FIGURE 2.

RIME reduces sensitivity to the cytotoxicity of CD8⁺ T cells by regulating immune checkpoint molecules. (A) Fluorescence in situ hybridization (FISH) assays showing the subcellular localization of RIME was both in the nucleus and cytoplasm of oesophageal squamous cell carcinoma (ESCC) cells. Scale bar, 2 μm. (B) Quantitative real‐time polymerase chain reaction (qRT‒PCR) analysis of RIME expression in the cytoplasmic and nuclear fractions of ESCC cells, which showed that RIME was localized both in the nucleus and cytoplasm. (C) qRT‒PCR analysis showed that RIME expression was significantly inhibited in RIME CRISPR KO cells. (D) Real‐time cell analyser was used to analyse the viability of ESCC cells alone or cocultured with human CD8⁺ T cells. RIME KO had a slight effect on ESCC cell viability but significantly enhanced the cytotoxicity of CD8⁺ T cells. (E, F) The cytotoxicity of CD8⁺ T cells to ESCC cells was determined by crystal violet staining, which showed that RIME KO rendered ESCC cells more sensitive to cytotoxic CD8⁺ T cells. (G) The LDH levels released from damaged cells were determined to quantify the cytotoxicity of CD8⁺ T cells. The LDH levels were significantly increased in the RIME KO group. (H) ELISA assays showed that the IFN‐γ levels in the coculture medium were significantly increased in the RIME KO group. (I, J) qRT‒PCR analysis showed that RIME KO significantly decreased multiple immune‐related genes in ESCC cells. (K, L) qRT‒PCR analysis showed that RIME KO resulted in a remarkable decrease of PD‐L1 and IDO‐1 expression in ESCC cells. (M–O) Flow cytometry and statistical analysis showed that RIME KO significantly decreased PD‐L1 and IDO‐1 expression, while RIME overexpression increased PD‐L1 and IDO‐1 expression. (P) Immunoblot analysis showed that RIME KO significantly decreased PD‐L1 and IDO‐1 protein levels in ESCC cells.