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Human T cells were labeled with eFluor 670 (eBioscience) and cultured (2 × 105 cells per well) with allogeneic moDC (5 × 104 cells per well) in flat-bottomed 96-well plates. moDCs were pre-activated with CD40 agonistic or control antibodies for 24 h prior to the addition of T cells. After 6 days of co-culture, supernatants were harvested and assessed for IFN-γ by ELISA (R&D Systems). To measure T-cell proliferation, cells were stained with anti-CD8 PE antibodies and T-cell divisions were evaluated using a MACS Quant® analyzer.
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