T cells use LFA-1 for transendothelial migration but not during capture and initial adhesion strengthening in healthy spinal cord microvessels in vivo","volume":"32","author":"Laschinger","year":"2002","journal-title":"Eur. J. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"S33","DOI":"10.1016\/S1465-3249(23)00182-2","article-title":"Off the Shelf, Allogeneic T regulatory cells show safety in Amyotrophic Lateral Sclerosis","volume":"25","author":"Shneider","year":"2023","journal-title":"Cytotherapy"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1016\/j.annonc.2021.12.003","article-title":"Management of adults and children receiving CAR T-cell therapy: 2021 best practice recommendations of the European Society for Blood and Marrow Transplantation (EBMT) and the Joint Accreditation Committee of ISCT and EBMT (JACIE) and the European Haematology Association (EHA)","volume":"33","author":"Hayden","year":"2022","journal-title":"Ann. Oncol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2667","DOI":"10.3324\/haematol.2020.254045","article-title":"Hematopoietic recovery and immune reconstitution after axicabtagene ciloleucel in patients with large B-cell lymphoma","volume":"106","author":"Strati","year":"2021","journal-title":"Haematologica"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2295","DOI":"10.1182\/blood-2017-06-793141","article-title":"Kinetics and biomarkers of severe cytokine release syndrome after CD19 chimeric antigen receptor-modified T-cell therapy","volume":"130","author":"Hay","year":"2017","journal-title":"Blood"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"664","DOI":"10.1158\/2159-8290.CD-16-0040","article-title":"Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia","volume":"6","author":"Teachey","year":"2016","journal-title":"Cancer Discov."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e003486","DOI":"10.1136\/jitc-2021-003486","article-title":"Time to evolve: Predicting engineered T cell-associated toxicity with next-generation models","volume":"10","author":"Donnadieu","year":"2022","journal-title":"J. Immunother. Cancer"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Zajkowska, M., and Mroczko, B. (2021). From Allergy to Cancer-Clinical Usefulness of Eotaxins. Cancers, 13.","DOI":"10.3390\/cancers13010128"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1007\/s13760-022-01984-3","article-title":"Eotaxin-1 (CCL11) in neuroinflammatory disorders and possible role in COVID-19 neurologic complications","volume":"122","author":"Nazarinia","year":"2022","journal-title":"Acta Neurol. Belg."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4589","DOI":"10.4049\/jimmunol.1301523","article-title":"Preclinical in vivo modeling of cytokine release syndrome induced by ErbB-retargeted human T cells: Identifying a window of therapeutic opportunity?","volume":"191","author":"Davies","year":"2013","journal-title":"J. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/s40164-020-00171-5","article-title":"Macrophage, the potential key mediator in CAR-T related CRS","volume":"9","author":"Hao","year":"2020","journal-title":"Exp. Hematol. Oncol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1808","DOI":"10.1002\/eji.201444960","article-title":"GM-CSF promotes migration of human monocytes across the blood brain barrier","volume":"45","author":"Vogel","year":"2015","journal-title":"Eur. J. Immunol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/s40364-018-0116-0","article-title":"Biomarkers of cytokine release syndrome and neurotoxicity related to CAR-T cell therapy","volume":"6","author":"Wang","year":"2018","journal-title":"Biomark. Res."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Hassan, G.S., Salti, S., and Mourad, W. (2022). Novel Functions of Integrins as Receptors of CD154: Their Role in Inflammation and Apoptosis. Cells, 11.","DOI":"10.3390\/cells11111747"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"871","DOI":"10.1002\/1529-0131(199905)42:5<871::AID-ANR5>3.0.CO;2-J","article-title":"Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera","volume":"42","author":"Vakkalanka","year":"1999","journal-title":"Arthritis Rheum."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1035","DOI":"10.1136\/gut.2003.026278","article-title":"The CD40\/CD40L costimulatory pathway in inflammatory bowel disease","volume":"53","author":"Danese","year":"2004","journal-title":"Gut"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2718","DOI":"10.1111\/j.1538-7836.2006.02206.x","article-title":"Soluble CD40 ligand in asthma patients during allergen challenge","volume":"4","author":"Kowal","year":"2006","journal-title":"J. Thromb. Haemost."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1038\/s41591-018-0041-7","article-title":"CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade","volume":"24","author":"Giavridis","year":"2018","journal-title":"Nat. Med."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/12\/14\/1880\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:13:48Z","timestamp":1760127228000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/12\/14\/1880"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,7,18]]},"references-count":42,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2023,7]]}},"alternative-id":["cells12141880"],"URL":"https:\/\/doi.org\/10.3390\/cells12141880","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,7,18]]}}}