Ab178572

In order to detect CD39 and CD73 co-expression by immunocytochemistry, fixed synovial cells were stained for these cell surface ectonucleotidases using the primary antibodies rabbit anti-CD39 (ab178572, Abcam, Cambridge, UK) and mouse anti-CD73 (ab133582, Abcam, Cambridge, UK). The expression of ADA, ENT1, ENT2, and ARs was investigated by immunohistochemical staining of synovial tissue samples using the primary antibodies rabbit anti-ADA (NBP1-90361, Novus Biologicals, Cambridge, UK), rabbit anti-ENT1 (NBP1-84838, Novus Biologicals, Cambridge, UK), rabbit anti-ENT2 (ab48595, Abcam, Cambridge, UK), rabbit anti-A₁AR (ab124780, Abcam, Cambridge, UK), rabbit anti-A_2AAR (ab260032, Abcam, Cambridge, UK), rabbit anti-A_2BAR (LS-C20310, LSBio, via Biozol, Eching, Germany), and rabbit anti-A₃AR (LS-A686, LSBio, via Biozol, Eching, Germany). After blocking (10% bovine serum albumine, 10% chicken serum, and 10% goat serum), cytospin or tissue slides were incubated with primary antibodies overnight at 4 °C. Primary staining was visualized using Alexa Fluor-labeled secondary antibodies (goat anti-rabbit Alexa Fluor 594 or goat anti-mouse or anti-rabbit Alexa Fluor 488; Thermo Fisher/Life technologies, Schwerte, Germany). Cell nuclei were counterstained with DAPI (Merck, Darmstadt, Germany). Slides without primary antibody served as negative controls (Supplementary Materials Figure S2).

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.