In line with this hypothesis, the IgM released from CpGPTO-stimulated B cells (14·6 ± 12 μg/ml) displayed unselective binding specificity, e.g. reactivity to lipopolysaccharide, pneumococcal polysaccharide, double-stranded DNA, 5-Fluoracil price single-stranded DNA or tetanus toxoid (Fig. 6b). To investigate

whether CpGPTO binds to autoantigens, we incubated HEp2G cells with supernatants from CpGPTO- or CD40L/rhIL-4-treated B cells or intravenous immunoglobulin G. Immunofluorescence microscopy showed binding of CpGPTO-induced immunoglobulin with a faint, mainly cytoplasmic staining pattern suggestive of low-degree autoreactivity (Fig. 6c). Hence, CpGPTO might preferentially target B cells expressing potentially polyreactive

IgM, which might belong to the IgM memory pool.[17] In B cells, internalization of antigen is mediated by the BCR. Recent studies suggested that physical linkage of a BCR antigen to a stimulatory nucleic acid represents the most efficient means to induce B-cell activation via TLR9.[9, 23, 24] This prompted us to ask whether CpGPTO trigger receptor selleck compound revision by simultaneously engaging BCR and TLR9 signalling in a B-cell subfraction. Notably, unmodified (phosphodiester) CpG ODN (CpGPO) lack mitogenicity (Fig. 7a), but the stimulatory activity of CpGPO was coupled to microspheres additionally Ribonucleotide reductase carrying a BCR stimulus [anti-human immunoglobulin F(ab′)2] (Fig. 7b). However, physical linkage of ODN did not waive the requirement for the TLR9-specific CpG-motif: F(ab′)2-coupled microspheres failed to induce proliferation in the absence of CpGPO or when CpGPO was substituted by a control GpCPO or a poly(T)2o-ODN (Fig. 7c). Next, we asked whether CpGPTO use BCR-dependent signalling. To answer this question, we stimulated B cells with CpGPTO in the presence or absence of inhibitors selectively targeting tyrosine kinases typically recruited upon BCR activation. In support of our hypothesis we found that CpGPTO-triggered B-cell proliferation was partially inhibited by the syk

kinase inhibitor R406 in a concentration-dependent manner (Fig. 7d). By contrast, proliferation was enhanced by 20 ± 0·6% when B cells were pretreated with the lyn inhibitor SU6656 (Fig. 7e), a finding well compatible with hyper-responsiveness of lyn–/– B cells.[25, 26] We concluded that, first, syk and lyn kinases participate in CpGPTO-mediated B-cell activation, and, second, CpGPTO either directly stimulate the BCR or bypass BCR signalling by recruiting molecules associated with proximal BCR signalling. To further investigate this question we sought to perform CpGPTO stimulation in the absence of the BCR. To this end we used plasmacytoid dendritic cells because they are characterized by TLR9 and a BCR-like signalosome.