M7531

Naive γδ T cells sorted from splenocytes of BALB/c mice were treated with AA (5 μM, catalog A5837; Sigma-Aldrich) using a mouse TCR γ/δ T cell Isolation Kit (Miltenyi Biotec) for 12 and 36 hours. Then, cells were washed, harvested, and stained with PE anti-CD39 mAb or intracellular stained with PE-Cy7 anti–IFN-γ mAb (1:20; catalog 505826; BioLegend) and detected by flow cytometry.
For the AA-induced CD39⁺γδ Treg–mediated CD8⁺ T cell suppression experiment, CD39⁺ γδ Tregs were sorted from above γδ T cells pretreated with AA (5 μM) and were then cocultured with αβ T cells labeled with CFSE (Invitrogen) at a 1:5 ratio for 2 days. αβ T cells were unlabeled by magnetic beads in the process of sorting γδ T cells. An anti-CD39 mAb (5 μg/mL; ab227840; Abcam) and an A2AR inhibitor, AZD4635 (5 μM; M7531; AbMole), were used for rescue experiments. Then, cells were washed, harvested, and stained with APC anti-CD8 mAb (1:20; catalog 100712; BioLegend). CFSE-low CD8⁺ T cells were detected by flow cytometry.

For the CD39⁺γδ Treg–mediated CD4⁺/CD8⁺ T cell suppression experiment, CD39⁺ γδ Tregs were sorted from tumors and paired normal tissues of patients with RSCRC/LSCRC and then were cocultured with allogeneic peripheral blood CFSE-labeled (5 μM; catalog 65-0850-84; Invitrogen) CD4⁺/CD8⁺ T cells at a 1:5 ratio in the presence of ImmunoCult Human CD3/CD28 T Cell Activator (catalog 10971; Stemcell Technologies) for 5 days. An anti-CD39 mAb (5 μg/mL; ab178572; Abcam) and an A2AR inhibitor, AZD4635 (5 μM; M7531; AbMole), were used for rescue experiments. CD4⁺/CD8⁺ T cells were separated by RosetteSep Human CD4⁺ T Cells Enrichment Cocktail (catalog 15062; Stemcell Technologies) and an EasySep Direct Human CD8⁺ T Cell Isolation Kit (catalog 19663; Stemcell Technologies). On day 5, cells were harvested and intracellularly stained with anti–IFN-γ mAb. CFSE-low cells and IFN-γ production were detected by flow cytometry. Peripheral blood samples of the healthy donor were collected in Tianjin Medical University Cancer Institute and Hospital.