Activated NKT cells inhibit autoimmune diabetes through tolerogenic recruitment of dendritic cells to pancreatic lymph nodes

NKT cell activation by alpha-galactosylceramide (alpha-GalCer) inhibits autoimmune diabetes in NOD mice, in part by inducing recruitment to pancreatic lymph nodes (PLNs) of mature dendritic cells (DCs) with disease-protective effects. However, how activated NKT cells promote DC maturation, and what downstream effect this has on diabetogenic T cells was unknown. Activated NKT cells were found to produce a soluble factor(s) inducing DC maturation. Initially, there was a preferential accumulation of mature DCs in the PLNs of alpha-GalCer-treated NOD mice, followed by a substantial increase in T cells. Adoptive transfer of a diabetogenic CD8 T cell population (AI4) induced a high rate of disease (75%) in PBS-treated NOD recipients, but not in those pretreated with alpha-GalCer (8%). Significantly, more AI4 T cells accumulated in PLNs of alpha-GalCer than PBS-treated recipients, while no differences were found in mesenteric lymph nodes from each group. Compared with those in mesenteric lymph nodes, AI4 T cells entering PLNs underwent greater levels of apoptosis, and the survivors became functionally anergic. NKT cell activation enhanced this process. Hence, activated NKT cells elicit diabetes protection in NOD mice by producing a soluble factor(s) that induces DC maturation and accumulation in PLNs, where they subsequently recruit and tolerize pathogenic T cells.     

IFN-gamma overcomes low responsiveness of myeloid dendritic cells to CpG DNA

Dendritic cells (DC) are professional APC that have an extraordinary capacity to prime naive T cells. It has been reported that human DC subsets express distinct toll-like receptor (TLR), which influences their function. In mice, we observed that plasmocytoid DC (pDC) express a higher level of TLR9 compared with myeloid DC (mDC) cultured with GM-CSF. However, we demonstrated that stimulation with IFN-gamma is capable of upregulating TLR9 expression in mDC to a level comparable with expression in pDC. Consistent with this observation, IL-12 p40 and IL-6 mRNA expression and IL-12 p70 secretion in response to CpG-oligodeoxynucleotides are enhanced in mDC pretreated with IFN-gamma compared with untreated cells. Therefore, TLR-mediated responses of DC subsets may be influenced not only by signals delivered by pathogens but also by regulatory signals from cytokines such as IFN-gamma.     

Suggestions to the hepatitis B vaccine events

<正>The media has reported several neonatal death events recently,and it is suspected that these events are related to improper vaccination.This is a deeply regrettable occurrence.Regarding the current situation,we suggest relevant national departments lead and organize experts in areas of clinical medicine,epidemiology,disease control,legal medicine,pharmacy,bio-product research and development,quality control and other related fields to establish a joint investigation team to conduct an investigation quickly and independently to determine the causes as soon as possible to reassure the public.Currently,the public is focused on whether the vaccine is safe.Therefore,it should be first made clear whether the deaths were caused by vaccination.     

The potent adjuvant activity of archaeosomes correlates to the recruitment and activation of macrophages and dendritic cells in vivo

The unique glycerolipids of ARCHAEA: can be formulated into vesicles (archaeosomes) with potent adjuvant activity. We studied the effect of archaeosomes on APCs to elucidate the mechanism(s) of adjuvant action. Exposure of J774A.1 macrophages to archaeosomes in vitro resulted in up-regulation of B7.1, B7.2, and MHC class II molecules to an extent comparable to that achieved with LPS. Similarly, incubation of bone marrow-derived DCs with archaeosomes resulted in enhanced expression of MHC class II and B7.2 molecules. In contrast, conventional liposomes made from ester phospholipids failed to modulate the expression of these activation markers. APCs treated with archaeosomes exhibited increased TNF production and functional ability to stimulate allogenic T cell proliferation. More interestingly, archaeosomes enhanced APC recruitment and activation in vivo. Intraperitoneal injection of archaeosomes into mice led to recruitment of Mac1alpha(+), F4/80(+) and CD11c(+) cells. The expression of MHC class II on the surface of peritoneal cells was also enhanced. Furthermore, peritoneal cells from archaeosome-injected mice strongly enhanced allo-T cell proliferation and cytokine production. The ability of archaeosome-treated APCs to stimulate T cells was restricted to Mac1alpha(high), B220(-) cells in the peritoneum. These Mac1alpha(high) cells in the presence of GM-CSF gave rise to both F4/80(+) (macrophage) and CD11c(+) (dendritic) populations. Overall, the activation of APCs correlated to the ability of archaeosomes to induce strong humoral, T helper, and CTL responses to entrapped Ag. Thus, the recruitment and activation of professional APCs by archaeosomes constitutes an efficient self-adjuvanting process for induction of Ag-specific responses to encapsulated Ags.     

GM-CSF plays a key role in zymosan-stimulated human dendritic cells for activation of Th1 and Th17 cells

In this study, we compared the effects of zymosan and LPS on human monocyte-derived dendritic cells. The specific effects of zymosan on the expression of several key cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins (IL-1α, IL-1β and IL-12 p70) were quite distinct from the effects of LPS. Unlike activation with LPS, DCs activated by zymosan expressed little or no IL-12 p70 due to lack of expression of the p35 subunit. However, treatment with zymosan resulted in a substantial increase in Th1 and Th17 cell-polarizing capacity of DCs. Furthermore, the GM-CSF secreted by zymosan-activated DCs enhanced IL-23 production, resulting in activation of a Th17 response. GM-CSF and IL-27, rather than IL-12 p70, were both major direct contributors to the activation of a Th1 response. This signaling mechanism is distinct and yet complementary to LPS-mediated T-cell activation. We suggest that this novel zymosan-induced GM-CSF-mediated signaling network may play a key role in regulating specific immune cell type activities.     

EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation

Dendritic filopodia are small protrusions on the surface of neuronal dendrites that transform into dendritic spines upon synaptic contact with axon terminals. The formation of dendritic spines is a critical aspect of synaptic development. Dendritic spine morphogenesis is characterized by filopodia shortening followed by the formation of mature mushroom-shaped spines. Here we show that activation of the EphB receptor tyrosine kinases in cultured hippocampal neurons by their ephrinB ligands induces morphogenesis of dendritic filopodia into dendritic spines. This appears to occur through assembly of an EphB-associated protein complex that includes focal adhesion kinase (FAK), Src, Grb2, and paxillin and the subsequent activations of FAK, Src, paxillin, and RhoA. Furthermore, Cre-mediated knock-out of loxP-flanked fak or RhoA inhibition blocks EphB-mediated morphogenesis of dendritic filopodia. Finally, EphB-mediated RhoA activation is disrupted by FAK knock-down. These data suggest that EphB receptors are upstream regulators of FAK in dendritic filopodia and that FAK-mediated RhoA activation contributes to assembly of actin filaments in dendritic spines.     

Immunostimulant patch containing heat-labile enterotoxin from Escherichia coli enhances immune responses to injected influenza virus vaccine through activation of skin dendritic cells

Vaccine strategies, such as influenza virus vaccination of the elderly, are highly effective at preventing disease but provide protection for only the responding portion of the vaccinees. Adjuvants improve the magnitude and rates of responses, but their potency must be attenuated to minimize side effects. Topical delivery of strong adjuvants such as heat-labile enterotoxin from Escherichia coli (LT) induces potent immune responses. We hypothesized that LT delivered alone in an immunostimulating (LT-IS) patch placed on the skin at the site of injection could augment the immune response to injected vaccines. This was based on the observation that topically applied LT induces migration of activated antigen-presenting cells (APCs) from the skin to the proximal draining lymph node (DLN), and that APCs loaded with antigen by injection in the same anatomical region also migrate to the same DLN. We observed that when influenza virus vaccine is injected and an LT-IS patch is placed to target the same DLN, the influenza virus antibody response is enhanced. Similarly, influenza virus-specific T cells isolated from the lungs show increased levels of gamma interferon and interleukin-4 production. An LT-IS patch placed near an injected vaccine also leads to increased levels of hemagglutination inhibition titers, enhanced mucosal immunoglobulin A responses, and enhanced antigen presentation. Although the mechanisms by which an LT-IS patch exerts its enhancing effects need further study, the enhanced immune responses, ability to safely use potent adjuvants, and simplicity of LT-IS patch application address an important unmet need and provide a new immune enhancement strategy.     

Synergism between curdlan and GM-CSF confers a strong inflammatory signature to dendritic cells

A simultaneous engagement of different pathogen recognition receptors provides a tailor-made adaptive immunity for an efficient defense against distinct pathogens. For example, cross-talk of TLR and C-type lectin signaling effectively shapes distinct gene expression patterns by integrating the signals at the level of NF-κB. In this study, we extend this principle to a strong synergism between the dectin-1 agonist curdlan and an inflammatory growth factor, GM-CSF. Both together act in synergy in inducing a strong inflammatory signature that converts immature dendritic cells (DCs) to potent effector DCs. A variety of cytokines (IL-1β, IL-6, TNF-α, IL-2, and IL-12p70), costimulatory molecules (CD80, CD86, CD40, and CD70), chemokines (CXCL1, CXCL2, CXCL3, CCL12, CCL17), as well as receptors and molecules involved in fugal recognition and immunity such as Mincle, dectin-1, dectin-2, and pentraxin 3 are strongly upregulated in DC treated simultaneously with curdlan and GM-CSF. The synergistic effect of both stimuli resulted in strong IκBα phosphorylation, its rapid degradation, and enhanced nuclear translocation of all NF-κB subunits. We further identified MAPK ERK as one possible integration site of both signals, because its phosphorylation was clearly augmented when curdlan was coapplied with GM-CSF. Our data demonstrate that the immunomodulatory activity of curdlan requires an additional signal provided by GM-CSF to successfully initiate a robust β-glucan-specific cytokine and chemokine response. The integration of both signals clearly prime and tailor a more effective innate and adaptive response against invading microbes and fungi.     

Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection

Type I inflammatory cytokines are essential for immunity to many microbial pathogens, including Toxoplasma gondii. Dendritic cells (DC) are key to initiating type 1 immunity, but neutrophils are also a source of chemokines and cytokines involved in Th1 response ignition. We found that T. gondii triggered neutrophil synthesis of CC chemokine ligand (CCL)3, CCL4, CCL5, and CCL20, chemokines that were strongly chemotactic for immature DC. Moreover, supernatants obtained from parasite-stimulated polymorphonuclear leukocytes induced DC IL-12(p40) and TNF-alpha production. Parasite-triggered neutrophils also released factors that induced DC CD40 and CD86 up-regulation, and this response was dependent upon parasite-triggered neutrophil TNF-alpha production. In vivo evidence that polymorphonuclear leukocytes exert an important influence on DC activation was obtained by examining splenic DC cytokine production following infection of neutrophil-depleted mice. These animals displayed severely curtailed splenic DC IL-12 and TNF-alpha production, as revealed by ex vivo flow cytometric analysis and in vitro culture assay. Our results reveal a previously unrecognized regulatory role for neutrophils in DC function during microbial infection, and suggest that cross-talk between these cell populations is an important component of the innate immune response to infection.     

Maturation of dendritic cells with lipopeptides that represent vaccine candidates for hepatitis C virus

The ability of antigens to elicit immune responses depends upon their initial recognition, uptake, processing and presentation by dendritic cells. This fact has been recognized by many workers and dendritic cells are now regarded as natural 'adjuvants' in the business of vaccine design. One way of persuading dendritic cells to become interested in foreign material is to decorate it with lipid moieties found in bacteria. This approach has been used in the context of synthetic peptide-based immunogens and depending on the nature of the epitopes included, can provide highly immunogenic structures capable of eliciting antibody or cytotoxic T cell responses. In this paper we describe the results of experiments in which the stimulatory effects of peptide-based vaccine candidates on human dendritic cells are examined. Our findings indicate that lipidated structures comprising vaccine target sequences of viral origin coupled to the synthetic lipid groups of bacteria are able to induce the maturation of dendritic cells, as measured by the expression of cell surface MHC class II molecules.     

重组CHO乙肝疫苗细胞培养收换液工艺改进

利用CHO细胞生产重组乙肝疫苗,在细胞培养收换液工艺中建立管道化生产线。采用大罐配成新鲜工作液,过滤除菌,经管道直接输入细胞培养瓶内;细胞培养上清的收集也经管道或直接倒入不锈钢罐内。工艺改进后,细胞原液收获率提高15％,配液污染率降低12％,同时降低了成本。     

In situ recruitment of antigen-presenting cells by intratumoral GM-CSF gene delivery

Proper antigen presentation is paramount to the induction of effective and persistent antitumor immune responses. In a murine model of hepatic metastasis of colon cancer, we found that the numbers of in situ mature dendritic cells (DCs) and macrophages in tumor-infiltrating leukocytes (TILs) were significantly increased in mice treated with the combination therapy of herpes simplex virus thymidine kinase, interleukin 2, and GM-CSF genes when compared with control groups without GM-CSF treatment. Significantly higher levels of IFN-, MIP-1, mIL-12, and GM-CSF were detected in the tumor after the combination therapy. T cells isolated from the combination therapy–treated mice exhibited higher ex vivo direct CTL activity than those from other treatment groups. Antigen-presenting cells (APCs) enriched from the TILs and liver of the combination therapy–treated mice induced higher levels of proliferation by the splenocytes from long-term surviving mice that had been cured of tumors at early time points (days 4 and 7) whereas significant APC activity was only observed in the spleen at the latter time point (day 7, 14) after the combination therapy. In contrast, APCs isolated from tk or tk + IL-2–treated mice did not induce any significant proliferation. Subcutaneous injection of fluorescence-labeled latex microspheres followed by the combination therapy showed a similar sequential trafficking of microspheres, day 4 after the combination therapy to tumor and day 14 to spleen. The results suggest that APCs recruited by intratumoral gene delivery of GM-CSF can capture antigens, mature to a stage suitable for antigen presentation, and subsequently migrate to the spleen where they can efficiently stimulate antigen-specific T cells.P.-Y. Pan and Y. Li contributed equally to this work.     

IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

High-dose intravenous immunoglobulin (IVIG) preparations are currently used for the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP). Although the mechanisms of IVIG efficacy remain enigmatic, some clinical and laboratory studies suggest that interaction of the Fc domain of IgG, especially the Fc domain of dimeric IgG, with its receptors (Fc gamma receptors; FcγRs) plays an essential role. In this study, IVIG was dimerized with chemical crosslinkers to augment its therapeutic efficacy. Dimerized IVIG was found to have a much higher affinity for FcγRs than monomeric IVIG. In a mouse ITP model, chemically dimerized IVIG abrogated the decrease in platelet numbers in the blood that was caused by an anti-platelet antibody at a dose that was one tenth of the required dose of IVIG. These results suggest that chemical dimerization of IVIG should greatly improve the efficacy of IVIG therapy of ITP.     

Leptin induces CD40 expression through the activation of Akt in murine dendritic cells

Increasing evidence suggests a regulatory role for leptin, an adipocyte-derived hormone, in immunity. Although recent studies indicated an essential role of leptin signaling in dendritic cell (DC) maturation, the molecular mechanisms by which leptin modulates DC functional maturation remained unclear. In this study, we showed that leptin induced CD40 expression in murine DC and significantly up-regulated their immunostimulatory function in driving T cell proliferation. Moreover, leptin markedly enhanced lipopolysaccharide-mediated DC activation. Using pharmacological inhibitors for Akt, STAT-1alpha, or NF-kappaB and the dominant negative forms of Akt and IkappaB kinase alpha/beta/gamma, as well as small interfering RNA for STAT-1alpha, we showed that Akt, STAT-1alpha, and NF-kappaB were important for the leptinor lipopolysaccharide-induced CD40 expression. Coimmunoprecipitation analysis revealed that leptin promoted immune complex formation between Akt and the IkappaB kinase subunits as well as STAT-1alpha. Blocking the activity of Akt demonstrated a crucial role for Akt in translocation of STAT-1alpha and NF-kappaB to the nucleus and activation of the CD40 promoter. Further analysis with chromatin immunoprecipitation assay confirmed that leptin recruited STAT-1alpha, NF-kappaBp65, and RNA polymerase II to the CD40 promoter and enhanced histone 4 acetylation in a time-dependent manner. Thus, our results have elucidated the molecular mechanisms underlying leptin-induced CD40 expression and DC maturation.     

Properties of recombinant hepatitis B vaccine

A large-scale purification method for hepatitis B surface antigen produced in a recombinant yeast (Saccharomyces cerevisiae) was established. The resulting HBsAg was greater than 99% pure and suitable for vaccine use. The yeast-derived HBsAg was structurally and biochemically similar to plasma-derived HBsAg. The anti-HBs antibody producing potency of the yeast-derived vaccine in mice was significantly higher than that of the plasma-derived vaccine. The yeast-derived vaccine induced protective antibody against hepatitis B virus of either adr or ayw subtype in a chimpanzee efficacy study. These observations demonstrate the usefulness of the yeast-derived vaccine as a second-generation hepatitis B vaccine.