Fig. 6: CD28 costimulation prevents CD73-mediated adenosine production by activated CD8⁺ T cells.

a P14 CD8⁺ T_eff cells were pre-cultured with CFSE-labeled activated (I) CD28WTCD8⁺; (II) CD28KOCD8⁺; (III) iCD73-pretreated CD28KOCD8⁺; (IV) CD28KOCD8⁺ T cells in the presence of A_2AR antagonist ZM241385 and A_2BR antagonist MRS1754, at a ratio of 3:1. At 40 h after co-culture, CFSE-negative P14 CD8⁺ T_eff in each group was sorted, restimulated with PMA/ionomycin, and subjected to intracellular staining of TNF, IFN-γ, perforin, and granzyme B. Number on the upper right indicates the percentage of the protein-positive population. The data presented are representative of one of the three independent experiments. b P14 CD8⁺ T cells treated with supernatants from activated CD28KOCD8⁺ (black), CD28WTCD8⁺ (red) or iCD73-pretreated CD28KOCD8⁺ (blue) cell cultures were added to Hepa 1.6.gp33 target at E:T ratios of 1:10, 1:3, and 1:1 (n = 3 each). P14 CD8⁺ T_eff cells without any treatment were used as control (E:T ratios of 1:10, n = 4; 1:3, n = 6; 1:1, n = 4) (gray). c Further analysis of tumor-killing at E:T ratio of 1:10 in b. (P14 CD8⁺ T_eff cells without any treatment, n = 4; the other groups, n = 3). d Survival of tumor-bearing mice. On day 8 after tumor implantation, tumor-bearing mice were intravenously administered P14 CD8⁺ T_eff cells that had been pre-cultured with: (I) activated CD28WTCD8⁺ (71.7% survival, n = 46, red); (II) activated CD28KOCD8⁺ (51.4% survival, n = 35, black), significantly reduced survival between days 30 and 48 compared to mice in group I, p < 0.01; (III) iCD73-pretreated activated CD28KOCD8⁺ (86.2% survival, n = 29, blue), (IV) activated CD28KOCD8⁺ T cells in the presence of ZM241385 (79.5% survival, n = 39, brown). Statistical evaluations were performed using the Student’s t-test with data expressed as the mean ± standard error of the mean (c). The ability of CD8⁺ T cells between two groups to in vitro kill tumor at different E:T ratio was analyzed by the general linear model (b). The survival difference between groups was analyzed by log-rank test (d). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not statistically significant.