Figure 5

Immunomax ® activates NF- k B via toll-like receptor 4. (A) NF-κ B activity was measured as NF-κ B-dependent SEAP reporter gene expression in HEK-Blue TLR null, 2, 3, 4, 5, 7, 8, or 9 cells incubated for 18 hours in the presence (5 μg/ml, ■) or absence (negative control, □) of Immunomax®. TNF-α (10 ng/ml), Pam₂CSK4 (1 μg/ml), poly I:C (10 μg/ml), LPS (1 μg/ml), flagellin (1 μg/ml), Imiquimod (1 μg/ml), CL097 (1 μg/ml) and ODN 2007 (10 μg/ml) were used as positive controls for TLR null, 2, 3, 4, 5, 7, 8 and 9 positive cells, respectively (gray square). Results are expressed as the fold-increase in NF-κ B reporter (SEAP) activity relative to intact (untreated) cells. Mean values from 3 independent experiments, each performed in duplicates are presented. (B) Immunomax® induces NF-κ B-dependent reporter (SEAP) gene expression in HEK-Blue TLR-4 cells in a dose-dependent manner. Intact HEK-Blue TLR-4 cells and HEK-Blue TLR null cells were used as negative controls. Results are expressed as the fold-increase in NF-κ B-dependent reporter (SEAP) activity in Immunomax® treated (□) relative to intact (untreated ■) cells. Mean values from 3 independent experiments, each performed in duplicate are presented. (C) NF-κ B activation in HEK-Blue TLR4 cells was analyzed according to the nuclear translocation of p65 subunit of NF-κ B. (D,E) Selective blockade of TLR4-signaling pathway abrogates the induction of DC activation by Immunomax®. (D) Addition of CLI-095 (10 μg/ml) to the BM-DC/Immunomax® culture significantly inhibits the number of BM-DC producing IL-12 cytokine. (E) CLI-095 added into the co-culture 4T1/BM-DC/NK cells inhibited the ability of Immunomax® to activate BM-DC/NK cell-mediated killing of 4T1 breast cancer cells.