α-ASGM-1 treatment predominantly targets NK cells but not activated T cells.

Splenocytes from naive mice or those from tumor-bearing mice 8 days after tumor injection, either immunocompetent or α-ASGM-1–treated, were collected. Splenocytes from immunocompetent tumor-bearing mice were restimulated in vitro 8 days after tumor injection with irradiated cancer cells for 3 days and subsequently treated with 1 μg/mL PMA and 1 ng/mL ionomycin for 5 hours, including GolgiPlug (BD Biosciences) during the final 4 hours. Viable cells were surface-labeled with α-CD8–PerCPCy5.5 and α-ASGM-1/FITC-conjugated α-rabbit IgG, fixed and permeabilized, stained intracellularly with α-IFN-γ–PE, and analyzed by FACS. The 2 upper panels represent total spleen cultures, the lower panel represents the gated IFN-γ⁺ population (indicated in red).