DC-mediated induction of AID. (A-B) Induction of AID mRNA in MM cells. (A) MM cell lines (U266, OPM2, ARK, CAG) were cocultured with monocyte-derived immature DCs (Mo-DCs) at tumor-to-DC ratio of 1:2. After overnight culture, tumor cells were re-isolated by magnetic bead or flow sorting, and expression of AID was analyzed by quantitative PCR. Data are normalized to the expression of the housekeeping gene GAPDH and expressed as fold change. To underestimate the fold induction of AID, the basal level of AID in MM cell lines cultured alone was deemed as detectable at 42 cycles, even when no signal was detected at 45 cycles. (*P < .05). Data are representative of ≥ 3 similar experiments. (B) MM cell lines (U266 and OPM2) were cocultured with or without DCs or freshly isolated CD14⁺ monocytes and analyzed for the induction of AID as in panel A. Data are representative of ≥ 3 similar experiments. (C) MM cell lines (U266 and OPM2) were cocultured with or without DCs or macrophages and analyzed for the induction of AID as in panel A. Data are representative of 3 similar experiments. (D-E) Detection of AID protein. (D) Lysates from MM cells either cultured alone or after exposure to DCs as in panel A were analyzed for the expression of AID protein by Western blot analysis with the use of an anti-AID Ab or β-actin as a loading control. Data are representative of ≥ 3 similar experiments. (E) The expression of AID in experiments in panel D was also analyzed by flow cytometry after permeabilization for intranuclear staining. Data are representative of ≥ 3 similar experiments. (F) Induction of AID by circulating human DC subsets. Circulating BDCA1⁺ mDCs or BDCA4⁺ pDCs were isolated by magnetic beads and cultured with U266 cells. Isolated tumor cells were analyzed for the induction of AID, as in panel A. (G) DC-mediated induction of AID in breast cancer cells. MCF-7 breast cancer cells were cultured alone or were re-isolated after exposure to DCs and analyzed for the expression of AID by quantitative PCR as in panel A.