CD4+ T cells were isolated from spleens of mice with a magnetic microbeads negative selection kit (EasySep Mouse CD4+ T Cell Isolation Kit; STEMCELL Technologies) and labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; CellTrace Proliferation Kit, Invitrogen) for 5 min in PBS containing 5% fetal bovine serum, at room temperature. After incubation, cells were washed twice with PBS, resuspended in RPMI-enriched medium (composition stated above), and activated with anti-CD3/anti-CD28 beads (Dynabeads, Gibco) in 96-well (U shape) plates (80 × 105 cells per well). After 72 hours, cells were collected and analyzed by flow cytometry.

Cytokine measurements. Isolated CD4+ T cells were incubated with anti-CD3/anti-CD28 beads for 48 hours. Six hours before analysis of TNF, IL-10, IFNγ, GzmB, perforin, IL-17a, IL-21, IL-2, and CCL5, cells were treated with brefeldin A (GolgiPlug, BD Biosciences) and cell culture (1 μl/ml, containing ~106 cells). The cells producing the cytokines were identified with flow cytometry (see the “Flow cytometry” section). Cells were cultured at 37°C and 5% CO2.

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