Figure 2.

IRF8 imprints constitutive IDO1 expression in cDC1

(A) ChIP-seq tracks display open chromatin areas and bindings of IRF8, BATF3, IRF4, H3K27ac, and H3K4me1 around Ido1 locus. Boxed areas at −126 bp or +495 bp from IDO1 TSS indicate regions assessed for enhancer activity.

(B and C) Flow cytometric analysis showing GFP-reporter activities (B) and quantification (C) in cDC1s and cDC2s expressing IDO1 −126 bp and + 495-bp enhancers (n = 7).

(D) FIMO analysis depicting p values of the two predicted AICEs (red boxes) in mouse Ido1 chr8: 25,694,453–25,713,138 (−126 bp from Ido1 TSS).

(E) IRF8 enrichment at the AICEs sequences of Ido1 promoter in sorted CCR7⁺ and CCR7⁻ cDC1 (n = 2).

(F) IDO1 expression (MFI) in CCR7⁺ cDC1s differentiated from Rosa26^Cas9−GFP/+ CD117^hi BM progenitors expressing scramble RNA or sgRNA(s) (black arrowheads) targeting Ido1 −126 bp AICE1, as depicted in the single-color histograms (n = 3).

(G and H) IDO1 expression by flow in WT and Batf3^−/− (H) and WT Irf8^VENUS+ and Batf3^−/−Irf8^VENUS+ cDC1 (n = 3).

(I) Tnf and Pdl1 mRNA expression in sorted CCR7⁺ cDC1 of indicated phenotypes (n = 4).

(J) PDL1, PDL2, CD40, CD80, and CD86 expressions by flow in CCR7⁺ cDC1 (n = 3).

(K) Sorted WT or Ido1^−/− cDC1 assayed for presentation to OT-I T cells in response to soluble OVA protein (n = 2).

(L) IFN-γ production in supernatants from (K) (n =2).

(M) Analysis of DTH is presented as change in footpad weight. n = 5 mice/group for (n = 2).

(N) H&E staining of mice footpad from mice in (M).

Data are shown as means ± SD. ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, ^∗∗∗∗ p < 0.0001, two-way ANOVA followed by Bonferroni multiple comparison test (C, E, F, J, K, and L) or Tukey’s multiple comparison test (M) and unpaired t test (I). Isotype control is shown as gray histogram. Please also see Figure S2.