Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy
Background 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...
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| Format: | Article |
| Language: | English |
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BMJ Publishing Group
2025-04-01
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| Series: | Journal for ImmunoTherapy of Cancer |
| Online Access: | https://jitc.bmj.com/content/13/4/e010684.full |
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| author | Li Zhang Sergio Rutella Jongbok Lee Ismat Khatri Yoosu Na Enoch Tin Karen Fang Michele Nawata Juan Arteaga Mark D Minden |
| author_facet | Li Zhang Sergio Rutella Jongbok Lee Ismat Khatri Yoosu Na Enoch Tin Karen Fang Michele Nawata Juan Arteaga Mark D Minden |
| author_sort | Li Zhang |
| collection | DOAJ |
| description | Background 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 γδ 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 Vγ9Vδ2 (Vδ2) TCR chain, we compared DNTs with donor-matched conventional Vδ2 T cells expanded with zoledronic acid.Methods Healthy donor-derived allogeneic DNTs and Vδ2 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 Vδ2 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 Vδ2 T-cells were explored using flow cytometry-based cytotoxicity assays, memory phenotyping, and xenograft models.Results Despite a shared Vδ2 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 Vδ2 T cells, including higher expression of genes identified in chimeric antigen receptor T cells that persist in patients with durable cancer-remission. Vδ2– DNTs exhibited strong persistence characteristics, and their presence promoted the cytotoxic capabilities of Vδ2+ 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 Vδ2 T cells in vitro and in vivo.Conclusions 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. |
| format | Article |
| id | doaj-art-e2d0d20d46134ee595d48b59971cf5a1 |
| institution | Kabale University |
| issn | 2051-1426 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | BMJ Publishing Group |
| record_format | Article |
| series | Journal for ImmunoTherapy of Cancer |
| spelling | doaj-art-e2d0d20d46134ee595d48b59971cf5a12025-08-20T03:52:06ZengBMJ Publishing GroupJournal for ImmunoTherapy of Cancer2051-14262025-04-0113410.1136/jitc-2024-010684Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapyLi Zhang0Sergio Rutella1Jongbok Lee2Ismat Khatri3Yoosu Na4Enoch Tin5Karen Fang6Michele Nawata7Juan Arteaga8Mark D Minden9Department of Medical Oncology, State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-sen University Cancer Center, Guangzhou, China1 John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, Nottinghamshire, UKToronto General Hospital Research Institute, Toronto, Ontario, CanadaToronto General Hospital Research Institute, Toronto, Ontario, CanadaUniversity Health Network, Toronto, Ontario, CanadaUniversity Health Network, Toronto, Ontario, CanadaUniversity Health Network, Toronto, Ontario, CanadaBiochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, CanadaBiochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, CanadaUniversity Health Network, Toronto, Ontario, CanadaBackground 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 γδ 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 Vγ9Vδ2 (Vδ2) TCR chain, we compared DNTs with donor-matched conventional Vδ2 T cells expanded with zoledronic acid.Methods Healthy donor-derived allogeneic DNTs and Vδ2 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 Vδ2 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 Vδ2 T-cells were explored using flow cytometry-based cytotoxicity assays, memory phenotyping, and xenograft models.Results Despite a shared Vδ2 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 Vδ2 T cells, including higher expression of genes identified in chimeric antigen receptor T cells that persist in patients with durable cancer-remission. Vδ2– DNTs exhibited strong persistence characteristics, and their presence promoted the cytotoxic capabilities of Vδ2+ 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 Vδ2 T cells in vitro and in vivo.Conclusions 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.https://jitc.bmj.com/content/13/4/e010684.full |
| spellingShingle | Li Zhang Sergio Rutella Jongbok Lee Ismat Khatri Yoosu Na Enoch Tin Karen Fang Michele Nawata Juan Arteaga Mark D Minden Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy Journal for ImmunoTherapy of Cancer |
| title | Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy |
| title_full | Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy |
| title_fullStr | Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy |
| title_full_unstemmed | Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy |
| title_short | Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy |
| title_sort | single cell rna sequencing of human double negative t cells reveals a favorable cellular signature for cancer therapy |
| url | https://jitc.bmj.com/content/13/4/e010684.full |
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