ICS was performed according to the method described in a previous report [22 (link)]. Briefly, CD8+ CTLs were subjected to the above-mentioned treatments for 48 h. Afterwards, Brefeldin A (10 μg/mL) was added for another 4 h of incubation. Then, the cells were stained with Live/Dead Fixable Red Stain dye (Thermo Scientific, USA) and fluorochrome-conjugated monoclonal antibodies for cell surface proteins, according to the manufacturer’s instructions. The stained cells were fixed and permeabilized using a fixation/permeabilization buffer kit (BD Biosciences, USA) and stained for intracellular cytokines. The stained cells were subjected to flow cytometric analysis.