Presentation Of Exogenous Antigens Research Articles

Carbon monoxide decreases endosome–lysosome fusion and inhibits soluble antigen presentation by dendritic cells to T cells.

Heme oxygenase-1 (HO-1) inhibits immune responses and inflammatory reactions via the catabolism of heme into carbon monoxide (CO), Fe(2+) , and biliverdin. We have previously shown that either induction of HO-1 or treatment with exogenous CO inhibits LPS-induced maturation of dendritic cells (DCs) and protects in vivo and in vitro antigen-specific inflammation. Here, we evaluated the capacity of HO-1 and CO to regulate antigen presentation on MHC class I and MHC class II molecules by LPS-treated DCs. We observed that HO-1 and CO treatment significantly inhibited the capacity of DCs to present soluble antigens to T cells. Inhibition was restricted to soluble OVA protein, as no inhibition was observed for antigenic OVA-derived peptides, bead-bound OVA protein, or OVA as an endogenous antigen. Inhibition of soluble antigen presentation was not due to reduced antigen uptake by DCs, as endocytosis remained functional after HO-1 induction and CO treatment. On the contrary, CO significantly reduced the efficiency of fusion between late endosomes and lysosomes and not by phagosomes and lysosomes. These data suggest that HO-1 and CO can inhibit the ability of LPS-treated DCs to present exogenous soluble antigens to naïve T cells by blocking antigen trafficking at the level of late endosome-lysosome fusion.

Immunodeficiency and autoimmunity in H2-O deficient mice (P5016)

Abstract HLA-DO/H2-O is a highly conserved non-polymorphic MHC class II-like molecule whose physiological function remains unknown. The cell maturation-dependent DO expression in B-lymphocytes and DCs suggests a role H2-O in promoting presentation of exogenous Ag by attenuating presentation of endogenous self-peptides. We report herein that H2-O-/- mice spontaneously develop high titers of IgG2a/c antinuclear antibodies (ANA) with specificity for dsDNA, ssDNA, and histones. Reconstitution of RAG1-/- mice with T and B cells from H2-O-/- or WT mice demonstrated that production of ANAs requires participation of CD4+ T cells from H2-O-/- mice. Bone marrow chimeras demonstrated that loss of H2-O expression in thymic epithelial cells did not induce ANAs, and that lack of H2-O expression in bone marrow-derived cells was sufficient to induce the autoimmune phenotype. Despite production of high titers of autoantibodies, H2-O-/- mice exhibit a delayed generation of humoral immunity to model antigens (OVA and KLH), affecting all major T-dependent Ig classes including IgG2a/c. Consistently, presentation of all the natural Ag epitopes by H2-O-/- APC was inefficient as compared to WT APC. Thus, H2-O promotes immunity towards exogenous Ag while preventing autoimmunity. We propose that H2-O, through spatially and temporally inhibiting H2-M, enhances presentation of exogenous antigen by preventing nascent/newly-synthesized class II molecules from forming stable complexes with endogenous self-peptides.

Metformin reduces gene expression of adipogenesis and inflammatory cytokines through the modulation of T cell priming in obese-induced Type II diabetic mice (P5063)

Abstract Inflammation is increasingly regarded as a key process underlying metabolic diseases in obese individuals where obese adipose tissue shows features characteristic of active local inflammation. Metformin is a biguanide derivative used in the treatment of T2D and one of the world's most widely prescribed drugs and have been suggested that metformin has anti-inflammatory properties. Here we show that metformin efficiently attenuates the metabolic disorder in DIO mice and suppresses MHC-restricted antigen presentation in dendritic cells. This study was to investigate the therapeutic effect of metformin on diabetic symptoms in DIO mice. Metformin decreased aipogenesis related gene, FAS, SCD-1, GPAT, SREBP1a, but also suppressed gene expression of innate cytokines than control mice. Metformin decreased both class I- and class II-restricted presentation of exogenous OVA in dendritic cells. Metformin also decreased expression of the co-stimulatory molecules, ICAM-1 and B7-1/-2, and MHC class I and II molecules but not phagocytic activity of DCs. Metformin decreased MHC class II-restricted exogenous antigen presentation in peritoneal macrophages in vivo. These results show that metformin attenuates immune responses followed by decreases MHC-restricted presentation of exogenous antigen in DCs via intracellular processing events. Theses finding suggest that modulation of the antigen presentation pathway could provide a novel means for regulating T cell responses in metabolic diseases.

Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire.

A catalytically inactive mutant of the deubiquitylase YOD-1 enhances antigen cross-presentation

AbstractAntigen presenting cells (APCs) that express a catalytically inactive version of the deubiquitylase YOD1 (YOD1-C160S) present exogenous antigens more efficiently to CD8+ T cells, both in vitro and in vivo. Compared with controls, immunization of YOD1-C160S mice led to greater expansion of specific CD8+ T cells and showed improved control of infection with a recombinant γ-herpes virus, MHV-68, engineered to express SIINFEKL peptide, the ligand for the ovalbumin-specific TCR transgenic OT-I cells. Enhanced expansion of specific CD8+ T cells was likewise observed on infection of YOD1-C160S mice with a recombinant influenza A virus expressing SIINFEKL. YOD1-C160S APCs retained antigen longer than did control APCs. Enhanced cross-presentation by YOD1-C160S APCs was transporter associated with antigen processing (TAP1)–independent but sensitive to inclusion of inhibitors of acidification and of the proteasome. The activity of deubiquitylating enzymes may thus help control antigen-specific CD8+ T-cell responses during immunization.

Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon

Major histocompatibility complex class II (MHCII) molecules are heterodimeric surface proteins involved in the presentation of exogenous antigens during the adaptive immune response. We demonstrate the existence of a fine level of regulation, coupling the transcription and processing of mRNAs encoding α and β chains of MHCII molecules, mediated through binding of their Untraslated Regions (UTRs) to the same ribonucleoproteic complex (RNP). We propose a dynamic model, in the context of the ‘MHCII RNA operon’ in which the increasing levels of DRA and DRB mRNAs are docked by the RNP acting as a bridge between 5′- and 3′-UTR of the same messenger, building a loop structure and, at the same time, joining the two chains, thanks to shared common predicted secondary structure motifs. According to cell needs, as during immune surveillance, this RNP machinery guarantees a balanced synthesis of DRA and DRB mRNAs and a consequent balanced surface expression of the heterodimer.

Abstract B72: CD8αα+ langerin+ dendritic cells maintain the effector CD8 T cell population after adoptive transfer.

Abstract Adoptive cell transfer (ACT) is a promising new area of anti-cancer therapy. The process involves the isolation of T cells from a patient's blood, in vitro stimulation of the T cells against cancer antigens, then re-infusion of the activated cells back into the patient. This process provides a population of effector CD8 T cells capable of recognizing and killing cancer cells. ACT is currently limited by the ability of the transferred cells to survive in high numbers for an extended period of time and therefore overcoming this problem is a focus to improve the efficacy of the treatment. The activation of cancer-specific CD8 T cells is dependent on the ability of dendritic cells (DCs) to acquire and present exogenous antigens on major histocompatibility complex class I (a process known as cross-presentation) for the stimulation of CD8 T cells. We have previously found a subset of splenic DCs expressing the CD8αα homodimer and the c-type lectin receptor langerin (CD207), that have a strong ability to activate CD8 T cells by cross-presentation. This DC subset is found in the marginal zone of the spleen, is highly efficient at phagocytosing apoptotic cells from the blood, and is believed to play an important role in scanning the blood for antigens. We therefore hypothesized that CD8αα+ langerin+ DCs influence the survival and expansion of adoptively transferred effector CD8 T cells in the blood. Within this project we have used langerin-diphtheria toxin receptor mice, in which all langerin-expressing cells are ablated in vivo upon injection of diphtheria toxin, to address the role of the CD8αα+ langerin+ DC subset in maintaining the CD8 T cell population after ACT. For this purpose, we challenged mice intravenously with the T cell thymoma EG7.OVA and adoptively transferred in vitro activated OVA-specific CD8 T cells (OT-I T cells) 12 days later with or without prior langerin-DC depletion. We found a larger population of OT-I T cells when these were transferred into an EG7.OVA bearing host when compared to mice without tumors. Interestingly, mice that were depleted of their CD8αα+ langerin+ DCs did not develop this increase in OT-I T cell number and their absence resulted in reduced survival. Our data therefore suggest that antigen presentation by CD8αα+ langerin+ DCs play a role in sustaining high numbers of transferred CD8 T cells after ACT, resulting in increased protection against EG7.OVA. Manipulations that increase the stimulatory capacity of this DC subset could be a way to increase the efficacy of ACT therapy. Citation Format: John David Gibbins, Ian Hermans, Troels Petersen. CD8αα+ langerin+ dendritic cells maintain the effector CD8 T cell population after adoptive transfer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B72.

The impact of sub-cellular localization and dose of tumor antigen on the efficiency cross-presentation

Event Abstract Back to Event The impact of sub-cellular localization and dose of tumor antigen on the efficiency cross-presentation Chidozie C. Anyaegbu1, Scott A. Fisher1, Bruce Robinson1 and Richard Lake1* 1 The University of Western Australia, School of Medicine and Pharmacology, Australia One of the most effective ways the immune system uses to detect tumor antigen is through a process termed Cross-presentation. In this process, antigen presenting cells capture, process, and present exogenous tumor antigens to CD8+ T cells, a key effector population. In order to induce an effective cross-presentation mediated anti-tumor immune response, it is crucial that the immune system is able to sense low antigen concentrations, typically present in distinct sub-cellular compartments within tumor cells. This study examines the efficiency of tumor antigen cross-presentation, and addresses the hypothesis that some sub-cellular compartments are better sources of tumor antigen. We compared tumor specific T cell responses elicited by mouse B16-F10 melanoma tumors stably expressing a model antigen (CL4 epitope of haemagglutinin), in cytoplasmic, secretory, or nuclear sub-cellular compartments within the tumor-cell. All three tumor cell-lines similarly expressed approximately 3.88 x 10-9 micromoles (µmol) of CL4-antigen per cell. Our results show that cross-presentation is remarkably sensitive to antigen dose, with a threshold of inoculated antigen (0.194 µmol) being required for cross-presentation induced proliferation of adoptively transferred, Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) labelled, CL4-T cell receptor transgenic CD8+ T cells. Furthermore, nuclear localized antigen was less efficiently cross-presented, with 40% proliferation of CD8+ CL4 T cells. By contrast, its cytoplasmic and secreted counterparts induced a near maximal 80% proliferation. Notably however, gemcitabine, an apoptosis inducing chemotherapy agent, was able to double the low cross-presentation efficiency of nuclear antigens, in an antigen dose dependent manner. Taken together, our data suggests that the dose and localization of antigen within a tumor cell can influence the robustness of anti-tumor T cell responses. Moreover, gemcitabine can enhance these responses by unmasking previously ignored, potentially novel nuclear antigens, like those that tend to arise in new tumor variants to which the host’s T cell repertoire is not tolerant. These findings imply that the dose and sub-cellular localization of antigen(s) should be considered when designing chemo-immunotherapy anti-tumor strategies. Figure 1 Acknowledgements Chidozie Anyaegbu is kindly supported by the University of Western Australia Research Training Scheme (RTS) Scholarship. This work is funded by the Australian government National Health and Medical Research Council's (NHMRC), Centres of Clinical Research Excellence (CCRE) grant held by NCARD. Keywords: Subcellular localization, Tumor antigens, cross-presentation, antigen processing and presentation, Cancer Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Anyaegbu CC, Fisher SA, Robinson B and Lake R (2013). The impact of sub-cellular localization and dose of tumor antigen on the efficiency cross-presentation. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00801 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Richard Lake, The University of Western Australia, School of Medicine and Pharmacology, Perth, Western Australia, 6009, Australia, richard.lake@uwa.edu.au Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Chidozie C Anyaegbu Scott A Fisher Bruce Robinson Richard Lake Google Chidozie C Anyaegbu Scott A Fisher Bruce Robinson Richard Lake Google Scholar Chidozie C Anyaegbu Scott A Fisher Bruce Robinson Richard Lake PubMed Chidozie C Anyaegbu Scott A Fisher Bruce Robinson Richard Lake Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

HSP90α deficiency does not affect immunoglobulin gene hypermutation and class switch but causes enhanced MHC class II antigen presentation

Heat shock protein 90 (HSP90) is a molecular chaperone required for efficient antigen presentation and cross-presentation. In addition, HSP90 was recently reported to interact with and stabilize the activation-induced cytidine deaminase (AID) and plays a critical role in immunoglobulin gene hypermutation and class switch recombination. In mice and humans, there are two HSP90 isoforms, HSP90α and HSP90β, but the in vivo role of each isoform remains largely unknown. Here we have analyzed humoral immune responses in HSP90α-deficient mice. We found that HSP90α deficiency did not affect AID protein expression. B cell development and maturation, as well as immunoglobulin gene hypermuation and class switch, occurred normally in HSP90α-deficient mice. However, antibody production to a T-dependent antigen was elevated in the mutant mice and this was associated with enhanced MHC class II antigen presentation to T helper cells by dendritic cells. Our results reveal a previously unidentified inhibitory role for HSP90α isoform in MHC class II antigen presentation and the humoral immune response. Along with our recent finding that HSP90α is required for antigen cross-presentation, these results suggest that HSP90α controls the balance of humoral and cellular immunity by dictating the fate of presentation of exogenous antigen.

Allergic Airway Inflammation in Mice Deficient for the Antigen-Processing Protease Cathepsin E

Background: Allergic asthma is a Th2-type chronic inflammatory disease of the lung. It is characterized by infiltration of eosinophils, neutrophils, mast cells and T lymphocytes into the airways. Th2 cytokines like interleukin (IL)-4, IL-5 and chemokines like eotaxin are increased in the asthmatic response. The processing and presentation of exogenous antigens is important in the sensitization to an allergen. Cathepsin E (Ctse) is an intracellular aspartic endoprotease which is expressed in immune cells like dendritic cells (DCs). It was found to play an essential role in the processing and presentation of ovalbumin (OVA). The aim of the present study was to investigate the inhibition of Ctse in two different experimental models of allergic airway inflammation. Methods: Ctse wild-type (Ctse^+/+) and Ctse-deficient (Ctse^–/–) bone marrow-derived DCs (BMDCs) were pulsed with OVA/OVA peptide and cocultured with OVA transgenic T II (OT II) cells whose proliferation was subsequently analyzed. Two different in vivo asthma models with Ctse^+/+ and Ctse^–/– mice were performed: an acute OVA-induced and a subchronic Phleum pratense-induced airway inflammation. Results: Proliferation of OT II cells was decreased when cocultured with BMDCs of Ctse^–/– mice as compared to cells cocultured with BMDCs of Ctse^+/+ mice. In vivo, Ctse deficiency led to reduced lymphocyte influx after allergen sensitization and challenge in both investigated airway inflammation models, compared to their control groups. Conclusion: Ctse deficiency leads to a reduced antigen presentation in vitro. This is followed by a distinct effect on lymphocyte influx in states of allergic airway inflammation in vivo.

Serpinb9 (Spi6)‐deficient mice are impaired in dendritic cell‐mediated antigen cross‐presentation

Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (GrB) that protects activated cytotoxic lymphocytes from apoptosis. We show here that the CD8(+) subset of splenic dendritic cells (DC), specialized in major histocompatibility complex class I (MHC I) presentation of exogenous antigens (cross-presentation), produce high levels of Sb9. Mice deficient in Sb9 are unable to generate a cytotoxic T-cell response against cell-associated antigen by cross-presentation, but maintain normal MHC-II presentation to helper T cells. This impaired cross-priming ability is autonomous to DC and is evident in animals deficient in both Sb9 and GrB, indicating that this role of Sb9 in DC is GrB-independent. In Sb9-deficient mice, CD8(+) DC develop normally, survive as well as wild-type DC after antigenic challenge, and exhibit unimpaired capacity to take up antigen. Although the core processing machinery is unaffected, Sb9-deficient DC appear to process antigen faster. Our results point to a novel, GrB-independent role for Sb9 in DC cross-priming.

Cross-presentation by dendritic cells

The presentation of exogenous antigens on MHC class I molecules, known as cross-presentation, is essential for the initiation of CD8(+) T cell responses. In vivo, cross-presentation is mainly carried out by specific dendritic cell (DC) subsets through an adaptation of their endocytic and phagocytic pathways. Here, we summarize recent advances in our understanding of the intracellular mechanisms of cross-presentation and discuss its role in immunity and tolerance in the context of specialization between DC subsets. Finally, we review current strategies to use cross-presentation for immunotherapy.

Adherent Human Alveolar Macrophages Exhibit a Transient Pro-Inflammatory Profile That Confounds Responses to Innate Immune Stimulation

Alveolar macrophages (AM) are thought to have a key role in the immunopathogenesis of respiratory diseases. We sought to test the hypothesis that human AM exhibit an anti-inflammatory bias by making genome-wide comparisons with monocyte derived macrophages (MDM). Adherent AM obtained by bronchoalveolar lavage of patients under investigation for haemoptysis, but found to have no respiratory pathology, were compared to MDM from healthy volunteers by whole genome transcriptional profiling before and after innate immune stimulation. We found that freshly isolated AM exhibited a marked pro-inflammatory transcriptional signature. High levels of basal pro-inflammatory gene expression gave the impression of attenuated responses to lipopolysaccharide (LPS) and the RNA analogue, poly IC, but in rested cells pro-inflammatory gene expression declined and transcriptional responsiveness to these stimuli was restored. In comparison to MDM, both freshly isolated and rested AM showed upregulation of MHC class II molecules. In most experimental paradigms ex vivo adherent AM are used immediately after isolation. Therefore, the confounding effects of their pro-inflammatory profile at baseline need careful consideration. Moreover, despite the prevailing view that AM have an anti-inflammatory bias, our data clearly show that they can adopt a striking pro-inflammatory phenotype, and may have greater capacity for presentation of exogenous antigens than MDM.

Role for Rab11a in mediating TLR dependent cross-presentation responses (106.17)

Abstract Toll-like receptor (TLR) signaling is critical for accelerated phagosome maturation and enhanced major histocompatibility complex class II (MHC II) presentation of phagocytosed antigens. Since cross-presentation is another consequence of phagosome maturation, we hypothesized that like MHC II presentation, the presentation of exogenous antigens on MHC class I (MHC I) by dendritic cells (DCs) is positively regulated by TLRs. Using antigen derived from bacteria, infected apoptotic cells or antigen-coated beads conjugated to a TLR ligand, our results show that cross-presentation is severely impaired when TLR signaling is abrogated in the cross-presenting DC or when TLR ligands are absent from phagocytic cargo. To delineate the mechanisms behind TLR enhancement of cross-presentation, we studied trafficking of MHC I in the presence or absence of TLR signaling. We observed recruitment of MHC I selectively to phagosomes containing TLR ligands along with the concomitant enrichment of the small guanosine tri-phosphatase (GTPase) Rab11a. In fact, Rab11a was crucial for TLR mediated enhancement of cross-presentation as selective knock-down of Rab11a abrogated the ability of DCs to cross-present antigen. Our results demonstrate a role for Rab11a in trafficking MHC I to phagosomes that engage TLR signaling, thus mediating enhanced cross-presentation of microbial antigens. Results from our studies have implications on anti-viral and anti-bacterial immunity as well as tumor immunotherapy.

Biodegradable polymer, PLGA, is an efficient vehicle for class II MHC-restricted antigen presentation and the induction of DC maturation (106.15)

Abstract The antigen presentation and maturation activities of antigen-encapsulated biodegradable nanoparticles were compared with that of antigen-antibody immune complexes. Dendritic cells (DCs) were allowed to uptake ovalbumin (OVA) via pinocytosis, phagocytosis or endocytosis, and then the levels of MHC-restricted OVA peptide presentation were measured using OVA specific CD4 and CD8 T cells. Poly(D,L-lactic acid-co-glycolic acid, PLGA)-encapsulated OVA (pOVA) and OVA-IgG complexes (OVA-IC) were equally efficient in inducing MHC class I and II-restricted OVA peptide presentation. Comparison of the amount of OVA peptide presentation showed that class II-restricted presentation pathways were robust process compared to class I-restricted presentation pathways. Maturation inducing activities of pOVA and OVA-IC were also examined. pOVA was as efficient as or better compared to OVA-IC in inducing the expression of cell surface molecules such as class II, CD80, CD86, and the secretion of cytokines IL-6, IL-1β and TNF-α. These results show that the biodegradable polymer, PLGA, is an efficient vehicle not only for class II-restricted antigen presentation but also for induction of DC maturation. The present study also shows that in exogenous antigen presentation by DCs, cross presentation is a much weaker phenomenon compared to MHC class II-restricted presentation.

IFN-α enhances cross-presentation in human dendritic cells by modulating antigen survival, endocytic routing, and processing

AbstractCross-presentation allows antigen-presenting cells to present exogenous antigens to CD8+ T cells, playing an essential role in controlling infections and tumor development. IFN-α induces the rapid differentiation of human mono-cytes into dendritic cells, known as IFN-DCs, highly efficient in mediating cross-presentation, as well as the cross-priming of CD8+ T cells. Here, we have investigated the mechanisms underlying the cross-presentation ability of IFN-DCs by studying the intracellular sorting of soluble ovalbumin and nonstructural-3 protein of hepatitis C virus. Our results demonstrate that, independently from the route and mechanism of antigen entry, IFN-DCs are extraordinarily competent in preserving internalized proteins from early degradation and in routing antigens toward the MHC class-I processing pathway, allowing long-lasting, cross-priming capacity. In IFN-DCs, both early and recycling endosomes function as key compartments for the storage of both antigens and MHC-class I molecules and for proteasome- and transporter-associated with Ag processing–dependent auxiliary cross-presentation pathways. Because IFN-DCs closely resemble human DCs naturally occurring in vivo in response to infections and other danger signals, these findings may have important implications for the design of vaccination strategies in neoplastic or chronic infectious diseases.

Analysis of HLA‐DM polymorphisms in the Chinese Han population

Non-classical human leukocyte antigen (HLA)-DM plays an important and unique role in the processing and presentation of exogenous antigens. Polymorphisms of certain genes and frequency of alleles in populations may indicate susceptibility to certain diseases. In this study, the analysis of HLA-DMA and HLA-DMB gene polymorphisms and haplotypes in the Chinese Han population was conducted to obtain population genetic data. HLA-DM typing has been performed previously by other groups by polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR-sequence-specific oligonucleotide probe techniques. In this study, we established a TaqMan PCR typing method as an alternative to these techniques to survey the frequency of DMA and DMB alleles in the population. Genotyping was conducted in 1000 unrelated individuals of Han nationality in South and North China using TaqMan PCR typing. Four different DMA alleles and six different DMB alleles were detected. All loci met the Hardy-Weinberg equilibrium principle that both allele and genotype frequencies in a population remain constant. We found that the DMA*01:01 (69.35%) and DMB*01:01 (52.5%) alleles were more frequent in Chinese Hans. Analysis of the haplotypes for two loci of DMA and DMB showed that a highly significant positive linkage disequilibrium (LD) presented for DMA*01:01-DMB*01:02, DMA*01:01-DMB*01:03, DMA*01:01-DMB*01:04, DMA*01:02-DMB*01:01, DMA*01:02-DMB*01:05, DMA*01:03-DMB*01:07, and DMA*01:04-DMB*01:01 haplotypes. Analysis of haplotypes for four loci associated with antigen processing (DMA-DMB-TAP1-TAP2) showed a highly significant LD in DMA*01:01-DMB*01:04-TAP1*02:01:01-TAP2*01:02, DMA*01: 02-DMB*01:05-TAP1*01:01-TAP2*01:01, and DMA*01:01-DMB*01:03-TAP1* 04:01-TAP2*01:01 haplotypes. The comparison between the Chinese Han population and non-Chinese populations showed that no significant differences were found at the HLA-DMA locus in the Chinese Han population compared with people of German nationality, whereas significant differences presented when compared with Turkish, American Caucasian, Japanese, French, and Italian nationalities. However, at the HLA-DMB locus, highly significant differences presented in the Chinese Han population compared with Germans and Italians. This study lays the foundations for further disease association analyses.

TLR and Nucleotide-Binding Oligomerization Domain-like Receptor Signals Differentially Regulate Exogenous Antigen Presentation

The effect of dendritic cell (DC) maturation on MHC class II-restricted Ag presentation is well studied, but less is known about the effects of DC maturation on MHC class I-restricted cross-presentation. We investigated the ability of mature DCs to present Ags from cells infected with HSV-1. Pretreatment with pure LPS increased cross-presentation in a manner dependent on both MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-β, whereas a similar dose of a less pure LPS preparation inhibited cross-presentation. The difference could not be attributed to differences in uptake or phenotypic maturation. The likely contaminant responsible for shutting down cross-presentation is peptidoglycan (PGN). Addition of PGN to pure LPS abrogated its ability to enhance cross-presentation. Direct activation of DCs with PGN inhibited cross-presentation through nucleotide-binding oligomerization domain-like receptor signaling. These results demonstrate that different maturation stimuli can have opposite impacts on the ability of DCs to cross-present viral Ags.

Antigen delivery for cross priming via the emulsion vaccine adjuvants

The function of emulsion adjuvants in vaccine antigen delivery remains unclear. To investigate the roles of emulsion adjuvants in cross presentation of exogenous antigens, a series of emulsions were prepared for both in vitro and in vivo studies. Bone marrow-derived dendritic cells (BMDCs) were treated with the adjuvants and analyzed by flow cytometry for the expression of costimulatory molecules. The activation of antigen-specific T cells in vitro was determined with B3Z cells. Antibody secretion in the draining lymph nodes of emulsion adjuvant-treated animals was measured by enzyme-linked immuno-spot (ELISPOT) assays, and antigen-specific proliferation of cells was conducted to examine the roles of emulsion adjuvants in antigen delivery. Data on phagocytosis of adjuvant-treated cells correlated well with the degree of cell death induced by the emulsion adjuvants. Significant inflammatory infiltration and cell death were observed in vivo at the adjuvant injection sites, as demonstrated by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Ovalbumin (OVA)-based ELISPOT assays showed that L121-adjuvant, containing Pluronic L121, induced the most significant cell death also stimulated the strongest antibody-producing response in the draining lymph nodes, consistent with the data on the proliferation of antigen-specific T cells and activation of B3Z cells in vitro. Results presented in this study have demonstrated the roles of emulsion adjuvants in induction of cell death and delivery of exogenous antigens for cross-priming, leading to stimulation of antigen-specific immune responses.

Activation of legumain involves proteolytic and conformational events, resulting in a context- and substrate-dependent activity profile.

Localized mainly to endo/lysosomes, legumain plays an important role in exogenous antigen processing and presentation. The cysteine protease legumain, also known as asparaginyl endopepetidase AEP, is synthesized as a zymogen and is known to undergo pH-dependent autoproteolytic activation whereby N-terminal and C-terminal propeptides are released. However, important mechanistic details of this pH-dependent activation as well as the characteristic pH activity profile remain unclear. Here, it is shown that all but one of the autocatalytic cleavage events occur in trans, with only the release of the C-terminal propeptide being relevant to enzymatic activity. An intriguing super-activation event that appears to be exclusively conformational in nature and enhances the enzymatic activity of proteolytically fully processed legumain by about twofold was also found. Accepting asparagines and, to lesser extent, aspartic acid in P1, super-activated legumain exhibits a marked pH dependence that is governed by the P1 residue of its substrate and conformationally stabilizing factors such as temperature or ligands. The crystallization and preliminary diffraction data analysis of active legumain are presented, which form an important basis for further studies that should clarify fundamental aspects of activation, activity and inactivation of legumain, which is a key target in (auto-)immunity and cancer.