(A) Flow cytometry analysis of CTLs in 2208L tumors treated for 7 days (n = 6). (B) Left: Representative images of CD8 immunostaining on sections of 2208L tumors treated for 7 days. Scale bar: 50 μm. Right: Quantification of CD8 staining (n = 12). For A and B, experiments were conducted 3 times. Data from 1 representative experiment are shown. (C) Log₂(fold change) of 2208L tumor volume (mean ± SD). Anti-CD8 was administered 24 hours before starting IACS-70654 treatment. Two-way ANOVA and Šidák’s multiple-comparison test were used (n = 5). (D) Quantification of CXCL10 level in 2208L tumor homogenate by cytokine/chemokine array (n = 3). Tumors were treated for 7 days. (E) Flow cytometry analysis of Tregs in 2208L tumors treated for 18 days (n = 5). For A, B, D, and E, a 2-tailed, unpaired Student’s t test was used. (F) Treatment schemes of IACS-70654 in combination with anti–PD-1 and DTX. DTX was administered at half of the clinically equivalent dose. After 27 days of treatment, only anti–PD-1 treatment was administered until all tumors reached the ethical endpoint (≥1500mm³). (G) Kaplan-Meier survival curves of 2208L tumor–bearing mice (vehicle and DTX + anti–PD-1 arms, n = 4; IACS-70654 and combination arms, n = 5). Log-rank test with Bonferroni’s correction was used. *P < 0.05. The experiment was conducted twice. Data from 1 representative experiment are shown. (H) Quantification of S100A8 immunostaining. Ordinary 1-way ANOVA and Šidák’s multiple-comparison test were used (vehicle and DTX + anti–PD-1 arms, n = 9; IACS-70654 and combination arms, n = 12). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. For A–E and H, data are presented as mean ± SD.