First, we aimed to identify molecular regulators of TRAIL expression. Second, we assessed whether type I buy FDA-approved Drug Library IFN-R signaling was the sole mediator of TRAIL induction upon pDC activation, or whether TLR7/9 triggering by itself could also lead to TRAIL induction. To identify molecules that mediate TRAIL expression in pDCs, we focused on the transcriptional regulator NGFI-A-binding protein

2 (NAB2) [14]. NAB2 is a regulator of the early growth response genes (EGR)-1, 2, and 3; transcription factors that mediate the expression of pro-apoptotic molecules as well as other genes [15-18]. NAB2 is rapidly induced upon a variety of extracellular stimuli, and it modulates in activated T-cell lines the expression of apoptotic molecules [19, 20]. We have recently shown that Nab2 blocks TRAIL induction in primary CD8+ T cells upon reactivation [21]. Furthermore, its homologous family member Nab1 inhibits TRAIL expression in intestinal epithelial cells upon bacterial infection by regulating the transcriptional

activity of EGR-1, 2, and 3 [14, 15]. In light of these findings, we set out to address whether NAB2 also regulates TRAIL in pDCs. Here, we show that NAB2 acts as a co-activator of TRAIL expression in TLR7/9-activated human pDCs. NAB2-mediated TRAIL expression depends on PI3K signaling, MG-132 cost and is independent of type I IFN-R engagement. Furthermore, our data provide evidence that optimal TRAIL induction in CpG-activated pDCs results from at least two distinct signaling pathways: (i) downstream of TLR9 signaling and regulated at least in part by NAB2, and (ii) through type I IFN-R signaling, independent of NAB2. The transcriptional regulator NAB2 is constitutively expressed Interleukin-2 receptor in neuronal and hematopoietic cells, and its expression levels increase upon activation [14, 20]. Here, we have analyzed NAB2 expression levels in primary human pDCs that were activated with the TLR9 agonist CpG A [22]. Interestingly, NAB2 mRNA and protein expression was increased by a -two- to sevenfold

(Fig. 1A, p < 0.05 and Supporting Information Fig. 1A) and was accompanied by the induction of TRAIL mRNA and protein (Fig. 1B; p = 0.02; [5]). In concordance with primary pDCs, the pDC-like cell line CAL-1 [23] also displayed increased NAB2 and TRAIL mRNA and protein levels in response to CpG B (Fig. 1C and D). Like primary pDCs, CAL-1 cells express TLR7 and TLR9, and upon CpG triggering rapidly produce IFN-β, IL-6, and TNF-α, and express CD40 and the IFN responsive protein MXA ([24]; Supporting Information Fig. 1B–E). Moreover, comparable to primary pDCs, CpG-activated CAL-1 cells effectively induced apoptosis in Jurkat cells in a TRAIL-dependent manner, as determined by AnnexinV and by activated Caspase-3 staining ([25]; Supporting Information Fig. 1F). This prompted us to use CAL-1 cells as a model system to further dissect the molecular regulation of TRAIL expression in pDCs. Not only TLR9 stimulation, but also TLR7 triggering with Imiquimod increased NAB2 levels in CAL-1 cells (Fig.