Differentiation of Naïve CD4+ T cells

Naïve CD4⁺ T cells were purified from the splenocytes of 6-7-week-old EGR2^-/-B6/lpr and control mice by using a mouse naïve CD4⁺ T cells isolation kit from Miltenyi Biotec as we previously reported (16 (link)). For Th1 differentiation, naïve CD4⁺ T cells (1.5x10⁶) were plated in 48-well plate and cultured with 2 μg/ml plate-bound anti-CD3 (clone 145-2C11, Bio X cell, Lebanon, NH, USA), 1 μg/ml soluble anti-CD28 (clone 37.51, Bio X cell), 5ng/ml IL-2 (eBioscience), 10ng/ml IL-12 (eBioscience) and 10 μg/ml anti-IL4 (Bio X cell) for 3 days. For Th17 differentiation, naïve CD4⁺ T cells (1.5x10⁶) were plated in 48-well plate and cultured with 2 μg/ml plate-bound anti-CD3, 1 μg/ml soluble anti-CD28, 20ng/ml IL-6, 3ng/ml hTGFβ1, 10 μg/ml anti-IFNγ (Bio X cell) and 10 μg/ml anti-IL4 for 5 days. The differentiated cells were stimulated with PMA (50 ng/ml), ionomycin (1 μg/ml) plus protein transporter inhibitor (BD GolgiPlug) for 5 hours, and then collected for intracellular flow cytometry analysis of IFNγ- and IL-17- expressing cells.