2.11. In vitro differentiation of CD4+T cells
Naïve CD4+ T cells were purified from spleens and lymph nodes of C57BL/6 mice by negative selection using T-cell enrichment columns (R&D Systems) and mouse CD4+ T cell isolation kits (Miltenyi Biotech, Auburn, CA, USA). In vitrodifferentiation of CD4+ T cells was performed as previously described (Kim et al., 2014; Lee et al., 2016). Briefly, cells were activated by plate-bound anti-CD3 antibody in a 96-well plate (BD Biosciences) and soluble anti-CD28 (2 µg/mL) antibody. The cells were differentiated under Th1 polarizing conditions (10 ng/mL IL-12 and 10 µg/mL anti-IL-4 antibody), Th2 polarizing conditions (20 ng/mL IL-4 and 10 µg/mL anti-IFN-γ antibody), or Th17 polarizing conditions (20 ng/mL IL-6, 10 ng/mL TGF-β, 10 µg/mL anti-IFN-γ antibody and 10 µg/mL anti-IL-4 antibody). All cytokines and antibodies used for CD4+ T cell differentiation were obtained from BD Biosciences.