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. 2023 Mar 3;42(3):112193. doi: 10.1016/j.celrep.2023.112193

Figure 4.

Figure 4

AHR-induced tolerogenic features are dependent on IL-10 and MTOR pathway inhibition

(A–D) Peripheral blood (PB) CD14+ monocytes were differentiated for 7 days in the presence of GM-CSF, IL-4 and IL-10 (DC-10), GM-CSF, and IL-4 without (DC) or with the AHR agonist ITE (30 μM) (AHRactDC) or the AHR inhibitor CH223191 (20 μM) (AHRinhDC).

(A) Experimental design.

(B) Heatmap displaying the median percentage of positive cells expressing the indicated marker in DC-10s, AHRactDCs, DCs, and AHRactDCs at 7 days of differentiation (n = 11–25). p values by Mixed effect model with Geisser-Greenhouse correction followed by Sidak’s multiple comparison test; only AHRinhDC vs. DC and AHRactDC vs. DC p values are shown, the complete list of significant p values is shown in Figure S4C.

(C) Short-term MLR of total CD4+ T cells stimulated with DC-10s, DCs, and AHRactDCs (n = 12). Percentages of proliferating (left) and CD25+HLA-DR+ activated (middle) CD4+ cells, and IFNγ production (right) in the culture supernatants of. p values by Wilcoxon matched pairs test.

(D) Short-term MLR of total (top panels) and naive (bottom panels) CD4+ T cells stimulated with DC-10s, DCs, and AHRinhDCs (n = 10). Percentages of proliferating (left) and of CD25+HLA-DR+ activated (middle) CD4+ cells; IFNγ production (right) in the culture supernatants. p values by Wilcoxon matched pairs test.

(E) DDIT4 expression by ddPCR in DCs and DC-10s expressed as ratio of DDIT4 molecules/mL to HPRT molecules/mL in arbitrary units (a.u.). p value by Wilcoxon matched pairs test (n = 11).

(F) Experimental design describing DC differentiation conditions and short-term MLR assay. Peripheral blood (PB) CD14+ monocytes were differentiated for 7 days in the presence of GM-CSF and IL-4 (DC), GM-CSF, IL-4, and IL-10 (DC-10) without or with AHR antagonist CH223191 (10 μM, AHRinh-loDC-10; 20 μM, AHRinhDC-10), MTOR activator MHY1485 (20 μM, MTORact-loDC-10; 50 μM, MTORactDC-10), or the combination of CH223191 (10 μM) and MHY1485 (20 μM) (AHRinh-lo/MTORact-loDC-10).

(G) Percentages of positive cells or relative fluorescence intensity (RFI) for the indicated markers in the indicated conditions gated on CD11c+ Live/Dead negative population (n = 6–12).

(H) Percentages of proliferating (left) and CD25+HLA-DR+ activated (middle) cells, and IFNγ production in the supernatant (right) of total CD4+ T cells stimulated with DC-10s, AHRinh-lo DC-10s, AHRinhDC-10s, MTORact-lo DC-10s, MTORactDC-10s, or AHRinh-lo/MTORact-lo DC-10s, relative to the results obtained with donor-matched DC-stimulated T, set at 100% (dotted line) (n = 6–15).

(I) Expression of AHR targets CYP1B1 (left) and AHRR (right) by ddPCR. Arbitrary units (a.u.) are expressed as ratio between molecules/μL of the target gene and molecules/μL of the reference HPRT gene. (G–I) Median, interquartile range and single values are displayed. (F–H) p values by Kruskal-Wallis test followed by Dunn’s multiple comparison test. See Figure S4.