Role In Antigen Presentation Research Articles

The Therapeutic Effect of Immune-Modifying Microparticles in an Acute Graft-Versus-Host Disease Model

Introduction Inflammatory monocytes are recruited to target organs during acute Graft versus Host Disease (aGVHD). As seen in other autoimmune disorders, inflammatory monocytes play an important role in antigen presentation and cytokine production. These actions allow for a sustained activation and proliferation signal to T-cells. Objective Previous studies have shown that IMP treatment in mouse models of colitis, encephalitis, myocardial infarction and peritonitis markedly reduced monocyte accumulation in the affected end-organs – promoting tissue repair; reducing disease symptoms and increasing survival. Therefore, our objective was to test clinical outcomes after IMP treatment in a mouse model of aGVHD. Methods Murine aGVHD model: BALB/c mice were given 800 cGy total body irradiation, irradiated BALB/c mice were transplanted with 5 × 106 C57BL/6 bone marrow cells and 1 × 106 C57BL/6 spleen cells via tail vein. IMP treatment: IMPs were made with PLGA (Phosphorex Inc, Hopkinton MA) was administered to the recipient mice (1.4 mg/kg body weight) by IV daily starting from day 5 to day 10 after bone marrow transplantation (BMT). PBS at the same volume was used as vehicle control. In vivo bioluminescence imaging: Mice were given an intraperitoneal injection of luciferin (150 mg/kg body weight) and then anesthetized and imaged using the IVIS Imaging system (Xenogen). Imaging data were analyzed and quantified with Living Image Software (Xenogen). Results IMP treated mice had significantly less severe acute GVHD symptoms (average score of 2.48) than the untreated BM+Sp group (average score 3.96) starting at the time of IMP treatment (days 5-10) and remained with significantly reduced symptoms for the 30 day course (Figure 1). IMP treatment also rescued BM+Sp mice from aGVHD associated mortality with a 30-day overall survival of 62% compared to 4% in the untreated BM+Sp group (Figure 2). Intestinal tissue from the IMP treated mice compared to the BM+Sp mice demonstrated less evidence of aGVHD (an average score of 1.25 and 2.75, respectively). Hepatic tissue from the IMP treated mice compared to the BM+Sp mice demonstrated less evidence of aGVHD (an average score of 1.5 and 2.42, respectively) (Figure 3). IMP treatment also significantly reduced INF-γ levels in the intestinal tissues of treated mice compared to untreated BM+Sp mice. In the mice infused with lymphoma cells (A20-luc), IMP treatment reduced aGVHD symptoms and death while preserving the GVL effect. (Figure 4) Conclusions Our results demonstrate that IMPs significantly reduce symptoms and mortality in a murine model of aGVHD while preserving GVL. The reduction in inflammatory monocytes with IMPs leads to a reduction in inflammatory cytokines, hepatic lymphocyte infiltration and intestinal mucosal denudation. These findings highlight the potential of IMP therapy as a specific and potentially safe treatment in acute GVHD.

HLA Class-II Expression in Human Tumors.

HLA class II molecules play a pivotal role in antigen presentation to T lymphocytes. This chapter analyzed the expression of these molecules in different human tumors and their role in cancer progression. The possible connection between tumor HLA class II expression and the pathogenesis of autoimmune diseases is discussed.

Samsum ant venom modulates the immune response and redox status at the acute toxic dose in vivo.

Background: Ant venoms express surface molecules that participate in antigen presentation involving pro- and anti-inflammatory cytokines. This work aims to investigate the expression of MHC-II, CD80 and CD86 on the polymorphonuclear cells (PMNs) in rats injected with samsum ant venom (SAV).Methods: Rats were divided into three groups - control, SAV-treated (intraperitoneal route, 600 μg/kg), and SAV-treated (subcutaneous route, 600 μg/kg). After five doses, animals were euthanized and samples collected for analysis.Results: The subcutaneous SAV-trated rats presented decreased levels of glutathione with increased cholesterol and triglyceride levels. Intraperitoneal SAV-treated animals displayed significantly reduced concentrations of both IFN-γ and IL-17 in comparison with the control group. However, intraperitoneal and subcutaneous SAV-treated rats were able to upregulate the expressions of MHC-II, CD80 and CD86 on PMNs in comparison with the control respectively. The histological examination showed severe lymphocyte depletion in the splenic white pulp of the intraperitoneal SAV-injected rats.Conclusion: Stimulation of PMNs by SAV leads to upregulation of MHC-II, CD 80, and CD 86, which plays critical roles in antigen presentation and consequently proliferation of T-cells. Subcutaneous route was more efficient than intraperitoneal by elevating MHC-II, CD80 and CD86 expression, disturbing oxidative stability and increasing lipogram concentration.

Maturation of dendritic cells by maitake α-glucan enhances anti-cancer effect of dendritic cell vaccination

Dendritic cells (DCs) play a primary role in antigen presentation to CD4+ and CD8+ T cells and induce acquired immune response against cancer cells. Therefore, determining positive modulators of DC activation to improve therapeutic approaches for cancer immunotherapy is greatly needed. In this study, we investigated the effect of maitake α-glucan YM-2A, isolated from Grifola frondosa, on the maturation and function of DCs and its adjuvant effect on a tumor-associated antigen (TAA)-loaded DC vaccine against murine tumor. We showed that YM-2A induced morphological changes and increased cell-surface maturation markers and cytokine production in DCs. In a mixed lymphocyte reactions assay, YM-2A-treated DCs increased proliferation and production of IFN-γ by allogeneic CD4+ and CD8+ T cells. These results indicate that YM-2A phenotypically and functionally activates DCs. Furthermore, YM-2A-treated TAA-loaded DC vaccine significantly reduced tumor growth and improved survival in two murine tumor models, CT-26 tumor-bearing BALB/c mice and B16 melanoma-bearing C57BL/6 mice. YM-2A-treated TAA-loaded DC vaccine increased splenic IFN-γ producing CD4+ and CD8+ T cells in CT-26 tumor-bearing BALB/c mice. Antibody neutralization studies indicated that YM-2A-induced DC maturation is mediated, in part, by the Dectin-1-dependent pathway. Overall, YM-2A-treatment with a TAA-loaded DC vaccine could be an excellent candidate for immunotherapy against cancer.

Invariant chain p41 mediates production of soluble MHC class II molecules

Major histocompatibility complex class II (MHC II) molecules are mainly expressed on antigen presentation cells and play an important role in immune response. It has been reported that MHC II molecules are also detected in serum as a soluble form (sMHC II molecules), and they are considered to be involved in the maintenance of self-tolerance. However, the mechanism by which sMHC II molecules are produced remains unclear. Invariant chain (Ii), also called CD74, plays an important role in antigen presentation of MHC II molecules. In the present study, we analyzed the role of Ii on the production of sMHC II molecules. We found that the amount of sMHC II molecules in serum was decreased in Ii-deficient mice compared to wild-type mice. sMHC II molecules were secreted from cells transfected with MHC II molecules and Ii but not from cells transfected with MHC II molecules alone. Moreover, isoform p41 of Ii-transfected cells induced more sMHC II molecules compared to isoform p31-transfected cells. The molecular weight of sMHC II molecules from MHC II and Ii p41-transfected cells was approximately 60 kDa, indicating that sMHC II molecules are a single heterodimer of α and β chains that is not associated with micro-vesicles. From the analysis of Ii-deletion mutants, we found that the luminal domain of Ii p41 is crucial for the production of sMHC II molecules. These results suggested that Ii has an important role in production of sMHC II molecules.

The Expression of CD83 Would be Increased in CD34-Positive Monocytes Detected in Peripheral Blood Mobilized By G-CSF in Humans

[Background]CD34-positive monocytes (CD34+mono) have recently been identified following mobilization by granulocyte-colony stimulating factor (G-CSF), and have been suggested to have a potential to modulate immune functions in animal models. However, the biological feature of CD34+mono in humans still remains unclear. Thus, we explored the difference between CD34+mono, CD34+cells, and monocytes through the analyses of gene expression profiles (GEP).[Methods]CD34+mono (Lin-CD34+CD33+CD14+CD11b+, Figure1), CD34+cells (Lin-CD34+CD33-CD14-CD11b- ), and monocytes (Lin-CD34-CD33+CD14+CD11b+) were directly sorted into tubes from cryopreserved grafts from three healthy donors. After the extraction of total RNA, microarray analyses were performed with GeneChip™ 3' IVT Pico Kit and analyzed according to the algorithm of Transcriptome Analysis Console (Thermo Fisher Scientific). Condition false discovery rate (FDR) F-test was used for filtering genes, and the threshold was <0.0005 for hierarchical clustering without considering fold changes. In addition, enrichment analyses were performed for gene ontology (GO) and pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG) through Database for Annotation, Visualization and Integrated Discovery (DAVID). The differentially expressed genes with <0.05 of F-test values and 2 or more fold changes in CD34+mono compared with CD34+cells and monocytes were selected for the enrichment analyses. Thereafter, the protein-protein interaction (PPI) network in CD34+mono was constructed through Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), which is a biological database. This study was approved by the institutional review board of Jichi Medical University and all subjects gave their written informed consent for the cryopreservation and analysis of the blood samples in accordance with the Helsinki Declaration.[Results]CD34+mono resembled con-mono in the appearance, although it might look slightly immature. In fact, the number of differential expressed genes was smaller in the pair of CD34+mono vs. monocytes (n=605) compared with the other pair (n=2136), suggesting that CD34+mono might stand nearby monocytes. However, a principle component analysis of GEP demonstrated that CD34+mono would be categorized into an independent cell type entity. In addition, the hierarchical clustering heat map suggested that CD34+mono would have the interim gene expressions between CD34+ cells and monocytes (Figure 2).GO analysis was also performed using the 203 genes which were differentially expressed in CD34+mono compared with CD34+cells and monocytes. The differentially expressed genes seemed to be significantly involved in the “immune system process” of biological process, followed by “locomotion”, “metabolic process” and “response to stimulus”, because pro-inflammatory genes in CD34+mono like IL6, CCL3, IL8, VEGFA, and IL1A were increased.In addition, PPI analyses indicated that 128 of the 203 differentially expressed genes in CD34+mono had close connections with each other. Especially, IL6, VEGFA, IL8, NFkB1, EGR1, CDKN1A (p21), and CYCS could be considered as a hub gene in CD34+mono. Of them, IL6, IL8, and VEGFA are considered to be pro-inflammatory cytokines. NFkB1, and EGR1 are transcriptional factors, and CDKN1A, and CYCS are considered to regulate cell cycle or apoptosis. Furthermore, simultaneously focusing on the difference in the fold changes of gene expressions between CD34+mono vs. monocytes, CD83 (a membrane protein and immunoglobulin superfamily that regulates antigen presentation) and FOSL1 (a kind of regulators of cell proliferation, differentiation, and transformation) were specifically increased in CD34+mono (Figure3).The increased expression of CD83 strongly suggests antigen presenting cells like mature dendritic cells. CD34+mono might be one of progenitors of dendritic cells, or might have a potential of antigen presentation itself.[Conclusion]In summary, the differentially expressed genes in CD34+mono would be mapped in the immune process, especially against infectious pathogens. In addition, the increased CD83 suggested that CD34+mono might play a role of antigen presentation in the immune response. Further investigations would be necessary to confirm the clinical roles of CD34+mono after transplantation. [Display omitted] DisclosuresNakasone:Phizer: Honoraria; Novartis: Honoraria; Kyowa Hakko Kirin: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen: Honoraria; Takeda: Honoraria. Kimura:Astellas: Honoraria; Pfizer: Honoraria; Sumitomo Dainippon Pharma: Honoraria; MSD: Other: Investigator in the institute; Nippon Kayaku: Honoraria; Celgene: Honoraria; Kyowa Hakko Kirin: Honoraria; Takeda: Honoraria. Kako:Takeda Pharmaceutical Company Limited.: Honoraria; Takeda Pharmaceutical Company Limited.: Honoraria; Celgene K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria. Kanda:Eisai: Consultancy, Honoraria, Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; MSD: Research Funding; Nippon-Shinyaku: Research Funding; Pfizer: Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; Taisho-Toyama: Research Funding; CSL Behring: Research Funding; Tanabe-Mitsubishi: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Ono: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.

Selective DNAM-1 expression on small peritoneal macrophages contributes to CD4+ T cell costimulation

Mouse peritoneal macrophages consist of two subsets: large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs), defined as CD11bhiF4/80hi and CD11b+F4/80lo cells, respectively. We reveal that SPMs, but not LPMs, have the ability to present antigens to naïve CD4+ T cells. Coculture of SPMs with naïve ovalbumin (OVA) specific CD4+ T cells (OT-II) in the presence of OVA peptide effectively induced CD4+ T cells priming. SPMs, but not LPMs, strongly express DNAM-1, an activating immunoreceptor. Although antigen uptake and processing were comparable between WT and DNAM-1-deficient SPMs, deficiency of DNAM-1 on SPMs or blockade of DNAM-1 and its ligand interaction impaired CD4+ T cells priming by SPMs. Furthermore, T and B cell responses in mediastinal lymph nodes of mice intraperitoneally immunized with trinitrophenyl (TNP)–OVA protein in Alum adjuvant were enhanced by intraperitoneally transferred wild-type, but not DNAM-1-deficient, SPMs. We propose that SPMs are functionally distinct from LPMs, and DNAM-1 plays a costimulatory role in antigen presentation by SPMs.

Intrahepatic Cross-Presentation and Hepatocellular Antigen Presentation Play Distinct Roles in the Induction of Hepatitis B Virus-Specific CD8+ T Cell Responses.

CD8+ T cells are the key cellular effectors mediating the clearance of hepatitis B virus (HBV) infections. However, early immunological events surrounding the priming of HBV-specific CD8+ T cell responses remain poorly understood. This study examined the importance of priming location and the relative contribution of endogenous antigen presentation by hepatocytes versus cross-presentation by bone marrow-derived cells to the induction of functional HBV-specific CD8+ T cell responses using the animal models of acute and chronic HBV infection. Functional HBV-specific CD8+ T cell responses could be induced to intrahepatically expressed HBV even when T cell homing to the lymphoid tissues was severely suppressed, suggesting that functional priming could occur in the liver. The expansion of HBV-specific CD8+ T cells was significantly reduced in the mice whose major histocompatibility complex (MHC) class I expression was mostly restricted to nonhematopoietic cells, suggesting the importance of cross-presentation by hematopoietic cells in the induction of HBV-specific CD8+ T cells. Strikingly, the expansion and cytolytic differentiation of HBV-specific CD8+ T cells were reduced even more severely in the mice whose MHC class I expression was restricted to hematopoietic cells. Collectively, these results indicate that cross-presentation is required but relatively inefficient in terms of inducing the cytolytic differentiation of HBV-specific CD8+ T cells by itself. Instead, the expansion and functional differentiation of HBV-specific CD8+ T cells are primarily dependent on hepatocellular antigen presentation.IMPORTANCE Hepatitis B virus (HBV) causes acute and chronic hepatitis. Approximately 260 million people are chronically infected with HBV and under an increased risk of developing cirrhosis and hepatocellular carcinoma. Host immune responses, particularly HBV-specific CD8+ T cell responses, largely determine the outcome of HBV infection. It is widely accepted that antigen inexperienced CD8+ T cells should be initially activated by professional antigen-presenting cells (pAPCs) in lymphoid tissues to differentiate into effector CD8+ T cells. However, this notion has not been tested for HBV-specific CD8+ T cells. In this study, we show that HBV-specific CD8+ T cell responses can be induced in the liver. Surprisingly, antigen presentation by hepatocytes is more important than cross-presentation by hematopoietic cells for the induction of HBV-specific CD8+ T cell responses. These results revealed a previously unappreciated role of antigen presentation by hepatocytes in the induction of HBV-specific CD8+ T cell responses.

Presence of increased inflammatory infiltrates accompanied by activated dendritic cells in the left atrium in rheumatic heart disease.

AimsLeft atrial (LA) structural remodelling develops in rheumatic heart disease (RHD) according to the disease severity of the mitral valve and the presence of atrial fibrillation. Sustained active inflammation has been previously reported in the LA of patients with RHD, suggesting a direct role of cell-mediated immunity in the pathogenesis of LA remodelling. Dendritic cells (DCs) have a major antigen-presenting role, and are known as crucial modulators of innate and adaptive immunity. We investigated whether DCs are involved in the pathogenesis of LA remodelling in RHD.Methods and resultsImmunohistochemical analyses were performed using antibodies to CD11c, CD209 and CD80 as markers of myeloid DCs, migratory-active DCs, mature DCs and infiltrated inflammatory cells including T lymphocytes (CD3) and M1 (CD68; pro-inflammatory profile) and M2 (CD163; pro-resolution profile) macrophages. Furthermore, tenascin-C, an extracellular matrix (ECM) protein that appears during ECM remodelling and inflammatory response, was examined. Infiltrated myeloid DCs, migratory-active DCs, mature DCs and other inflammatory infiltrates including T lymphocytes and M1 and M2 macrophages, were significantly higher in the RHD group than the non-RHD group. The positive area fraction for tenascin-C was significantly higher in the RHD group than in the non-RHD group.ConclusionOur histological findings suggest that inflammation may persist long after a bout of rheumatic fever, ultimately leading to ECM remodelling. We identified and quantitatively assessed several subsets of DCs and other immunocompetent cells, and our results indicated that activation of DCs has some role in persistence of LA inflammation in patients with chronic RHD.

Epithelial MHC Class II Expression and Its Role in Antigen Presentation in the Gastrointestinal and Respiratory Tracts.

As the primary barrier between an organism and its environment, epithelial cells are well-positioned to regulate tolerance while preserving immunity against pathogens. Class II major histocompatibility complex molecules (MHC class II) are highly expressed on the surface of epithelial cells (ECs) in both the lung and intestine, although the functional consequences of this expression are not fully understood. Here, we summarize current information regarding the interactions that regulate the expression of EC MHC class II in health and disease. We then evaluate the potential role of EC as non-professional antigen presenting cells. Finally, we explore future areas of study and the potential contribution of epithelial surfaces to gut-lung crosstalk.

A Novel Antigen-Sampling Cell in the Teleost Gill Epithelium With the Potential for Direct Antigen Presentation in Mucosal Tissue.

In mammals, M cells can take up antigens through mucosal surfaces of the gut and the respiratory tract. Since M cells are deficient of lysosomes and phagosomes, the antigens are directly delivered to the mucosa-associated lymphoid tissue (MALT) without degradation. In teleost fish, the entire body surface (gills, skin, and intestinal system) is covered by mucus; however, specific antigen-sampling cells have not yet been identified in their mucosal tissues. Here, we show that two phenotypes of antigen-sampling cells take up antigens through epithelial surfaces of the rainbow trout gill. One phenotype of antigen-sampling cells has features of monocyte/macrophage/dendritic cell-type cells; they have large vacuoles in the cytoplasm and express PTPRC (CD45), CD83, IL-1β, and IL-12p40b. The second phenotype exhibits similar characteristics to mammalian M cells; the corresponding cells bind the lectin UEA-1 but not WGA and show expression of M cell marker gene Anxa5. In contrast to mammalian M cells, teleost M-type cells were found to exhibit small vacuoles in their cytoplasm and to express almost all genes related to the “phagosome”, “lysosome,” and “antigen processing and presentation” pathways. Furthermore, MHC class II was constitutively expressed on a fraction of M-type cells, and this expression was significantly increased after antigen uptake, suggesting that the MHC class II is inducible by antigen stimulation. Here, we suggest that teleost M-type cells play a role in the phylogenetically primitive teleost immune system, similar to bona-fide M cells. In addition, the presence of MHC class II expression suggests an additional role in antigen presentation in the gills, which are an organ with high T cell abundance, especially in interbranchial lymphoid tissue. The present results suggest an unconventional antigen presentation mechanism in the primitive mucosal immune system of teleosts, which generally lack highly organized lymphoid tissues. Moreover, the results of this work may be valuable for the development of mucosal vaccines that specifically target M-type cells; mucosal vaccines significantly reduce working costs and the stress that is usually induced by vaccination via injection of individual fish.

Host genetic factors affecting hepatitis B infection outcomes: Insights from genome-wide association studies.

The clinical outcome of hepatitis B virus (HBV) infection depends on the success or failure of the immune responses to HBV, and varies widely among individuals, ranging from asymptomatic self-limited infection, inactive carrier state, chronic hepatitis, cirrhosis, hepatocellular carcinoma, to liver failure, depending on the success or failure of immune response to HBV. Genome-wide association studies (GWAS) identified key genetic factors influencing the pathogenesis of HBV-related traits. In this review, we discuss GWAS for persistence of HBV infection, antibody response to hepatitis B vaccine, and HBV-related advanced liver diseases. HBV persistence is associated with multiple genes with diverse roles in immune mechanisms. The strongest associations are found within the classical human leukocyte antigen (HLA) genes, highlighting the central role of antigen presentation in the immune response to HBV. Associated variants affect both epitope binding specificities and expression levels of HLA molecules. Several other susceptibility genes regulate the magnitude of adaptive immune responses, determining immunity vs tolerance. HBV persistence and nonresponse to vaccine share the same risk variants, implying overlapping genetic bases. On the other hand, the risk variants for HBV-related advanced liver diseases are largely different, suggesting different host-virus dynamics in acute vs chronic HBV infections. The findings of these GWAS are likely to pave the way for developing more effective preventive and therapeutic interventions by personalizing the management of HBV infection.

Th17 activation by dendritic cells stimulated with gamma-irradiated Streptococcus pneumoniae

Dendritic cells (DCs) play an important role in antigen presentation, which is an essential step for the induction of antigen-specific adaptive immunity. Inactivated bacterial whole cell vaccines have been widely used to prevent many bacterial infections because they elicit good immunogenicity due to the presence of various antigens and are relatively inexpensive and easy to manufacture. Recently, gamma-irradiated whole cells of nonencapsulated Streptococcus pneumoniae were developed as a broad-spectrum and serotype-independent multivalent vaccine. In the present study, we generated gamma-irradiated S. pneumoniae (r-SP) and investigated its capacity to stimulate mouse bone marrow-derived DCs (BM-DCs) in comparison with heat-inactivated and formalin-inactivated S. pneumoniae (h-SP and f-SP, respectively). r-SP showed an attenuated binding and internalization level to BM-DCs when compared to h-SP or f-SP. r-SP weakly induced the expression of CD80, CD83, CD86, MHC class I, and PD-L2 compared with h-SP or f-SP. Furthermore, r-SP less potently induced IL-6, TNF-α, and IL-23 expression than h-SP or f-SP but more potently induced IL-1β expression than h-SP or f-SP in BM-DCs. Since Th17-mediated immune responses are known to be important for the protection against pneumococcal infections, r-SP-primed DCs were co-cultured with splenocytes or splenic CD4+ T cells. Interestingly, r-SP-sensitized BM-DCs markedly induced IL-17A+ CD4+ T cells whereas h-SP- or f-SP-sensitized BM-DCs weakly induced them. Collectively, these results suggest that r-SP could be an effective pneumococcal vaccine candidate eliciting Th17-mediated immune responses by stimulation of DCs.

Inhibitors of elastase stimulate murine B lymphocyte differentiation into IgG- and IgA-producing cells.

It is well established that dendritic cells and macrophages play a role in antigen presentation to B and T cells and in shaping B and T cell responses via cytokines they produce. We have previously reported that depletion of neutrophils improves the production of mucosal IgA after sublingual immunization with Bacillus anthracis edema toxin as adjuvant. These past studies also demonstrated that an inverse correlation exists between the number of neutrophils and production of IgA by B cells. Using specific inhibitors of elastase, we addressed whether the elastase activity of neutrophil could be the factor that interferes with production of IgA and possibly other immunoglobulin isotypes. We found that murine splenocytes and mesenteric lymph node cells cultured for 5 days in the presence of neutrophil elastase inhibitors secreted higher levels of IgG and IgA than cells cultured in the absence of inhibitors. The effect of the inhibitors was dose-dependent and was consistent with increased frequency of CD138+ cells expressing IgG or IgA. Finally, neutrophil elastase inhibitors increased transcription of mRNA for AID, IL-10, BAFF and APRIL, factors involved in B cell differentiation. These findings identify inhibitors of elastase as potential adjuvants for increasing production of antibodies.

Sustained cross-presentation capacity of murine splenic dendritic cell subsets in vivo.

An exclusive feature of dendritic cells (DCs) is their ability to cross‐present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen‐antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α+ DCs showed sustained MHC class I‐peptide specific CD8+ T‐cell activation for more than 4 days. CD8α− DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α− DCs were able to present antigen in MHC class II to specific CD4+ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T‐cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T‐cell activation and have distinct roles in antigen presentation to specific T cells in vivo.

The C-terminal extension landscape of naturally presented HLA-I ligands

HLA-I molecules play a central role in antigen presentation. They typically bind 9- to 12-mer peptides, and their canonical binding mode involves anchor residues at the second and last positions of their ligands. To investigate potential noncanonical binding modes, we collected in-depth and accurate HLA peptidomics datasets covering 54 HLA-I alleles and developed algorithms to analyze these data. Our results reveal frequent (442 unique peptides) and statistically significant C-terminal extensions for at least eight alleles, including the common HLA-A03:01, HLA-A31:01, and HLA-A68:01. High resolution crystal structure of HLA-A68:01 with such a ligand uncovers structural changes taking place to accommodate C-terminal extensions and helps unraveling sequence and structural properties predictive of the presence of these extensions. Scanning viral proteomes with the C-terminal extension motifs identifies many putative epitopes and we demonstrate direct recognition by human CD8+ T cells of a 10-mer epitope from cytomegalovirus predicted to follow the C-terminal extension binding mode.

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo.

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP−/−) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty virions may allow us to rationally design effective strategies to prevent elimination of AAV transduced target cells by capsid specific CD8+ T cells.

Identification of a Potential Common Ancestor for Mammalian Cross-Presenting Dendritic Cells in Teleost Respiratory Surfaces

Dendritic cells (DCs) are highly specialized antigen-presenting cells that bridge innate and adaptive immune responses in vertebrates, being key modulators in the initiation of specific responses. Although teleost fish present the main elements of a fully developed adaptive immune system, not many studies have focused on identifying specific DC subsets in teleost species. Previous work from our group identified in rainbow trout (Oncorhynchus mykiss) skin a DC subpopulation co-expressing CD8α and major histocompatibility complex II β on the cell surface. Interestingly, these CD8+ DCs expressed common unique markers of mammalian cross-presenting DCs, a DC subset with an important role in antigen presentation and activation of CD8+ T cytotoxic lymphocytes. In this study, we have identified a similar DC subset in rainbow trout gills that also transcribes molecules uniquely expressed on diverse mammalian cross-presenting DC populations such as CD8, CD103, CD141, Batf3, IFN regulatory protein 8, and toll-like receptor 3. Hence, we have undertaken a broad phenotypic and functional characterization of this new DC subset that includes the confirmation of novel capacities for DCs in teleost, such an IgM-binding capacity and responsiveness to CD40 ligand. Furthermore, our results show that in gills, this DC subset shows some different phenotypic and functional characteristics when compared with their homologs in the skin, suggesting an adaptation of the cells to different mucosal tissues or different maturation status depending on their location. Our findings contribute to increase our knowledge on fish cross-presenting DCs, an important cell population to take into account for the future design of mucosal vaccination strategies.

HSC extrinsic sex-related and intrinsic autoimmune disease–related human B-cell variation is recapitulated in humanized mice

AbstractB cells play a major role in antigen presentation and antibody production in the development of autoimmune diseases, and some of these diseases disproportionally occur in females. Moreover, immune responses tend to be stronger in female vs male humans and mice. Because it is challenging to distinguish intrinsic from extrinsic influences on human immune responses, we used a personalized immune (PI) humanized mouse model, in which immune systems were generated de novo from adult human hematopoietic stem cells (HSCs) in immunodeficient mice. We assessed the effect of recipient sex and of donor autoimmune diseases (type 1 diabetes [T1D] and rheumatoid arthritis [RA]) on human B-cell development in PI mice. We observed that human B-cell levels were increased in female recipients regardless of the source of human HSCs or the strain of immunodeficient recipient mice. Moreover, mice injected with T1D- or RA-derived HSCs displayed B-cell abnormalities compared with healthy control HSC-derived mice, including altered B-cell levels, increased proportions of mature B cells and reduced CD19 expression. Our study revealed an HSC-extrinsic effect of recipient sex on human B-cell reconstitution. Moreover, the PI humanized mouse model revealed HSC-intrinsic defects in central B-cell tolerance that recapitulated those in patients with autoimmune diseases. These results demonstrate the utility of humanized mouse models as a tool to better understand human immune cell development and regulation.

The major histocompatibility complex class I immunopeptidome of extracellular vesicles

Extracellular vesicles (EVs) are released by most cell types and have been associated with multiple immunomodulatory functions. MHC class I molecules have crucial roles in antigen presentation and in eliciting immune responses and are known to be incorporated into EVs. However, the MHC class I immunopeptidome of EVs has not been established. Here, using a small-scale immunoisolation of the antigen serotypes HLA-A*02:01 and HLA-B*27:05 expressed on the Epstein-Barr virus-transformed B cell line Jesthom and MS of the eluted peptides from both cells and EVs, we identified 516 peptides that bind either HLA-A*02:01 or HLA-B*27:05. Of importance, the predicted serotype-binding affinities and peptide-anchor motifs did not significantly differ between the peptide pools isolated from cells or EVs, indicating that during EV biogenesis, no obvious editing of the MHC class I immunopeptidome occurs. These results, for the first time, establish EVs as a source of MHC class I peptides that can be used for the study of the immunopeptidome and in the discovery of potential neoantigens for immunotherapies.