Plasmacytoid dendritic cells (pDCs) sense viral and microbial DNA through endosomal Toll-like receptors to produce type 1 interferons. pDCs do not normally respond to self-DNA, but this restriction seems to break down in human autoimmune disease by an as yet poorly understood mechanism. Here we identify the antimicrobial peptide LL37 (also known as CAMP) as the key factor that mediates pDC activation in psoriasis, a common autoimmune disease of the skin. LL37 converts inert self-DNA into a potent trigger of interferon production by binding the DNA to form aggregated and condensed structures that are delivered to and retained within early endocytic compartments in pDCs to trigger Toll-like receptor 9. Thus, our data uncover a fundamental role of an endogenous antimicrobial peptide in breaking innate tolerance to self-DNA and suggest that this pathway may drive autoimmunity in psoriasis.

Although plasmacytoid dendritic cells (pDCs) respond to virus replication in a non-specific fashion by producing large amounts of type I interferon, a rapid, direct role of pDCs in activating antiviral lymphocytes is less clear. Here we showed that pDCs possess the capacity to rapidly initiate antigenspecific antiviral CD8 + T cell responses. Following virus exposure, pDCs efficiently and rapidly internalized exogenous viral antigens and then presented those antigens on major histocompatibility complex (MHC) class I to CD8 + T cells. Processing of exogenous antigen occurred within endocytic organelles and did not require transit of antigen to the cytosol. Intracellular stores of MHC class I partially colocalized with transferrin receptor and internalized transferrin in endosomes, suggesting that such recycling endosomes are sites of peptide loading onto MHC class I or peptide transit. These data demonstrate that pDCs utilize ready-made stores of MHC class I to rapidly present exogenous antigen to CD8 + T cells.The high rate of viral replication represents a considerable challenge for the immune response. Human DCs can be classified into two major cell subsets, myeloid and plasmacytoid DCs, both of which are critical for the initiation of viral immune responses 1,2 . The presentation of viral peptides to CD4 + and CD8 + T cells is mediated by major histocompatibility complex (MHC) class II molecules and class I, respectively. DC maturation leads to a variety of changes including, activation induced antigen processing and increased surface expression of MHC class I and class II 3,4 . Exogenous antigen presentation on MHC class II molecules by myeloid DC (mDCs) is a rapid and coordinated process 5 . Pools of pre-synthesized MHC class II are stored in late endosomal and lysosomal compartments, which are loaded in an activationdependent manner and translocated to the cell surface 6-10 . NIH Public Access RESULTS pDCs drive CD8 + T cell proliferationHealthy donor pDCs and mDCs were purified from blood by negative depletion followed by direct cell sorting for mDCs (HLA-DR high CD11c high) and pDCs (HLA-DR high CD123 high). Their APC function was assessed by their ability to induce the proliferation of allogeneic (allo) CD4 + and CD8 + T cells. pDCs demonstrated no allo-stimulatory capacity for either CD4 + or CD8 + T cells, while mDCs showed low stimulatory capacity (data not shown). Upon activation with influenza virus, both pDCs and mDCs induced strong proliferation of allo-CD4 + and CD8 + T cells. Influenza virus-treated pDCs induced stronger allo-proliferation of CD8 + T cells when compared to their myeloid counterpart (Fig. 1a,b, upper panels). Reactivation of the pDC-expanded CD8 + T cells with anti-CD3 and anti-CD28 led to high levels of IFN-γ secretion, indicating the acquisition of effector function (data not shown). In contrast, influenza virus-treated mDCs induced a higher proliferation of allo-CD4 + T cells (Fig. 1a,b, bottom panels) indicating that the difference in CD8 + T cell proliferation wa...

Dendritic cells (DCs) can capture extracellular antigens and load resultant peptides on to MHC class I molecules, a process termed cross presentation. The mechanisms of cross presentation remain incompletely understood, particularly in primary human DCs. One unknown is the extent to which antigen delivery to distinct endocytic compartments determines cross presentation efficiency, possibly by influencing antigen egress to the cytosol. We addressed the problem directly and quantitatively by comparing the cross presentation of identical antigens conjugated with antibodies against different DC receptors that are targeted to early or late endosomes at distinct efficiencies. In human BDCA1 ؉ and monocyte-derived DCs, CD40 and mannose receptor targeted antibody conjugates to early endosomes, whereas DEC205 targeted antigen primarily to late compartments. Surprisingly, the receptor least efficient at internalization, CD40, was the most efficient at cross presentation. This did not reflect DC activation by CD40, but rather its relatively poor uptake or intraendosomal degradation compared with mannose receptor or DEC205. Thus, although both early and late endosomes appear to support cross presentation in human DCs, internalization efficiency, especially to late compartments, may be a negative predictor of activity when selecting receptors for vaccine development.

Human BDCA3 DCs are superior to BDCA1 DCs at antigen cross presentation when delivered to late endosomes and lysosomes but not when delivered to early endosomes.

Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was ∼300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection.

Protein-coding genes of trypanosomes are mainly transcribed polycistronically and cleaved into functional mRNAs in a process that requires trans splicing of a capped 39-nucleotide RNA derived from a short transcript, the spliced-leader (SL) RNA. SL RNA genes are individually transcribed from the only identified trypanosome RNA polymerase II promoter. We have purified and characterized a sequence-specific SL RNA promoterbinding complex, tSNAP c , from the pathogenic parasite Trypanosoma brucei, which induces robust transcriptional activity within the SL RNA gene. Two tSNAP c subunits resemble essential components of the metazoan transcription factor SNAP c , which directs small nuclear RNA transcription. A third subunit is unrelated to any eukaryotic protein and identifies tSNAP c as a unique trypanosomal transcription factor. Intriguingly, the unusual trypanosome TATA-binding protein (TBP) tightly associates with tSNAPc and is essential for SL RNA gene transcription. These findings provide the first view of the architecture of a transcriptional complex that assembles at an RNA polymerase II-dependent gene promoter in a highly divergent eukaryote.

Epstein Barr virus (EBV) infection expands CD8+ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8+ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8+ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8+ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development.

Immune responses to Epstein–Barr virus (EBV) infection synergize with the main genetic risk factor HLA‐DRB1*15:01 (HLA‐DR15) to increase the likelihood to develop the autoimmune disease multiple sclerosis (MS) at least sevenfold. In order to gain insights into this synergy, we investigated HLA‐DR15 positive human immune compartments after reconstitution in immune‐compromised mice (humanized mice) with and without EBV infection. We detected elevated activation of both CD4+ and CD8+ T cells in HLA‐DR15 donor‐reconstituted humanized mice at steady state, even when compared to immune compartments carrying HLA‐DRB1*04:01 (HLA‐DR4), which is associated with other autoimmune diseases. Increased CD8+ T cell expansion and activation was also observed in HLA‐DR15 donor‐reconstituted humanized mice after EBV infection. Despite this higher immune activation, EBV viral loads were less well controlled in the context of HLA‐DR15. Indeed, HLA‐DR15‐restricted CD4+ T cell clones recognized EBV‐transformed B cell lines less efficiently and demonstrated cross‐reactivity toward allogeneic target cells and one MS autoantigen. These findings suggest that EBV as one of the main environmental risk factors and HLA‐DR15 as the main genetic risk factor for MS synergize by priming hyperreactive T‐cell compartments, which then control the viral infection less efficiently and contain cross‐reactive CD4+ T cell clones.