TCR Reporter Assay

Specific TCR combinations were tested for their resultant induction of NFAT-driven luciferase output using a previously described protocol (23). For peptide presentation, single antigen lines for the relevant HLA allele were cultured and passaged at least once at 0.3×10⁶cells/mL before peptide pulse in RPMI supplemented with 10% FBS, 1% L-glutamine and 1% penicillin streptomycin. Cells were washed with RPMI (no phenol red) supplemented with 10% FBS and 1% L-glutamine and resuspended at 1×10⁶ cells/mL, with 50,000 cells added to each well of a U-bottom 96 well plate. Peptides at a final concentration of 30ng/µL (with serial 10-fold dilutions) were added and incubated overnight at 37°C and 5% CO₂ in a final volume of 70µL. After 24 hours, transfected Jurkat E6.1 cells were counted and resuspended at 1×10⁶ cells/mL, with 50,000 cells being added to target cells in a final volume of 100µL. Cells were co-cultured for six hours and transferred to a white Cliniplate (ThermoScientific) prior to the addition of 100µL Bright-Glo (Promega) with an incubation of 10 minutes in the dark at room temperature. Luciferase output was measured on a DTX 880 multimode detector (Beckman Coulter) using 3000ms integration. PMA/Ionomycin was used as a positive control with four negative controls: 1) media only; 2) Jurkat only; 3) Jurkat and APCs; and 4) Jurkat and peptide. All conditions were in duplicate.