Contrary to other activation signals that we applied, poly(I:C) did not tolerize MoDCs to LPS-induced activation and the pre-treatment with IFN-γ, although it did not activate DCs between day 0 and 2, synergized strongly with a later LPS signal (Fig. 1B, left panel). The inability of early-stage MoDCs that develop in the presence of various activation signals to respond to further TLR ligation is in line with previous data obtained with macrophages or DCs 9 and we Protein Tyrosine Kinase inhibitor showed here that synergistic activation signals do not rescue the cells from functional exhaustion. In addition, we showed the complete lack of inflammatory cytokine gene expression in LPS-tolerized MoDCs in response to further

stimuli, suggesting a major impairment of the signaling cascade that leads to DC activation. In order to search for molecular mechanisms responsible for DC inactivation by chronic

stimulatory signals we compared the gene expression pattern of MoDCs that developed for 2 days in the presence or absence of LPS using check details the Illumina microarray technology and a TLR-pathway focused PCR array (Fig. 2A and Supporting Information Fig. 2). Interestingly, the majority of TLR pathway-associated genes were unaffected by the presence of LPS measured by both technologies, suggesting no major alteration in the expression of the pathway components required for DC activation (Supporting Information Fig. 2). We observed a significant upregulation of potential DC inhibitory factors in response to 2-day exposure to LPS. These included SOCS2 and SOCS3, known regulators of TLR pathways12, the ITIM-containing receptor LILRB2 implicated in DC exhaustion by CD8+ suppressor T cells 23 and the molecules S100A8 and S100A9 that might inhibit DC differentiation and contribute to the development of myeloid suppressor cells in tumor tissues 24. The expression of CD150 (SLAM) molecules, which potently inhibit the CD40L-induced DC activation 25, was also induced

in the presence of LPS. Other known inhibitory factors, including ATF3, SOCS1, STAT3, TGF-β or IRAK-M, were expressed similarly in LPS-treated or control samples. Increased gene expression of the cytokine IL-10 was detected by PCR array in MoDCs cultured for 2 days in the presence of LPS (2.1- to next 9.5-fold upregulation by LPS, n=3) and confirmed by ELISA (Fig. 2B). Expression of miR146a and miR155 were upregulated by LPS added at day 2 to MoDCs (Fig. 2C) in line with previous findings 15, 16. However miR146a levels were only minimally elevated and miR155 was not affected in MoDCs cultured for 2 days in the presence of LPS as compared with non-treated cells, suggesting a time-limited functionality of these microRNAs in LPS-activated DCs. In order to better understand which DC modulatory factors might participate in DC exhaustion by persistent activation signals we analyzed the expression kinetics of a wide range of potential inhibitory factors in MoDCs developing in the presence or absence of LPS. As shown by Fig.