American Association for Cancer Research

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Figure S1-S8 from Eradication of Hepatocellular Carcinoma by NKG2D-Based CAR-T Cells

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posted on 2023-04-04, 00:40 authored by Bin Sun, Dong Yang, Hongjiu Dai, Xiuyun Liu, Ru Jia, Xiaoyue Cui, Wenxuan Li, Changchun Cai, Jianming Xu, Xudong Zhao

Figure S1. NKG2DL levels in 50 pairs of normal tissues (N) and liver cancers (T). Data was obtained from The Cancer Genome Atlas (TCGA) database. Statistical differences were analyzed using paired Student's t-tests, *P {less than or equal to} 0.05, **P {less than or equal to} 0.01, ***P {less than or equal to} 0.001. Figure S2. Immunohistochemical analysis was performed to detect the expression of MICA and ULBP2 in Normal (N1-N4), Cirrhosis (C1-C7) and tumors (T) tissue, scale bar = 100 μm. Figure S3. T cells were isolated from peripheral blood using the RosetteSep{trade mark, serif} Human T Cell Enrichment Cocktail and the cell-surface expression of CD3 was analyzed by flow cytometry. The RAJI cell line was used as a negative control. Figure S4. SMMC7721 cells were cells infected with plko-shcoo2, plko-shMICA, and plko-shULBP2 lentivirus. Quantitative RT-PCR analysis showing the efficiency of MICA and ULBP2 knockdown in SMMC7721 cells. The shMICA-2# and shULBP2-2# was used to construct SMMC7721-shMICA and SMMC7721-shULBP2 cell lines, respectively. shCOO2 was used as negative control. The data are presented as the means {plus minus} SD. Figure S5. Growth curves of T cells isolated from healthy donor, patient 1 and patient 2. The data are presented as the means {plus minus} SD. P< 0.05 for the indicated cells. Figure S6. Flow cytometry analysis of the expression of CD19 and NKG2D CAR in transduced T cells isolated from healthy donor, patient 1 and patient 2. Figure S7. Proportions of CD4+ and CD8+ T cells in NTD, CD19-BBz CAR-T, and NKG2D-BBz CAR-T cells by flow cytometry. Figure S8. Proliferation (A) and apoptosis (B) of CD19-BBz CAR-T and NKG2D-BBz CAR-T cells at day 11 after transduction. The data are presented in as the means {plus minus} SD, ns, not significant.


National Natural Science Foundation of China



Despite the great success of chimeric antigen receptor T (CAR-T)–cell therapy in the treatment of hematologic malignancies, CAR-T–cell therapy is limited in solid tumors, including hepatocellular carcinoma (HCC). NK group 2 member D (NKG2D) ligands (NKG2DL) are generally absent on the surface of normal cells but are overexpressed on malignant cells, offering good targets for CAR-T therapy. Indeed, analysis of The Cancer Genome Atlas and HCC tumor samples showed that the expression of most NKG2DLs was elevated in tumors compared with normal tissues. Thus, we designed a novel NKG2D-based CAR comprising the extracellular domain of human NKG2D, 4-1BB, and CD3ζ signaling domains (BBz). NKG2D-BBz CAR-T cells efficiently killed the HCC cell lines SMMC-7721 and MHCC97H in vitro, which express high levels of NKG2DLs, whereas they less efficiently killed NKG2DL-silenced SMMC-7721 cells or NKG2DL-negative Hep3B cells. Overexpression of MICA or ULBP2 in Hep3B improved the killing capacity of NKG2D-BBz CAR-T cells. T cells expressing the NKG2D-BBz CAR effectively eradicated SMMC-7721 HCC xenografts. Collectively, these results suggested that NKG2D-BBz CAR-T cells could potently eliminate NKG2DL-high HCC cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for patients with NKG2DL-positive HCC.

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