Rosettesep human cd4 t cells enrichment cocktail

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.

Fresh human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque gradient centrifugation, and plated in serum-free medium for 3 hr at 37°C. The non-adherent cells were discarded and adherent monocytes were cultured in RPMI (Gibco) with 10% FCS (Sigma) and recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) (100 ng/ml, Gibco). Cell differentiation to macrophages was completed after for 4 to 6 days, and cells were treated as indicated at day seven prior to infection.
Primary CD4+ T cells were isolated from human blood by Ficoll-Paque gradient centrifugation and negative selection (RosetteSep Human CD4+ T Cells Enrichment Cocktail, StemCell Technologies). Primary CD4+ T cells and PBMCs were activated with Phytohemagglutinin-L (2 μg/ml, Sigma) for 48 hr, cultured in the presence of interleukin-2 (50 U/ml, PeproTech), and treated as indicated for 16 hr prior to infection.