Fig. 4: Oral administration of hexa-acylated LPS enhances the efficacy of anti-PD-1 therapy.

a–d, Wild-type, TLR2^−/− or TLR4^−/− NF-kB reporter THP-1 (human monocyte/macrophage) cells were incubated with the indicated doses of hexa- or penta-acylated LPS for 24 h. Data are from two biological replicates per concentration of LPS and are representative of at least three experimental replicates. a, NF-kB activation in response to 1 ng ml⁻¹ hexa-acylated LPS or 1 µg ml⁻¹ of penta-acylated LPS (Hexa LPS WT versus TLR4^−/−, *P = 0.0274). b, NF-kB activation dose–response with the indicated doses of hexa- or penta-acylated LPS. c, IL-1β (left) and IL-6 (right) secretion by THP-1 cells after 24 h treatment with the indicated doses of LPS, shown as fold increase over media controls. d, NF-kB activation with the indicated combined doses of hexa- and penta-acylated LPS (1 ng Hexa versus +10, +100 and +1,000 ng Penta, ****P < 0.0001). e, MC38 colorectal adenocarcinoma cells were subcutaneously implanted into wild-type mice, and measurements were taken at indicated time points post implantation. Indicated animals were treated with anti-PD-1 i.p. and/or oral hexa- or penta-acylated LPS or control drinking water starting at day 10 after tumour implantation (anti-PD-1 versus Isotype ctrl, ***P = 0.0002; anti-PD-1 versus anti-PD-1 + Hexa, *P = 0.0318). Data shown were pooled from 2 experimental repeats with n = 10 mice per group. f, Flow cytometry measurements of IFNγ (anti-PD-1 versus Isotype ctrl, *P = 0.0325; anti-PD-1 versus anti-PD-1 + Hexa, *P = 0.0423) and TNF (anti-PD-1 versus anti-PD-1 + Hexa, *P = 0.0352) producing CD8⁺ T cells infiltrating tumours of mice in e with n = 5 mice per group. Brown–Forsythe and Welch ANOVA comparing all to WT in a. One-way ANOVA with a Dunnett’s multiple comparisons test in d. Two-way ANOVA comparing all to anti-PD-1 in e and Brown–Forsythe with post hoc Dunnett’s test comparing all to anti-PD-1 in f, with bars and error representing mean and s.e.m.

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