{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T02:36:05Z","timestamp":1788834965801,"version":"build-2803163510"},"reference-count":50,"publisher":"BMJ","issue":"4","license":[{"start":{"date-parts":[[2025,4,16]],"date-time":"2025-04-16T00:00:00Z","timestamp":1744761600000},"content-version":"unspecified","delay-in-days":15,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"Alberta Cancer Foundation Start-up Fund","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}]},{"name":"Canadian Cancer Society Impact Grant","award":["#704121"],"award-info":[{"award-number":["#704121"]}]},{"DOI":"10.13039\/501100000024","name":"Canadian Institutes of Health Research","doi-asserted-by":"publisher","award":["#191854"],"award-info":[{"award-number":["#191854"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000024","name":"Canadian Institutes of Health Research","doi-asserted-by":"publisher","award":["#419699"],"award-info":[{"award-number":["#419699"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Princess Margaret Cancer Centre Innovation Acceleration Fund","award":["IAF #2023-003"],"award-info":[{"award-number":["IAF #2023-003"]}]}],"content-domain":{"domain":["bmj.com"],"crossmark-restriction":true},"short-container-title":["J Immunother Cancer"],"accepted":{"date-parts":[[2025,4,2]]},"published-print":{"date-parts":[[2025,4]]},"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Allogeneic double-negative T-cell (DNT) therapy has emerged as a novel, off-the-shelf cellular treatment with clinical feasibility, safety, and promising efficacy against leukemia. However, the biology of DNTs is less well characterized, and how DNT therapy distinguishes from conventional \u03b3\u03b4 T-cell therapy remains unclear. Collectively, this hinders our ability to bolster DNT functionalities in cancer therapy. Here, we performed single-cell RNA sequencing with in vitro and in vivo functional analysis on DNTs. As a significant proportion of DNTs express V\u03b39V\u03b42 (V\u03b42) TCR chain, we compared DNTs with donor-matched conventional V\u03b42 T cells expanded with zoledronic acid.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>Healthy donor-derived allogeneic DNTs and V\u03b42 T cells were expanded ex vivo. Single-cell RNA sequencing analysis was performed on both cellular products to identify the transcriptional landscape and inferred cellular interactions within DNTs, followed by comparisons with donor-matched V\u03b42 T cells. Unique cellular subsets found only in DNTs were depleted to identify their contributions to the overall efficacy of DNTs against acute myeloid leukemia. The anti-leukemic activity and in vivo persistence of DNTs and V\u03b42 T-cells were explored using flow cytometry-based cytotoxicity assays, memory phenotyping, and xenograft models.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Despite a shared V\u03b42 expression between cellular products, we identified unique cellular compositions in DNTs that contribute to distinct transcriptional and cellular communication patterns relative to the donor-matched V\u03b42 T cells, including higher expression of genes identified in chimeric antigen receptor T cells that persist in patients with durable cancer-remission. V\u03b42\n                      <jats:sup>\u2013<\/jats:sup>\n                      DNTs exhibited strong persistence characteristics, and their presence promoted the cytotoxic capabilities of V\u03b42\n                      <jats:sup>+<\/jats:sup>\n                      DNTs in repeated stimulation assays. This unique genetic signature and diverse cellular composition of DNTs resulted in better overall ex vivo expansion, prolonged persistence, and superior anti-leukemic activity compared with V\u03b42 T cells in vitro and in vivo.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>These results highlight the unique transcriptional, cellular, and functional profile of human DNTs and support the continued clinical investigation of allogeneic DNT therapy. The data also provide a reference gene signature that may help improve the efficacy of other types of allogeneic adoptive cellular therapies.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1136\/jitc-2024-010684","type":"journal-article","created":{"date-parts":[[2025,4,17]],"date-time":"2025-04-17T20:15:21Z","timestamp":1744920921000},"page":"e010684","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":7,"title":["Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy"],"prefix":"10.1136","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7838-0400","authenticated-orcid":false,"given":"Enoch","family":"Tin","sequence":"first","affiliation":[{"name":"University Health Network, Toronto, Ontario, Canada"},{"name":"Immunology, University of Toronto, Toronto, Ontario, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ismat","family":"Khatri","sequence":"additional","affiliation":[{"name":"University Health Network, Toronto, Ontario, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-6662-7872","authenticated-orcid":false,"given":"Karen","family":"Fang","sequence":"additional","affiliation":[{"name":"University Health Network, Toronto, Ontario, Canada"},{"name":"Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoosu","family":"Na","sequence":"additional","affiliation":[{"name":"University Health Network, Toronto, Ontario, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michele","family":"Nawata","sequence":"additional","affiliation":[{"name":"Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada"},{"name":"Arnie Charbonneau Cancer Institute, Calgary, Alberta, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Arteaga","sequence":"additional","affiliation":[{"name":"Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada"},{"name":"Arnie Charbonneau Cancer Institute, Calgary, Alberta, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark D","family":"Minden","sequence":"additional","affiliation":[{"name":"University Health Network, Toronto, Ontario, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1970-7375","authenticated-orcid":false,"given":"Sergio","family":"Rutella","sequence":"additional","affiliation":[{"name":"John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jongbok","family":"Lee","sequence":"additional","affiliation":[{"name":"Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada"},{"name":"Arnie Charbonneau Cancer Institute, Calgary, Alberta, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Zhang","sequence":"additional","affiliation":[{"name":"University Health Network, Toronto, Ontario, Canada"},{"name":"Immunology, University of Toronto, Toronto, Ontario, Canada"},{"name":"Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"239","published-online":{"date-parts":[[2025,4,17]]},"reference":[{"key":"2026052108353443000_13.4.e010684.1","doi-asserted-by":"crossref","unstructured":"Dance A . 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Allogeneic double\u2010negative T cell therapy for relapsed acute myeloid leukemia patients post allogeneic hematopoietic stem cell transplantation: A first\u2010in\u2010human phase I study. 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