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.