Moderate expression of CD39 in GPC3-CAR-T cells shows high efficacy against hepatocellular carcinoma.

CD39 serves as a crucial biomarker for neoantigen-specific CD8⁺ T cells and is associated with antitumor activity and exhaustion. However, the relationship between CD39 expression levels and the function of chimeric antigen receptor T (CAR-T) cells remains controversial. This study aimed to investigate the role of CD39 in the functional performance of CAR-T cells against hepatocellular carcinoma (HCC) and explore the therapeutic potential of CD39 modulators, such as mitochondrial division inhibitor-1 (mdivi-1), or knockdown CD39 through short hairpin RNA. Our findings demonstrated that glypican-3-CAR-T cells with moderate CD39 expression exhibited a strong antitumor activity, while high and low levels of CD39 led to an impaired cellular function. Methods modulating the proportion of CD39 intermediate (CD39^int)-phenotype CAR-T cells such as mdivi-1 and CD39 knockdown enhanced and impaired T cell function, respectively. The combination of mdivi-1 and CD39 knockdown in CAR-T cells yielded the highest proportion of infiltrated CD39^int CAR-T cells and demonstrated a robust antitumor activity in vivo. In conclusion, this study revealed the crucial role of CD39 in CAR-T cell function, demonstrated the potential therapeutic efficacy of combining mdivi-1 with CD39 knockdown in HCC, and provided a novel treatment strategy for HCC patients in the field of cellular immunotherapy.