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. 2020 Oct 16;11:577823. doi: 10.3389/fimmu.2020.577823

Figure 6.

Figure 6

Inhibition of human TLR4 by tetra- and tri-acylated components of MPL adjuvant. Synthetic versions of several major components of MPL adjuvant® were tested for inhibitory activity using human TLR4 HEK-Blue reporter cells. (A) MPL adjuvant® components ML3, ML4A, ML4B, ML5A and ML6 were diluted in half-log steps in a mixture containing a fixed concentration of PHAD (1 μM) and added to human TLR4 HEK-Blue reporter cells for 22 h. Values shown are averages ± SD of secreted alkaline phosphatase from two independent experiments, each performed in triplicate and normalized by setting 100% = PHAD alone (1 μM). (B, C) MPL adjuvant® components ML3 and ML4A were diluted from a peak dose of 31.6 μM (or a peak dose of 0.03 μM Lipid IVa as antagonist control) in half-log steps in a mixture containing a fixed concentration of PHAD (30 nM) or 3D-6A-PHAD (300 nM), respectively. Values shown are averages ± SD of secreted alkaline phosphatase from two independent experiments normalized by setting 100% = PHAD or 3D-6A-PHAD alone, respectively. Shaded regions indicate 99% confidence intervals within which the true population means should occur 99% of the time. Calculated IC50PHAD values (95% CI) were: ML3, 1.32 μM (1.08–1.67); ML4A, 0.40 μM (0.27–0.62); and Lipid IVa: 0.19 nM (0.15–0.23).