Single-cell transcriptome analysis of human natural killer cells derived from umbilical cord blood in response to the BCL-2 inhibitor venetoclax

Natural killer (NK) cell-based immunotherapy holds promise for cancer treatment, but its efficacy is still limited, necessitating the development of new strategies. Here, we report an unexpected discovery that venetoclax, the first FDA-approved BCL-2 inhibitor, acts as an immunometabolic modulator to potentiate adoptive NK cell immunotherapy against acute myeloid leukemia (AML). Venetoclax directly activates human NK cells, boosting their cytotoxicity against AML both in vitro and in vivo, independent of BCL-2 inhibition. Notably, we identify a distinct CD161low CD218b+ NK cell subpopulation exhibiting the most pronounced proportional increase and transcriptomic changes upon venetoclax treatment.Venetoclax enhances NK cell binding avidity and lytic granule polarization during immunological synapse (IS) formation.Furthermore, venetoclax promotes mitochondrial respiration and ATP synthesis via the NF-?B pathway, facilitating IS formation and effector function in human NK cells.Our findings establish venetoclax as a multifaceted immunometabolic modulator that enhances NK cell function, providing new insights for augmenting NK cell-mediated cytotoxicity in cancer immunotherapy. Overall design: In order to explore the regulatory role of venetoclax in the killing function of NK cells, we isolated and purified NK cells derived from umbilical cord blood from 4 donors and treated them with venetoclax for 18 hours.For the scRNA-seq analysis of human cord blood NK ,pool 4 cells from the control group in one tube and 4 cells from the Venetoclax-treated group in another tube. We then counted and resuspended the pooled cells at a concentration of 1000 cells/µL, targeting an estimated 6000 cells per library, following the instructions of the Single Cell 3' Solution v3 Kit (10X Genomics). Single-cell libraries were constructed strictly according to the manufacturer's standard protocol. Libraries were sequenced on the Illumina NovaSeq 6000 System.