Requirement of immobilization of CD137 ligand agonists.

<p>SGH-MM5 (A and B) or SGH-MM6 (C and D) cells at a density of 10⁶ cells/ml were cultured on uncoated plates (PBS), or on plate-bound Fc, CD137-Fc, mouse IgG (MOPC21) or anti-CD137 ligand antibody (clones 5F4 and C65-485) or on uncoated plates (PBS), or to which Fc or CD137-Fc proteins were added soluble at 10 µg/ml. (A) Percentage of dead cells (left panel) and number of total live cells (right panel) were determined after 24 h via trypan blue staining. (B) Extent of apoptosis of cells in (A) was determined by Annexin V and 7-AAD staining. (C) Percentages of dead cells were determined at indicated times via trypan blue staining. (D) IL-8 concentrations in 24 h cell supernatants as determined by ELISA. Depicted are means ± standard deviations of triplicate measurements. * p<0.05.</p

CD137 inhibits proliferation and induces cell death of MM but not of non-MM cells.

<p>Cells were cultured on plate-bound Fc or CD137-Fc protein or on uncoated plates (PBS). (A) After indicated times proliferation was determined via ³H-thymidine incorporation. (B) Cell viability was determined after 24, 48, 72 and 96 h via trypan blue staining. Depicted are means ± standard deviations of triplicate measurements. * p<0.05. This experiment is representative of three independent experiments with similar results.</p

CD137 ligand is expressed by B cell lymphoma and myeloma cell lines.

<p>Cells were stained by PE-conjugated monoclonal antibodies against CD137 (clone 4B4-1), or anti-CD137 ligand (clone 4B1-436), (open curves) or their isotype control (MOPC-21), (filled curve).</p

CD137-induced MM cell death is not inhibited by IL-6 or IL-2.

<p>SGH-MM6 cells at a density of 1.2×10⁶ cells/ml were cultured on plate-bound Fc or CD137-Fc protein or on uncoated plates (PBS), and IL-6 (1 ng/ml) or IL-2 (100 units/ml) were added. Cell viability was determined after 24 h via trypan blue staining. Depicted are means ± standard deviations of percentages live cells from triplicate measurements. * p<0.05. This experiment is representative of three independent experiments with similar results.</p

Early activation of the classical NF-κB pathway is initiated upon CD137 ligand signaling.

<p>SGH-MM5, SGH-MM6 and RPMI 8226 cells were treated with plate-bound Fc or CD137-Fc. (A) Total protein was extracted at indicated times. NF-κB signaling proteins were detected by immunoblotting. (B) Cells were treated with CD137-Fc for indicated times and nuclear extracts were isolated and subjected to NF-κB transcription factor assay analysis. Data is represented by the average of triplicates within the experiment and is representative of two independent experiments. P values were calculated using pair wise t-test comparing time zero to time 60 min. * p<0.05. (C) Cells were treated with plate bound Fc or CD137-Fc protein for six hours following which total RNA was extracted. Real time RT-PCR was performed on both human IκBα and IL-6 transcripts. RT-PCR data is represented by fold change as calculated using the 2^ΔΔCT method where GADPH served as a reference gene, and where each data point was performed in triplicate. (D) Cells were cultured for 48 h on plates with immobilized Fc (white open curve) or CD137-Fc protein (grey filled curve), and then stained with 50 nM DiOC₆ and analyzed by flow cytometry. Cells with no DiOC₆ added were used as background control (hatched).</p

CD137 induces apoptosis in the MM cell lines.

<p>(A) SGH-MM5 cells at a density of 10⁶ cells/ml were cultured on plate-bound Fc or CD137-Fc protein or on uncoated plates (PBS). After 24 h the cells were stained with Annexin V and 7-AAD. Similar results were obtained for the other MM cell lines. (B) Cells from (A) were stained with Acridine Orange (green) and Ethidium Bromide (red). Photographs were taken at a magnification of 40×. (C) CD137-Fc treated SGH-MM5 cells of B at a magnification of 200×. (D) Caspase 3 activity was determined 6 h after exposure of SGH-MM5 and RPMI 8226 cells to immobilized Fc or CD137-Fc protein. These experiments are representative of three independent experiments with similar results.</p