Antigen-Specific T Cell Activation by DCs

Primary splenic DCs were purified as previously described^{43 (link)}. Activated primary DCs were incubated for 14–16 h in the presence of 0.5 µM CpG Type B 1688 (Geneworks). Unstimulated DCs were pulsed with 100 μg OVA (Worthington) together with 5 µM CpG, and activated splenic DCs pulsed with 100 μg OVA for 45 minutes at 37 °C. Excess OVA was removed by washing. Both unstimulated and activated DCs were pulsed with BP or BP-OVA beads that remained in culture. 5 × 104 CTV-labelled purified OT-I or OT-II T cells were cultured in vitro with unstimulated or activated DCs in RPMI 1640 supplemented with 10% FBS, 100 IU/mL penicillin (MPU) and 100 μg/ml streptomycin (MPU). Cells were incubated for 60 - 64 h (37 °C, 10% CO₂). T cells were stained with mAbs specific for: CD4 (GK1.5, FITC, 1:500, 100406), CD8 (53-6.7, FITC, 1:400, WEHI), TCRVα2 (B20.1, APC, 1:800, WEHI) and Ly5.1 (A20.1, PE-Cy7, 1:200, 110730) (Biolegend). T cell proliferation was analyzed using flow cytometry and CTV dilution. Counting beads (Accucheck, Thermo Fischer) were included to enable determination of the number of proliferating T cells.