rATG-induced apoptosis of primary myeloma-cell isolates from bone marrow aspirates. (A) CD138⁺-expressing cells were selected by immunomagnetic bead affinity columns from fresh normal (N), fresh patient with myeloma (P), or previously frozen (F) Ficoll density gradient-prepared bone marrow aspirates, with each data point composed of 2 replicates. Cells were incubated with 100 μg/mL rATG or unimmunized rabbit IgG serum in complement-inactivated free medium for 18 hours, and apoptosis measured by annexin V⁺TOPRO-3⁺ staining. (B) Effect of IL-6, bone marrow stromal secreted factors (SS), or contact (SC) on rATG-induced apoptosis. Myeloma cell lines or human primary myeloma cells (hMc) were incubated with rATG alone (□) or in combination with 5 ng/mL IL-6 (○) and 50 ng/mL IL-6 (•), or cultured in direct contact (SC; ♦)or in a transwell culture system sharing media but no cell-cell contact (SS; ⋄) with human primary bone marrow stromal cells. While IL-6 and soluble stromal-cell factors had no effect on rATG-induced apoptosis, stromal-cell contact modestly reduced rATG-induced apoptosis for NCI-H292, MC-CAR, and the U266 myeloma cell lines (brackets). (C) Incubation of myeloma cell lines or primary myeloma cells from bone marrow aspirates with rATG (250 μg/mL; □) and dexamethasone (100 μg/mL;▵), sirolimus (10 ng/mL; •), or bortezomib (10 nM; ♦) did not significantly augment rATG-induced apoptosis. (D) Sirolimus does not augment rATG-induced apoptosis of the myeloma cell line RPMI-8226. RPMI-8226 cells were incubated with clinically relevant concentrations of sirolimus and increasing concentrations of rATG. Data points represent 2 independent experiments.