Anti pd 1 antibody clone eh12.2h7

Human T-lymphocytes were isolated from healthy donors using RosetteSep Human T-cell Enrichment Cocktail (STEMCELL Technologies). Lymphocytes were cultured in a cell culture plate coated with anti-human CD3 antibody (OKT3; eBiosciences, San Diego, CA, USA) and human CD28 antibody (BioLegend, San Diego, CA, USA). The proliferation of lymphocytes was examined by the BrdU incorporation assay (Cell Proliferation ELISA kit, Roche, Basel, Switzerland). Anti-PD-1 antibody (clone EH12.2H7) and isotype-matched IgG were obtained from BioLegend.

One day before co-culture, lymphocytes were thawed and cultured in lymphocyte medium supplemented with 200 IU/ml IL-2 overnight at 37℃. Lymphocyte medium consisted of RPMI-1640 (Wako)/AIM V(Gibco) supplemented with 12.5 mM HEPES, 2-Mercapto-ethanol and 5% human AB serum. Tumor cells were stimulated overnight with 200 IU/ml of human recombinant IFNγ to facilitate HLA expression. 96-well U-bottom plates were coated with 5 μg/ml anti-CD28 (clone CD28.2) and kept overnight at 4 ℃. On the next day, tumor cells were dissociated to single cells with TrypLE (Gibco) and resuspended in lymphocytes medium. Lymphocytes were seeded at a density of 10⁵ cells/well (total 1 × 10⁶ cells) and co-cultured with tumor cells at a 20:1 ratio (lymphocytes: cancer cells, respectively). Co-culture was kept in the presence of 200 IU/ml IL-2 and 10 μg/ml anti-PD-1 antibody (clone EH12.2H7, Biolegend). Half of the medium was replaced two to three times per week. 1 week after the co-culture, lymphocytes were collected, counted, and replated at the concentration of 10⁵ cells/well with fresh tumor cells [24 (link)].