Figure 6. Modulation of the AHR pathway influences the tolerogenic profile of tolDCs.

(A) Box plots of relative expression of individual genes performed by RT-qPCR of mRNA in HD tolDCs versus tolDCs + FICZ. P values from Wilcoxon’s tests are shown. n = 4–6 depending on the gene, 2 independent experiments. (B) Before-after scatter bar plot showing flow cytometry data relative to the percentage of CD83⁺CD86⁺, CD14⁺, CCR7⁺, or HLA-DR⁺ cells among tolDCs, tolDCs + FICZ, and tolDCs + CH223191. P values from repeated-measures 1-way ANOVA with multiple comparisons are shown. n = 6 in each sample group. (C) Before-after scatter bar plot representing the effect of FICZ agonist on production of IL-6, IL-12p70, and IL-1β cytokines by tolDCs. FICZ was added at day 0 and day 4 of differentiation of tolDCs, with a final concentration of 18 μM. P values from Wilcoxon’s tests are shown. n = 9 in each sample group. (D) Proliferation of allogeneic peripheral mononuclear cells cocultured with HD tolDCs and tolDCs differentiated in the presence of either FICZ (HD tolDCs + FICZ) or CH223191 (HD tolDCs + CH223191). Inhibition of proliferation was assessed as percentage of Violet 450–positive lymphocytes and calculated using mDC-induced proliferation as reference for each sample by the following formula: (mDCs – tolDCs)/mDCs, obtaining the percentage of reduction of proliferation of tolDCs in reference to the donor-matched mDCs. P values from repeated-measures 1-way ANOVA with multiple comparisons are shown. n = 8 in each sample group. (E) Quantification of pH, glucose, and lactate concentration on day 6 cell culture supernatants. P values from repeated-measures 1-way ANOVA with multiple comparisons are shown. n = 8 in each sample group.