Antigen uptake and presentation by dendritic leukocytes.

Dendritic leukocytes are required for the initiation of T cell mediated immune responses. These cells, in different stages of maturation, are distributed throughout lymphoid and most non-lymphoid tissues. Immature cells may be localized predominantly within non-lymphoid tissues, and are responsible for endocytosis and processing of antigens. Inflammatory mediators are thought to promote their maturation and migration, via lymph and blood, into secondary lymphoid tissues. Here the mature cells present foreign peptide-MHC complexes to T cells, and deliver unique signals for T cell activation (immunostimulation). Within the thymus, however, these cells may be important for inducing T cell tolerance to self peptide-MHC complexes.     

DEC-205 mediates antigen uptake and presentation by both resting and activated human plasmacytoid dendritic cells

DEC-205 is a type I C-type lectin receptor (CLR) that is expressed on various APC subsets and has been suggested to bind necrotic and apoptotic cells. Here we study DEC-205 characteristics in plasmacytoid DCs (pDCs) obtained from healthy individuals and assess its ability to mediate antigen presentation by isolating sufficient numbers of pDCs from apheresis material obtained from stage III/IV melanoma patients. The results demonstrate that DEC-205 is expressed on human pDCs. Internalization of DEC-205 after antibody ligation is clathrin- and dynamin-dependent as it is blocked by hypertonic shock or by inhibition of dynamin activity. Antibody targeting to DEC-205 does not affect TLR-induced expression levels of co-stimulatory and MHC molecules, but clearly impairs TLR-induced IFN-α secretion by 40%. We observed that TLR-mediated signaling increases DEC-205 expression levels without affecting receptor internalization. Moreover, human pDCs retained the capacity to present antigens via DEC-205 following TLR activation.     

Ethanol inhibits antigen presentation by dendritic cells

Previous studies suggest that altered virus-specific T-cell responses observed during chronic ethanol exposure may be due to abnormal functioning of dendritic cells (DCs). Here we explored the effects of ethanol on exogenous antigen presentation by DCs. BALB/c, C57BL/6, and CBA/caj mice were fed ethanol or an isocaloric control diet for 8 weeks. The splenic DC population was expanded using an Flt3L expression plasmid via tail vein injection. DCs were purified and assessed for antigen presentation and processing and for peptide-major histocompatibility complex class I and II (MHCI and MHCII) formation on the cell surface. Interleukin-2 (IL-2) was measured as an indicator of antigen-specific T-cell activation by DCs in coculture. Antigen processing and peptide-MHCII complexes were evaluated by flow cytometry. We observed that ethanol not only suppressed allogeneic peptide presentation to T cells by DCs but also altered presentation of exogenous ovalbumin (OVA) peptide 323-339 to an OVA-specific DO11 T-cell line as well as to OVA-sensitized primary T cells. Smaller amounts of peptide-MHCII complexes were found on the DCs isolated from the spleens of ethanol-fed mice. In contrast to MHCII presentation, cross-presentation of exogenous OVA peptide via MHCI by DCs remained intact. More importantly, ethanol-exposed DCs had reduced B7-DC and enhanced ICOS-L (inhibitory) costimulatory molecule expression. Ethanol inhibits exogenous and allogeneic antigen presentation and affects the formation of peptide-MHCII complexes, as well as altering costimulatory molecule expression on the cell surface. Therefore, DC presentation of peptides in a favorable costimulatory protein environment is required to subsequently activate T cells and appears to be a critical target for the immunosuppressive effects of ethanol.     

Three-day dendritic cells for vaccine development: Antigen uptake,processing and presentation

Background

Antigen-loaded dendritic cells (DC) are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility.

Methods

In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date.

Results

Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivt)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivt RNA.

Conclusions

This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivt RNA as sources of specific antigen.

     

Steroids inhibit uptake and/or processing but not presentation of antigen by airway dendritic cells.

Recent studies from our laboratory indicate that local and (particularly) systemic steroids can modulate the traffic of dendritic cells (DC) through resting and inflamed airway epithelial tissues. The present report focuses upon the T-cell activating properties of DC, which are controlled by granulocyte-macrophage colony-stimulating factor (GM-CSF) signals, and in particular the question of whether the DC-stimulating effects of GM-CSF are susceptible to regulation by steroids. We present evidence that while dexamethasone inhibited GM-CSF-dependent uptake and/or processing of exogenous antigen by DC, it was ineffective in blocking the presentation of preprocessed self antigen to alloreactive T cells in a one-way mixed lymphocyte reaction (MLR). Associated GM-CSF-induced up-regulation of major histocompatability complex (MHC) class II and CTLA4 ligand expression by DC were also unaffected by dexamethasone phosphate (DX), reinforcing the view that the inhibitory effects of steroids on the T-cell activating functions of DC are restricted to steps upstream from presentation of processed antigen to the T-cell receptor (TCR). These findings have potentially important implications in relation to the use of topical steroids in the treatment of atopic asthma, a disease in which local T-cell activation in airway tissue is a key pathogenic factor, and which furthermore is characterized by intense production of GM-CSF within the airway epithelium.     

Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells

Dendritic cells (DC) efficiently take up antigens by macropinocytosis and mannose receptor-mediated endocytosis. Here we show that endocytosis of mannose receptor-antigen complexes takes place via small coated vesicles, while non-mannosylated antigens were mainly present in larger vesicles. Shortly after internalization the mannose receptor and its ligand appeared in the larger vesicles. Within 10 min, the mannosylated and non-mannosylated antigens co-localized with typical markers for major histocompatibility complex class II-enriched compartments and lysosomes. In contrast, the mannose receptor appeared not to reach these compartments, suggesting that it releases its ligand in an earlier endosomal structure. Moreover, we demonstrate that mannosylation of protein antigen and peptides resulted in a 200–10 000-fold enhanced potency to stimulate HLA class II-restricted peptide-specific T cell clones compared to non-mannosylated peptides. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DC which may be applicable in vaccine design.     

CpG oligodeoxynucleotides enhance FcgammaRI-mediated cross presentation by dendritic cells

Dendritic cells (DC) can trigger naive CD8(+) T cell responses by their capacity to cross-present exogenous antigens via the major histocompatibility complex class I pathway. The myeloid class I IgG receptor, FcgammaRI (CD64), is expressed on DC, and in vivo targeting of antigens to FcgammaRI induces strong humoral and cellular immune responses. We studied the capacity of human FcgammaRI (hFcgammaRI) to facilitate DC-mediated cross presentation and T cell activation, and assessed the effect of CpG oligodeoxynucleotides on this process. We generated hFcgammaRI expressing immature DC from hFcgammaRI transgenic and immature DC from non-transgenic mice. Antigens were targeted to Fcgamma receptors as ovalbumin immune complexes, or selectively to hFcgammaRI via ovalbumin-CD64 mAb fusion proteins. Co-incubation of immature DC with CpG ODN led to markedly increased MHC class I presentation of FcgammaR-targeted antigens. When OVA was selectively targeted to hFcgammaRI, few differences were observed between Tg and NTg DC. However, upon co-incubation with CpG ODN, hFcgammaRI-triggered cross presentation was enhanced. These results document the capacity of hFcgammaRI on DC to trigger cross presentation via MHC class I upon co-culture with CpG ODN.     

Phagocytosis and antigen presentation in dendritic cells

Summary:  Like macrophages and neutrophils, dendritic cells (DCs) are considered professional phagocytes. Even if the three cell types phagocytose parasites, bacteria, cell debris, or even intact cells very efficiently, the functional outcomes of the phagocytic event are quite different. Macrophages and neutrophils scavenge and destroy phagocytosed particles, a critical step in innate immunity. DCs, in contrast, have developed means to 'preserve' useful information from the ingested particles that serve to initiate adaptive immune responses. Thus, both phagosomal degradation and acidification are much lower in DCs than in macrophages or neutrophils. Reduced degradation results in the conservation of antigenic peptides and in their increased presentation on major histocompatibility complex class I and II molecules. In this article, we review the mechanisms that control this delicate equilibrium between phagosomal degradation/cytotoxicity and antigen presentation in the different families of phagocytes.     

Surface expression of transglutaminase 2 by dendritic cells and its potential role for uptake and presentation of gluten peptides to T cells

Celiac disease is a chronic small intestinal inflammation driven by gluten-reactive T cells of the intestinal mucosa. These T cells are HLA-DQ2 or -DQ8 restricted, and predominantly recognize gluten peptides that are deamidated by the enzyme transglutaminase 2 (TG2). Our recent results strongly suggest that duodenal CD11c(+) dendritic cells (DC) are directly involved in T cell activation in the celiac lesion. The aim of this study was to investigate whether surface-associated TG2 could be involved in receptor-mediated endocytosis of gluten peptides, a process that may contribute to the preferential recognition of deamidated peptides. We found that both monocyte-derived DC and local CD11c(+) DC in the duodenal mucosa expressed cell surface-associated TG2. As phenotypic characterization of CD11c(+) DC in the celiac lesion suggests that these cells may be derived from circulating monocytes, we used monocyte-derived DC in functional in vitro studies. Using a functional T cell assay, we obtained evidence that cell surface-associated TG2 is endocytosed by monocyte-derived DC. However, we were unable to obtain evidence for a role of surface TG2 in the loading and subsequent generation of deamidated gluten peptides in these cells.     

Pathways utilized by dendritic cells for binding, uptake, processing and presentation of antigens derived from HIV-1

The outcome following HIV infection depends on the nature and durability of the HIV-specific T cell response induced initially. The activation of protective T cell responses depends upon dendritic cells (DC), antigen-presenting cells which have the capacity to process and present viral antigens. DC pulsed with aldrithiol-2-inactivated HIV and delivered in vivo were reported to induce immune responses and promote virologic control in chronically HIV-1-infected subjects. To gain an understanding of this phenomenon, we characterized the steps involved in the presentation of antigens derived from aldrithiol-2-treated vs. infectious HIV-1 by DC. Antigen presentation, on both MHC class I and II, was independent of DC-specific ICAM-3-grabbing integrin, DEC-205 and macrophage mannose receptor, C-type lectins expressed by the DC. Inhibitor studies showed that presentation on MHC class I was dependent on viral fusion in a CD4/coreceptor-dependent manner, both at the cell surface and within endosomes, and access to the classical endosomal processing pathway. MHC class II presentation of HIV-associated antigens was dependent on active endocytosis, probably receptor-mediated, and subsequent degradation of virions in acidified endosomes in the DC. Our study brings forth new facts regarding the binding, uptake, and processing of chemically inactivated virions leading to efficient antigen presentation and should aid in the design of more effective HIV vaccines.     

骨髓来源的原代树突状细胞对细菌颗粒抗原交叉呈递的动力学研究

目的研究小鼠骨髓细胞来源的树突状细胞对细菌颗粒抗原交叉递呈的动力学特点。方法取小鼠骨髓细胞,采用GM-CSF诱导培养。用倒置显微镜观察培养过程中细胞形态,用流式细胞仪进行细胞纯度和表型测定。采用第7天未成熟的DC细胞进行细菌抗原的交叉递呈能力检测和动力学分析。结果小鼠骨髓细胞经GM-CSF诱导培养,第7天获得大量具有典型形态和免疫表型的树突状细胞,并且在细菌脂多糖刺激后,其表型也发生特征性变化表现在共刺激分子CD80由刺激前的27.7%上调到71.6%,CD86从刺激前的36.0%上调到80.3%,DEC205从刺激前的6.5%上调到26.2%。同时该细胞吞噬低剂量的细菌抗原即可有效活化T细胞,其动力学曲线为:1～3h,该细胞抗原递呈能力呈对数增加,3～8hDC抗原递呈能力进入一个平台期,10～12h,抗原递呈能力又迅速增强。结论采用GM-CSF可以从小鼠骨髓中获得大量纯度高并且具有典型免疫表型的DC,该DC细胞能有效的递呈外源性细菌抗原,并具有特殊的抗原递呈动力学曲线。     

Antigen presentation by murine peritoneal cavity macrophage-derived dendritic cells.

Murine peritoneal cavity macrophage derived dendritic cells (PEC-DC) generated using early growth factors, interleukin 4 and granulocyte-macrophage colony-stimulating factor followed by maturation in interferon-gamma plus either, Toxoplasma lysate antigen (TLA) or lipopolysaccharide, bind TLA by a nonspecific mechanism and continue to express major histocompatibility complex class II antigens after 24 h of culture in vitro. Moreover, the proliferation of CD3+ spleen T cells from mice immunized with Toxoplasma gondii homogenate, induced by PEC-DC-mediated antigen presentation was statistically significant and of consistent amplitude. This accessory function of PEC-DC is antigen specific.     

Allergen presentation by epidermal Langerhans' cells from patients with atopic dermatitis is mediated by IgE.

To investigate the role of IgE-bearing Langerhans' cells (LC) from atopic dermatitis (AD) patients in antigen presentation, IgE+LC and non-IgE bearing LC (IgE-LC) from AD patients were investigated for their antigen-presenting capacity and compared to antigen-presenting cells (APC) from peripheral blood. The T-cell response to Candida albicans, using IgE+LC from AD patients as APC, was in the same range as with IgE-LC. Also, the T-cell response to Candida with autologous APC from peripheral blood did not significantly differ between these groups. In contrast to this finding, the T-cell response to house dust allergen (HDA) was dependent on the type of APC used. If non-T cells from peripheral blood were used as APC, both AD patients and controls responded to HDA. However, when LC were used as APC, a T-cell response to HDA was only observed in the presence of IgE+LC. IgE-LC from AD patients or LC from normal controls were unable to present HDA. Preincubation of IgE+LC with anti-IgE or anti-kappa/lambda antibodies inhibited HDA-induced T-cell proliferation, whereas the response to Candida was not affected. These in vitro results, which demonstrate the necessity of cell-bound IgE on LC for the presentation of aero-allergens, strongly correlate with the in vivo presence of a positive delayed patch reaction to the same antigens. When the LC of a patient appeared to be IgE-, the in vitro proliferative response as well as, in most cases, the in vivo patch test reaction to the same antigens was negative. In conclusion, these experiments demonstrate that there are at least two different mechanisms by which LC capture antigens for antigen presentation. In one of them cell-bound IgE, as can be demonstrated on LC from AD patients, plays a crucial role. The binding and presentation of HDA by APC from peripheral blood can take place independently of cell-bound IgE. Candida can be presented by both types of APC in the absence of IgE.     

Processing and presentation of intact hen egg-white lysozyme by dendritic cells.

Dendritic cells in lymphoid tissues are of key importance as highly specialized antigen-presenting cells for the induction of T lymphocyte responses. Conflicting results have been published regarding antigen processing of intact proteins by dendritic cells. We now report that highly purified dendritic cells isolated from H-2k mouse spleens very efficiently generated immunogenic fragments of intact hen egg-white lysozyme (HEL) protein to present to an I-Ak-restricted T hybridoma cell line, specific for HEL peptide 46-61. Dendritic cells required 100 times less HEL protein than lipopolysaccharide-induced B cell blasts for effective presentation. Uptake of 125I-labeled HEL protein by dendritic cells and inhibition of presentation of HEL protein by chloroquine treatment was observed. This indicates an endocytotic process and the involvement of acidified compartments. Since the supernatant of dendritic cells, that were incubated with intact HEL protein, contained immunogenic fragments, further evidence for processing of HEL protein by dendritic cells was obtained. When HEL protein was covalently coupled to beads, dendritic cells were not able to ingest these beads, but could still process HEL protein for presentation. This suggests cell surface processing of HEL protein, although internalization of HEL protein released from the beads cannot be excluded. Taken together, these data show that H-2k dendritic cells are capable of processing and presenting intact HEL protein.