Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy

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Tin, E, Khatri, I, Fang, K, Na, Y, Nawata, M, Arteaga, J, Minden, MD, Rutella, S ORCID: https://orcid.org/0000-0003-1970-7375, Lee, J and Zhang, L, 2025. Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy. Journal for ImmunoTherapy of Cancer, 13 (4): e010684. ISSN 2051-1426

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Official URL: https://doi.org/10.1136/jitc-2024-010684

Abstract

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.

Item Type:	Journal article
Publication Title:	Journal for ImmunoTherapy of Cancer
Creators:	Tin, E., Khatri, I., Fang, K., Na, Y., Nawata, M., Arteaga, J., Minden, M.D., Rutella, S., Lee, J. and Zhang, L.
Publisher:	BMJ
Date:	April 2025
Volume:	13
Number:	4
ISSN:	2051-1426
Identifiers:	Number Type 10.1136/jitc-2024-010684 DOI 2431857 Other
Rights:	© Author(s) (or their employer(s)) 2025. Published by BMJ Group. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
Divisions:	Schools > School of Science and Technology
Record created by:	Melissa Cornwell
Date Added:	28 Apr 2025 09:09
Last Modified:	28 Apr 2025 09:09
URI:	https://irep.ntu.ac.uk/id/eprint/53468