Fig. 7

CARHSP1 knockdown stimulates anti-tumor T cell immunity. (A) Analysis of cell surface PD-1 protein using flow cytometry in Jurkat cells after the treatment with CD3/CD28/CD2 T Cell Activator (25 μL/mL) for 24 h (n = 3, mean ± SD). (B, C) T cell-mediated tumor cell killing assay in 22Rv1 and PC-3 cells with stable depletion of CARHSP1. Activated Jurkat cells were co-cultured with control or CARHSP1-depleted PCa cells at the T cell to tumor cell ratio of 10:1. After 24 h interaction, cell death of both PCa cells (B) and Jurkat cells (C) in the PCa cell-Jurkat cell co-culture systems were analyzed by PI staining followed by flow cytometry. The quantitative ratio of dead cells is showed by the bar graph (n = 3, mean ± SD). (D) The secretion of IFN-γ, IL-2, and TNF-α by Jurkat cells after co-cultured with PC-3 cells transfected with shCAR or shCon for 24 h, as detected by RT-qPCR (n = 3, mean ± SD). 18 S was used as an internal loading control. (E) The levels of IFN-γ produced by Jurkat cells after co-cultured with PC-3 cells for 24 h, as detected by ELISA (n = 3, mean ± SD). (F) Apoptosis of Jurkat cells after treatment with IL-17 A (20 ng/mL), as detected by flow cytometry assay (n = 3, mean ± SD). (G) T cell-mediated tumor cell killing assay in 22Rv1 and PC-3 cells with stable depletion of CARHSP1 after treatment with IL-17 A (20 ng/mL). Activated Jurkat cells were co-cultured with control or CARHSP1-depleted PCa cells as described above. Simultaneously treatment with IL-17 A (20 ng/mL) for 24 h, cell death of both PCa cells in the PCa cell-Jurkat cell co-culture systems was analyzed by PI staining followed by flow cytometry. (n = 3, mean ± SD). **p < 0.01; ***p < 0.001; ns, not significant