Lymphoid biased progenitors in BM express high levels of TLR9 and respond to CpG in culture. BM fractions were isolated as described in “Isolation of cell populations and flow cytometry” before analysis by quantitative RT-PCR. (A) The results are representative of 2 independent experiments and are normalized to β-actin and compared with pDC, the subset with highest expression. Normal Lin⁻IL-7Rα⁺c-kit^loSca1⁺ CLPs were sorted and treated in serum-free, stromal-free cultures for 2 or 48 hours with 0.6 μg/mL CpG. The cultures were washed and then incubated for an additional 8 days with stem cell factor, FL, and IL-7 before flow cytometric analyses. Whereas almost pure populations of B220⁺CD19⁺ lymphocytes were present in control cultures, CD19⁻ cells emerged as a result of extended CpG treatment (B, left panel). B220^+/− CD19⁻ cells were gated (B, right panel) and further analyzed to reveal B220⁺CD19⁻CD11c⁺CD11b⁻ pDCs and/or NK-like IKDCs, and B220⁻ CD19⁻CD11c⁺CD11b⁺ cDCs. Sorted CLPs were placed in limiting dilution stromal-cell free, serum-free cultures with and without 0.6 μg/mL of CpG for 48 hours. They were washed and then returned to culture for an additional 8 days before flow cytometry analysis. (C) Individual wells were scored as being positive for CD19⁺ B lineage and/or CD11c⁺ DCs.