Clone jes6 1a12

T lymphocytes from spleens were isolated from naïve C57BL/6N mice as previously described^{19 (link)}. 500,000 cells/well were cultured in a 24-wells plate for five days at 37 °C in an atmosphere of 5% CO₂, in X-vivo medium (Lonza, LOT 8MB036) supplemented with 1% penicillin–streptomycin (Lonza 09-757F). Cells were activated with plate-bound anti-CD3 (5 µg/ml; BioLegend; clone 17A2) and soluble anti-CD28 (2.5 µg/ml; BioLegend; clone 37.51). After 2 h preincubation with or without 5 µM SB-505124 (Sigma-Aldrich), cells were differentiated into Th17 cells with αIL-2 (10 µg/ml; BioLegend; clone JES6-1A12), IL-1β (10 ng/ml; BioLegend), IL-6 (50 ng/ml; ITK diagnostics), IL-23 (10 ng/ml; ITK diagnostics) and increased concentrations of TGF-β1 (0.05, 0.5, 1 or 10 ng/ml, BioLegend). After five days of differentiation, the supernatant was collected for cytokine measurement and the cells were processed for RNA isolation (n = 3 biological replicates from one mouse) or flow cytometry (n = 3 mice).

Nunc F96 Micro Well PolySorp Black (Thermo Fisher) microplates were coated over night at 4°C with 1 µg/mL rat anti‐mouse IL2 capture antibody (Biolegend, Clone JES6‐1A12) in sodium carbonate buffer (50 mM Na₂CO₃, 50 mM NaHCO₃, pH 9.6), washed twice with PBS‐T (0.05% Tween20 in PBS), and blocked with 0.2% gelatin in PBS. Plates were washed five times with PBS‐T. Culture supernatants were added and incubated overnight at 4°C. Plates were washed five times and incubated with 0.25 µg/mL anti‐mouse IL2‐biotin detector antibody (Biolegend, Clone JES6‐5H4) for 2 h at room temperature. After five more wash steps with PBS‐T, 0.1 µg/mL DELFIA europium (Eu)‐labeled streptavidin (PerkinElmer) diluted in IL‐2 assay buffer (PerkinElmer) was added and incubated for 45 min at room temperature. The reaction was enhanced with DELFIA enhancement solution (PerkinElmer). After 10 min of incubation at room temperature, fluorescence values were measured by using 320 nm excitation and 615 nm emission fluorescence filters in a spectrofluorometer (Tecan, Infinite M1000 Pro). IL‐2 secretion curves were analyzed and AUC values calculated by GraphPad Prism Version 6. TCR clones with AUC values above 0.5 (50× increased in comparison to negative controls; 0.01 ± 0.003) were considered OVA reactive.

For MHC I, MHC II, and IL-2 neutralization experiments, CD8+ and CD4+ T cells (in a 1:1 ratio) were prestimulated with anti-CD3 antibody and recombinant PD-L1-Fc chimera for 24 h. Subsequently, T cells and Panc02-OVA cells were cocultured at a 10:1 effector to target cell ratio. Anti-mouse MHC class I antibody (10 μg/ml, clone M1/42.3.9.8, InVivoMAb), anti-mouse MHC class II antibody (10 μg/ml, clone M5/114.15.2, eBioscience) and LEAF purified anti-mouse IL-2 antibody (10 μg/ml, clone JES6-1A12, BioLegend) were added during prestimulation and co-culture. Supernatants were analyzed for IFN-γ by ELISA.