Fig 2: Tumor-bearing mice were treated with IL-18BP, tumor-bearing mice treated with phosphate buffered saline (PBS) as negative control. Anti-IL-18 treatment significantly reduced IL-18 level (a) corresponding to the MDSC reduction, both in the blood and tumor (b). Anti-IL-18 treatment also decreased protein expression of iNOS and Arg-1 in the tumor lysates (c). Data were expressed as mean ± SD (n = 5). *p < 0.05, **p < 0.01 vs negative control.

Fig 3: Changes in PPTs and the amount of IL-18 produced after administration of Brilliant Blue G (BBG) and IL-18 binding protein (IL-18BP). The PPTs (a) and quantities of IL-18 (b) in non-stimulated muscles, stimulated muscles of non-treated mice, the saline group, and the BBG group seven days after initiating electrical stimulation are shown. The PPTs increased, and the amount of IL-18 decreased significantly in the BBG group compared with that in the saline group. No significant differences were observed between the saline group and the stimulated muscles of non-treated mice. Time course of the PPT changes in the IL-18BP group and the saline group (c). In the IL-18BP group, PPTs significantly increased compared with those in the saline group; †p < 0.05, significantly different from the IL-18BP group; §p < 0.05, significantly different from the pre-stimulation values (day 0). The protein concentrations of protein of all samples used for ELISA were equalized to 1.3 mg/ml.

Fig 4: Single-cell resolved cell-cell interaction analysis shows that CD40ag combines with ICB to activate a coordinated cytokine-chemokine network between DCs, macrophages, and T cells.(A) UMAP embeddings of macrophages, DC subsets, and T cell subsets in gene expression space (left); and of corresponding NICHES interactomes (center) colored by broad cell type pair and (right) grouped into nhoods for differential abundance testing and colored by cluster for downstream analyses. Interactomes across experimental conditions (Control, ICB lo, ICB lo + CD40ag) are combined into a single visualization.(B) Heatmap depicting per-row scaled interaction scores for selected differentially predicted ligand-receptor axes (y-axis) across clusters 9, 15, 14, 13, 16, and 1 and compared to all non-differentially abundant neighborhoods (i.e., NS) (x-axis). The key above the heatmap distinguishes which cell type sender-receiver pairs are involved in each highlighted cluster.(C-D) UMAP embeddings as in (A, left) colored by (C) mregDCs (left) and Tregs (right) predicted to interact in clusters 13 and 15, (D, top) macrophages (left) and T cells (right) predicted to interact in clusters 1 and 14, and by (D, bottom) Tregs (left) and macrophages (right) predicted to interact in cluster 9.(E) Schematic illustrating the timeline of serum collection for peripheral blood cytokine and chemokine (C/C) profiling from ICB lo + CD40ag-treated YR1.7 tumor-bearing WT C57Bl/6J mice which had been previously treated with IL-12 and/or IL-18 blockade. Created with BioRender.com.(F) Hierarchical clustering of average C/C data from YR1.7 tumor-bearing mice either left untreated (n=3), or treated with ICB lo + CD40ag (n=2, abbreviated Tx in figure), Tx + IL18BP-Fc (n=2), Tx + anti-IL12 Ab (n=3), or Tx + anti-IL12 Ab + IL18BP-Fc (n=3). C/Cs were grouped into 6 modules for downstream analyses.(G) Example plots of peripheral blood cytokine expression levels for GM-CSF, IL-15, IL-10, IFNg, and CXCL9, from samples shown in (F). Summary data is presented as mean ± SEM. *p < 0.05, **p < 0.01 by ordinary one-way ANOVA with Dunnett’s multiple comparisons testing (calculated in GraphPad). Only showing significant results for comparisons between Tx and other conditions.