Impaired cytokine induction and partner interactions in IRF8^R294C DCs. (A) BMDCs from IRF8^WT and IRF8^R294C mice were stimulated with indicated TLR ligands. IL12p40 and IFNα transcripts (measured 6 hours after stimulation) and proteins (measured 24 hours after stimulation) were measured by quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Error bars represent SD. (B) BM cells from IRF8 KO mice were transduced with pMSCV vectors for IRF8^WT or IRF8^R294C, and expression of B220 and CD8α (24 hours after CpG stimulation) was detected by flow cytometry. (C) EMSA analysis: in vitro–transcribed and – translated proteins from control pcDNA, IRF8^WT, IRF8^R294C, and IRF8^R289E vectors (lanes 1–4) were mixed with the indicated in vitro–transcribed and – translated partner proteins and ³²p-labeled ISRE (for IRF2) and EICE (for PU.1 and SpiB). Asterisks indicates IRF8-partner complexes. Specificity of mobility shifts was verified by adding excess unlabeled probes (lane 5), which removed the shifted band or by adding antibodies for IRF8 (lane 6) or partner proteins (lane 7), which “supershifted” the band mobility. Lane 8 contained partner proteins without IRF8, which produced no shifted band. (D) Cystatin C transcript expression was tested for IRF8^WT and IRF8^R294C BMDCs (left) by quantitative reverse-transcription (RT)–PCR. ChIP analysis was performed for the Csc3 promoter for binding of IRF8 or PU.1 in above DCs. Normal rabbit IgG was used as a control. Data represent the average of 3 determinations (± SD). (E) Diagram of IRF8^R294C-directed DC development. The mutation abolishes the development of CD8α⁺ DCs, without affecting that of pDCs. The mutation results in increased CD4⁺ DCs and pDCs. The impaired ability to interact with partner proteins may partly account for the differential effect of this mutation on DC subset development.