The immune cell populations were subjected to surface staining for flow cytometry analysis. The used antibodies are as follows: PE‐conjugated anti‐CD3ε (Biolegend), PerCP‐conjugated anti‐CD4 (BD Pharmingen), PerCP‐conjugated anti‐CD8α (BD Pharmingen), FITC‐conjugated anti‐NK1.1 (Biolegend), FITC‐conjugated anti‐CD11b (Biolegend), PE‐conjugated anti‐CD103 (Biolegend), APC‐conjugated anti‐CD11c (BD Pharmingen), PerCP‐conjugated anti‐F4/80 (Biolegend), or FITC‐conjugated anti‐Gr‐1 (Biolegend).
For analyzing cytokine secretion, splenocytes were inoculated into a 12‐well plate at 4 × 106/well and incubated with 10 μg/mL CAIX protein at 37°C and 5% CO2 for 3 days, then added 500 ng/mL Ionomycin (Sigma‐Aldrich) and 50 ng/mL PMA (Sigma‐Aldrich) and 5 ng/mL Brefeldin A (BFA, eBioscience) to incubate for 5 hours. Then cells were collected and operated extracellular staining with anti‐mouse PerCP‐conjugated anti‐CD8α and intracellular staining with following anti‐mouse antibodies: APC‐conjugated anti‐IFN‐γ (BD Pharmingen), PE‐conjugated anti‐IL‐2 (BD Pharmingen), FITC‐conjugated anti‐TNF‐α (BD Pharmingen). The data acquired from BD FACSCanto II (BD Biosciences) in FACSDiva software were analyzed by FlowJo software (Tree Star Inc).
For analyzing cytokine secretion, splenocytes were inoculated into a 12‐well plate at 4 × 106/well and incubated with 10 μg/mL CAIX protein at 37°C and 5% CO2 for 3 days, then added 500 ng/mL Ionomycin (Sigma‐Aldrich) and 50 ng/mL PMA (Sigma‐Aldrich) and 5 ng/mL Brefeldin A (BFA, eBioscience) to incubate for 5 hours. Then cells were collected and operated extracellular staining with anti‐mouse PerCP‐conjugated anti‐CD8α and intracellular staining with following anti‐mouse antibodies: APC‐conjugated anti‐IFN‐γ (BD Pharmingen), PE‐conjugated anti‐IL‐2 (BD Pharmingen), FITC‐conjugated anti‐TNF‐α (BD Pharmingen). The data acquired from BD FACSCanto II (BD Biosciences) in FACSDiva software were analyzed by FlowJo software (Tree Star Inc).