Alteration of T-cell subsets and cytokine production in NICD1 mice. (A) Intracellular flow cytometric analysis of splenic CD3⁺CD4⁺ T cells from NICD1 mice and controls. T cells were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin for 5 hours before analysis. (B) Intracellular flow cytometric analysis of splenic CD3⁺CD8⁺ T cells from NICD1 mice and controls. (C) Intracellular flow cytometric analysis of splenic B220^midCD49b⁺ NK cells from NICD1 mice and controls. In panels A-C, the mean percentage of cells from 3 to 4 individual mice per group is shown. Data in the bar graphs are expressed as the mean and SEM. *P < .05, **P < .01, and ***P < .001 (Student t test). (D) Expression of mRNA encoding FOXP3, GATA3, and TBX21 by splenic CD3⁺CD4⁺ T cells isolated from NICD1 mice and controls, as evaluated by quantitative PCR. Expression levels are expressed relative to those detected in CD4⁺ T cells from control mice. Data in the bar graphs are expressed as the mean and SEM from 4 individual mice per group. *P < .05, **P < .01, and ***P < .001 (Tukey-Kramer test). n.s., not significant.