6A). Alternatively, differential TRAIL expression could result from stochastic cell

activation, and only continuous BAY 73-4506 or additional triggering allows for optimal TRAIL expression of the whole pDC population. In support of this, unmanipulated CAL-1 cells also displayed a broad spectrum of TRAIL expression at 4 h post CpG activation and 6 h post Imiquimod triggering, when the cells were not fully activated yet (Fig. 2B and Supporting Information Fig. 6B). As TRAIL expression in pDCs results both from type I IFN-R signaling and from TLR signaling (Fig. 1; [13]), we addressed whether these two signaling pathways act separately and/or cooperate to induce optimal TRAIL expression. CpG triggering — that elicits both TLR signaling and IFN-R signaling — results in lower selleck screening library TRAIL levels in CAL-1-NAB2E51K cells than in CAL-1-NAB2, or CAL-1-EV cells (Fig. 5; top panel). To dissect the contribution of TLR signaling versus IFN-R signaling, we activated CAL-1 cell variants with CpG, while blocking

type I IFN-R signaling with the vaccinia virus-encoded type I IFN decoy receptor B18R [28-30]. Blocking type I IFN-R signaling resulted in reduced TRAIL levels in CAL-1-EV and CAL-1-NAB2 cells (Fig. 5, middle panel) that were comparable to suboptimal activation conditions (i.e., at 4 h post CpG activation, Fig. 3C). Remarkably, addition of B18R completely abolished TRAIL expression in CpG-activated CAL-1-NAB2E51K cells (Fig. 5, middle panel), indicating that both TLR signaling through PI3K/NAB2 and type I IFN-R signaling contribute to optimal TRAIL expression. Of note, all three cell variants expressed high levels of TRAIL when stimulated solely via type I IFN-R with recombinant IFN-β (Fig. 5; bottom panel). Together,

these data imply that (1) NAB2-dependent TRAIL induction occurs downstream of TLR engagement, independently of type I IFN-R signaling, and that (2) the remaining TRAIL expression upon CpG stimulation in CAL-1-NAB2E51K cells possibly resulted from type I IFN-R signaling. Here, we have identified NAB2 as a novel transcriptional regulator of TRAIL in pDCs. We show that NAB2-mediated TRAIL expression is dependent on TLR-mediated PI3K signaling, and independent of type I IFN-R signaling. In addition, our results reveal that TRAIL induction in pDCs can occur at least via Calpain two independent signaling pathways: (i) downstream of TLR signaling and at least in part mediated by NAB2, and (ii) downstream of type I IFN-R signaling, independently of NAB2. As both pathways must be blocked to completely abolish TRAIL induction in pDCs (Fig. 5), our data show that these two signaling pathways independently induce TRAIL, and suggest that they act in concert to achieve full TRAIL expression. Recent data have indicated that TRAIL induction upon TLR7 triggering can occur independently of type I IFN stimulation [13, 31].