Objective Plasmacytoid dendritic cells (pDCs) were previously thought to be the major source of type I interferon in SLE. BDCA-2 appears to be exclusively expressed on pDCs in fresh blood and has been investigated as a therapeutic target. However, we showed that pDCs lose their immunogenic functions prior to onset of SLE[PMID: 33262343]. Here, we aimed to characterise pDC marker expression on myeloid lineage cells.

Methods Following density gradient separation, PBMCs were cultured in RPMI. At 0 and 24h, cells were stained for CD3, CD19, CD14, CD16, CD11c, HLA-DR, CD123, BDCA-2, BDCA-4 and ILT7 and analysed by flow cytometry. We next performed an in vitro M1 macrophage differentiation. Monocytes were purified by negative selection and cultured with GM-CSF for 5 days. On day 6, IFN-γ and LPS were added and cells analysed by flow cytometry. For M2 macrophages, monocytes were cultured with M-CSF for 5 days and IL-4 and IL-13 added on day 6.

Results For PBMCs, at 0 hours, apart from pDCs, no monocyte subset or myeloid DCs expressed BDCA-2, BDCA-4, CD123 or ILT7. However, at 24h, each of these markers was expressed by at least one myeloid subset, predominantly by classical and intermediate monocytes. M1 macrophages (CD14+HLA-DR+CD80+CD206+) expressed BDCA2 and BDCA4. M2 macrophages (CD14+CD16+CD80dimCD206+CD163high) expressed BDCA4.

Conclusions BDCA-2, BDCA4 and CD123 are not specific to pDCs and are also expressed on monocyte subsets upon differentiation. Therefore these surface markers cannot reliably identify pDCs in immunohistochemistry studies. Since monocyte subsets are key responder cells to IFN- I, with pathogenic roles in SLE, the efficacy of therapies targeting these markers may be explained by their effects on other myeloid cells and not pDCs. This is important for the safety of these therapies as well as their potential indications beyond the IFN-I mediated diseases.