At the peak of EAE disease, the mice were sacrificed and CD4+ T cells were purified from spleens by CD4+ T cell isolation kit (Miltenyi, Auburn, CA). The purified T cells (2.5 × 106 cells/ml) were cultured in 96-well round-bottom microculture plates (Falcon Labware, Oxnard, CA) in RPMI-complete media containing RPMI 1640 (Life Technologies, Gaithersburg, MD), 10% FBS, and 100 μg/ml streptomycin and penicillin (Atlanta Biologicals Norcross, GA), 1 mM glutamine, 1 mM nonessential amino acids, and 50 μΜ 2-mercaptoethanol (Sigma-Aldrich).
For skewing of different CD4+ T cell subsets and their expansion, the isolated CD4+ T cells were stimulated with PLP139–151 (5 μg/ml) with IL-2 (10ng/ml) for TH0, IL-2 (10ng/ml), rhlL12p35 (10ng/ml), and anti-IL-4 (1μg/ml) for TH1, or rmlL12/23p40 homodimer (10ng/ml), anti-IFN-γ 1(μg/ml), anti-IL-4 (1 μg/ml) for TH17. All cytokines and antibodies were purchased from BD Biosciences (San Diego, CA). Following stimulation, the cells were harvested for adoptive transfer of EAE disease and the culture supernatants were collected for analysis of IFN-γ, IL-17, and IL-10 expression by ELISA (BioLegend Cat# 430802, 432505, and 431411; San Diego, CA).
For skewing of different CD4+ T cell subsets and their expansion, the isolated CD4+ T cells were stimulated with PLP139–151 (5 μg/ml) with IL-2 (10ng/ml) for TH0, IL-2 (10ng/ml), rhlL12p35 (10ng/ml), and anti-IL-4 (1μg/ml) for TH1, or rmlL12/23p40 homodimer (10ng/ml), anti-IFN-γ 1(μg/ml), anti-IL-4 (1 μg/ml) for TH17. All cytokines and antibodies were purchased from BD Biosciences (San Diego, CA). Following stimulation, the cells were harvested for adoptive transfer of EAE disease and the culture supernatants were collected for analysis of IFN-γ, IL-17, and IL-10 expression by ELISA (BioLegend Cat# 430802, 432505, and 431411; San Diego, CA).