Figure 3.

Bone marrow monocytes stimulated with TLR8 ligands produce myeloma growth factors. (A) 100 000 sorted CD14^high and CD16⁺CD14^dim blood monocytes were cultured with 100 ng/mL LPS, 1 μg/mL CL075 or medium alone for 12 h in 100 μL medium in a 96 well plate. TNFα was measured in the supernatant by ELISA. (B,C) CD16⁺CD14^dim bone marrow monocytes from myeloma patients produce cytokines after stimulation with TLR8 agonist. Monocytes were immunomagnetically purified from bone marrow of myeloma patients as described. A total of 200,000 cells were stimulated with 1 μg/mL TLR8 agonist (CL075) or medium for 24 h before harvesting the supernatant, (B) TNFα secretion in the supernatant of monocytes from three representative patients out of eight analyzed. The monocytes were enriched from bone marrow as described in the Materials and Methods and stained with anti CD16 and anti CD14 after enrichment (patient 1: 9.3% CD16⁺CD14^dim, patient 2: 10.3% CD16⁺CD14^dim, patient 3: 13.8% CD16⁺CD14^dim). (C) CD16⁺CD14^dim monocytes produce myeloma growth factors after TLR8 stimulation. (D) IL6 and CCL3 in the supernatant from one representative patient out of eight analyzed. Cytokines were detected using Multiplex (27 plex BioRad). Figures shows mean and SEM of triplicate wells from each patient analyzed (D).