Fig. 6: The anti-tumor effects of ML364 combined with anti-PD-1 therapy require both macrophages and CD8⁺ T cells.

a A schematic treatment plan for immunocompetent C57BL/6J mice bearing sgControl- or sgCd47-LLC tumors. Mice were subcutaneously implanted with 2 × 10⁶ sgControl or sgCd47-LLC cells and treated with a control vehicle, USP2 inhibitor (ML364, 5 mg/kg, daily for 12 doses), anti-PD-1 mAb (100 μg per mouse, every three days for 4 treatments), or combined treatment, respectively. i.p., intraperitoneal; s.c., subcutaneous. b, c Tumor volume in each treatment group was measured using calipers, and the tumor growth curve was subsequently plotted (b). At the study endpoint, the weight of LLC tumors was recorded (c). n = 5 mice per group. d The body weight of C57BL/6J mice was monitored throughout the duration of the indicated treatment. n = 5 mice per group. e A schematic treatment plan for CD8 T cell or macrophage deletion experiments. Mice were randomly divided into five groups: (1) vehicle, (2) ML364 & PD-1 mAb (MP), (3) MP & CD8 deletion (αCD8), (4) MP & macrophage deletion (clodronate liposomes, CL), and (5) MP & αCD8 & CL. On day 5 post-tumor implantation, mice in MP were treated with a combination of ML364 (5 mg/kg) daily for 12 treatments and anti-PD-1 mAb (100 μg per mouse) every 3 days for 4 doses. After grouping as indicated, mice received αCD8 antibody (200 μg per mouse) every 3 days for 4 doses, starting three days before tumor implantation. Macrophage deletion by CL was administered with 200 μl per mouse every 3 days for 4 doses, starting on the day of tumor implantation. i.p., intraperitoneal; s.c., subcutaneous. f, g Tumor volume for each treatment group was measured using calipers, and the tumor growth curve was plotted (f). The weight of LLC tumors was measured at the endpoint (g). n = 5 mice per group. h, i Quantification of CD8⁺ represented as percentage of CD3⁺ T cells (h) and F4/80⁺ macrophages represented as percentage of CD11b⁺ cells (i) in subcutaneous LLC tumors derived from C57BL/6J mice treated with indicated reagents. For (c) and (g–i), unpaired two-tailed Student’s t-test. n = 5 mice per group. Two-way ANOVA for (b, d, and f). Data are shown as the mean ± SD. P < 0.05 was considered statistically significant. Source data are provided as the Source Data file.