Extended Data Fig. 5: The Irf8 + 32^H enhancer drives hybrid DC development independently of the Irf8 + 41 kb enhancer.

(a) Diagram illustrating the deletion of the Irf8 + 41 kb enhancer on the Irf8 + 32^H/H background using CRISPR/Cas9. (b) Sequence of a 658 bp region surrounding the Irf8 + 41 kb enhancer. Nucleotides deleted in both the Irf8 + 41^−/− line²³ and the Irf8 + 32^H/H + 41^−/− line are highlighted in red. Additional nucleotides deleted only in the Irf8 + 32^H/H + 41^−/− line highlighted in blue. sgRNAs target sites are underlined. (c) Representative flow cytometry plots of BM cells stained for pre-cDC1 (upper panel), and splenocytes stained for cDCs (lower panel) from WT, Irf8 + 41^−/− (+41^−/−), Irf8 + 32^H/+ + 41^−/− (+32^H/+ + 41^−/−), and Irf8 + 32^H/H + 41^−/− (+32^H/H + 41^−/−) mice. Chromatin phasing diagrams are displayed above the plots for context. Pre-gates: lin⁻ SiglecH⁻ Flt3⁺ cells for BM, and B220⁻ SiglecH⁻ CD11c⁺ MHCII⁺ cells for spleen. (d) Frequencies of pre-cDC1s as a percentage of lin⁻ SiglecH⁻ BM cells across the indicated genotypes. Data are pooled from three independent experiments (n = 4 for WT, 5 for +41^−/−, 5 for +32^H/+ + 41^−/−, and 4 for +32^H/H + 41^−/− mice). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. (e) Frequencies of splenic cDC1s, cDC2s and hybrid DCs across the indicated genotypes. Data are pooled from six independent experiments. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. (f) IRF8 expression in cDC2s and hybrid DCs from the indicated genotypes as determined by intracellular staining. Data shown are representative of two similar experiments. Data in d and e are presented as mean values +/− SD. P values are indicated above the graphs.