The presence of B cells and plasma cells in the normal human thymii. Ten pediatric thymii were stained by hematoxylin and eosin, and by immunohistochemistry for the T-cell-specific marker CD3, the pan-B-cell markers CD79α and CD20, and the plasma cell-specific marker CD138. Representative photomicrographs from a single thymus are shown. The CD20⁺ cells appeared to cluster around the Hassel corpuscles (asterisk), while the CD138-staining plasma cells were seen in the medullary areas (arrow). Micrographs are all × 40, except as noted.

Delineation of apoptotic pathways triggered by rATG. Myeloma cell lines and primary myeloma cells were incubated with 250 μg/mL unimmunized rabbit IgG (filled areas) or rATG (open areas) and assayed at 18 hours. Representative histograms are shown (from n = 5 experiments/cell type). Loss of mitochondrial-membrane potential was a prominent feature of all cell lines with the exception of U266, which exhibited primarily caspase-3 activation. Caspase-3 activation was seen to varying degrees in all cell lines and primary cells. All myeloma cell lines and primary cells exhibited subdiploid DNA fragmentation.

rATG-induced apoptosis in myeloma cell lines. (A) To determine the effective dose that renders apoptotic induction, different concentrations of rATG were added to myeloma cells. ARH-77 cells were isolated by Ficoll-Paque centrifugation, and 10⁵ cells/well were placed in 48-well flat-bottom plates. Cells were incubated with various concentrations of rATG (0.0001-1 μg/mL) at 37°C for 18 hours and assayed with annexin/TOPRO. Incubation of myeloma cells at increasing concentrations of rATG demonstrated a progression from live (annexin^neg TOPRO^neg) to apoptotic (annexin^pos TOPRO^neg), and finally to late apopototic/necrotic (annexin^pos TOPRO^pos) phases. (B) Concentration-dependent lysis of human myeloma cell lines. Multiple cell lines were incubated with rATG at varying concentrations for 18 hours in complement-free medium, and apoptosis was measured by annexin V binding. Data points are from n = 5 independent experiments. (C) Absence of lot-to-lot variation in apoptotic potential of rATG. MC-CAR myeloma cells were incubated with rATG for 18 hours; apoptosis was assessed by annexin V staining in 2 independent experiments for each data point. Five lots of rATG were tested.

Identification of rATG-binding targets by competitive inhibition of binding. NCI-H929 myeloma cells were incubated with control rabbit IgG (□) or rATG (▪) for 1 hour at 37°C followed by incubation on ice with the monoclonal antibody noted to assess for blockade of rATG binding. Myeloma cell lines and naive and CD40L-activated B cells were used as targets based on expression of the antigen of interest: RPMI-866 (IgG1, CD38), NCI-H929 (HLA-ABC, CD16, CD32, CD64, CD126 [IL-6R]), naive human B cells (CD52), CD40L-activated B cells (HLA-DR, CD19, CD20, CD27, CD30, CD40, CD80, CD95), and human myeloma cells (CD38, CD138). Significant inhibition of rATG binding to numerous B-cell-specific surface proteins from all stages of B-cell development was observed. Error bars denote 1 SD. The table shows the average fraction of myeloma cell lines and cells from 3 patients expressing the noted surface marker. Relative level of common expression of each marker among all cell types is summarized at the bottom of the table.

Effects of complement upon rATG-induced cell death. (A) Addition of complement augments the ability of rATG to kill CD138⁺ myeloma cells. Once isolated by Ficoll centrifugation and washed, 10⁵ cells/well were incubated with rIgG control, rATG alone, or rATG plus rabbit complement. Compared with rATG alone, rATG with added complement significantly increased death of CD40L-stimulated B cells, H-Sultan, NCI-H929, RPMI-8226, MC-CAR, and U266 cell lines. However, it had no effect on CHO or ARH-77 cell lines. Error bars indicate the mean ± standard deviation. (B) rATG F(ab′)₂ fragment activity was assessed by reconstituting with anti-FcR-specific monoclonal antibodies. CD40L-stimulated human B-cell blasts or myeloma cell lines (10⁵ cells/well) were incubated with 250 μg/mL rATG F(ab′)₂ fragments alone or coincubated with either human Fc fragments, anti-CD32, or anti-CD64 (♦). The response is measured against the intact rATG molecule (▪). Controls without rATG F(ab′)₂ but with unimmunized rIgG, Fc fragments, anti-CD32, or anti-CD64 are also shown (⋄). rATG F(ab′)₂ fragment with or without human Fc fragment showed a weaker apoptotic induction potential than intact rATG (P < .05, all samples). For CD40L sBc, RPMI-8226, and NCI-H929 cells, rATG F(ab′)₂ fragments coincubated with anti-CD32 or anti-CD64 increased apoptosis of myeloma cells, although still somewhat less than intact rATG. No effect was observed on U266 cells.

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.