Major murine IFN-α–producing DC subset expresses a CD11c^low, B220⁺ phenotype and lacks CD8α expression.

(A) Staining of total splenocytes derived from Flt3L+GM-CSF–treated mice show that, in addition to myeloid CD11c⁺CD11b⁺ DC, a subset of CD11c⁺DC coexpresses the B220⁺ antigen. This population is distinct from CD11c⁺CD8α⁺ lymphoid DCs. (B) The CD11c⁺CD11b⁻ DC subset was further subdivided using antibodies to CD8α-PE and B220-APC. Total CD11c⁺ cells were isolated from the spleens of Flt3L+GM-CSF–treated mice using CD11c-conjugated magnetic beads. FITC-conjugated antibodies to CD11b, CD3, and sIg were used to exclude myeloid DCs, T cells and B cells in FACS sorting. Sorted cells were typically more than 96% pure. (C) Sorted splenic CD11c⁺B220⁺ DCs were cultured for 24 hours alone or with HSV, SAC, CD40 antibodies, or 2 μg/mL CpG (ODN). Supernatants were harvested and examined for their IFN-α and IL-12 content by specific ELISA. Major HSV-responsive, IFN-α₂–producing cells were found in the CD11c⁺B220⁺ subset. These cells exhibited the capacity to produce IFN-α and IL-12 in response to SAC. (D) CD11c⁺CD8α⁺ lymphoid DCs did not produce IFN-α on stimulation with HSV, but SAC induced low levels of IFN-α and high levels of IL-12. At least 5 experiments performed gave comparable results.