Furthermore, the doxycycline-induced proliferative expansion of pre-B- and immature B-cell pools is reversible upon termination of Pim1/Myc overexpression. To test whether Pim1/Myc overexpression could also induce propagation of mature B cells

after in vivo maturation, 5×106 Pim1/Myc double-transgenic pre-BI cells were transplanted into sublethally irradiated Rag1−/− mice. After 2–4 weeks, expression of Pim1 and Myc was switched on in these matured cells by feeding doxycycline. Two and four weeks after the start of doxycycline Epacadostat mw treatment, mice were sacrificed and B-lineage cells of the BM (1 femur, 1 tibia), spleen and peritoneal cavity were analyzed by FACS (see Fig. 4A for an outline of the experiment). As shown in Fig. 4B, total B-cell numbers did not increase in BM, spleen or peritoneum when overexpression of Pim1 and Myc in B cells was induced 2 weeks after transplantation. Furthermore, there was no change in the percentage of immature, CD93+ cells versus mature, CD93−IgM+ cells in the spleen (Fig. 4C) and peritoneum. Transplantation of Pim1/Myc transgenic pre-B cells into sublethally irradiated Rag1−/− mice in the absence of doxycycline allows the development of CD93+IgM+ immature and of CD93−IgM+ mature B cells in the spleen of the transplanted mice. Thus, four weeks after maturation of these

transplanted cells, IgM+ cells were prepared from the spleens of these mice by magnetic MACS beads Z-VAD-FMK mouse (see Fig. 5A for an outline of the experiment). The cells, which had a purity of ∼97%, were induced for 18 h with doxycycline or left in medium alone. Then, RNA was prepared, transcribed into cDNA, and cDNA levels of Pim1, Myc and Gapdh were analyzed by semiquantitative PCR (Fig. 5B). The results of the RT-PCR analyses show that doxycycline successfully induced the overexpression of transgenic Pim1 and Myc in IgM+ sorted splenic B cells ex vivo. However, in spite of the induced overexpression of both oncogenes, there were no significant changes in the survival and proliferation of IgM+ or CD19+ splenic B cells (Fig. 5C). CFSE labeling of splenic CD19+ B

cells cultured for 4 days Thiamine-diphosphate kinase in the absence of mitogens revealed that overexpression of Pim1 and Myc did not induce cell cycle entry in these cells (Fig. 5D). Next, we tested these resting, mature B cells for their capacities to proliferate in response to polyclonal B-cell activators. Hence, the sorted IgM+ splenic B cells were cultured either in the absence or in the presence of polyclonal B-cell activators such as CD40-specific mAbs, IL-4, IL-5, IgM-specific mAbs, LPS, or combinations thereof. To rule out the possibility that the purification step with anti-IgM antibodies had an anergizing or otherwise detrimental effect on the sorted B cells, the experiment was repeated with CD19+ MACS-sorted B cells and erythrocyte-depleted total splenic cells. Data in Fig.