T2 Cell-Jurkat Co-Culture Assay

The stimulation assay was done as previously described(62 (link)). The assay was setup in 96-well clear round bottom microplates (Corning) with a volume of 200μl during all incubation steps. T2 cells expressing HLA-A*02:01 were plated at a density of 50,000 cells/well in IMDM media (Thermo Fisher Scientific) supplemented with 10% FBS (R&D Systems) and 100U/ml of penicillin-streptomycin and pulsed with 100mM of the test peptide for 3h at 37°C. The cells were then washed and co-cultured with Jurkat cells expressing an exogenous TCR of interest (100,000 cells/well) in RPMI media (Thermo Fisher Scientific) supplemented with 10% FBS (R&D Systems) and 100U/ml of penicillin-streptomycin for 16h. Next day, the cells were washed with FACS buffer and stained with anti-CD3 (APC, clone OKT3) and anti-CD69 (PE, clone FN50) antibodies for 20 mins at 4°C. Cells were washed, resuspended in FACS buffer and analyzed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific). The data was analyzed using FlowJo (v10) software.