Impaired Th2 response in viperin^−/− naive CD4⁺ T cells cultured under suboptimal Th2 conditions. (A) Naive CD4⁺ T cells were stimulated under optimal Th2-skewed conditions for 2 weeks. Differentiated cells were restimulated with 5 μg/mL plate-bound CD3/CD28 for 6 hours for intracellular production of IL-4 and IFN-γ (left panel), or for 24 hours for the amount of cytokines secreted into the supernatants (right panel; data shown is a restimulated culture after 1 week of differentiation). Data are representative of 2 independent experiments. The numbers in quadrants indicate the percentage of each quadrant. (B) Naive T cells were cultured with 5 μg/mL anti-CD3 and 1 μg/mL anti-CD28 in the presence of 30 U/mL IL-2 and a graded dose of IL-4 for 5 days. RNA expression of Th2 cytokines was measured with real-time RT-PCR. (C) Naive T cells were stimulated as in panel B for 5 days, washed, counted, and then restimulated with 10 μg/mL anti-CD3 for 48 hours. The culture supernatants were then assayed for cytokine production measurement. (D) IFN-γ production cultured as in panels B and C was analyzed. Data shown in panels B-D are representative of 3 independent experiments. Results are expressed as the mean value of triplicate determinations plus or minus SD. *P < .05; **P < .01, WT versus KO.