The Cellular Redox Environment Alters Antigen Presentation

Cysteine-containing peptides represent an important class of T cell epitopes, yet their prevalence remains underestimated. We have established and interrogated a database of around 70,000 naturally processed MHC-bound peptides and demonstrate that cysteine-containing peptides are presented on the surface of cells in an MHC allomorph-dependent manner and comprise on average 5–10% of the immunopeptidome. A significant proportion of these peptides are oxidatively modified, most commonly through covalent linkage with the antioxidant glutathione. Unlike some of the previously reported cysteine-based modifications, this represents a true physiological alteration of cysteine residues. Furthermore, our results suggest that alterations in the cellular redox state induced by viral infection are communicated to the immune system through the presentation of S-glutathionylated viral peptides, resulting in altered T cell recognition. Our data provide a structural basis for how the glutathione modification alters recognition by virus-specific T cells. Collectively, these results suggest that oxidative stress represents a mechanism for modulating the virus-specific T cell response.     

组织蛋白酶S在抗原提呈过程中的作用

效应T细胞激活需要两个或以上的信号诱导.抗原肽结合MHC-Ⅱ分子的溶酶体途径（第一信号）包括两个重要的步骤：不变链（invariant chain Ii）降解和抗原加工.目前所知的溶酶体蛋白酶中,组织蛋白酶S（cathepsin S,cat S）被认为以非冗长的作用参与了上述两步过程,其还参与了降解细胞外基质（extracellular matrix,ECM）的过程.因此被认为在调节免疫、自身免疫性疾病、抗肿瘤免疫、组织重塑等方面是具有潜力的研究靶点.     

GoldCLIP: Gel-omitted Ligation-dependent CLIP

Protein–RNA interaction networks are essential to understand gene regulation control.Identifying binding sites of RNA-binding proteins(RBPs) by the UV-crosslinking and immunoprecipitation(CLIP) represents one of the most powerful methods to map protein–RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method(Gold CLIP) that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein(PTB), a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. Gold CLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.     

Application of Liposomes in Antigen Presentation and Cytokine Delivery

Abstract

Introduction

Ever since the liposome has been proposed as an antigen carrier or vaccine adjuvant to enhance immune responses of various vaccines (1), a great deal of effort has been made to understand the physical and chemical properties of the liposome membranes that modulate the potency of liposomal adjuvants [for review, see (2)]. While no generally consistent conclusion can be drawn for all vaccine antigens, the role of lipid fluidity in liposome adjuvanticity has been investigated extensively. Kinsky (3) showed that trinitrophenyl (TNP)-sensitized liposomes composed primarily of gelphased lipids [defined by a gel-to-liquid phase-transition temperature (Tc) higher than 37°C] were more potent in eliciting B cell response. In this study, TNP is a lipid membrane-bound antigen. However, membrane fluidity does not appear to play a role in adjuvanticity with a water-soluble antigen. Six et al. (4) showed, using the water-soluble adenovirus type 5 hexon, that liposomes made of gel-phased lipids – distearoyl phosphatidylcholines (PC) (Tc = 57°C) and dipalmitoyl PC (Tc = 41 °C) - produced similar adjuvant effects in responders compared to liposomes made of liquid-phased lipids – dimyristoyl PC (Tc = 23°C) and dioleoyl PC (Tc = -22°C). Other experimental results regarding membrane fluidity and the adjuvanticity of various lipid compositions and protein antigens (5-8) yielded conflicting conclusions. These inconsistent results may have arisen from the differences in the studied protein antigen and from the unique interaction between the antigen and lipid membrane. Overall, liposome adjuvant studies to date have concentrated on the role of the physical characteristics of liposome membranes in potentiating immune interactions and paid limited attention to the physiological constraint and immune recognition and interaction at the cellular and molecular levels. With the recent advances in our understanding of the cellular and molecular mechanisms of immune regulation, one can now rationally design strategies to deliver antigen and cytokines to selective sites or cells involved in immune potentiation. In the following sections, we will present our observations about such strategies for the delivery of antigens with antigen-presenting liposomes (APLs) targeted to macrophages and the use of liposomes to deliver cytokines for the enhancement of antigen-dependent T and B cell growth.     

Virus-Mediated Suppression of the Antigen Presentation Molecule MR1

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DNA疫苗抗原提呈的传感器分子研究新进展

DNA疫苗进入细胞后,除了转译成蛋白质抗原,通过MHC分子进行内源性或外源性抗原提呈外,近年来还发现可直接与相应的被称为核酸传感器分子,如TLR9、DAI、AIM2、STING、DDX41解旋酶和RNA聚合酶Ⅲ等结合,继而激活不同的免疫信号通路.基于DNA疫苗的传感器分子和信号通路研制免疫佐剂,可有效增强DNA疫苗的免疫原性.     

Septate and paranodal junctions: kissing cousins

A series of recent publications significantly advances our knowledge about the evolution and composition of septate junctions in arthropod and chordate species. These papers indicate that insect septate junctions share several structural and functional components with paranodal junctions, which join myelinating glial cells to axons in the vertebrate nervous system, and that both probably evolved from a common ancestral precursor.     

Use of Antiviral T-Lymphocyte Clones to Characterize Antigen Presentation and T-Lymphocyte Subsets

The ability to isolate homogeneous populations of antiviral T lymphocytes from immune mice has led to insight into a variety of areas in cellular immunology. It has permitted the characterization of the distinct pathways of antigen processing and presentation to CD8⁺, Class I MHC-restricted and CD4⁺, Class II MHC-restricted cytolytic T lymphocytes as well as the identification of antigenic epitopes for T lymphocytes. In addition,in vivoeffector function of CD8⁺and CD4⁺cytolytic T-cell clones in protection from lethal viral pneumonia in a murine model of influenza virus infection has been demonstrated. Since the identification of CD4⁺T-lymphocyte helper subsets based on the lymphokine profiles of clonal populations, much interest has been focused on the role of specific cytokines in ultimately determining the effector functions of those cells. The protocol presented in this paper has been used to isolate Th1 and Th2 clones in a viral infectious disease model that has enabled us to further investigate the role of specific cytokines in controlling viral infection.     

Mass Spectrometry of Human Leukocyte Antigen Class I Peptidomes Reveals Strong Effects of Protein Abundance and Turnover on Antigen Presentation

HLA class I molecules reflect the health state of cells to cytotoxic T cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (p < 0.0001). When analyzing overpresented proteins – those with at least fivefold higher density score than expected for their abundance – we noticed that they are degraded almost 3 h faster than similar but nonpresented proteins (top 20% abundance class; median half-life 20.8h versus 23.6h, p < 0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain, and nucleosomal proteins are particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our data set. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides. Remarkably, we identified five mutated peptides from a human colon cancer cell line, which have very recently been predicted to be HLA-I binders. Altogether, we demonstrate the usefulness of combining MS-analysis with immunogenesis prediction for identifying, ranking, and selecting peptides for therapeutic use.The highly polymorphic Human Leukocyte Antigen class I (HLA-I)¹ genes are encoded by three loci (HLA-A, B, and C) in a gene-rich region on chromosome 6. They produce up to six unique cell surface receptors that bind and present the so-called HLA class I peptidome, which consists of peptides derived from proteolysis of intracellular proteins. Their function is to reflect the health state of the body''s cells to CD8+ cytotoxic T cells. During thymic maturation T cells that react to self-peptides are eliminated (1), leaving T cells with the capability to recognize peptides from viruses and bacteria. This recognition is interpreted as a danger signal, leading to removal of infected cells. Transformed, preneoplastic and cancer cells also tend to display atypical self-peptides from mutated or excessively expressed self-proteins, known as tumor associated antigens (TAAs). Although HLA-I molecules are indispensable in prevention of disease, they also pose a substantial health problem by causing allergies (2), life-threatening autoimmune diseases (3), and the often fatal rejection of donor organs because of recognition of both major and minor histocompatibility antigens (4).Finding the rules for peptide generation and selection is regarded as the most important open issue in the field of HLA-I biology by leading experts (5). Although the antigen presentation pathway is well characterized, it is still unclear how basic properties such as protein abundance, turnover, and subcellular localization influence and shape the HLA-I presented peptidome (6–10). One expectation is that protein abundance should correlate with presentation (11), but previous studies have reported conflicting and contradicting results that mostly argue against a strong link (6, 7, 10, 12, 13). It is also not fully understood why only some HLA-sampled self-peptides from cancer antigens spontaneously activate T cells, whereas others do not.The majority of HLA-I peptides are derived from proteasomal degradation (5). Although the proteasome generates an excess of peptides, only some have the required sequence motifs for HLA binding, resulting in a selective sampling of available peptides (14). The presented peptides are typically nine amino acids long, but the length can range from eight to 15. The high degree of genetic variance of HLA-I receptors translates into allele-specific peptide-binding motifs defined by anchor positions, which are usually the second and the last positions in a peptide (15). Each cell has around 200,000 cell-surface-expressed HLA complexes, which bind about 10,000 unique peptide sequences (16). The affinity of a peptide toward the presenting HLA molecule does not correlate strongly with its immunogenicity, and neither does the number of presented HLA complexes (17). Instead, the most robust predictor of peptide immunogenicity appears to be the number of potential reactive T-cell clones (17–19).The longer the source protein, the higher the chances it will contain sequences that fit to a certain HLA motif, which would inflate the representation of longer proteins regardless of biological role. Furthermore, some HLA-I peptide sequences can be mapped to multiple proteins, potentially causing a problem in determining the number of observed HLA peptides per protein (13). This illustrates that careful accounting of the potentially and actually presented HLA peptides is important in properly delineating trends in propensity of peptide presentation.In cancer immunotherapy, T cells can be directed against tumors, based on the pattern of cancer associated HLA peptides. Therefore, there is great interest in determining the identity of these immunogenic peptides. Bioinformatic methods that attempt to predict HLA peptides of cancer proteins of interest are easily accessible and most commonly used. They typically score sequences with respect to proteasomal degradation, transport into the ER via the transporter associate with antigen processing (TAP) and binding to different HLA-I alleles (20). However, their precision success is modest (21, 22). The second approach is to directly capture the naturally presented peptides using mass spectrometry; however, this requires the relevant biological sample and sophisticated instruments and workflows, which have become accessible only recently for large-scale work (23–28). Although identification of cancer associated HLA peptides by MS, if performed stringently, establish the in vivo existence of the peptide, it still does not guarantee that it will elicit a potent T-cell response, which is required for further development into therapeutics (29). Therefore, like in the case of in silico predicted peptides, the immunogenicity of the peptides must in any case be tested empirically.We here present a rich and high confidence HLA-I peptidome, established by applying state-of-the-art mass-spectrometric techniques on a collection of seven cell lines. We investigate how abundance affects the propensity of proteins to be presented as measurable HLA peptides and whether or not there are specific protein classes that are overrepresented even independent of abundance. Likewise, we explore how to use in silico immunogenicity tools on the set of identified HLA peptides from cancer-associated proteins, with a view to select vaccine candidates.     

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.     

Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells

Virus-specific CD8⁺ T cells (T_CD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector T_CD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated T_CD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the T_CD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines.