Ril 1β

CD4⁺ T cells were isolated from the lymphoid tissues of 6–8 wk old mice using EasySep kits (StemCell Technologies) according to the manufacturer’s instructions, except that CD25⁺ nTregs and γδ T cells were also depleted during the process. Purified CD4⁺ T cells were cultured in 6-well plates coated with anti-CD3 (5µg/ml, clone 2c11, BioLegend) and anti-CD28 (5µg/ml, clone 37.51, BioLegend) with complete RPMI-1640 medium for 5 days. For Th1 differentiation, the cultures were supplemented with anti-IL-4 (Frederick National Laboratory) (10µg/ml), rIL-12 (R&D Systems) (5ng/ml), and rIL-2 (Frederick National Laboratory) (200units/ml). For Th17 differentiation, the cultures were supplemented with anti-IL-4 (10µg/ml), anti-IFNγ (10µg/ml, purified in house), rTGFβ1 (2ng/ml), rIL-6 (20ng/ml), rIL-1β (10ng/ml), and rIL-23(5ng/ml) (BioLegend). After 5 days of culture, the cells were rested in complete medium containing rIL-7 (10ng/ml, Frederick National Laboratory) for 2 days before purification and cell transfer.

Mice were anesthetized with isofluorane and intrapharyngeally administered 50pg of rIL-1β (Biolegend) in 20uL of cold PBS on days 1, 3, 5, and 7 post-infection for the caspase-block experiments, or days 4, 5, 6, and 7 for the experiments involving transfer of irradiated thymocytes.