Ex vivo pretreatment with venetoclax boosts antileukemic efficacy of therapeutic γδ T cells in AML models
Xingchi Chen, Lin Zhang, Bingbing Yan, Yinqiang Sui, Weiwei Ma, Hui Zhao, Yining Wang, Kepeng Yang, Jiewen Ma, Baolin Tang, Yonghui Zhang, Xiaoyu Zhu
Xingchi Chen, Lin Zhang, Bingbing Yan, Yinqiang Sui, Weiwei Ma, Hui Zhao, Yining Wang, Kepeng Yang, Jiewen Ma, Baolin Tang, Yonghui Zhang, Xiaoyu Zhu
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Research Article Hematology Immunology

Ex vivo pretreatment with venetoclax boosts antileukemic efficacy of therapeutic γδ T cells in AML models

Abstract

Ex vivo engineering strategies for adoptive αβ T cell therapies increasingly use pharmacological modulation to improve survival, expansion, and antitumor activity. Short-term exposure to the B cell lymphoma-2 (BCL-2) inhibitor venetoclax during αβ T cell manufacturing enhances apoptotic priming and effector persistence, suggesting a route to strengthen other T cell lineages. γδ T cells share cytotoxic properties with αβ T cells but recognize targets independently of major histocompatibility complex and show low alloreactivity, supporting off-the-shelf use in acute myeloid leukemia (AML). Whether such conditioning benefits γδ T cells was unknown. Here, we show that ex vivo venetoclax pretreatment enhances the antileukemic efficacy of therapeutic γδ T cells and chimeric antigen receptor (CAR) γδ T cells. Venetoclax-pretreated γδ T cells displayed increased cytotoxicity and proliferation with reduced exhaustion, yielding superior control of AML blasts and xenografts. These functional gains coincided with elevated mitochondrial content and a fatty acid oxidation metabolic profile. In vivo, venetoclax-pretreated γδ T cells achieved durable disease suppression, and the same conditioning improved CAR γδ T cell efficacy. Together, these results show that short-term BCL-2 inhibition enhances γδ T cell cytotoxicity and persistence. Incorporating venetoclax pretreatment into γδ T cell manufacturing may improve therapeutic efficacy and inform next-generation γδ T cell therapies for AML.

Authors

Xingchi Chen, Lin Zhang, Bingbing Yan, Yinqiang Sui, Weiwei Ma, Hui Zhao, Yining Wang, Kepeng Yang, Jiewen Ma, Baolin Tang, Yonghui Zhang, Xiaoyu Zhu

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Figure 6

Venetoclax promotes mitochondrial abundance and FAO–OXPHOS–linked metabolism in γδ T cells.

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Venetoclax promotes mitochondrial abundance and FAO–OXPHOS–linked metabo...
(A) Transmission electron microscopy of γδ T cells pretreated with dimethyl sulfoxide (DMSO) or venetoclax. White arrows indicate mitochondria. Scale bars: 2 μm. Mitochondrial number was quantified from more than 3 randomly selected cells per group (n = 3 donors). (B) Mitochondrial DNA content in DMSO- and venetoclax-pretreated γδ T cells (ven-γδ T cells), measured by qPCR for mitochondrially encoded cytochrome c oxidase I (MT-CO1), as a marker of mitochondrial DNA content, relative to β-actin (n = 5 donors). (C) Mitochondrial mass assessed by MitoTracker Green flow cytometry (n = 5 donors). (D) Confocal images and 3D renderings of DMSO- and ven-γδ T cells. Images are representative of 3 independent donor experiments. Red, γδ TCR; blue, Draq5; green, MitoTracker Green. Scale bars: 5 μm. (E) Heatmap of differentially abundant metabolites (P < 0.05) identified by untargeted metabolomics using γδ T cells from 5 donors. Abundances are Z-score–normalized. (F) Pathway enrichment by MetaboAnalyst; main pathways with a P value < 0.05 are shown. (G) Fold-changes of FAO-related genes in ven-γδ T cells versus DMSO-γδ T cells, normalized to β-actin (≥3 donors, triplicate measurements). (H) CPT1A protein by immunoblot (representative of 3 donors) with pooled quantification (n = 6 donors). Mean ± SD. Data were analyzed using Wilcoxon’s matched pairs test. (I) OCR curves after DMSO, venetoclax, etomoxir (Eto) plus DMSO, or venetoclax plus etomoxir (5 μM, 18 hours) treatment, with basal and maximal OCR quantified. Data are representative of 3 independent donor experiments. Oligo, oligomycin; Rot/AA, rotenone and antimycin A; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. (J) Cytotoxicity of γδ T cells after the indicated treatments (n = 3). Data represent mean ± SD. Statistical analyses were performed using unpaired, 2-tailed Student’s t test (A and G), paired 2-tailed Student’s t test (B and C), and repeated measures 1-way ANOVA with Tukey’s multiple comparisons test (I and J). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.