Cell-cycle response of primary MM cells to APRIL and BAFF. (A) Freshly purified CD138⁺ MM cells were incubated in 20% MM patient plasma (CM) with or without APRIL (200 ng/mL) and analyzed for cell-cycle response as for Figure 1. Results are percentage of CD138⁺ cells in S/G₂M. Three representative cases showing time course of APRIL-induced cell-cycle progression of CD138⁺ MM cells in the presence of CM. Cases 1 and 13 harbor the t(14;16) translocation, whereas case 12 expressed cyclin D2 in the absence of an IgH translocation (control indicates CM alone; APRIL, CM + APRIL, all 3 cases with circulating plasma cells). (B-C) Representative examples of cell-cycle responses to cytokines in primary MM cells, incubated in CM alone (control) or with APRIL, BAFF (both 200 ng/mL), IGF-1 (500 ng/mL), or IL-6 (100 ng/mL). FACS plots show the S/G₂M fraction of CD138⁺ cells after 72 hours of culture in CM with or without growth factors compared with freshly isolated cells (0 hours). (B) Cyclin D2–expressing case with t(14;16) (case 1, Table 1). (C) Cyclin D1–expressing case with t(11;14) (case 20, Table 1). (D) Effect of APRIL (200 ng/mL) or BAFF (200 ng/mL) on cell-cycle progression of CD138⁺ MM cells cultured in CM. Results are mean ± SEM (percentage of CD138⁺ cells in S/G₂M). Fourteen patients expressed cyclin D1 (cases 16-29, Table 1) and 12 patients expressed cyclin D2 (cases 1-12, 7 with circulating plasma cells, Table 1). The proliferative fraction at the time of isolation (0 hours) is shown for comparison. (E) The cell-cycle response to APRIL in cyclin D2–expressing CD138⁺ cells harboring IgH translocations (n = 8, 5 with circulating plasma cells) compared with cells without IgH translocations (n = 7, 3 with circulating plasma cells; cases 1-15, Table 1), Data are mean ± SEM. (F) CD138-selected cells from patient 3 (expressing cyclin D2 with t(14;16) and with circulating plasma cells) were cultured in RPMI with (CM⁺) or without plasma (CM⁻) alone or with APRIL or BAFF (as indicated) for 72 hours and pulsed with ³H-thymidine for the last 2 hours. 0 Hrs indicates pulsed with ³H-thymidine for 2 hours immediately after selection. One representative case: Data are mean ± SD of triplicates. (G) Left panel: Dose-response to APRIL in one representative patient. CD138⁺ purified cells (patient 7) were cultured with increasing concentrations of APRIL for 72 hours and pulsed with ³H-thymidine for the last 2 hours. Data are mean ± SD of triplicates. Right panel: TACI-Fc blocks APRIL-induced proliferation. TACI-Fc (10 μg/mL) was added to CD138-selected MM cells before culture for 72 hours, and DNA synthesis assessed in panel F. Data are mean ± SD of triplicates (patient 7).