Plasmacytoid dendritic cells (pDCs) are the most powerful type I interferon (IFN-I) producing cells, playing important roles in defense against pathogens, tumor immune-surveillance and autoimmune diseases. Therefore, understanding pDC regulatory pathways to ultimately manipulate them holds great promise for immune-based therapeutics to attenuate several human illnesses. Here we investigated the role of Src Family Kinases (SFKs), a family of non-receptor tyrosine kinases, in pDC response to toll like receptor (TLR) stimulation. We observed for the first time that pDCs from either Fyn or Lyn deficient mice exhibited impaired IFN-I and pro-inflammatory cytokine production after TLR7 and TLR9 stimulation, whereas no difference was observed in conventional DCs. Consistently, treatment with either a pan-SFK inhibitor or a selective dual BCR-ABL/LYN inhibitor (i.e. Bafetinib) profoundly ablated TLR-induced IFN-I production in both a human pDC cell line (CAL-1) and in primary pDCs from human peripheral blood. Furthermore, genetic ablation of LYN by CRISPR/Cas9 in CAL-1 cells resulted in diminished TLR responses. Mechanistically, we showed that the inhibition of SFK activity by Bafetinib reduced the TLR-induced activation of IKK\u3b1/\u3b2, ERK1/2, P38 and IRF7, key elements downstream pDC TLR signaling, as well as those of MTOR and BCAP, an adaptor protein that bridges PI3K and TLR pathways. Taken together, our data indicate that SFKs, particularly LYN, promote pDC IFN-I and pro-inflammatory cytokine production and suggest that SFK inhibitory drugs, such as Bafetinib, could be considered for attenuating pDC response in autoimmune diseases
