Antigen-Specific T Cell Responses in Mice

C57Bl/6J and P2rx7^−/− mice were immunized subcutaneously at the tail base with 20 µg OVA_323-339 peptide (Anaspec, Fremont, CA) emulsified in CFA (BD Biosciences) or 20 µg KLH in CFA in indicated experiments. Some groups were treated with the selective P2X7R antagonist, A740003 (50 μg/kg diluted in PBS/0.55% DMSO; Tocris, Minneapolis, MN) or the vehicle control, i. p every other day for 7 days (De Marchi et al., 2019 (link)). On day 7, inguinal lymph nodes were collected and processed into single cell suspensions. For determination of IL-17A⁺ and IFNγ⁺ CD45.1⁺ OT-II CD4⁺ T cell percentages, cells were restimulated with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of GolgiPlug for 4 h then stained for flow cytometric analysis. For detection of cytokines in supernatants, cells were restimulated with cognate antigens (OVA, 10 μg/ml and KLH 50 μg/ml) for 3 days then supernatants were collected and IL-17A and IFNγ levels were assessed by ELISA, performed according to manufacturer’s instructions (Biolegend).