Additional file 1 of B7H3-targeting chimeric antigen receptor modification enhances antitumor effect of Vγ9Vδ2 T cells in glioblastoma

Additional file 1: Figure S1. Hematoxylin and eosin staining of xenograft tumor sample from NSG mouse. Magnification 20 ×. Scale bar = 50 µm. Figure S2. A Flow cytometry analysis was conducted to detect the expression of BTN2A1 and BTN3A1 in GBM cell lines. B Car-B7H3-γδT cells were obtained by transfecting Vγ9Vδ2 T cells with plasmids containing scFv of the anti-B7-H3 antibody. Flow cytometry analysis was used to determine the purity of Car-B7H3-γδT cells. C Flow cytometry analysis was performed to determine B7-H3 expression in U87-MG, TJ905, and HTB15 cells. The protein expression of B7-H3 was 92.6%, 91.8%, and 93.2% in U-87MG, TJ905, and HTB15, respectively. D Vγ9Vδ2 T or Car-B7H3-γδT cells were incubated with U-87MG-Luc, TJ905-Luc, or HTB15-Luc cells at different effective target (E: T) ratios of 0:1, 0.5:1, 1 :1, or 3:1 in the presence of IL-2. The luciferase activity was measured to determine the cell viability of GBM cells at 18–20 h. E The culture medium was collected. ELISA was performed to measure IFN-γ and TNF-α levels. Data are expressed as the mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001; n = 3. Figure S3. Hematoxylin and eosin staining of the liver, lung, ovary, brain, spleen, kidney, stomach, heart, and uterus tissue samples from NSG mice after injection with PBS A or high-dose (5 × 106 cells/5 μL PBS) Vγ9Vδ2 T cells B. Magnification 10 ×. Figure S4. Evaluation of Vγ9Vδ2 T and Car-B7H3-γδT cell toxicity. A The body weights of mice were measured at different time points after intraventricular injection of control (PBS), low-dose of Vγ9Vδ2 T cells (5 × 105 cells/5 μL PBS), or high-dose of Vγ9Vδ2 T cells (5 × 106 cells/5 μL PBS). B The body weights of GBM tumor-bearing mice were measured at different time points after intraventricular injection. C Cytokine multiplex assay was carried out to examine mouse serum cytokine alterations at 7 days after T cell therapy. Z-score of each cytokine was calculated as the mean fluorescence intensity. A heatmap of the z-scores ranging from − 3 to 3 was generated. Table S1. Basic characteristics of all PTC patients. Table S2. A Primary antibody used in Multiplex staining. B Experimental conditions and procedures of Multiplex staining of TCRδ2 panel. C. Experimental conditions and procedures of Multiplex staining of B7H3 panel. Table S3. Relationship between different grades of glioma and different responses to Vγ9Vδ2 T therapy.