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.