In vitro CD4+ T cell differentiation

Naïve CD4⁺ T cells were isolated from spleen and LNs using Miltenyi mouse naïve CD4 T cell isolation kits (>97% purity). Naive CD4⁺ T cells (2 × 10⁵) were co-cultured at a ratio of 1:5 with mitomycin-treated T-depleted splenocytes as APCs (1 × 10⁶) in IMDM media supplemented with 10% FBS, Penicillin/Streptomycin, L-Glutamine, 2-mercaptoethanol, 1 μg/ml anti-CD3 and 3 μg/ml anti-CD28 in 48-well plates for 72 hours. T_H1 conditions included 20 ng/ml IL-12 and 20 μg/ml anti-IL-4. T_H2 conditions included 40 ng/ml IL-4 and 20 μg/ml anti-IL-12. T_FH conditions included 100 ng/ml IL-6, 50 ng/ml IL-21, 20 μg/ml anti-IFNγ, 10 μg/ml anti-IL-4 and 10 μg/ml anti-IL-12 and 20 μg/ml anti-TGFβ. All antibodies were purchased from BioXcell and all cytokines were purchased from Peprotech. For intracellular cytokine analysis, cells were restimulated at the end of the 72h culture period with 50 ng/mL PMA and 500 ng/mL Ionomycin in the presence of GolgiStop (1/2000) for 4 hours. Cells were surfaced stained and fixed with 4% paraformaldehyde, and intracellular staining was performed in the presence of 0.5% Triton X-100, 0.1% BSA in PBS.