Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-alphabeta receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

Despite differences between dendritic cells and Langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime T cells

As human papillomavirus-like particles (HPV-VLP) represent a promising vaccine delivery vehicle, delineation of the interaction of VLP with professional APC should improve vaccine development. Differences in the capacity of VLP to signal dendritic cells (DC) and Langerhans cells (LC) have been demonstrated, and evidence has been presented for both clathrin-coated pits and proteoglycans (PG) in the uptake pathway of VLP into epithelial cells. Therefore, we compared HPV-VLP uptake mechanisms in human monocyte-derived DC and LC, and their ability to cross-present HPV VLP-associated antigen in the MHC class I pathway. DC and LC each took up virus-like particles (VLP). DC uptake of and signalling by VLP was inhibited by amiloride or cytochalasin D (CCD), but not by filipin treatment, and was blocked by several sulfated and non-sulfated polysaccharides and anti-CD16. In contrast, LC uptake was inhibited only by filipin, and VLP in LC were associated with caveolin, langerin, and CD1a. These data suggest fundamentally different routes of VLP uptake by DC and LC. Despite these differences, VLP taken up by DC and LC were each able to prime naive CD8(+) T cells and induce cytolytic effector T cells in vitro. (C) 2004 Elsevier Inc. All rights reserved.

T cells signaled by NF-kappa B- dendritic cells are sensitized not anergic to subsequent activation

Paradoxically, while peripheral self-tolerance exists for constitutively presented somatic self Ag, self-peptide recognized in the context of MHC class II has been shown to sensitize T cells for subsequent activation. We have shown that MHC class II(+)CD86(+)CD40(-) DC, which can be generated from bone marrow in the presence of an NF-kappaB inhibitor, and which constitutively populate peripheral tissues and lymphoid organs in naive animals, can induce Ag-specific tolerance. In this study, we show that CD40(-) human monocyte-derived dendritic cells (DC), generated in the presence of an NF-kappaB inhibitor, signal phosphorylation of TCRzeta, but little proliferation or IFN-gamma in vitro. Proliferation is arrested in the G(1)/G(0) phase of the cell cycle. Surprisingly, responding T cells are neither anergic nor regulatory, but are sensitized for subsequent IFN-gamma production. The data indicate that signaling through NF-kappaB determines the capacity of DC to stimulate T cell proliferation. Functionally, NF-kappaB(-)CD40(-)class II+ DC may either tolerize or sensitize T cells. Thus, while CD40(-) DC appear to prime or prepare T cells, the data imply that signals derived from other cells drive the generation either of Ag-specific regulatory or effector cells in vivo.

Interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity

Dendritic cells (DCs) regulate various aspects of innate immunity, including natural killer (NK) cell function. Here we define the mechanisms involved in DC - NK cell interactions during viral infection. NK cells were efficiently activated by murine cytomegalovirus ( MCMV) - infected CD11b(+) DCs. NK cell cytotoxicity required interferon-alpha and interactions between the NKG2D activating receptor and NKG2D ligand, whereas the production of interferon-gamma by NK cells relied mainly on DC-derived interleukin 18. Although Toll-like receptor 9 contributes to antiviral immunity, we found that signaling pathways independent of Toll-like receptor 9 were important in generating immune responses to MCMV, including the production of interferon-alpha and the induction of NK cell cytotoxicity. Notably, adoptive transfer of MCMV-activated CD11b(+) DCs resulted in improved control of MCMV infection, indicating that these cells participate in controlling viral replication in vivo.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-B-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.

The colony-stimulating factor 1 receptor is expressed on dendritic cells during differentiation and regulates their expansion

The lineage of dendritic cells (DC), and in particular their relationship to monocytes and macrophages, remains obscure. Furthermore, the requirement for the macrophage growth factor CSF-1 during DC homeostasis is unclear. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced GFP in cells of the myeloid lineage, we determined that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation. Furthermore, plasmacytoid DC and all CD11c(high) DC subsets are reduced by 50-70% in CSF-1-deficient osteopetrotic mice, confirming that CSF-1 signaling is required for the optimal differentiation of DC in vivo. These data provide additional evidence that the majority of tissue DC is of myeloid origin during steady state and supports a close relationship between DC and macrophage biology in vivo.

Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFN gamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1 beta, IL-6, and prostaglandin E-2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFN gamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFN gamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFN gamma secretion. Thus, MoDCs generated ill SFM efficiently stimulate T-cell IFN gamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.

Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and anti-viral immunity

The mechanisms responsible for the immunosuppression associated with sepsis or some chronic blood infections remain poorly understood. Here we show that infection with a malaria parasite (Plasmodium berghei) or simple systemic exposure to bacterial or viral Toll-like receptor ligands inhibited cross-priming. Reduced cross-priming was a consequence of downregulation of cross-presentation by activated dendritic cells due to systemic activation that did not otherwise globally inhibit T cell proliferation. Although activated dendritic cells retained their capacity to present viral antigens via the endogenous major histocompatibility complex class I processing pathway, antiviral responses were greatly impaired in mice exposed to Toll-like receptor ligands. This is consistent with a key function for cross-presentation in antiviral immunity and helps explain the immunosuppressive effects of systemic infection. Moreover, inhibition of cross-presentation was overcome by injection of dendritic cells bearing antigen, which provides a new strategy for generating immunity during immunosuppressive blood infections.

IL10 and IL12B polymorphisms each influence IL-12p70 secretion by dendritic cells in response to LPS

Dendritic cells (DC) are the main producers of the cytokine IL-12p70, through which they play a direct role in the development of IFN-gamma-secreting Th1 cells, costimulation of CTL differentiation and NK-cell activation. In contrast, IL-10, which is also produced by DC, negatively regulates IL-12 production. IL-12p70 production varies widely between individuals, and several polymorphisms in the gene encoding IL-12p40 (IL12B) have been identified that influence susceptibility and severity of infectious, autoimmune and neoplastic disease. Here we show that polymorphisms not only of IL12B, but also in the IL10 promoter, influence IL-12p70 secretion by monocyte-derived DC in response to LPS. Although IL12B promoter homozygotes were prone to making more IL-12p70, presence of the IL10 high genotype restricted IL-12p70 production in these individuals. These observations provide a further genetic control of IL-12p70 regulation and emphasize the complexity of production of this cytokine. They also suggest genotypes that might influence the outcome of DC immunotherapy.

RNA Loading of Leukemic Antigens into Cord Blood–Derived Dendritic Cells for Immunotherapy

The manipulation of dendritic cells (DCs) ex vivo to present tumor-associated antigens for the activation and expansion of tumor-specific cytotoxic T lymphocytes (CTLs) attempts to exploit these cells’ pivotal role in immunity. However, significant improvements are needed if this approach is to have wider clinical application. We optimized a gene delivery protocol via electroporation for cord blood (CB) CD34+ DCs using in vitro–transcribed (IVT) mRNA. We achieved > 90% transfection of DCs with IVT-enhanced green fluorescent protein mRNA with > 90% viability. Electroporation of IVT-mRNA up-regulated DC costimulatory molecules. DC processing and presentation of mRNA-encoded proteins, as major histocompatibility complex/peptide complexes, was established by CTL assays using transfected DCs as targets. Along with this, we also generated specific antileukemic CTLs using DCs electroporated with total RNA from the Nalm-6 leukemic cell line and an acute lymphocytic leukemia xenograft. This significant improvement in DC transfection represents an important step forward in the development of immunotherapy protocols for the treatment of malignancy.

Expression of multilectin receptors and comparative FITC-dextran uptake by human dendritic cells

Dendritic cells (DC) are potent antigen-presenting cells and understanding their mechanisms of antigen uptake is important for loading DC with antigen for immunotherapy. The multilectin receptors, DEC-205 and macrophage mannose receptor (MMR), are potential antigen-uptake receptors; therefore, we examined their expression and FITC-dextran uptake by various human DC preparations. The RT-PCR analysis detected low levels of DEC-205 mRNA in immature blood DC, Langerhans cells (LC) and immature monocyte-derived DC (Mo-DC), Its mRNA expression increased markedly upon activation, indicating that DEC-205 is an activation-associated molecule. In Mo-DC, the expression of cell-surface DEC-205 increased markedly during maturation. In blood DC, however, the cell-surface expression of DEC-205 did not change during activation, suggesting the presence of a large intracellular pool of DEC-205 or post-transcriptional regulation. Immature Mo-DC expressed abundant MMR, but its expression diminished upon maturation. Blood DC and LC did not express detectable levels of the MMR, FITC-dextran uptake by both immature and activated blood DC was 30- to 70-fold less than that of LC, immature Mo-DC and macrophages. In contrast to immature Mo-DC, the FITC-dextran uptake by LC was not inhibited effectively by mannose, an inhibitor for MMR-mediated FITC-dextran uptake. Thus, unlike Mo-DC, blood DC and LC do not use the MMR for carbohydrate-conjugated antigen uptake and alternative receptors may yet be defined on these DC. Therefore, DEC-205 may have a different specificity as an antigen uptake receptor or contribute to an alternative DC function.