Anti pd l1 clone 29e 2a3

Arterial CD8⁺ T cells were cultured (5×10⁵ per well) in the presence of 10 μg/ml anti-Tim-3 (clone F38-2E2, BioLegend), anti-PD-L1 (clone 29E.2A3, BioLegend), anti-Tim-3 plus anti-PD-L1, or isotype control. After 48 h, the culture supernatant was collected and further analyzed by FCM.
For intracellular cytokine analysis, brefeldin A (a Golgi inhibitor) (10 mg/ml, Biolegend, USA) was used for 4h (at the end of the culture) to block the secretion of cytokines into the media after the activation of cells by phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, 12h) and ionomycin (1μg/ml, 12h). Cells were harvested and analyzed by FCM for intracellular cytokine production.

To measure cytotoxicity in the allogeneic assays, purified activated CD4+ or CD8+ T-cells from HLA-A*02:01-negative donors were incubated with HLA-A*02:01-positive PDAC cell lines at a ratio of 2:1. Where indicated, soluble LAG-3 (#2319-L3-050; R&D Systems), anti-LAG-3 (clone 17B4; Novus Biologicals; #NBP1-97657), anti-PD-L1 (clone 29E.2A3; #329716; BioLegend) or a combination of anti-LAG-3 and anti-PD-L1 antibodies or soluble LAG-3 and anti-PD-L1 were added at 10 μg/mL. After overnight incubation, the cells were stained with BV421-conjugated anti-HLA-A2 (#740082; BD), Calcein-AM and Ethidium Homodimer 1 (#L3224; Invitrogen) to determine cell death. Target PDAC cells were gated on HLA-A2 positive cells. The cytotoxicity of peptide-stimulated CD4+ T-cells on Capan-1 cells was determined through measurement of lactate dehydrogenase (LDH) released into the culture medium with the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (#G1780; Promega). The assay was carried out according to the manufacturer’s instructions. Results are expressed as relative cytotoxicity, that is, cell death of Capan-1 cells by peptide-stimulated CD4+ T-cells in relation to unstimulated CD4+ T-cells.