Defective Th response on ablation of Bach1. WT or Bach1^−/− mice were immunized subcutaneously with 100 μg of OVA/CFA. One month after immunization, the animals were boosted with 100 μg in IFA. Splenocytes were isolated, equal numbers were plated and cultured with OVA (10 μg/mL). (A) Bach1^−/− splenocytes are defective in T-cell activation. After 2 days of OVA treatment, splenocytes were subjected to flow cytometric analysis to determine the percentage of T cells that expressed the activation marker (CD25) after OVA induction. The graph shows the average percentage of CD3ϵ⁺ CD25⁺ population in the splenocytes. P < .05. KO indicates knockout. (B) Bach1^−/− splenocytes are defective in antigen-specific–stimulated proliferation. Splenocytes were pulsed with tritiated (³H) thymidine for 16-24 hours. The incorporated radiolabeled thymidine in the cells was quantified in scintillation fluid and measured as counts per minute (CPM). The time on the x-axis indicates the hours of OVA stimulation, which also includes the ³H thymidine pulse time. Two-way ANOVA of WT OVA versus Bach1^−/− OVA, P < .001. (C) Impaired OVA-induced T-cell expansion on deletion of Bach1. Splenocytes were cultured for 60 hours, and flow cytometry was used to quantify the percentage of CD3ϵ T cells. (D-H) Defective production of T-cell cytokines on OVA restimulation in Bach1^−/− splenocytes. After OVA restimulation of culture splenocytes for 3 days, ELISAs were performed with the supernatant fluid to measure cytokine production. Three independent experiments were performed, and each experiment contained 4-12 mice per genotype. WT OVA versus Bach1^−/− OVA, P < .05. All plots are expressed with SEM.