Adenosine Generated by Regulatory T Cells Induces CD8⁺ T Cell Exhaustion in Gastric Cancer through A2aR Pathway

Figure 3

Treg induces apoptosis and inhibits proliferation of CD8⁺ T cells. (a, b) Bead-purified peripheral single CD8⁺ T cells were stained with 1.5 μM CFSE and incubated at 37°C for 15 min. Subsequently, the cells were washed twice with phosphate-buffered saline. CFSE-labeled CD8⁺ T cells (10⁵ cells/well) were incubated in 6-well plates with Tregs at the ratio of 1 : 1; MRS1754 (A_2bR antagonist; 200 nM) and ZM241385 (A_2aR antagonist; 1 μM) were added to CD8⁺ T cells 30 min before adding Tregs. CD8⁺ T cells without Treg coculture and treated with vehicle were set as control. OKT-3 (2 μg/ml), soluble anti-CD28mAb (2 μg/ml; Miltenyi), and IL-2 (150 IU/ml) were added to induce Treg proliferation. After 5 days of coculturing, apoptotic CFSE-stained CD8⁺ T cells were quantified using Annexin V Apoptosis Detection Kit. (c) The cell proliferation index of CFSE-stained CD8⁺ T cells was calculated based on the reduction of CFSE-positive cells (, , and vs Tregs). In ANOVA, all experiments are repeated at least five times.

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