Fig. 2

Endocannabinoid AEA inhibits function and expansion of CD8⁺ T cells. Mice bearing MC38 (a) or B16 (b) tumors were treated with DMSO, AEA, PD-1 antibody, or AEA plus PD-1 antibody on day 10 after tumor inoculation. Tumor volumes were measured every other day (two-way ANOVA, mean ± SEM, *P < 0.05, and **P < 0.01). Wild-type CD8⁺ T cells were stimulated with 5 μg/ml plate-bound anti-CD3 and anti-CD28 antibodies, and were incubated with different concentrations of endocannabinoid AEA simultaneously for 48 h. c The proliferation of CD8⁺ T cells was measured by CFSE dilution. d, e The production of IFN-γ and TNF-α cytokines in CD8⁺ T cells were detected by intracellular staining (mean ± SD, *P < 0.05, **P < 0.01). Statistical significance was assessed by ordinary one-way ANOVA. Data are representative of three independent experiments. B16-OVA tumors were established subcutaneously in 6–10 weeks old C57BL/6J mice 10 days before adoptive cell transfer of 1× 10⁶ OT-I T cells (CD45.1⁺) and AEA was intraperitoneally injected on day 12, 14, and 16. f Tumors were measured every 2 days and the volume was calculated. Data in bar graphs represent mean ± SEM, three independent experiments were performed. g, h The frequencies and numbers of OT-I T cells in tumors and the production of IFN-γ in OT-I T cells from tumor after in vitro activation with PMA and Ionomycin were shown. i Kaplan–Meier estimates of overall survival comparing high to low levels of AEA in serum of lung cancer patients measured by ELISA. Data are shown as mean ± SEM; log-rank test. j Representative IHC images of CNR2^high and CNR2^low tumor sections stained with CNR2 (left). Scale bars correspond to 100 μm. Kaplan–Meier estimates of patients’ overall survival comparing high to low expression of CNR2 (right). Statistical significance was assessed by the log-rank (Mantel–Cox) test of survival curve.