Abstract: Objective To study the effect of carboxyamidotriazole (CAI) on CD8⁺T cells, and to explore the critical mechanism of CAI in reinforcing cytotoxicity of activated T cells. Methods CD8⁺T cells of mouse were isolated by immunomagnetic beads and divided into control group, CAI group, ZK756326 (Ca²⁺ activator) group and CAI+ZK756326 group. Flow cytometry was used to detect the level of intracellular free calcium ions in CD8⁺T cells; Enzyme-linked immunosorbent assay was used to detect the expression of intracellular calcineurin (CaN); Immunoflu- orescence staining was used to detect NFAT2 nuclear transportation; Chromatin immunoprecipitation-qPCR was used to detect the expression of PD-1 regulated by NFAT2. Mice spleen cytotoxic T lymphocytes (CTLs) were isolated and examined for the expression of PD-1 in CD8⁺T cells by flow cytometry. Results Intracellular Ca²⁺ concentration in CD8⁺T cells significantly decreased in the CAI group (P＜0.001), while intracellular Ca²⁺ concentration increased in the CAI+ZK756326 group(P＜0.01); CAI significantly decreased the content of calcineurin phosphatase in CD8⁺T cells (P＜0.001);CAI significantly inhibited NFAT2 nuclear transportation (P＜0.001) and significantly inhibited PD-1 transcription process depend on NFAT2 (P＜0.001); CAI significantly reduced the proportion of PD-1⁺CD8⁺T cells in mice spleen CTLs cells (P＜0.001). Conclusions CAI inhibits NFAT2 nuclear translocation by decreasing the level of calcium ions in the CD8⁺ T cells and inhibiting the expression of calcineurin, thereby reducing the expression of PD-1 in CD8⁺ T cells, which facilitated immunotherapy intervention.

Key words: carboxyamidotriazole, CD8⁺T cells, Ca²⁺, activated T cell nuclear factor 2, PD-1