Impaired T-cell responses in C57bl/6 recipient animals reconstituted with BMCs from Bach1^−/− donors. Lethally irradiated C57bl/6 recipients reconstituted with BMCs from WT or Bach1^−/− donors were immunized with OVA/CFA, and splenocytes were cultured in the presence or absence of OVA. After 3 days in culture, splenocytes were subjected to (A) flow cytometric analyses (see also supplemental Figure 1A) to quantify the percentage of CD4⁺ cells that expressed the CD25 T-cell activation marker and (B) the supernatant fluids were examined for the amount of IFNδ with the use of ELISAs. Student t test P < .05 for the OVA-treated samples. (C) Total splenocytes from WT or Bach1^−/− animals were stimulated with concanavalin A (ConA) and pulsed with tritiated thymidine for the last 16-22 hours. (D) The amount of IL-2 secreted by T cells in total splenocytes after stimulation with ConA was determined by ELISA. (E-F) WT and Bach1^−/− mice were immunized with OVA/CFA and boosted with OVA/IFA. Splenic CD4⁺ T cells from these animals were purified 2 weeks later and cocultured with naive total WT splenocytes in the absence or presence of OVA for 72 hours. The percentage of activated CD4⁺ T cells (CD4⁺ CD25⁺) was measured by flow cytometry, (E) and the amount of secreted IL-2 was quantified by ELISA (F). Splenocytes from animals that had undergone immunization with OVA/CFA were subjected to flow cytometric analyses to examine the percentage of B cells (G), macrophages (H), and DCs (I). See also supplemental Figure 1B, C, and D for flow cytometric plots for panels G, H, and I, respectively. The data represent averages of ≥ 3 mice per group.