Sandra S. Diebold

Toll-like receptor 3 promotes cross-priming to virus-infected cells.

Cross-presentation of cell-associated antigens plays an important role in regulating CD8+ T cell responses to proteins that are not expressed by antigen-presenting cells (APCs). Dendritic cells are the principal cross-presenting APCs in vivo and much progress has been made in elucidating the pathways that allow dendritic cells to capture and process cellular material. However, little is known about the signals that determine whether such presentation ultimately results in a cytotoxic T cell (CTL) response (cross-priming) or in CD8+ T cell inactivation (cross-tolerance). Here we describe a mechanism that promotes cross-priming during viral infections. We show that murine CD8α+ dendritic cells are activated by double-stranded (ds)RNA present in virally infected cells but absent from uninfected cells. Dendritic cell activation requires phagocytosis of infected material, followed by signalling through the dsRNA receptor, toll-like receptor 3 (TLR3). Immunization with virus-infected cells or cells containing synthetic dsRNA leads to a striking increase in CTL cross-priming against cell-associated antigens, which is largely dependent on TLR3 expression by antigen-presenting cells. Thus, TLR3 may have evolved to permit cross-priming of CTLs against viruses that do not directly infect dendritic cells.

Viral infection switches non-plasmacytoid dendritic cells into high interferon producers

Type I interferons (IFN-I) are important cytokines linking innate and adaptive immunity. Plasmacytoid dendritic cells make high levels of IFN-I in response to viral infection and are thought to be the major source of the cytokines in vivo. Here, we show that conventional non-plasmacytoid dendritic cells taken from mice infected with a dendritic-cell-tropic strain of lymphocytic choriomeningitis virus make similarly high levels of IFN-I on subsequent culture. Similarly, non-plasmacytoid dendritic cells secrete high levels of IFN-I in response to double-stranded RNA (dsRNA), a major viral signature, when the latter is introduced into the cytoplasm to mimic direct viral infection. This response is partially dependent on the cytosolic dsRNA-binding enzyme protein kinase R and does not require signalling through toll-like receptor (TLR) 3, a surface receptor for dsRNA. Furthermore, we show that sequestration of dsRNA by viral NS1 (refs 6, 7) explains the inability of conventional dendritic cells to produce IFN-I on infection with influenza. Our results suggest that multiple dendritic cell types, not just plasmacytoid cells, can act as specialized interferon-producing cells in certain viral infections, and reveal the existence of a TLR-independent pathway for dendritic cell activation that can be the target of viral interference.

Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8α+ DC correlates with unresponsiveness to imidazoquinolines

Toll‐like receptors (TLR) recognize microbial and viral patterns and activate dendritic cells (DC). TLR distribution among human DC subsets is heterogeneous: plasmacytoid DC (PDC) express TLR1, 7 and 9, while other DC types do not express TLR9 but express other TLR. Here, we report that mRNA for most TLR is expressed at similar levels by murine splenic DC sub‐types, including PDC, but that TLR3 is preferentially expressed by CD8α+ DC while TLR5 and TLR7 are selectively absent from the same subset. Consistent with the latter, TLR7 ligand activates CD8α– DC and PDC, but not CD8α+ DC as measured by survival ex vivo, up‐regulation of surface markers and production of IL‐12p40. These data suggest that the dichotomy in TLR expression between plasmacytoid and non‐plasmacytoid DC is not conserved between species. However, lack of TLR7 expression could restrict the involvement of CD8α+ DC in recognition of certain mouse pathogens.

Nr4a1-Dependent Ly6C(low) Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal

Summary The functions of Nr4a1-dependent Ly6Clow monocytes remain enigmatic. We show that they are enriched within capillaries and scavenge microparticles from their lumenal side in a steady state. In the kidney cortex, perturbation of homeostasis by a TLR7-dependent nucleic acid “danger” signal, which may signify viral infection or local cell death, triggers Gαi-dependent intravascular retention of Ly6Clow monocytes by the endothelium. Then, monocytes recruit neutrophils in a TLR7-dependent manner to mediate focal necrosis of endothelial cells, whereas the monocytes remove cellular debris. Prevention of Ly6Clow monocyte development, crawling, or retention in Nr4a1−/−, Itgal−/−, and Tlr7host−/−BM+/+ and Cx3cr1−/− mice, respectively, abolished neutrophil recruitment and endothelial killing. Prevention of neutrophil recruitment in Tlr7host+/+BM−/− mice or by neutrophil depletion also abolished endothelial cell necrosis. Therefore, Ly6Clow monocytes are intravascular housekeepers that orchestrate the necrosis by neutrophils of endothelial cells that signal a local threat sensed via TLR7 followed by the in situ phagocytosis of cellular debris.

Mannose polyethylenimine conjugates for targeted DNA delivery into dendritic cells

Cell surface-bound receptors represent suitable entry sites for gene delivery into cells by receptor-mediated endocytosis. Here we have taken advantage of the mannose receptor that is highly expressed on antigen-presenting dendritic cells for targeted gene transfer by employing mannosylpolyethylenimine (ManPEI) conjugates. Several ManPEI conjugates were synthesized and used for formation of ManPEI/DNA transfection complexes. Conjugates differed in the linker between mannose and polyethylenimine (PEI) and in the size of the PEI moiety. We demonstrate that ManPEI transfection is effective in delivering DNA into mannose receptor-expressing cells. Uptake of ManPEI/DNA complexes is receptor-specific, since DNA delivery can be competed with mannosylated albumin. Additionally, incorporation of adenovirus particles into transfection complexes effectively enhances transgene expression. This is particularly important for primary immunocompetent dendritic cells. It is demonstrated here that dendritic cells transfected with ManPEI/DNA complexes containing adenovirus particles are effective in activating T cells of T cell receptor transgenic mice in an antigen-specific fashion.

Microbial Recognition Via Toll-Like Receptor-Dependent and -Independent Pathways Determines the Cytokine Response of Murine Dendritic Cell Subsets to CD40 Triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8α+ and CD8α−CD4− DC to make IL-12 p70. In contrast, exposure of CD8α+, CD4+ and CD8α−CD4− DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.

Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides

Toll‐like receptor 7 (TLR7) mediates innate responses by responding to viral RNA in endocytic compartments. However, the molecular pattern recognised by TLR7 and whether it differs between RNA of viral and self origin remains unclear. Here, we identify nucleic acids that act as TLR7 agonists for mouse and human cells. We show that uridine and ribose, the two defining features of RNA, are both necessary and sufficient for TLR7 stimulation, and that short single‐stranded RNA (ssRNA) act as TLR7 agonists in a sequence‐independent manner as long as they contain several uridines in close proximity. Consistent with the notion that TLR7 lacks specificity for sequence motifs, we show that it is triggered equally efficiently by viral or self RNA delivered to endosomes. Our results support the notion that TLR7 recognises uracil repeats in RNA and that it discriminates between viral and self ligands on the basis of endosomal accessibility rather than sequence.

Efficient Gene Delivery into Human Dendritic Cells by Adenovirus Polyethylenimine and Mannose Polyethylenimine Transfection

Gene-modified human dendritic cells (DCs) were generated by transfection with adenovirus polyethylenimine DNA (Ad/PEI/DNA) and mannose polyethylenimine DNA (ManPEI/DNA) complexes. Ad/PEI/DNA complexes have plasmid DNA bound to adenovirus particles by PEI and deliver DNA into cells via the adenovirus infection route. Such transfection complexes yield high transduction levels and sustained expression of luciferase and green fluorescent protein reporter genes and were almost as effective as recombinant adenovirus vectors. ManPEI/DNA complexes rely on uptake by receptor-mediated endocytosis via mannose receptor, which is highly expressed on DCs. While gene delivery by ManPEI/DNA complexes was less efficient than by Ad/PEI transfection, incorporation of adenovirus particles in ManPEI/DNA transfection complexes further enhanced transduction efficiencies and transgene expression. We also demonstrate that Ad/PEI-transfected DCs are competent in stimulating T cell proliferation in allogeneic and autologous mixed lymphocyte reactions, and in activating T cells from T cell receptor (TCR)-transgenic mice in an antigen-specific manner. Thus, the present study establishes the following relative order of transduction efficiencies of viral and nonviral gene delivery systems for primary human DCs: recombinant adenovirus > Ad/PEI = Ad/ManPEI > ManPEI > PEI. Ad/PEI and ManPEI transfection modes represent particularly versatile transduction systems for DCs, with ManPEI being built up exclusively of synthetic compounds.

Recognition of viral single-stranded RNA by Toll-like receptors ☆

The Toll-like receptors (TLR), mediating innate immune activation upon recognition of viral nucleic acids, represent promising targets for the development of adjuvants. Therefore, there is great interest in unraveling the underlying mechanisms of ligand recognition. Studies aiming to identify which sequences, nucleic acid modifications and molecular moieties of viral nucleic acids trigger or inhibit TLR activation have allowed insights into this subject, yet there are still many aspects of innate recognition of viral nucleic acids which are only partially understood. This review discusses our current understanding of TLR-mediated recognition of viral single-stranded RNA (ssRNA) by TLR7 and TLR8. Oligoribonucleotides (ORN) and small immune response modifiers such as imidazoquinolines with agonist function have served as tools to study ligand recognition. In addition, there is increasing evidence that TLR-mediated recognition of mammalian ssRNA triggers innate immune activation and plays a role in autoimmunity. Thus the development of suitable TLR7 and TLR8 antagonists could pave the way for therapeutic intervention of particular autoimmune diseases.

Plasmacytoid dendritic cell-derived type I interferon is crucial for the adjuvant activity of Toll-like receptor 7 agonists

There is a high demand for the development of adjuvants that induce cytotoxic T lymphocytes, which are crucial for the elimination of intracellular pathogens and tumor cells. Toll-like receptor (TLR) agonists are prime candidates to fulfill this role because they induce innate immune activation and promote adaptive immune responses. The successful application of the TLR7 agonist R837 for treatment of basal cell carcinoma shows the potential for exploiting this pathway in tumor immunotherapy. Imidazoquinolines like R837 and stimulatory ssRNA oligonucleotides both trigger TLR7-mediated immune activation, but little is known about their comparative ability to promote immunity induction. We investigated differences in innate immune activation and adjuvant activity between the imidazoquinoline R848 and the ssRNA TLR7 agonist polyUs21. In contrast to R848, polyUs21 induced detectable levels of intracellular interferon-alpha (IFN-alpha) in plasmacytoid dendritic cells (PDCs). In immunization studies, only polyUs21 led to robust priming of type 1 T helper cells and cytotoxic T lymphocytes, and it was more efficient in inducing antitumor immunity than R848. Notably, exogenous IFN-alpha augmented the adjuvant activity of R848, whereas depletion of PDC abrogated the adjuvanticity of polyUs21. This study, therefore, identifies sufficient IFN-alpha production by PDC as an important determinant of vaccine efficacy.