4.8. In Vitro Antigen Presentation and T Cell Activation

BMDCs (1.0 × 10⁵) were mixed with OT-II CD4+ T cells (5.0 × 10⁵) on 96-well plate in cell culture medium. OVA peptide (OVA_323–339: 10 ug/mL) was added into cultures and then samples were treated with single or combination TLR ligands (LPS or IMQ LPS or IMQ+LTA, LPS). The ligand concentrations were LPS: 100 ng/mL, IMQ: 10 μg/mL and LTA: 5 ug/mL. Some cultures were treated with LTA only or vehicle control (no-Ag negative control). The culture was incubated at 37 °C for 72 h. At the last 5 h, the cells were re-stimulated with PMA (500 ng/mL) and ionomycin (1 ug/mL) in the presence of GolgiStop^TM. The IFN-γ+CD4+ T (Th1) cells and CD69 expression in Th1 cell populations were analyzed by flow cytometry.