Cell activation cocktail including brefeldin a

Spleen cells were harvested by passing the single-cell suspensions through 70-μm pore-sized cell strainers (BD Falcon, Durham, NC). For detection of myeloid-derived suppressor cells (MDSCs), splenocytes were stained with FITC-conjugated anti-CD11b (eBioscience), percpcy5.5-conjugated anti-Gr-1 (Biolegend, San Diego, CA), and PE-CY7 anti-CD244 (Biolegend) antibodies. To examine the expression of INF-γ and CD107a in CD8 + T cells, both CD8 + T lymphocytes from the in vitro co-culture and the isolated splenocytes were stimulated with a cell activation cocktail (including Brefeldin A; Biolegend) according to the manufacturer's instructions and incubated at 37°C for 6 h before staining. The cells were stained with Brilliant Violet 510-conjugated anti-CD3 (Biolegend) and Alexa Fluor 488-conjugated anti-CD8a (Biolegend), followed by intracellular staining with APC-conjugated anti-interferon γ (anti-IFN-γ; eBioscience) and PE-conjugated anti-CD107a (eBioscience). To examine the expression of programmed death 1 (PD-1) in CD8 + T lymphocytes in vitro, the cells were stained with Alexa Fluor 488-conjugated anti-CD8a and Brilliant Violet 421-conjugated anti-CD279 (PD-1; Biolegend). Cells were analyzed using an LSR Fortessa (BD Biosciences, San Jose, CA) flow cytometer, and the data were analyzed using FlowJo software (BD Life Sciences, Franklin Lakes, NJ).