Intracellular Toll-like receptor 3 (TLR3), TLR7, and TLR9 localize in endosomes and recognize single-stranded RNA and nucleotides from viruses and bacteria. This interaction induces their conformational changes resulting in the production of proinflammatory cytokines and upregulation of cell surface molecules. TLR9 requires a proteolytic cleavage for its signaling. Here, we report that myeloid and plasmacytoid dendritic cells (DCs) deficient for the asparagine endopeptidase (AEP), a cysteine lysosomal protease, showed a decrease in the secretion of proinflammatory cytokines in response to TLR9 stimulation in vitro and in vivo. Upon stimulation, full-length TLR9 was cleaved into a 72 kDa fragment and this processing was strongly reduced in DCs lacking AEP. Processed TLR9 coeluted with the adaptor molecule MyD88 and AEP after size exclusion chromatography. When expressed in AEP-deficient DCs, the 72 kDa proteolytic fragment restored TLR9 signaling. Thus, our results identify an endocytic protease playing a critical role in TLR processing and signaling in DCs.

Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge.

A recent report demonstrated that free human T-cell leukemia virus 1 (HTLV-1) could infect plasmacytoid dendritic cells (pDCs). The major role of pDCs is to secrete massive levels of interferon-␣ (IFN-␣) upon virus exposure; however, the induction of IFN-␣ by HTLV-1 remains unknown. We demonstrate here that cellfree HTLV-1 generated a pDC innate immune response by producing massive levels of IFN-␣ that were inhibited by anti-HTLV-1 antibodies. HTLV-1 induced costimulatory molecules and rapid expression of the apoptotic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Furthermore, HTLV-1 stimulated pDC-induced apoptosis of CD4 ؉ T cells expressing DR5, transforming pDCs into IFN-producing killer pDCs. We also observed that an endosomal acidification inhibitor and a Toll-like receptor-7 (TLR7)-specific blocker drastically inhibited pDC response to HTLV-1. Three-dimensional microscopy analysis revealed that unstimulated pDCs were "dormant" IFNproducing killer pDCs with high levels of intracellular TRAIL that could be rapidly mobilized to the surface in response to TLR7 activation. Inhibition of viral degradation in endosomes by chloroquine maintained viral integrity, allowing virus detection by 3-dimensional microscopy. We demonstrate that pDCs respond to cell-free HTLV-1 by producing high levels of IFN-␣ and by mobilizing TRAIL on cell surface after TLR7 triggering. This is the first demonstration of an innate immune response induced by free HTLV-1. (Blood. 2010;115:2177-2185) IntroductionHuman T-cell leukemia virus 1 (HTLV-1), the first characterized human retrovirus, 1 has been identified as the causative agent for adult T-cell leukemia/lymphoma (ATLL) 2,3 and HTLV-1-associated myelopathy/tropical spastic paraparesis, 4 uveitis, and infective dermatitis in children. 5 HTLV-1 virions infect CD4 ϩ T cells, which represent the main target for HTLV-1 infection in peripheral blood. HTLV-1-associated diseases occur after long periods of virus latency. 6 For years it has been thought that unlike other retroviruses, free virions were poorly infectious. 7 However, Jones et al 8 recently reported that freshly isolated myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) are efficiently and productively infected by cell-free HTLV-1. Furthermore, infected mDCs and pDCs were able to transfer virions to autologous CD4 ϩ T cells, clearly demonstrating that cell-free HTLV-1 can be infectious and target DCs. 8 pDCs participate in innate and adaptive immunity, 9,10 are located in blood and lymphoid organs, 10,11 and produce up to 1000-fold more interferon-␣ (IFN-␣) than other cell types in response to virus exposure. 12 Three molecules have been characterized for HTLV-1 entry into cells, heparan sulfate proteoglycans 13 and BDCA-4 (also called neuropilin-1) 14 for the initial virus binding to target cells 15 and glucose transporter 1 for the postattachment and the viral fusion. 16,17 Interestingly, BDCA-4 is expressed by mDC and T cells 18,19 but cells expressing the greatest level of BDCA...