Fig. 3. Direct and abscopal therapeutic effects and immunomodulation by GSNO and aCTLA-4 combination therapy.

a Tumor model and treatment schedule. 1^o and 2^o tumors were formed in C57Bl/6 mice by inoculation of 10⁵ B16F10-OVA cells in 30 μL saline on day 0 and day 4, respectively. GSNO (480 μg kg⁻¹) in 30 μL saline was intratumorally treated on day 7, and aCTLA-4 (100 μg mouse⁻¹) in 30 μL saline was intraperitoneally administered on day 8, 11, and 14. Blood was harvested from the facial vein on day 13 for the profiling of blood immune cells. b Average and individual volumes of 1^o (directly injected) tumors. c Average and individual volumes of 2^o (uninjected) tumors. d Relative body weight changes post treatment. e Kaplan–Meier survival curves. f–i Relative blood abundance of f CD45⁺, g CD45⁺CD3⁺CD4⁺ T (CD4⁺ T), LAG-3⁺ CD4⁺ T, PD-1⁺ CD4⁺ T, and CD45⁺CD3⁺CD4⁺CD25⁺Foxp3⁺ (T_reg), h CD45⁺CD3⁺CD8⁺ T (CD8⁺ T), CD25⁺ CD8⁺ T, LAG-3⁺ CD8⁺ T, PD-1⁺ CD8⁺ T, and tetramer⁺ CD8⁺ T, and i CD45⁺CD3^-NK1.1⁺ (NK) and CD45⁺CD3⁺NK1.1⁺ (NKT). Data are presented as individual biological replicates and mean ± SEM. b–e n = 5 for Control, Control+aCTLA-4, and GSNO + aCTLA-4, and n = 6 for GSNO. f–i n = 5 for Control, Control+aCTLA-4, and GSNO + aCTLA-4, and n = 4 for GSNO. *****p < 0.0001, ****p < 0.001, ***p < 0.01, **p < 0.05, and *p < 0.1. Exact p-values for b, c and e–i are reported in Supplementary Table 1 and 2. b, c ANOVA using linear mixed-effects model. d Two-way ANOVA using Tukey post-hoc statistical hypothesis. e Log-rank using Mantel–Cox statistical hypothesis by comparing the GSNO + aCTLA-4 with control groups. f–i One-way ANOVA using Tukey post-hoc statistical hypothesis. j Proposed actions of combinational use of GSNO and aCTLA-4 on immune response. Blue arrows indicate the mechanism associated with GSNO. Red arrows and crosses represent mechanisms associated with aCTLA-4. Source data are available in a Source Data file.