Autologous T-Cell Cytotoxicity Assay

Thawed autologous PHA-blasts were cultured in RPMI1640 medium containing 10% FBS and 100 U/mL IL-2 in 24-well plate for 1 to 3 days prior to use in a cytotoxic assay to evaluate autoimmune effect by activated autologous T cells. Collected PHA-blasts were stained as target cells with 2.5 μM of CellTrace Violet (CTV) dye (ThermoFisher Scientific, C34557) for flow cytometry analysis. T cells with peptide-loaded DCs or non-peptide loaded DCs were cultured for at least 2 h prior to the assay. Those T cells were co-cultured with stained PHA-blasts in the effector to target cells (E:T) ratio of 20:1 in 6-well plate at 37 °C for 4 h^{21 (link)}. PHA-blasts without T cells were also incubated in the same manner to detect basal cell death for the calculation of specific cell lysis by T cells. After the incubation, cells were stained with eBioscience Fixable Viability Dye (FVD) eFluor 780 (Thermo Fisher Scientific, 65–0865) and fixed with 4% formaldehyde, followed by filtering through 70 μm and 40 μm cell strainers. Single cell suspension was transferred to FACS tube and stored at 4 °C protected from light until use in flow cytometry analysis. Percentages of dead PHA-blast cells (CTV-positive/FVD-positive) were measured by BD LSRFortessa flow cytometry. The percentage of specific lysis was calculated by the same equation used for cytotoxicity assay^{58 (link)}.