Antigen Presentation Process Research Articles

Glycolipid sensing and innate immunity in paracoccidioidomycosis.

Distinct glycolipid profiles are described in microorganisms, which have been shown to modulate the innate immune system. We tested the hypothesis that glycosphingolipids from Paracoccidioides brasiliensis have immunomodulatory properties on monocytes and dendritic cells of two groups of healthy individuals, one cured of paracoccidioidomycosis in the past (CUR-I) and the other nonexposed to P. brasiliensis (HNE-I). Two classes of glycosphingolipids purified from yeast cells were evaluated: a neutral glycosphingolipid, monohexosylceramide (CMH), and acidic glycosylinositolphosphorylceramides (GIPCs). Both glycosphingolipids affected the functioning of innate immunity cells, interfering with the antigen presenting process: P. brasiliensis yeast cells phagocytosis, IL-10 secretion, and costimulatory molecules and recognition receptors expression by monocytes were altered, while dendritic cell antigen presentation to autologous T cells was markedly down-modulated as shown by reduced T-cell proliferative responses. The mechanisms by which CMH and GIPCs exert their effects differ since the target cells did not always respond similarly to the challenge with the glycosphingolipids. Moreover, CUR-I and HNE-I presented different responses to the glycosphingolipids. Differences not only in the glycosphingolipid structure (such as the polar head group or the ceramide moiety), but also in the innate immunity properties of CUR-I and HNE-I, may underlie these differences and contribute to individual's susceptibility or resistance to develop paracoccidioidomycosis.

Dissecting a highly immunogenic antigen presentation process triggered by a natural pathogenic antigen: listeriolysin O (APP3P.104)

Abstract Listeriolysin O (LLO) is the major immunogen and virulence factor secreted by L. monocytogenes. Peptides processed from the LLO are presented at low nanomolar concentration to CD4 T cells. The mechanism for the efficiency of this antigen presentation process remains unknown and may provide novel insight into antigen processing and presentation. We tested LLO mutants with impaired molecular properties, such as membrane binding, cytolytic activity, and catabolism. All LLO variants triggered T cell responses at low nanomolar concentrations. Because LLO presentation was independent of any known activity intrinsic to LLO, we investigated cellular mechanisms that may increase its antigenicity. All LLO mutants were taken up into cells at high amounts in large undefined vesicles. Based on this, we hypothesized that bulk protein uptake of LLO could cause a novel cellular stress mechanism that would explain its immunogenicity. Endoplasmic reticulum stress (ERS) is a cellular response that deals with large amounts of unfolded protein. We investigated if the entry of LLO into APC triggered the ERS. Treatment with nanomolar concentrations of LLO caused an increase in Chop and Gadd34 expression, as well as other canonical markers of the ERS. However, it did not induce Xbp1 splicing or the ER-chaperone response. In conclusion, LLO may be activating the Perk pathway leading to an ERS in APC. The biological and antigen presentation outcomes of this response remain under investigation.

Dietary plant extracts modulate gene expression profiles in ileal mucosa of weaned pigs after an Escherichia coli infection1

This study was conducted to characterize the effects of infection with a pathogenic F-18 Escherichia coli and 3 different plant extracts on gene expression of ileal mucosa in weaned pigs. Weaned pigs (total = 64, 6.3 ± 0.2 kg BW, and 21-d old) were housed in individual pens for 15 d, 4 d before and 11 d after the first inoculation (d 0). Treatments were in a 2 × 4 factorial arrangement: with or without an F-18 E. coli challenge and 4 diets (a nursery basal, control diet [CON], 10 ppm of capsicum oleoresin [CAP], garlic botanical [GAR], or turmeric oleoresin [TUR]). Results reported elsewhere showed that the plant extracts reduced diarrhea in challenged pigs. Total RNA (4 pigs/treatment) was extracted from ileal mucosa of pigs at d 5 post inoculation. Double-stranded cDNA was amplified, labeled, and further hybridized to the microarray, and data were analyzed in R. Differential gene expression was tested by fitting a mixed linear model in a 2 × 4 factorial ANOVA. Bioinformatics analysis was conducted by DAVID Bioinformatics Resources 6.7 (DAVID; National Institute of Allergy and Infectious Diseases [NIAID, NIH], http://david.abcc.ncifcrf.gov). The E. coli infection altered (P < 0.05) the expression of 240 genes in pigs fed the CON (148 up- and 92 down-regulated). Compared with the infected CON, feeding CAP, GAR, or TUR altered (P < 0.05) the expression of 52 genes (18 up, 34 down), 117 genes (34 up- and 83 down-regulated), or 84 genes (16 up- and 68 down-regulated), respectively, often counteracting the effects of E. coli. The E. coli infection up-regulated (P < 0.05) the expression of genes related to the activation of immune response and complement and coagulation cascades, but down-regulated (P < 0.05) the expression of genes involved in protein synthesis and accumulation. Compared with the CON, feeding CAP and GAR increased (P < 0.05) the expression of genes related to integrity of membranes in infected pigs, indicating enhanced gut mucosa health. Moreover, feeding all 3 plant extracts reduced (P < 0.05) the expression of genes associated with antigen presentation or other biological processes of immune responses, indicating they attenuated overstimulation of immune responses caused by E. coli. These findings may explain why diarrhea was reduced and clinical immune responses were ameliorated in infected pigs fed plant extracts. In conclusion, plant extracts altered the expression of genes in ileal mucosa of E. coli-infected pigs, perhaps leading to the reduction in diarrhea reported previously.

Antigen Presentation and T Cell Response in Umbilical Cord Blood and Adult Peripheral Blood

The efficiency of the immune response is well-known to be decreased in the perinatal period compared to adulthood. Several factors may play a role in this finding, including immaturity of adaptive immune responses, as well as alterations in the prevalence and functionality in elements of humoral and cellular immune reactions compared to adult-type immunity. The process of antigen presentation and adequate T cell function are cornerstone features in coordinating the immune response already at this early age. Over the recent decades, several studies have revealed remarkable details that contribute to these alterations. However, many aspects of the exact mechanisms are still not fully understood. In this review, we aim to summarize current knowledge of studies of altered cell prevalence and functionality that contribute to differences of antigen presentation and the T cell immune response between the perinatal and adult periods. Decreased level of antigen presentation, lower expression of costimulatory molecules, lower Th1 and Th17 response, and deficient function of regulatory elements are the most important differences in CB compared with adult peripheral blood. These differences are of practical importance from two distinct aspects. First, the decreased efficiency of the immune response plays an important role in the development of several diseases affecting preterm and term neonates, as well as in a higher incidence of infections compared to adults. Second, umbilical cord blood (UCB)-derived hematopoietic stem cells are widely used in the treatment of different hematological and immunological disorders. The prevalence of graft-versus-host disease (GVHD) is lower upon UCB-derived stem cell transplantation compared to adult peripheral blood or bone marrow-derived stem cells. Therefore, the deeper understanding of the mechanisms contributing to a decreased T cell response is of importance in improving therapeutic efficiency in related disorders.

Tumor immunotherapy based on tumor-derived heat shock proteins (Review)

Heat shock proteins (HSPs), the most important type of molecular chaperone, are expressed in all eukaryotic cells and have multiple functions, including the folding and unfolding of other proteins and peptides, the transport of proteins and peptides and the support of antigen presentation processes. Due to these important properties, the use of HSPs has been explored as a promising tumor immunotherapy strategy. It has been previously demonstrated that HSP peptide complex (HSP.PC) derived from tumors is the immunogenic entity that elicits powerful antitumor immune responses. Previous animal studies and phase III clinical trials have demonstrated the efficacy, safety and feasibility of HSP-based tumor vaccines. However, the limitations are also apparent and specific alternatives have been developed. The present review focused on the history of HSP-based immunotherapy, the mechanism of its immunogenicity and the previous efforts to promote the efficacy. The current review may be useful for antitumor studies based on the tumor-derived HSPs.

Multifunctional Nanorods Serving as Nanobridges To Modulate T Cell-Mediated Immunity

Electrodeposited nanorods serving as multivalent bridges were fabricated and surface-decorated with ligands for immune cells. Gold and nickel solutions were sequentially electrodeposited on nanoporous anodized disc templates and the template was dissolved to retrieve bisegmented nanorods with different lengths. Gold and nickel segmented nanorods were surface-immobilized with mannose and RGD peptides to prepare immune-cell recruiting nanorods. Surface-functionalization of nanorods were confirmed by fluorescence-labeling of each ligands and confocal microscopy. Dendritic cells and T cells were co-incubated with the surface-functionalized nanorods, and the proximity between the nanorods and the immune cells was visualized by variable pressure scanning electron microscopy and confocal microscopy. The long nanorods were associated with the immune cells, whereas the shorter nanorods were rather endocytosed by cells, suggesting a feasibility of the longer nanorods as bridging for the cells. Cytokine releases from the immune cells were monitored by cultivating lipopolysaccharide-activated dendritic cells with T cells. Interleukine-2 and interferon-γ release profiles showed a strong correlation with the length of the nanorod, where the 4 μm nanorods induced the highest levels of cytokine release compared to 1 or 2 μm nanorods. Thus, we concluded that the proximity of the immune cells increased by bridging the immune cells with the nanobridging system, which subsequently increased cytokine release by facilitating the antigen presentation process.

A fat story—antigen presentation by butyrophilin 3A1 to γδ T cells

A fat story—antigen presentation by butyrophilin 3A1 to γδ T cells

Inflammatory mediators from monocytes down-regulate cellular proliferation and enhance cytokines production in patients with polar clinical forms of Chagas disease

Exposure to Trypanosoma cruzi parasites induces monocytes and macrophages to produce various endogenous mediators, including prostaglandins and cytokines. To clarify the involvement of monocytes as an important source of inflammatory mediators in Chagas disease patients, we evaluated PBMC before and after depletion of adherent cells (monocytes) from patients with indeterminate (IND) and cardiac (CARD) clinical forms and from non-infected individuals (NI). We demonstrated that after the partial depletion of adherent cells, production of PGE2 was slightly decreased in patients with Chagas disease. Inhibition of the cells by indomethacin increased the proliferation in PBMC cells from patients after antigen stimulation. Pro-inflammatory cytokines as IL-2 and IFN-γ also had a greater decrease after partial depletion of adherent cells in both clinical forms of Chagas disease. IL-10 and IL-5 levels were also reduced after partial depletion of adherent cells both in IND and CARD patients. In addition, we evaluated the APC potential of B cells and observed that the MHCII and CD80 molecules had an increased expression after partial depletion of most monocytes in all groups. Thus, inflammatory mediators produced by monocytes seem to be important to modulate immune responses in Chagas disease by regulating the processes of inflammation and antigen presentation.

HLA-DO as the Optimizer of Epitope Selection for MHC Class II Antigen Presentation

Processing of antigens for presentation to helper T cells by MHC class II involves HLA-DM (DM) and HLA-DO (DO) accessory molecules. A mechanistic understanding of DO in this process has been missing. The leading model on its function proposes that DO inhibits the effects of DM. To directly study DO functions, we designed a recombinant soluble DO and expressed it in insect cells. The kinetics of binding and dissociation of several peptides to HLA-DR1 (DR1) molecules in the presence of DM and DO were measured. We found that DO reduced binding of DR1 to some peptides, and enhanced the binding of some other peptides to DR1. Interestingly, these enhancing and reducing effects were observed in the presence, or absence, of DM. We found that peptides that were negatively affected by DO were DM-sensitive, whereas peptides that were enhanced by DO were DM-resistant. The positive and negative effects of DO could only be measured on binding kinetics as peptide dissociation kinetics were not affected by DO. Using Surface Plasmon Resonance, we demonstrate direct binding of DO to a peptide-receptive, but not a closed conformation of DR1. We propose that DO imposes another layer of control on epitope selection during antigen processing.

Endoplasmic Reticulum Aminopeptidase-1 Functions Regulate Key Aspects of the Innate Immune Response

Endoplasmic reticulum aminopeptidase-1 (ERAP1) is a multifunctional, ubiquitously expressed enzyme whose peptide-trimming role during antigen processing for presentation by MHC I molecules is well established, however, a role for ERAP1 in modulating global innate immune responses has not been described to date. Here we demonstrate that, relative to wild type mice, mice lacking ERAP1 exhibit exaggerated innate immune responses early during pathogen recognition, as characterized by increased activation of splenic and hepatic NK and NKT cells and enhanced production of pro-inflammatory cytokines such as IL12 and MCP1. Our data also revealed that ERAP1 is playing a critical role in NK cell development and function. We observed higher frequencies of terminally matured NK cells, as well as higher frequencies of licensed NK cells (expressing the Ly49C and Ly49I receptors) in ERAP1-KO mice, results that positively correlated with an enhanced NK activation and IFNγ production by ERAP1-KO mice challenged with pro-inflammatory stimuli. Furthermore, during pathogen recognition, ERAP1 regulates IL12 production by CD11c+ DCs specifically, with increases in IL12 production positively correlated with an increased phagocytic activity of splenic DCs and macrophages. Collectively, our results demonstrate a previously unrecognized, more central role for the ERAP1 protein in modulating several aspects of both the development of the innate immune system, and its responses during the initial stages of pathogen recognition. Such a role may explain why ERAP1 has been implicated by GWAS in the pathogenesis of autoimmune diseases that may be precipitated by aberrant responses to pathogen encounters.

Adipose Tissue Macrophages Are Innate to the Immunological Awareness of Adipose Tissue

The antigen-presentation process constitutes a fundamental basis of the functional immune system in vertebrates. The late Ralph Steinman, who discovered antigen-presenting dendritic cells, commented about the immune system that, “it is diverse beyond compare, tolerant without fail, and capable of behaving appropriately with a myriad of infections and other challenges ” (1). Significant progress has been made in understanding the classical immune response to infections and rare failings of tolerance to self (2). However the underlying mechanisms of how the immune system processes and responds to “ other challenges,” such as sensing and storage of caloric excess in adipose tissue and the integration of immune system with metabolism, remains largely unknown. Multiple studies during the past decade have identified visceral adipose tissue as an important site of residence of leukocytes, including cells of the innate (macrophages and neutrophils) and adaptive immune system (T and B cells) (3–5). It is clear that adipose tissue is a major endocrine organ that controls energy homeostasis. In addition, the presence of a significant number of hematopoietic cells in adipose tissue suggests that immune cells may impart unique immunological properties to the adipose tissue (4). For example, 1 g of enzymatically dispersed adipose tissues can contain up to 5 million stromal vascular fraction (SVF) cells, and after exclusion of adipocytes, ∼50–65% of SVF cells are leukocytes (6). Considering that in severe obesity in humans, the total fat content can constitute up to 50% of the total body mass, adipose tissue thus represents an uncharacterized immunological organ. For such an immunological characterization, specific cells in adipose tissue must be able to capture, process, and present antigens to T cells and mount a functional immunological response. In this issue, Morris et al. (7) further the hypothesis that adipose tissue is immunologically aware by providing tantalizing …

Leishmania Evades Host Immunity by Inhibiting Antigen Cross-Presentation through Direct Cleavage of the SNARE VAMP8

During phagocytosis, microorganisms are taken up by immune cells into phagosomes. Through membrane-trafficking events mediated by SNARE proteins, phagosomes fuse with lysosomes, generating degradative phagolysosomes. Phagolysosomes contribute to host immunity by linking microbial killing within these organelles with antigen processing for presentation on MHC class I or II molecules to Tcells. We show that the intracellular parasite Leishmania evades immune recognition by inhibiting phagolysosome biogenesis. The Leishmania cell surface metalloprotease GP63 cleaves a subset of SNAREs, including VAMP8. GP63-mediated VAMP8 inactivation or Vamp8 disruption prevents the NADPH oxidase complex from assembling on phagosomes, thus altering their pH and degradative properties. Consequently, the presentation of exogenous Leishmania antigens on MHC class I molecules, also known as cross-presentation, is inhibited, resulting in reduced Tcell activation. These findings indicate that Leishmania subverts immune recognition by altering phagosome function and highlight the importance of VAMP8 in phagosome biogenesis and antigen cross-presentation.

Complement opsonization of HIV-1 results in a different intracellular processing pattern and enhanced MHC class I presentation by dendritic cells.

Induction of optimal HIV-1-specific T-cell responses, which can contribute to controlling viral infection in vivo, depends on antigen processing and presentation processes occurring in DCs. Opsonization can influence the routing of antigen processing and pathways used for presentation. We studied antigen proteolysis and the role of endocytic receptors in MHC class I (MHCI) and II (MHCII) presentation of antigens derived from HIV-1 in human monocyte-derived immature DCs (IDCs) and mature DCs, comparing free and complement opsonized HIV-1 particles. Opsonization of virions promoted MHCI presentation by DCs, indicating that complement opsonization routes more virions toward the MHCI presentation pathway. Blockade of macrophage mannose receptor (MMR) and β7-integrin enhanced MHCI and MHCII presentation by IDCs and mature DCs, whereas the block of complement receptor 3 decreased MHCI and MHCII presentation. In addition, we found that IDC and MDC proteolytic activities were modulated by HIV-1 exposure; complement-opsonized HIV-1 induced an increased proteasome activity in IDCs. Taken together, these findings indicate that endocytic receptors such as MMR, complement receptor 3, and β7-integrin can promote or disfavor antigen presentation probably by routing HIV-1 into different endosomal compartments with distinct efficiencies for degradation of viral antigens and MHCI and MHCII presentation, and that HIV-1 affects the antigen-processing machinery.

Production of LacZ Inducible T Cell Hybridoma Specific for Human and Mouse gp10025–33 Peptides

Identification and quantification of immunogenic peptides and tumor-derived epitopes presented on MHC-I molecules are essential for basic studies and vaccines generation. Although lymphocytes derived from transgenic mice can serve as sensitive detectors of processes of antigen presentation and recognition, they are not always available. The use of cell lines might be extremely useful. In this study, we generated a lacZ inducible CD8+ hybridoma (BUSA14) capable of recognizing both human and mouse gp10025–33 melanoma antigens presented on dendritic and tumor cell lines. This hybridoma expresses a variety of membranal T cell markers and secretes IL-2 and TNFα. Thus, BUSA14 offers a quantifiable, cheap and straightforward tool for studying peptide presentation by MHC-I molecules on the cell surface.

DOα−β+ expression in favor of HLA-DR engagement in exosomes

The expression of DOβ and not DOα, in addition to the high intracellular DR, low DM levels and absence of surface DR expression in K562 and HL-60 cells introduce alternative regulatory pathways in DR trafficking and consequently the antigen presentation process. The present study attempted to define the naturally occurring DOα negative state and explain the role of DOβ in the intracellular DR accumulation in K562 and HL-60 cells. Despite the absence of DOα, the DOβ chain was detected in the endosomal compartments. The lack of DOα was found to be partially responsible for the absence of DR from the cell membrane since stable K562-DOα transfectants allowed expression of membrane DR. This expression could be significantly increased upon DM induction by IFN-γ, indicating that DM was another limiting factor for the migration of DR to the cell surface of K562 and HL-60 cells. Furthermore, intracellular DR co-localized with the exosome specific marker CD9, while culture supernatants were shown to contain exosome-engaged and exosome free DR activity as evaluated by SDS-page followed by western blot, ELISA and transmission electron microscopy analysis. These findings indicated that in DOα−β+ cells, DR molecules were programmed to secretion rather than surface expression. The presented results provide novel regulatory processes as to DR trafficking, avoiding expression to the cell surface.

Abstract B4: Tumor-educated CD11c+/CD11bint/Gr-1- regulatory dendritic cells show a mutated pattern of trafficking molecules implicated in cytokine secretion.

Abstract Dendritic cells (DCs) play a pivotal role in regulating innate and adaptive immunity and are key elements in the immunological response against tumors. Several specialized features make DCs uniquely efficient in priming anti-cancer T cell responses. DCs possess specialized pathways to acquire tumor antigens from apoptotic and necrotic cells and to present them to tumor specific T cells. Efficient T cell activation depends on the density of T cell receptors (TCR) ligands and co-stimulatory molecules on the cell surface, and on secretion of cytokines that determine T cell differentiation. It is well documented that DCs properties are suppressed by the tumor microenvironment and many evidences suggest that tolerogenic DCs may be a crucial element in the development of tumor-mediated immune anergy. The tumor microenvironment suppresses DCs functions by targeting different steps in the antigen presentation process, such as phagocytosis, antigen processing and presentation. Suppression also causes an important skewing in the pattern of secreted cytokines. In particular, how secretion of inflammatory cytokines becomes impaired in tumor-exposed DCs remains unclear. We have recently undergone a study to identify the trafficking proteins SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) implicated in secretion of cytokines in DCs and to examine whether they are modified by the tumor microenvironment. Using imaging and functional assays we identified the SNARE VAMP7 as a specific mediator of the secretion of interleukine-12 (IL-12), the major inflammatory cytokine implicated in T cell priming. Interestingly, depletion of VAMP7 does not affect secretion of the inhibitory cytokine IL-10, indicating that distinct secretory pathways direct the release of mediators with opposite cellular functions. We next analyzed lung DCs in healthy mice and in mice bearing Lewis lung carcinoma (LLC). We observed a consistent tumor-induced differentiation of the subpopulation CD11chigh/CD11b-/Gr-1- into a CD11bint phenotype, which was also confirmed in vitro by co-culturing conventional DCs on LLC monolayer. Both in vitro and in vivo, these cells show a normal maturation capacity and expression of surface co-stimulatory molecules, but a strong impairment in inflammatory cytokines secretion, in particular IL-12, upon restimulation with TLR agonists. We isolated CD11chigh/CD11b-/int/Gr-1- cells to high purity to analyze the expression of trafficking proteins. We found that tumor-exposed DCs downregulate, both at mRNA and protein level, the SNAREs VAMP7 and VAMP3, and Rab27. In addition, VAMP7 in tumor-educated DCs displays a different molecular weight suggesting post-transcriptional modification or expression of a different isoform. These preliminary observations suggest that regulation/modification of trafficking proteins in DCs may represent a novel pathway targeted during suppression. Ongoing studies aim at extending the analysis to other trafficking proteins potentially involved in modulation of cytokine secretion. Citation Format: Giulia Chiaruttini, Irene Soncin, Jennifer L. Stow, Andrew A. Peden, Federica Benvenuti. Tumor-educated CD11c+/CD11bint/Gr-1- regulatory dendritic cells show a mutated pattern of trafficking molecules implicated in cytokine secretion. [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 B4.

HLA-DM Focuses on Conformational Flexibility Around P1 Pocket to Catalyze Peptide Exchange

Peptides presented by major histocompatibility complex class II (MHCII) molecules to CD4+ T cells play a central role in the initiation of adaptive immunity. This antigen presentation process is characterized by the proteolytic cleavage of foreign and self proteins, and loading of the resultant peptides onto MHCII molecules. Loading and exchange of antigenic peptides is catalyzed by a non-classical MHCII molecule, HLA-DM. The impact of HLA-DM on epitope selection has been appreciated for a long time. However, the molecular mechanism by which HLA-DM mediates peptide exchange remains elusive. Here, we review recent efforts in elucidating how HLA-DM works, highlighted by two recently solved co-structures of HLA-DM bound to HLA-DO (a natural inhibitor of HLA-DM), or to HLA-DR1 (a common MHCII). In light of these efforts, a model for HLA-DM action in which HLA-DM utilizes conformational flexibility around the P1 pocket of the MHCII-peptide complex to catalyze peptide exchange is proposed.

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.

Differential expression of the costimulatory molecules CD86, CD28, CD152 and PD-1 correlates with the host-parasite outcome in leprosy

Leprosy is a spectral disease exhibiting two polar sides, namely, lepromatous leprosy (LL) characterised by impaired T-cell responses and tuberculoid leprosy in which T-cell responses are strong. Proper T-cell activation requires signalling through costimulatory molecules expressed by antigen presenting cells and their ligands on T-cells. We studied the influence of costimulatory molecules on the immune responses of subjects along the leprosy spectrum. The expression of the costimulatory molecules was evaluated in in vitro-stimulated peripheral blood mononuclear cells of lepromatous and tuberculoid patients and healthy exposed individuals (contacts). We show that LL patients have defective monocyte CD86 expression, which likely contributes to the impairment of the antigen presentation process and to patients anergy. Accordingly, CD86 but not CD80 blockade inhibited the lymphoproliferative response to Mycobacterium leprae. Consistent with the LL anergy, there was reduced expression of the positive signalling costimulatory molecules CD28 and CD86 on the T-cells in these patients. In contrast, tuberculoid leprosy patients displayed increased expression of the negative signalling molecules CD152 and programmed death-1 (PD-1), which represents a probable means of modulating an exacerbated immune response and avoiding immunopathology. Notably, the contacts exhibited proper CD86 and CD28 expression but not exacerbated CD152 or PD-1 expression, suggesting that they tend to develop a balanced immunity without requiring immunosuppressive costimulatory signalling.

Role of bombesin receptor activated protein in the antigen presentation by human bronchial epithelial cells

Bombesin receptor activated protein (BRAP) was identified in a bacterial two-hybrid screen for proteins interacting with bombesin receptor subtype-3 (BRS-3). We found that BRAP is widely expressed in the airway epithelium of human lungs and may play a role during the stress response of lung epithelium. In this work, we explored the potential roles of BRAP in the antigen presenting function of human bronchial epithelial cells (HBECs). Overexpression of a BRAP recombinant protein in a human bronchial epithelial cell line resulted in a reduction of FITC-OVA uptake by HBECs, which indicated that the antigen uptake ability is inhibited. The analysis of the protein expression of surface molecules including B7 homologs and the major histocompactability complex (MHC) class II molecules showed that the expression levels of HLA-DR and B7DC increased while the levels of B7-H1 and B7.2 decreased. Since those surface molecules are all related to antigen presenting process, the altered expression pattern of those molecules provides further evidence showing that BRAP overexpression leads to a change in antigen presenting function of HBECs. Moreover, overexpression of BRAP in HBECs caused a decrease of co-cultured lymphocytes proliferation and a changed pattern of cytokines produced by lymphocytes in the presence of FITC-OVA, which indicated that changes in the maturation pattern and functions of co-cultured lymphocytes were induced by BRAP overexpression. Overall, our results suggested that overexpression of BRAP may play a role during the antigen presenting process of bronchial epithelium by inhibiting the antigen uptake ability of bronchial epithelial cells.