Fig. 4.

CD8⁺NKT-like cells exert their cytotoxicity via granule exocytosis pathway. a CD8⁺NKT-like cells, NK cells, and NK1.1⁻CTLs were gated from in vitro-activated lymphocytes and their expression of cytotoxicity-associated molecules was detected by flow cytometry. b OT-I mouse-derived CD8⁺NKT-like cells and EL4-OVA8 cells were co-cultured at an E:T ratio of 5:1 with or without 50 μg/mL of anti-FasL, 50 μg/mL anti-TRAIL, 50 μM granzyme B inhibitor Z-AAD-CMK, or 100 μg/mL of anti-IFN-γ. After 24 h, cytotoxicity against target cells was evaluated. c OT-I mouse-derived CD8⁺NKT-like cells and OVA_257–264-loaded/unloaded MDSCs were co-cultured at an E:T ratio of 10:1 with or without 50 μg/mL anti-FasL, 50 μg/mL anti-TRAIL, or 50 μM granzyme B inhibitor Z-AAD-CMK. After 24 h, 7-AAD-negative live MDSCs were collected and counted by flow cytometry. Their cytotoxicity against target cells was evaluated and shown as a histogram. d Granzyme B expression was evaluated by in situ anti-granzyme B fluorescent antibody staining (green) in the co-culture system of CD8⁺NKT-like and B16 cells (red) during different killing stages. Nucleus was stained with Hoechst 33,342 (blue). These experiments were repeated thrice