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

doi:10.1007/s11684-024-1071-9

Front. Med.

2024, Vol. 18

Issue (4) : 708-720 https://doi.org/10.1007/s11684-024-1071-9

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

Fan Zou^1,⁵, Jialiang Wei³, Jialang Zhuang^6,⁷, Yafang Liu², Jizhou Tan⁴(

), Xianzhang Huang²(

), Ting Liu²(

)

¹. Guangdong Cardiovsacular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Science, Guangzhou 510080, China
². The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Department of Laboratory Medicine/State Key Laboratory of Traditional Chinese Medicine Syndrome, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou 510120, China
³. Department of Medical Oncology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
⁴. Department of Stomatology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China
⁵. Medical Research Institute, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Science), Southern Medical University, Guangzhou 510080, China
⁶. School of Food and Drug, Shenzhen Polytechnic University, Shenzhen 518055, China
⁷. Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518055, China

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Abstract

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.

Keywords CD39 CAR-T cells mdivi-1 hepatocellular carcinoma antitumor activity

Corresponding Author(s): Jizhou Tan,Xianzhang Huang,Ting Liu

Just Accepted Date: 10 May 2024 Online First Date: 05 June 2024 Issue Date: 30 August 2024

Cite this article:

Fan Zou,Jialiang Wei,Jialang Zhuang, et al. Moderate expression of CD39 in GPC3-CAR-T cells shows high efficacy against hepatocellular carcinoma[J]. Front. Med., 2024, 18(4): 708-720.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-024-1071-9
https://academic.hep.com.cn/fmd/EN/Y2024/V18/I4/708

Fig.1 CD39^int CD8⁺ CAR-T cells presented an antitumor activity in vivo. (A) In vivo experimental design. GPC3⁺ Huh7 cell lines at 1 × 10⁶ cells were inoculated s.c. into NSG mice. Fourteen days later, GPC3-CAR-T cells were infused via tail injection, n = 6. On day 14, tumors were resected, followed by digestion and FACS analysis. (B,C) Flow cytometry for analyzing the CD39^hi/int/- subsets gated from tumor-infiltrating GPC3-CAR-T cells. The frequency of IFN-γ⁺ from each subset is revealed. Statistical data is also shown. All data are the mean of three independent experiments performed in duplicate. Mean ± SEM, unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig.2 CD39 knockdown compromised cytotoxicity and proliferation but reduced apoptosis in CAR-T cells in vitro. (A) Schematic of the lentiviral vectors carrying a GPC3-specific CAR moiety and a cluster of sh-CD39. (B) In vitro experimental design. GPC3-CAR-T and shCD39-GPC3-CAR-T cells were separately cocultured and stimulated by targeted antigen rhGPC3 protein. T cells were harvested and tested by flow cytometry to analyze the CAR-T function. (C) Bar plots revealing the proportion of CD39^-/int/hi expression in GPC3-CAR-T and shCD39-GPC3-CAR-T cells. (D) Bar plots demonstrating the expression of exhaustion markers, including PD-1, Tim-3, and Lag-3, in GPC3-CAR-T and shCD39-GPC3-CAR-T cells. (E) Flow analysis and bar plot presenting the proportion of IFN-γ⁺ GPC3-CAR-T and shCD39-GPC3-CAR-T cells. (F) Flow analysis and bar plot showing the proportion of Ki67⁺ GPC3-CAR-T and shCD39-GPC3-CAR-T cells. (G) Flow analysis and bar plot indicating the level of apoptosis in GPC3-CAR-T and shCD39-GPC3-CAR-T cells in the presence of TNF-a and FasL (100 ng/mL). All data are the mean of three independent experiments performed in duplicate. Mean ± SEM, unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig.3 Mdivi-1 promoted the cytokine secretion, proliferation, and cytotoxicity but induced exhaustion and apoptosis in CAR-T cells in vitro. (A) Three candidate chemical compounds that potentially affect CD39 expression. (B) IC50 of three compounds in GPC3-CAR-T cells. (C) Bar plots showing the proportion of CD39^int CAR-T cells with different dosages of compound stimulation. (D) In vitro experimental design. CD8⁺ CAR-T cells were sorted into CD39^hi, CD39^int, and CD39^- subpopulations using a CD39 flow cytometry-based sorting method. Then, the sorted CAR-T cells were stimulated in the presence or absence of 50 µM mdivi-1 and incubated for 24 h. Afterward, the cells were labeled with the same flow cytometry antibodies and analyzed. The flow analysis revealed that mdivi-1 reverted the CD39^- CAR-T cells into the CD39^int CAR-T cell population. (E) In vitro experimental design. GPC3-CAR-T cells in the presence or absence of 50 µM mdivi-1 were stimulated by targeted antigen rhGPC3 protein. T cells were harvested and tested by flow cytometry to analyze the function of GPC3-CAR-T cells. (F) Bar plots revealing the proportion of CD39^-/int/hi expression in GPC3-CAR-T cells in the presence or absence of 50 µM mdivi-1. (G) Bar plots demonstrating the expression of exhaustion markers, including PD-1, Tim-3, and Lag-3, in GPC3-CAR-T cells in the presence or absence of 50 µM mdivi-1. (H) Expression of INF-γ and Ki-67 of GPC3-CAR-T cells in the presence or absence of 50 µM mdivi-1. (I) Level of apoptosis of GPC3-CAR-T cells in the presence or absence of 50 µM mdivi-1 treated with TNF-a and FasL (100 ng/mL). Bar plots showing the statistical data of apoptotic assay. All data are the mean of three independent experiments performed in duplicate. Mean ± SEM, unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig.4 shCD39-CAR-T cells combined with mdivi-1 exhibited a synergistic effect and enhanced the antitumor activity in the HCC organoid model. (A) GPC3-CAR-T and shCD39-GPC3-CAR-T cells cocultured with HCC tumor organoids for 24 h in the presence or absence of 50 µM mdivi-1. CAR-T cells were labeled with CellTrace Far Red (red), and apoptotic cells were labeled with caspase3/7 probe (green). A real-time Biotech imaging system was used to take 1 picture per hour. Scale bar = 300 mm. (B) Summary of quantitative statistics. Apoptotic cells (green) were calculated using the Spot function of Imaris software on the basis of the size and intensity threshold. The initial number of infiltrating GPC3-CAR-T cells at 0 h was defined as the base point. (C) Flow analysis revealing the proportion of CD39 and IFN-γ expression in CAR-T cells. (D) Bar plots showing the statistical data of the percentage of CD39^int/hi CD8⁺ GPC3-CAR-T cells. (E) Bar plots presenting the proportion of IFN-γ⁺ GPC3-CAR-T and shCD39-GPC3-CAR-T cells in the presence or absence of 50 µM mdivi-1. (F) Bar plots demonstrating the proportion of Ki67⁺ GPC3-CAR-T and shCD39-GPC3-CAR-T cells in the presence or absence of 50 µM mdivi-1. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). All data are means ± SEMs.

Fig.5 shCD39-CAR-T cells combined with mdivi-1 exhibited a potent antitumor activity by increasing the proportion of infiltrated CD39^int CAR-T cells. GPC3⁺ Huh7 cell lines were inoculated s.c. into NSG mice at a concentration of 1 × 10⁶ cells. After 14 days, CAR-T cells were infused via tail injection, and mdivi-1 was administered via intraperitoneal injection at a dose of 20 mg/kg. (A) Representative photo of the tumor (n = 6). (B) Line chart depicting the trend in tumor volume (n = 6). (C) Dot plots showing the tumor weight on day 40 (n = 6). (D) Tumor-infiltrating CAR-T cells analyzed by flow cytometry. The percentage of CD39^int/hi CAR-T cells in tumor of different groups is shown (n = 6). (E) Flow analysis revealing the proportion of CD39 and IFN-γ expression in CAR-T cells. (F) Tumor-infiltrating CAR-T cells analyzed by flow cytometry. The percentages of IFN-γ⁺ of CD39^int/hi CAR-T cells of different groups are presented (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). All data are means ± SEMs.

Fig.6 shCD39-GPC3-CAR-T combined with mdivi-1 exerted strong infiltration and cytotoxicity. (A) Immunofluorescence staining from tumors of the Huh7 cell injection model treated with GPC3-CAR-T cells. Scale bar = 20 μm. (B) Dot plots representing infiltrated CAR-T cells and GZMB⁺ CAR-T cells. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). All data are means ± SEMs.

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