嗜酸性粒细胞可作为抗原呈递细胞

目的探讨ＨＬＡ－ＤＲ＋嗜酸性粒细胞（ＥＯＳ）在体外培养条件下作为抗原呈递细胞（ＡＰＣ）将破伤风类毒素抗原（ＴＴ）呈递给Ｔ淋巴细胞的能力。方法新分离的ＥＯＳ经人重组粒细胞－巨噬细胞集落刺激因子刺激２４小时,以诱导ＨＬＡ－ＤＲ的表达,然后将其暴露于不同浓度的ＴＴ,检测ＨＬＡ－ＤＲ＋ＥＯＳ对自身Ｔ细胞增殖反应的影响,以评价ＥＯＳ呈递抗原的能力。结果ＨＬＡ－ＤＲ＋ＥＯＳ于ＴＴ存在时可以明显促进Ｔ细胞的增殖反应,并与ＴＴ的浓度呈剂量相关性。而抗ＨＬＡ－ＤＲ单克隆抗体则可以明显地抑制ＥＯＳ的抗原呈递过程。结论人ＥＯＳ可以摄入和处理抗原并能将其传递给自身Ｔ细胞,而ＥＯＳ呈递抗原的过程具有明显的ＨＬＡ－ＤＲ依赖性。     

Distribution of dendritic cells in normal human salivary glands

Dendritic cells (DC) are believed to contribute to development of autoimmune sialadenitis, but little is known about their distribution in normal salivary glands. In this study, DC were identified and their distribution was determined in normal human parotid and submandibular glands. For light microscopy, salivary gland sections were stained with H&E or immunocytochemically using antibodies to DC markers. Transmission electron microscopy (TEM) was used to evaluate the ultrastructural characteristics of DC. In H&E sections, elongated, irregularly shaped nuclei were occasionally seen in the striated and excretory duct epithelium. Immunolabeling with anti-HLA-DR, anti-CD11c and anti-S100 revealed DC with numerous processes extending between ductal epithelial cells, often close to the lumen. Morphometric analyses indicated that HLA-DR-positive DC occupied approximately 4-11% of the duct wall volume. Similar reactive cells were present in acini, intercalated ducts and interstitial tissues. TEM observations revealed cells with indented nuclei containing dense chromatin, pale cytoplasm with few organelles, and lacking junctional attachments to adjacent cells. These results indicate that DC are abundant constituents of normal human salivary glands. Their location within ductal and acinar epithelium suggests a role in responding to foreign antigens and/or maintaining immunological tolerance to salivary proteins.     

Cloned human T cells synthetize Ia molecules and can function as antigen presenting cells

TNP-specific proliferative cloned human T cell lines were investigated for their synthesis and cell surface expression of HLA-DR molecules and for their capacity to function as antigen presenting cells. Utilizing radioactive amino acid precursors for metabolic labeling, these studies demonstrated endogenous synthesis of HLA-DR molecules by cloned T cells, which by two-dimensional gel electrophoresis were similar to HLA-DR molecules expressed by B cells and monocytes. Moreover, when TNP-modified, the irradiated cloned T cells functioned very effectively to stimulate TNP-specific proliferation by cloned responders; when unmodified they were potent stimulators of allogeneic mixed leukocyte responses. Thus, for haptens covalently attached to cell membrane proteins and for allogeneic HLA antigens, Ia⁺ cloned T cells can function as effectively for antigen presentation and T cell activation as other Ia⁺ populations to which such properties have been ascribed.     

Regulation of primary HIV-1 isolate replication in dendritic cells

The potential role of dendritic cells (DC) in the immunopathology of human immunodeficiency virus 1 (HIV-1) disease remains controversial. This study examines replication of a panel of HIV-1 strains (both laboratory adapted and primary) within DC, in the context of the well-established monocyte-DC and monocyte-macrophage transition. Viral replication was assessed by p24 ELISA assay. All strains of HIV-1 tested replicated in DC. Only CCR5-tropic virus replicated in macrophages. Lipopolysaccharide (LPS) induced DC maturation (as reflected in altered cell phenotype) and at the same time diminished the ability of DC to support HIV-1 replication. In contrast the presence of activated T cells, which had been fixed to prevent them acting as a site for viral replication, enhanced the ability of the DC to support viral replication, as has been reported previously for macrophages. Thus cells that are DC by phenotype, but are not activated, act as the optimum reservoir for HIV-1 replication. If this form of DC is present in peripheral tissues, this will be permissive for amplification of the in vivo viral load at sites where there are few responder cells available, and hence contribute to the persistent immunopathology.     

Central and overlapping role of Cathepsin B and inflammasome adaptor ASC in antigen presenting function of human dendritic cells

Inflammasomes are increasingly implicated in regulating immunity, but how their activation relates to function of human dendritic cells (DCs) is unknown. Here we show that DC maturation stimuli lead to rapid activation of caspase-1 in human monocyte-derived DCs. RNAi mediated inhibition of the inflammasome component ASC leads to marked inhibition of the capacity of lipopolysachharide (LPS)-matured DCs to stimulate antigen-specific T cells. RNAi mediated inhibition of Cathepsin B (CatB) also similarly inhibits the capacity of human DCs to stimulate immunity. The defective ability of ASC or CatB deficient DCs to stimulate T cells is independent of inflammasome-mediated processing of inflammatory cytokines and also includes DCs loaded with pre-processed peptide. Gene expression profiles of ASC or CatB deficient human DCs show marked overlap with downregulation of genes implicated in DC function. These data demonstrate an important role for ASC and CatB in regulating function of human DCs with overlapping effects on gene expression.     

Binding of live conidia of Aspergillus fumigatus activates in vitro-generated human Langerhans cells via a lectin of galactomannan specificity

Aspergillus fumigatus is the most common aetiological fungus responsible for human pulmonary aspergilloses. This study investigated the primary contact between Langerhans cells (LC), corresponding to dendritic cells present in pulmonary mucosa and live conidia of A. fumigatus. LC play a key role in antigen presentation for initiation of the primary T cell response. In vitro-generated LC (iLC) were differentiated from cultured human cord blood CD34+ cells and incubated at 4 degrees C or 37 degrees C with fluorescein-isothiocyanate (FITC)-stained conidia or control latex beads. In vitro, conidia were shown by microscopy and cytometry to adhere to iLC in a dose- and time-dependent manner. This adhesion was not limited to iLC because interstitial dendritic and other cells also fluoresced in the presence of conidia-FITC. A lectin other than mannose receptor-type lectin was demonstrated to be responsible of conidial binding. Inhibition of binding was observed with heterologous galactomannan and EDTA, indicating a C-lectin-like receptor with galactomannan structure specificity. After binding only a few conidia were internalized in acidic vesicles, as indicated by the cessation of conidial fluorescence. Conidial binding was followed by activation and maturation of iLC, suggesting that LC present in the lung may play a role in cellular host defence against aspergilloses.     

Artificial human antigen‐presenting cells are superior to dendritic cells at inducing cytotoxic T‐cell responses

Peptide recognition through the MHC class I molecule by cytotoxic T lymphocytes (CTLs) leads to the killing of cancer cells. A potential challenge for T‐cell immunotherapy is that dendritic cells (DCs) are exposed to the MHC class I–peptide complex for an insufficient amount of time. To improve tumour antigen presentation to T cells and thereby initiate a more effective T‐cell response, we generated artificial antigen‐presenting cells (aAPCs) by incubating human immature DCs (imDCs) with poly(lactic‐co‐glycolic) acid nanoparticles (PLGA‐NPs) encapsulating tumour antigenic peptides, followed by maturation with lipopolysaccharide. Tumour antigen‐specific CTLs were then induced using either peptide‐loaded mature DCs (mDCs) or aAPCs, and their activities were analysed using both ELISpot and cytotoxicity assays. We found that the aAPCs induced significantly stronger tumour antigen‐specific CTL responses than the controls, which included both mDCs and aAPCs loaded with empty nanoparticles. Moreover, frozen CTLs that were generated by exposure to aAPCs retained the capability to eradicate HLA‐A2‐positive tumour antigen‐bearing cancer cells. These results indicated that aAPCs are superior to DCs when inducing the CTL response because the former are capable of continuously presenting tumour antigens to T cells in a sustained manner. The development of aAPCs with PLGA‐NPs encapsulating tumour antigenic peptides is a promising approach for the generation of effective CTL responses in vitro and warrants further assessments in clinical trials.     

Cross-presentation of a human tumor antigen delivered to dendritic cells by HSV VP22-mediated protein translocation

Dendritic cells (DC) capture antigens from apoptotic and/or necrotic tumor cells and cross-present them to T cells, and various ways of delivering tumor antigens to DC in vitro and in vivo are being pursued. Since fusions of antigenic proteins with the HSV integument protein VP22 are capable of intercellular trafficking, this approach has been exploited for delivery of antigens to antigen-presenting cells. Adenoviral vectors were used to express the tumor-associated-but-self-antigen MART-1 fused to HSV VP22 in MART-1-negative A375 melanoma cells and in DC. When expressed in A375 cells and allowed to spread to DC across a transwell barrier, the VP22-MART-1 fusion protein localized to both early and late endosomal structures of the DC. The DC loaded with the VP22-MART-1 fusion by intercellular trafficking efficiently presented the MART-1(27-35) epitope to MART-1(27-35)-specific CTL. Furthermore, transloaded DC were capable of expanding the population of MART-1(27-35)-specific CTL. Thus, a tumor antigen acquired by intercellular trafficking can be cross-presented by DC. This experimental approach should therefore be useful not only for studying the mechanism of cross-presentation but also for vaccine development.     

Discordant effects of interleukin-2 on viral and immune parameters in human immunodeficiency virus-1-infected monocyte-derived mature dendritic cells

Use of interleukin-2 (IL-2) in the immunotherapy of human immunodeficiency virus (HIV) has frequently resulted in the restoration of CD4 lymphocyte counts but not of virus-specific responses. We reasoned that the absence of reconstituted functional immune parameters could be related to the inability of IL-2 to correct HIV-induced dysfunctions in antigen-presenting cells. In this study, we used in vitro-differentiated monocyte-derived macrophages (MDMs) and mature dendritic cells (MDDCs), acutely infected with primary HIV-1 isolates, to analyse the effects of IL-2 on virus replication, co-receptor expression, and cytokine or chemokine release. Stimulation of MDMs with IL-2 had no measurable effect on HIV-1 replication, on cytokine secretion, or on CD4 and CXCR4 gene expression. Moreover, although a significant down-regulation of CCR5 mRNA expression could be repeatedly detected in MDMs, this IL-2-mediated effect was not of substantial magnitude to affect virus replication. On the other hand, IL-2 stimulation of MDDCs dramatically increased HIV-1 replication and this effect was highly evident on low-replicating, CXCR4-dependent isolates. Nevertheless, the HIV-enhancing activity of IL-2 in MDDCs was not accompanied by any measurable change in cytokine or chemokine release, in virus receptor and co-receptor mRNA accumulation, or in the surface expression of a battery of receptors implicated in virus entry, cell activation or costimulatory function. Taken together, these findings point to a role for IL-2 in inducing virus purging from dendritic cell reservoirs but indicate no relevant potential of the cytokine in restoring defective elements of innate immunity in HIV infection.     

Natural mannosylation of HIV-1 gp120 imposes no immunoregulatory effects in primary human plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) play a vital role in activation of anti-HIV-1 immunity, and suppression of pDCs might mitigate immune responses against HIV-1. HIV-1 gp120 high-mannose has been attributed immunosuppressive roles in human myeloid DCs, but no receptors for high-mannose have so far been reported on human pDCs. Here we show that upon activation with HIV-1 or by a synthetic compound triggering the same receptor in human pDCs as single-stranded RNA, human pDCs upregulate the mannose receptor (MR, CD206). To examine the functional outcome of this upregulation, inactivated intact or viable HIV-1 particles with various degrees of mannosylation were cultured with pDCs. Activation of pDCs was determined by assaying secretion of IFN-alpha, viability, and upregulation of several pDC-activation markers: CD40, CD86, HLA-DR, CCR7, and PD-L1. The level of activation negatively correlated with degree of mannosylation, however, subsequent reduction in the original mannosylation level had no effect on the pDC phenotype. Furthermore, two of the infectious HIV-1 strains induced profound necrosis in pDCs, also in a mannose-independent manner. We therefore conclude that natural mannosylation of HIV-1 is not involved in HIV-1-mediated immune suppression of pDCs.     

Granuloma cells in chronic inflammation express CD205 (DEC205) antigen and harbor proliferating T lymphocytes: Similarity to antigen‐presenting cells

Granulomas are classified as immune or foreign body granulomas. Of these, the immune granulomas, a hallmark of granulomatous inflammation, are closely related to cell‐mediated immune responses. The aim of the present study is to characterize immune granuloma cells in 33 patients with granulomatous inflammation focusing on the expression of CD205 (DEC205), a cell surface marker of antigen presenting cells, and their spatial relationship to T cells. CD205 was frequently expressed by immune granuloma cells, in contrast to foreign body granuloma cells that lacked CD205 expression. T cells were not only distributed in a lymphocyte collar around the granuloma, but also present among the granuloma cells (termed ‘intra‐granuloma T cells’). Intra‐granuloma T cells stained positive for Ki‐67 (median positivity = 9.4%) by double immunostaining for CD3 and Ki‐67. This indicated the presence of proliferative stimuli within the granuloma that could activate the intra‐granuloma T cells. The labeling index of Ki‐67 in intra‐granuloma T cells was significantly higher than that of T cells in the lymphocyte collar (P < 0.0001) or T cells in the T cell zone (paracortex) of chronic tonsillitis or reactive lymphadenitis (P = 0.002). These data indicate a close similarity between immune granulomas and antigen presenting cells.     

The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells

Dendritic cells, in contrast to B lymphocytes, must be able to efficiently internalize a diverse array of antigens for processing and loading onto major histo-compatibility complex (MHC) class II molecules. Here we characterize the mannose receptor pathway in dendritic cells and show that mannose receptor-mediated uptake of antigens results in a ～ 100-fold more efficient presentation to T cells, as compared to antigens internalized via fluid phase. Immunocytochemistry as well as subcellular fractionation revealed the localization of the mannose receptor and MHC class II molecules in distinct subcellular compartments. The mannose receptor thus functions in rapid internalization and concentration of a variety of glycosylated antigens that become available for processing and presentation. This may contribute to the unique capacity of dendritic cells to generate primary T cell responses against infectious agents.     

TLR signaling in human antigen‐presenting cells regulates MR1‐dependent activation of MAIT cells

Mucosal‐associated invariant T (MAIT) cells are an abundant innate‐like T lymphocyte population that are enriched in liver and mucosal tissues. They are restricted by MR1, which presents antigens derived from a metabolic precursor of riboflavin synthesis, a pathway present in many microbial species, including commensals. Therefore, MR1‐mediated MAIT cell activation must be tightly regulated to prevent inappropriate activation and immunopathology. Using an in vitro model of MR1‐mediated activation of primary human MAIT cells, we investigated the mechanisms by which it is regulated. Uptake of intact bacteria by antigen presenting cells (APCs) into acidified endolysosomal compartments was required for efficient MR1‐mediated MAIT cell activation, while stimulation with soluble ligand was inefficient. Consistent with this, little MR1 was seen at the surface of human monocytic (THP1) and B‐cell lines. Activation with a TLR ligand increased the amount of MR1 at the surface of THP1 but not B‐cell lines, suggesting differential regulation in different cell types. APC activation and NF‐κB signaling were critical for MR1‐mediated MAIT cell activation. In primary cells, however, prolonged TLR signaling led to downregulation of MR1‐mediated MAIT cell activation. Overall, MR1‐mediated MAIT cell activation is a tightly regulated process, dependent on integration of innate signals by APCs.     

Apoptosis and antigen receptor function in T and B cells following exposure to herpes simplex virus

T cells are an essential component of the immune response against herpes simplex virus (HSV) infection. We previously reported that incubation of T cells with HSV-infected fibroblasts inhibits subsequent T cell antigen receptor signal transduction. In the current study, we found that incubation of T cells with HSV-infected fibroblasts also leads to apoptosis in exposed T cells. Apoptosis was observed in Jurkat cells, a T cell leukemia line, and also in CD4(+) cells isolated from human peripheral blood mononuclear cells. Direct infection of these cells with HSV also resulted in apoptosis. Clinical isolates of both HSV type 1 and 2 induced apoptosis in infected T cells at comparable levels to cells infected with laboratory strains of HSV, suggesting an immune evasion mechanism that may be clinically relevant. Further understanding of these viral immune evasion mechanisms could be exploited for better management of HSV infection.     

Increased serum transforming growth factor-beta1 in human colorectal cancer correlates with reduced circulating dendritic cells and increased colonic Langerhans cell infiltration

Cancer-related cytokines may interfere with the differentiation and migration of dendritic cells (DCs) and with the associated up-regulation of co-stimulatory molecules in vitro. We determined whether cytokines affected the distribution and activation of DCs in patients with colorectal cancer by measuring the levels of serum cytokines [transforming growth factor (TGF)-beta1 and vascular endothelial growth factor (VEGF)], DC numbers and phenotype from peripheral blood and mesenteric lymph nodes draining the cancer, and the infiltration of DCs into colorectal cancer. A significant increase in the serum level of TGF-beta1 correlated with a significant reduction in the level of circulating DCs in cancer patients that was associated with an increased infiltration of Langerhans cells into colorectal mucosa. The prevalence but not intensity of co-stimulatory molecule expression in circulating and mesenteric lymph node DCs was reduced in patients with colorectal cancer compared to patients with inflammatory bowel conditions. There was no correlation between co-stimulatory molecule expression and serum TGF-beta1. Thus the circulating DC depletion in colorectal cancer could be explained by a TGF-beta1-related DC redistribution from the circulation into the colorectal cancer and adjacent mucosa where DC levels were increased. There was an impairment of DC activation within colorectal cancer that was not related to serum level of cytokines.     

Caveolin-1 is related to lipid droplet formation in hepatic stellate cells in human liver

Caveolins (CAVs) regulate intracellular cholesterol transport by a complex process involving caveolae, endoplasmic reticulum (ER), and the Golgi network. Hepatic stellate cells (HSCs) are the central site for retinoid storage in the liver and indeed the entire body. Herein, we attempted to elucidate the ultrastructural localization and expression of caveolin-1 (CAV-1) in human HSCs during the progression of liver cirrhosis (LC). Normal and hepatitis C-related cirrhotic liver samples were prepared using a modified perfusion-fixation method to fix organelle structures and molecules in their in vivo positions, and examined using immunoelectron microscopy. In control liver specimens, CAV-1 was minimally associated with low electron density lipid droplets (LDs) segregated around zones 1―2, and specifically associated with membranes surrounding LDs. CAV-1 was segregated in high-density LDs, consistent with the formation of membrane-enclosed lipid-rich vesicular structures, as well as caveolae on plasma membranes around zones 2―3. In cirrhotic liver specimens, CAV-1 molecules were inserted into the cytoplasmic leaflets of ER membranes for transportation to LDs. Thus, CAV-1 transport to LDs might represent an intracellular pathway from the ER in cirrhotic liver tissue.     

Role of B cells as antigen-presenting cells in vivo revisited: antigen-specific B cells are essential for T cell expansion in lymph nodes and for systemic T cell responses to low antigen concentrations.

Studies in B cell-deficient mice generated by continuous injection of anti-mu antibodies (muSM) showed that T cell priming in lymph nodes was dependent on antigen presentation by B cells. This concept has recently become controversial since a wide range, from complete deficiency to near normal T cell responses, was reported in studies carried out with B cell-deficient mice generated by gene disruption (muMT). In this study we show that in the absence of B cells, T cell responses are greatly reduced in all the available muMT mouse strains although responses in muMT of the C57BL/6 background (which were used for most studies with muMT) were much more variable and could reach up to 42% of control. In contrast, T cell responses in muMT --> F(1) bone marrow chimeras which have the same phenotype as muMT were totally impaired, suggesting a principle difference between mice developing without B cells (muMT mice) and muSM which are made B cell deficient only after birth. Normal T cell priming was completely restored by reconstitution of muMT and muMT --> F(1) mice with syngeneic B cells. Interestingly, only B cell populations containing antigen-specific B cells were capable of reconstituting T cell responses. Monoclonal B cells taken from Ig transgenic mice could not reconstitute responses to an irrelevant antigen. We also found that B cells were also required for systemic T cell priming when antigen concentrations were limiting but were not required for priming (for T cell help) when mice were immunized with a high antigen dose.     

“Double‐duty” conventional dendritic cells in the amphibian Xenopus as the prototype for antigen presentation to B cells

Two populations of dendritic cells (DCs) are found in mammals, one derived from hematopoietic precursors (conventional/cDC), and another derived from mesenchymal precursors, the follicular DC (FDC); the latter is specialized for antigen presentation to B cells, and has only been definitively demonstrated in mammals. Both cDC and FDC are necessary for induction of germinal centers (GC) and GC‐dependent class switch recombination (CSR) and somatic hypermutation (SHM). We demonstrate that in Xenopus, an amphibian in which immunoglobulin CSR and SHM occur without GC formation, a single type of DC has properties of both cDC and FDC, including high expression of MHC class II for the former and display of native antigen at the cell surface for the latter. Our data confirm that the advent of FDC functionality preceded emergence of bona fide FDC, which was in turn crucial for the development of GC formation and efficient affinity maturation in mammals.     

LPS priming in early life decreases antigen uptake of dendritic cells via NO production

Immunological mechanisms of hygiene hypothesis are expected to develop a novel strategy for allergy prevention. Although a large number of studies has investigated the relation between allergies and infection, little is known about the influence of the exposure to infections on antigen uptake by dendritic cells (DCs). In this study, we examined the effect of lipopolysaccharide (LPS) priming in early life on the antigen uptake ability of DCs by using an original mouse model. LPS priming in juvenile mice decreased the migration of antigen-capturing CD11c⁺ cells in the lymph nodes, but not in aged mice. Besides, the bone marrow-derived DCs (BMDCs) from juvenile LPS-primed mice had the poor antigen uptake ability, and constitutively produced NO through the inducible nitric oxide synthase (iNOS). Interestingly, the LPS priming-induced poor antigen uptake of BMDCs was mimicked by the NO donor, and recovered by the iNOS inhibitor. Additionally, LPS priming in juvenile mice prevented the allergic reactions, but not in aged mice.Our results suggested that an exposure to infections in early life prevents allergy through the alteration of the BM cells fate that is to induce the differentiation of BM cells into inhibitory DCs such as NO-producing DCs.