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. 2024 Apr 9;12(6):687–703. doi: 10.1158/2326-6066.CIR-23-0706

Figure 4.

Figure 4. Mouse and human IL18 pathway share similar biological properties. A, IL18 and IL18BP levels were measured in sera and TDS taken from patients with cancer and tumor-bearing mice using ELISA kits (mouse sample information is indicated in Supplementary Table S9). B, Mouse T cells were purified from mouse tumors and matched spleens (n = 16) and stained for IL18Rα expression by flow cytometry. Each dot represents one mouse. C, Affinity of mouse IL18 to mouse IL18BP measured by KinExA. Two curves with different mouse IL18BP concentrations were run and analyzed using n-curve analysis to determine the Kd. Y-axis represents the free IL18BP. Free fraction of mouse IL18BP is measured pre-equilibrium, and the signal is a function of time and concentration of the tittered IL18. D, Affinity of anti-mouse IL18BP Ab to mouse IL18BP measured by surface plasmon resonance (SPR). Different colors represent the different concentrations of mouse IL18BP (0.125–256 nmol/L). E and F, Mouse cell lines (E) and mouse splenocytes, purified from mouse spleens (F), were stained with anti-mouse IL18BP Ab. Staining was analyzed by flow cytometry. G, Mouse CD3+ T cells were isolated from mouse splenocytes, activated with anti-CD3 and anti-CD28 and incubated with IL12 (2 ng/mL), and IL18:IL18BP (0.5 ng/mL:2 μg/mL) preformed complexes before addition of anti-mouse IL18BP Ab (10 μg/mL). Following the 24-hour culture, supernatant was collected for IFNγ secretion analysis. The median is depicted by a short black line in the violin plots. Bar graph shows the mean ± SEM; ***, P < 0.001 and ****, P < 0.0001 by two-tailed t test or by one-way ANOVA followed by two-tailed t test.

Mouse and human IL18 pathway share similar biological properties. A, IL18 and IL18BP levels were measured in sera and TDS taken from patients with cancer and tumor-bearing mice using ELISA kits (mouse sample information is indicated in Supplementary Table S9). B, Mouse T cells were purified from mouse tumors and matched spleens (n = 16) and stained for IL18Rα expression by flow cytometry. Each dot represents one mouse. C, Affinity of mouse IL18 to mouse IL18BP measured by KinExA. Two curves with different mouse IL18BP concentrations were run and analyzed using n-curve analysis to determine the Kd. Y-axis represents the free IL18BP. Free fraction of mouse IL18BP is measured pre-equilibrium, and the signal is a function of time and concentration of the tittered IL18. D, Affinity of anti-mouse IL18BP Ab to mouse IL18BP measured by surface plasmon resonance (SPR). Different colors represent the different concentrations of mouse IL18BP (0.125–256 nmol/L). E and F, Mouse cell lines (E) and mouse splenocytes, purified from mouse spleens (F), were stained with anti-mouse IL18BP Ab. Staining was analyzed by flow cytometry. G, Mouse CD3+ T cells were isolated from mouse splenocytes, activated with anti-CD3 and anti-CD28 and incubated with IL12 (2 ng/mL), and IL18:IL18BP (0.5 ng/mL:2 μg/mL) preformed complexes before addition of anti-mouse IL18BP Ab (10 μg/mL). Following the 24-hour culture, supernatant was collected for IFNγ secretion analysis. The median is depicted by a short black line in the violin plots. Bar graph shows the mean ± SEM; ***, P < 0.001 and ****, P < 0.0001 by two-tailed t test or by one-way ANOVA followed by two-tailed t test.