Fig. 6: IL-18 treatment leads to the upregulation of A_2AR which consequently limits anti-tumor immunity.

A CD8⁺ T cells were isolated from C57BL/6 A_2AR eGFP or C57BL/6 WT mice and stimulated with plate-bound anti-CD3 and soluble anti-CD28 (2 μg/ ml) mAbs plus indicated cytokines (100 IU/ ml IL-2, or 50 ng/ ml other cytokines) for 72hrs. Data represent the mean from 2 individual technical replicates. B AT3 ova tumor-infiltrating lymphocytes were isolated and cultured overnight with indicated cytokines (50 ng/ ml). Data represent the mean ± SD of 3 technical replicates from a representative experiment of n = 3. C A_2AR GFP splenocytes were treated for 3 days with anti-CD3/ anti-CD28 and, where indicated, IL-18 (10 ng/ ml). Histogram overlay of concatenated samples (left) or mean ± SD of 2 (Naïve) or 3 (activated) replicate samples from a representative experiment of n = 3 (right). D Human NK cells were stimulated with 20 IU/ml IL-2 (control) or IL-2 and IL-18 (50 ng/ ml) for 16 h. Expression of A_2AR mRNA relative to GAPDH housekeeping gene. Data represent the relative expression (±SD) of triplicate samples. E–J C57BL/6 A_2AR eGFP mice (E, F), C57BL/6 WT mice or A_2AR^-/- mice (G, H) were injected with 1 × 10⁶ MC38 mCherry or MC38 mCherry-IL-18 expressing tumor cells. E, F Expression of PD-1 and A_2AR (GFP) by indicated subsets. Flow cytometry plots of concatenated samples from a representative experiment. F Data represented as the mean ± SEM of 12 (MC38 Ctrl) or 14 (MC38 IL-18) mice per group pooled from 2 experiments. Data were normalized relative to the average MFI of the relevant cell population in control tumors within each experiment. G Tumor growth represented as the mean ± SEM pooled from 2 representative experiments (n = 15 WT MC38, 17 WT MC38-IL-18, l0 A_2AR KO MC38, 9 A_2AR KO MC38-IL-18) H Survival of mice with 100mm² tumor size used to designate survival. (n = 15 WT control, 16 WT IL-18, 7 A_2AR KO control, 8 A_2AR KO IL-18). I C57BL/6 WT mice or A_2AR^−/− mice were injected with 1.5 × 10⁶ tumor cells and where indicated treated with anti-CD8 or anti-GM-1, n = 5–13 per group. J At day 12 post-tumor inoculation 1 µg IL-18 was administered intra-tumorally at 17 and 2 h prior to analysis of GFP expression. Data are represented as the mean ± SEM of 11 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 unpaired two-sided t test (F, J), Two-way ANOVA (G) or log-rank (H) statistical test. Source data are provided as a Source Data file.