T cell enrichment columns

B16 cells were obtained from the American Type Culture Collection (ATCC). MC38-OVA cells were generously provided by yue zhang. Cancer cells were cultured in DMEM/H(HyClone) supplemented with 10%FBS(Gibico) and 100 U/ml penicillin/streptomycin. CD8⁺T lymphocytes were isolated from spleens of wild type mice using T-cell enrichment columns (Stem Cell). To activate spleen or CD8⁺T cells, the cells were seeded in 96-well plate with OVA-derived peptide, control H-2Kb RAHYNIVTF peptides, or gp100 peptide. For T cell suppression assays, CD8⁺ T cells were labeled with CFSE and cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. And T cells were cocultured at 2:1 ratios with spleen-derived CD45⁺Ter119⁺CD71⁺cells (CD45⁺EPC) in 96-well flat-bottom plates. Seventy-two hours later, cells were analyzed by flow cytometry. In some coculture experiments, 10ug/ml OVA/H2Kb or gp100 peptides was seeded for the cell activation, and CD45⁺ EPCs were treated for 30 min before and during coculture with 300 mM apocynin to inhibit NADPH oxidase.

Splenocytes were collected by mechanical disruption. Cell suspensions were passed through 70-μm cell strainers and then washed and resuspended in staining buffer. For the isolation of spleen CD45⁺Ter119⁺CD71⁺cells(CD45⁺EPC), cells were stained with anti-CD45 monoclonal antibody (mAb),anti-Ter119 (mAb), and anti-CD71 (both from Biolegend). Then, CD45⁺EPC were sorted using the BD FACSAsia II Special Order System. For spleen CD8⁺ T cell isolation, CD8⁺T lymphocytes were isolated from spleens of wild type mice using T-cell enrichment columns (Stem Cell). For all sorted samples, a purity of greater than 95% was achieved.