Anti cd8α clone 53 6.7

Mice were subcutaneously inoculated with 3 × 10⁶ tumor cells for survival and treatment analysis and 5 × 10⁶ tumor cells for immune profiling. Intravenous injection was performed with 2 × 10⁵ tumor cells. Intratracheal inoculation was conducted as follows. Mice were anesthetized and supported on an intubation stand (Natsume). A 22-G catheter (TERUMO) was inserted into the trachea. A cell suspension containing 1 × 10⁶ cells in 50-μl sterile PBS was pipetted into the catheter and inhaled by mice. For CD8⁺ T cell depletion experiments, anti-CD8α (clone 53-6.7) (100 μg per mouse) (Bio X Cell) and isotype control antibody (rat IgG2a, Bio X Cell) (100 μg per mouse) were injected intraperitoneally at days −1, 3, 6, and 10 after tumor inoculation. For anti–PD-1 antibody treatment, anti–PD-1 antibody (clone RMP1–14) (200 μg per mouse) (Bio X Cell) and isotype control antibody (rat IgG2a, Bio X Cell) (200 μg per mouse) were intraperitoneally injected twice a week from day 3 to day 10. Recombinant mouse Sema4A-Fc (50 μg per mouse) was purchased from Oriental Yeast and intravenously injected at days 3, 7, and 10 after tumor inoculation. Tumor size was measured using calipers, and tumor volume was calculated using the following formula: (major axis × minor axis²) × 0.5.