Ova257 264 peptide

OVA specific CD8⁺ cytolytic T-cells were generated by incubating isolated splenocytes from OT-1 mice with SIINFEKL peptide of OVA (OVA_257–264) at 10 ng/mL for 5–7 days. In certain cases, naive OT-1 T-cells were isolated from spleens using T-cell enrichment columns (R&D) and subjected to nuclear transfection (see method below). In the case of purified naive T-cells, the thymoma EL4 cell line was used to present OVA_257–264 to primary T-cells. EL4 cells were incubated with 10 nM OVA_257–264 peptide (Bachem) for 1 h at 37°C and treated with mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) (final concentration of 10 µg/mL) prior to mixing with primary T-cells by coculturing at a ratio of 1:5 of EL4 and T-cells to generate cytotoxic T-cells. In either case, CTLs were generated in the presence or absence of SMI and/or anti-CD28 or CTLA-4-Ig (inhibitors/Abs added simultaneously with OVA-stimulation for 5–7 days) prior to washing and analysis by FACs, PCR, or cytotoxicity assays. Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 50 mM beta-mercaptoethanol, sodium pyruvate, 2 mM l-glutamine, 100 U/ml penicillin, and streptomycin (GIBCO).

The following antibodies were used in experiments: InVivo anti-PD-1 (CD279, J43) (BioXCell), Elraglusib (9-ING-41) kindly provided by Actuate Therapeutics Ltd and OVA_257-264 peptide (Bachem Ag). Conjugated antibodies for flow cytometry: anti-CD8α (clone, 53–6.7), anti-CD3 (eBioscience) and CD279 (clone EH12.2H7) (BioLegend). Antibodies for IHC: anti-PD-1 (Cell Signaling Technology), and anti-CD8 (Abcam).

Stimulations were performed using 10 nM OVA_257–264 peptide (Bachem), anti-CD28 (clone PV1, bioXpress), and CTLA-4 IgG Fusion Protein (BD Pharmingen) where stated. SMI (GSK-3 inhibitor) was obtained from Abcam plc. and suspended in DMSO to give a stock solution of 25 mM and diluted to a concentration of 10 μM in vitro. Fluorescently labeled Abs to GZMB, PD-1, and Lamp-1 (CD107a) were obtained from Biolegend.