CD4+ T cells were activated with plate-bound
α-CD3 (3.75 μg/mL; Immunotech) and soluble α-CD28
(1 μg/mL; Immunotech) in X-VIVO 20 serum-free medium (Lonza).
X-VIVO 20 medium was supplemented withl -glutamine (2 mM,
Sigma-Aldrich) and antibiotics (50 U/mL penicillin and 50 μg/mL
streptomycin; Sigma-Aldrich). Th17 cell differentiation was induced
using a cytokine cocktail of IL-6 (20 ng/mL; Roche), IL-1β (10
ng/mL), and TGF-β (10 ng/mL) in the presence of the neutralizing
Ab anti-IFN-γ (1 μg/mL) and anti-IL-4 (1 μg/mL)
to block Th1 and Th2 polarization, respectively. For the control cells
(Th0), CD4+ T cells were TCR-stimulated with α-CD3
and α-CD28 in the presence of neutralizing Ab. All cytokines
and neutralizing Ab were purchased from R&D Systems, unless otherwise
stated. All cultures were maintained at 37 °C in a humidified
atmosphere of 5% (v/v) CO2/air.
α-CD3 (3.75 μg/mL; Immunotech) and soluble α-CD28
(1 μg/mL; Immunotech) in X-VIVO 20 serum-free medium (Lonza).
X-VIVO 20 medium was supplemented with
Sigma-Aldrich) and antibiotics (50 U/mL penicillin and 50 μg/mL
streptomycin; Sigma-Aldrich). Th17 cell differentiation was induced
using a cytokine cocktail of IL-6 (20 ng/mL; Roche), IL-1β (10
ng/mL), and TGF-β (10 ng/mL) in the presence of the neutralizing
Ab anti-IFN-γ (1 μg/mL) and anti-IL-4 (1 μg/mL)
to block Th1 and Th2 polarization, respectively. For the control cells
(Th0), CD4+ T cells were TCR-stimulated with α-CD3
and α-CD28 in the presence of neutralizing Ab. All cytokines
and neutralizing Ab were purchased from R&D Systems, unless otherwise
stated. All cultures were maintained at 37 °C in a humidified
atmosphere of 5% (v/v) CO2/air.