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      Development of a novel immunoproteasome digestion assay for synthetic long peptide vaccine design

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          Abstract

          Recently, many autologous tumor antigens have been examined for their potential use in cancer immunotherapy. However, the success of cancer vaccines in clinical trials has been limited, partly because of the limitations of using single, short peptides in most attempts. With this in mind, we aimed to develop multivalent synthetic long peptide (SLP) vaccines containing multiple cytotoxic T-lymphocyte (CTL) epitopes. However, to confirm whether a multivalent vaccine can induce an individual epitope-specific CTL, the only viable screening strategies currently available are interferon-gamma (IFN-μ enzyme-linked immunospot (ELISPOT) assays using human peripheral blood mononuclear cells, or expensive human leukocyte antigen (HLA)-expressing mice. In this report, we evaluated the use of our developed murine-20S immunoproteasome (i20S) digestion assay, and found that it could predict the results of IFN-μ ELISPOT assays. Importantly, the murine-i20S digestion assay not only predicted CTL induction, but also antitumor activity in an HLA-expressing mouse model. We conclude that the murine-i20S digestion assay is an extremely useful tool for the development of “all functional” multivalent SLP vaccines.

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          Cancer immunotherapy: moving beyond current vaccines.

          Great progress has been made in the field of tumor immunology in the past decade, but optimism about the clinical application of currently available cancer vaccine approaches is based more on surrogate endpoints than on clinical tumor regression. In our cancer vaccine trials of 440 patients, the objective response rate was low (2.6%), and comparable to the results obtained by others. We consider here results in cancer vaccine trials and highlight alternate strategies that mediate cancer regression in preclinical and clinical models.
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            Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function

            Interleukin (IL)-21 is the most recently recognized of the cytokines that share the common cytokine receptor γ chain (γc), which is mutated in humans with X-linked severe combined immunodeficiency. We now report that IL-21 synergistically acts with IL-15 to potently promote the proliferation of both memory (CD44high) and naive (CD44low) phenotype CD8+ T cells and augment interferon-γ production in vitro. IL-21 also cooperated, albeit more weakly, with IL-7, but not with IL-2. Correspondingly, the expansion and cytotoxicity of CD8+ T cells were impaired in IL-21R−/− mice. Moreover, in vivo administration of IL-21 in combination with IL-15 boosted antigen-specific CD8+ T cell numbers and resulted in a cooperative effect on tumor regression, with apparent cures of large, established B16 melanomas. Thus, our studies reveal that IL-21 potently regulates CD8+ T cell expansion and effector function, primarily in a synergistic context with IL-15.
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              Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity.

              Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit β5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of β5c/X but not β5i, thereby explaining the selectivity of PR-957 for β5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders. Copyright © 2012 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysis
                Role: Formal analysis
                Role: Formal analysis
                Role: Formal analysis
                Role: Formal analysis
                Role: Project administration
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                3 July 2018
                2018
                : 13
                : 7
                : e0199249
                Affiliations
                [001]Discovery and Preclinical Research Division, Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, Japan
                Monash University, AUSTRALIA
                Author notes

                Competing Interests: All of the authors are salaried employee of the Taiho Pharmaceutical Co. Ltd.However, this does not alter our adherence to PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0003-0158-1743
                Article
                PONE-D-17-42577
                10.1371/journal.pone.0199249
                6029771
                29969453
                c75a2e94-aa1e-48c2-bd6e-efc62acd7081
                © 2018 Wada et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 4 December 2017
                : 4 June 2018
                Page count
                Figures: 5, Tables: 2, Pages: 15
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100009954, Taiho Pharmaceutical;
                All of the authors are salaried employee of the Taiho Pharmaceutical Co. Ltd. However, the funder provided support in the form of salaries for all authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the “author contributions” section.
                Categories
                Research Article
                Research and Analysis Methods
                Immunologic Techniques
                Immunoassays
                Enzyme-Linked Immunoassays
                Medicine and Health Sciences
                Infectious Diseases
                Infectious Disease Control
                Vaccines
                Research and Analysis Methods
                Experimental Organism Systems
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