Coculture with DCs downmodulates MM proliferation and enhances survival. (A) Proliferation. K562 were cultured in media alone (K562m; Km), or differentiated with PMA (K562p or Kp) or PMA plus TNF-α (K562 P+T or Kpt), irradiated, and cocultured with 8226 (right panel) or U266 (left panel). Proliferation was measured by thymidine incorporation and expressed as means ± SD of triplicate wells. Data are representative of 4 independent experiments. Compared with coculture with undifferentiated K562 (K562m): *P = .001; **P < .001; ⁺P = .004; ⁺⁺P = .001. (B) Cell-cycle analysis. CSFE-labeled 8226 were cocultured at a 1:1 ratio (2 × 10⁴ cells) with either undifferentiated K562 (top) or K562 differentiated with PMA (K562-DC; bottom) for 24 hours, permeabilized, and stained with PI. Cell-cycle analysis of CSFE⁺ cells was conducted on a BD LSR1; percentage of cells in G₀/G₁, S, and G₂/M are shown to the right. Data are representative of 3 independent experiments. (C) MM viability following coculture with DCs. CFSE-labeled 8226 were cocultured with irradiated PMA-differentiated K562 at a 1:1 ratio. After 24 hours, the cultures were stained with PI and analyzed by flow cytometry. Data are representative of 3 independent experiments. (D) CD28-Ig blockade. DCs were differentiated from KG1 using PMA, irradiated, and cocultured with 8226. CD28-Ig was added to the DCs 1 hour before addition of MM cells. Proliferation was measured by thymidine incorporation. Data are representative of 2 experiments. *P = .01 compared with 8226 plus DCs. (E) iDCs versus mDCs derived from normal monocytes. To generate iDCs, monocytes were cultured in GM-CSF and IL-4 for 8 days. To generate mDCs, TNF-α was added for the last 4 days of culture. These cells were then irradiated and cocultured with myeloma cells at the ratios indicated. Proliferation was measured by thymidine incorporation. Data are representative of 2 independent experiments. Compared with 8226 alone: *P = .006; **P = .007; ⁺P = .006. (F) Protection against dex-induced cell death. 8226 were cultured alone, or 1:1 with irradiated, undifferentiated K562 (DC precursors) or K562-derived DCs (DC), treated for 72 hours with 100 μM dex, and analyzed by FACS for 7AAD (dead cells) and CD28 expression (MM cells). Percentage viable refers to myeloma cell viability. Data are representative of 2 independent experiments.