CD103^hi iCD103-DCs develop independent of monocytes through Batf3- and Irf8-sensitive pathways and express CD8α/CD103 DC signature genes. (A) Relative expression of transcription factors by GM-DCs, FL-DCs, and iCD103-DCs. (B-C) iCD103-DCs generated from wild-type (WT) and (B) BXH2 (Irf8^mut) or (C) Batf3^−/− (Batf3KO) mice were analyzed by flow cytometry. The upper left plots show frequencies of CD11c⁺B220^– cDCs and the lower left plots show frequencies of CD103^hiClec9A⁺ DCs among cDCs (representative of 3 independent experiments). Quantifications of DC yields relative to WT cells are shown on the right for 3 independent experiments. NS, not statistically significant; **P < .01, ***P < .0001; paired Student t test. (D-E) GM-DCs, FL-DCs, and iCD103-DCs generated from LysM-Cre × Rosa26-RFP or Rosa26-RFP control mice (Ctrl) were analyzed for RFP expression by flow cytometry. One of 2 independent experiments, each involving 3 mice per genotype, is shown. (D) Representative plots compare CD11c vs RFP expression among CD11c⁺B220^– BMDC subsets. (E) Summarized percentages of RFP⁺ DCs among CD11c⁺ GM-DCs, CD11c⁺B220^–SIRPα^– CD8α-like FL-DCs, CD11c⁺B220^–SIRPα⁺ CD11b-like FL-DCs, CD11c⁺B220⁺ FL-pDCs, CD11c⁺B220^–SIRPα⁺CD103^– CD11b-like iCD103-DCs, and CD11c⁺B220^–SIRPα^–CD103^hi CD8α-like iCD103-DCs. (F-H) Relative mRNA expression from FACS-sorted CD103^hi and CD103^– iCD103-DCs by Agilent microarray analysis. The expression of published gene signatures for pDCs, CD8⁺/CD103⁺ DCs, CD8^– DCs, and macrophages was compared. See also supplemental Table 1. (F) Fraction of signature genes (black) upregulated ≥threefold in CD103⁺ vs CD103^– iCD103-DCs (left; pDC: 4.3%, CD8⁺/CD103⁺: 82.1%, CD8^–: 0%, MF: 0.9%) or upregulated ≥threefold in CD103^– vs CD103⁺ iCD103-DCs (right; pDC: 32.3%, CD8⁺/CD103⁺: 0%, CD8^–: 71.4%, MF: 47.4%). (G) Degree of overexpression (red) or underexpression (blue) for all genes of the CD8⁺/CD103⁺ DC signature in CD103⁺ vs CD103^– iCD103-DCs (equal expression in white). (H) Signatures of pDCs, CD8^– DCs, and macrophages as in (G). (I) Relative expression levels of Itgae, Tlr3, and Xcr1 among BMDCs. (J) GM-DCs or iCD103-DCs were stimulated for 24 hours with poly(inosine:cytidine) (pI:C) or CpG. IL-12/23p40 was quantified in the supernatant by enzyme-linked immunosorbent assay. Mean + SD (n = 3) is shown for 1 of 2 independent experiments. ND, not detectable. Data from (A) and (I) are combined from 2 independent experiments. Values are normalized to Hprt and GM-DCs.