Figure 3.

Pre–B cell receptor activation induces expression of BCL6 via down-regulation of IL-7 responsiveness. IL-7–dependent pre–B cells were transduced with a retroviral vector encoding a constitutively active STAT5 mutant (STAT5-CA-GFP; Onishi et al., 1998) or an empty vector control (GFP). (A) GFP-expressing transduced cells were sorted, subjected to IL-7 withdrawal, and analyzed by Western blotting for expression of BCL6 and tyrosine phosphorylation of STAT5 using β-actin as a loading control (n = 3). (B) Mouse B cell precursors were isolated by B220⁺ MACS from freshly harvested bone marrow cells. Freshly isolated bone marrow B cell precursors and IL-7–dependent pre–B cell cultures were treated with 10 µg/ml of a neutralizing anti–IL-7 antibody overnight and were subjected to Western blot analysis (three experiments were performed). (C) Bone marrow B cell precursors from RAG2^−/− tTA/μ chain–transgenic mice are unable to express an endogenous μ chain but carry a functionally prerearranged μ chain under control of tetO sequences (Hess et al., 2001). These mice express a tTA under control of endogenous μ chain regulatory elements, and withdrawal of tetracycline results in activation of μ chain expression (routinely performed quality control). The effect of tetracycline-inducible activation of μ chain expression on BCL6 expression and STAT5 tyrosine phosphorylation was determined by Western blotting (n = 3; right). (D) IL-7–dependent pre–B cells lacking the pre–B cell receptor–related linker molecule SLP65 were transduced with retroviral expression vectors encoding either SLP65-GFP or GFP alone. Surface expression levels of IL-7Rα chain were measured by flow cytometry (the experiment was performed twice). The histogram shows the IL-7Rα levels in transduced GFP⁺ (green) and untransduced (gray) cells.