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Molecular Pathways for Immune Recognition of Preproinsulin Signal Peptide in Type 1 Diabetes

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Molecular Pathways for Immune Recognition of Preproinsulin Signal Peptide in Type 1 Diabetes Diabetes Volume 67, April 2018 687 Molecular Pathways for Immune Recognition of Preproinsulin Signal Peptide in Type 1 Diabetes Deborah Kronenberg-Versteeg,1,2 Martin Eichmann,1 Mark A. Russell,3 Arnoud de Ru,4 Beate Hehn,5 Norkhairin Yusuf,1 Peter A. van Veelen,4 Sarah J. Richardson,3 Noel G. Morgan,3 Marius K. Lemberg,5 and Mark Peakman1,2 Diabetes 2018;67:687–696 | https://doi.org/10.2337/db17-0021 The signal peptide region of preproinsulin (PPI) contains In type 1 diabetes, the pathological process of immune- epitopes targeted by HLA-A-restricted (HLA-A0201, A2402) mediated destruction of insulin-producing b-cells leads to IMMUNOLOGY AND TRANSPLANTATION cytotoxic T cells as part of the pathogenesis of b-cell de- insulin deficiency and hyperglycemia (1). Multiple arms of struction in type 1 diabetes. We extended the discovery of the immune system are likely to contribute to this tissue- the PPI epitope to disease-associated HLA-B*1801 and damaging process, with strong indications that CD8+ cyto- HLA-B*3906 (risk) and HLA-A*1101 and HLA-B*3801 (pro- toxic T lymphocytes (CTLs) are a dominant killing pathway. tective) alleles, revealing that four of six alleles present Evidence includes data from preclinical models showing de- epitopes derived from the signal peptide region. During pendence of disease development on intact CD8/MHC class cotranslational translocation of PPI, its signal peptide is I mechanisms (2), supported by compelling findings in hu- cleaved and retained within the endoplasmic reticulum man studies, including the existence of high-risk polymor- (ER) membrane, implying it is processed for immune phic HLA class I genes (3); enrichment of effector CTLs recognition outside of the canonical proteasome-directed specificforb-cell targets in the circulation in new-onset pathway. Using in vitro translocation assays with specific disease (4,5); recapitulation of b-cell killing by patient- inhibitors and gene knockout in PPI-expressing target derived preproinsulin (PPI)-specificCTLsinvitro(4,6); cells, we show that PPI signal peptide antigen processing CD8 T-cell dominance of islet infiltrates in patients, includ- requires signal peptide peptidase (SPP). The intramem- ing the presence of CD8s bearing receptors specificfor brane protease SPP generates cytoplasm-proximal epito- b-cell autoantigens (7,8); hyperexpression of HLA class I, pes, which are transporter associated with antigen processing both at the RNA and protein levels, in residual insulin- (TAP), ER-luminal epitopes, which are TAP independent, containing islets (ICIs) in type 1 diabetes pancreatic tissue each presented by different HLA class I molecules and (9); and recent success in halting b-cell loss using immuno- N-terminal trimmed by ER aminopeptidase 1 for optimal therapy targeted at effector CD8 T cells (10). presentation. In vivo, TAP expression is significantly The potential for the immunological dialogue between upregulated and correlated with HLA class I hyperexpression CTLs and b-cells to be a key component of the development in insulin-containing islets of patients with type 1 diabetes. of type 1 diabetes has led several groups to focus on the Thus, PPI signal peptide epitopes are processed by SPP relevant molecular interactions that govern this interface, and loaded for HLA-guided immune recognition via path- including studies of HLA class I gene polymorphisms carry- ways that are enhanced during disease pathogenesis. ing modified risk of disease (HLA-A*0201,-A*1101,-A*2402, 1Department of Immunobiology, Faculty of Life Sciences and Medicine, King’s Received 5 January 2017 and accepted 10 January 2018. College London, London, U.K. This article contains Supplementary Data online at http://diabetes 2 ’ National Institute for Health Research, Biomedical Research Centre at Guy s and .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0021/-/DC1. St. Thomas’ Hospital Foundation Trust and King’s College London, London, U.K. D.K.-V. and M.E. contributed equally to this work. 3Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, U.K. D.K.-V. is currently affiliated with the Wellcome Trust – Medical Research Council 4Department of Immunohematology and Blood Transfusion, Leiden University Cambridge Stem Cell Institute, University of Cambridge, Cambridge, U.K. Medical Center, Leiden, the Netherlands © 2018 by the American Diabetes Association. Readers may use this article as 5Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Al- long as the work is properly cited, the use is educational and not for profit, and the liance, Heidelberg, Germany work is not altered. More information is available at http://www.diabetesjournals Corresponding author: Deborah Kronenberg-Versteeg, [email protected]. .org/content/license. 688 Preproinsulin Processing for Immune Recognition Diabetes Volume 67, April 2018 -B*1801,-B*3801,and-B*3906 [3,11,12]) and antigenic tar- RESEARCH DESIGN AND METHODS gets within b-cells recognized by CTLs. These have espe- Cell Lines and Epitope Discovery cially focused on PPI, which is considered a primary target K562 cells transfected with HLA-A*1101, HLA-B*1801, in b-cell autoimmunity because anti-insulin autoantibodies HLA-B*3801,orHLA-B*3906 and PPI cDNAs (K562-HLA- fi are frequently the rst disease manifestation in high-risk PPI cells) were generated as described previously (4). Ex- children (12). pression of HLA class I was confirmed by flow cytometry For immune recognition, processing and presentation of (anti-HLA-ABC antibody W6/32; Serotec, Oxford, U.K.). peptides by MHC class I commonly results from degrada- Proinsulin was detected in supernatants by ELISA (DRG tion of proteins by the proteasome (13), generating cytosolic International, Marburg, Germany). Subsequently, ;1010 – peptides of 8 16 amino acids (14), which are transported cells from each cell line were harvested and immunoaffinity into the endoplasmic reticulum (ER) lumen via the trans- purification of HLA-A1101, HLA-B1801, HLA-B3801, and porter associated with antigen processing (TAP) (15), part HLA-B3906, peptide extraction and nano high-performance of the MHC class I peptide loading complex (16). ER ami- liquid chromatography–mass spectrometry performed as nopeptidase (ERAP) 1 trims peptides to a suitable length described elsewhere (4,6,27,28). for MHC loading, if required (17), and peptide-MHC complexes are delivered to the cell surface for immune rec- Inhibitors, RNA Interference and T-Cell Clone Activation ognition. Proteasome-independent, cytoplasm-based nonca- K562-A24-PPI cells were transfected twice within 48 h with nonical antigen presentation pathways have also been 20 nmol/L small interfering RNAs (Applied Biosystems, described (18). Whereas these and the canonical route col- Foster City, CA) targeting B2M (s1852), ERAP1 (s28618), lectively require TAP to delivery peptides into the ER, signal ERAP2 (s34520), TAP1 (s13778, s13780), and TAP2 (s13781, peptide epitopes from secretory proteins may be MHC- s13782, s13783) using Lipofectamine RNAiMAX (Invitrogen, loaded independently of TAP (19) after signal peptidase Paisley, U.K.). Knockdown was assessed by RT-PCR us- cleavage and intramembrane proteolysis by signal peptide ing TaqMan-specific primers and relative cDNA content peptidase (SPP) (20–22). Interestingly, peptides originating normalized to GAPDH gene expression. CD8 T-cell clones from the ER luminal side of the signal peptide can access for PPI3–11-HLA-A2402 and PPI15–24-HLA-A0201 (4,6) were the peptide loading complex directly (23–25), whereas cocultured with K562-HLA-PPI target cells at indicated epitopes close to the cytosol may require proteasomal trim- effector-to-target ratios (4 h), and response was measured ming and TAP for entry into the ER (26). However, the as degranulation (CD107a expression [29]) or macrophage extent to which signal peptides and these noncanonical inflammatory protein 1b (MIP-1b) release (R&D Systems, epitope-generation pathways fuel immune recognition of Minneapolis, MN). single antigens and play a role in physiological responses Site-Directed Mutagenesis remains unclear. To alter single or multiple amino acids (highlighted in Sup- This study was motivated by the need for a better un- plementary Table 1), PPI-containing plasmid pcDNA3/PPI derstanding of autoantigen processing for immune recog- was amplified using altered primers (PPI9P→L, CCC→CTC; nition of b-cells via these different routes, which could PPI12A→L, GCG→CTG; PPI15A→L, GCC→CTC; with 18- to provide novel insights into disease pathogenesis and high- 20-nucleotide overhang preceding and following the mu- light pathways susceptible to therapeutic manipulation. We tatedsite)andPfuTurboDNAPolymeraseAD(Agilent previously reported that the predominant epitope species Technologies, Santa Clara, CA); sequences were confirmed presented to CTLs by human cell lines cotransfected with before use. the INS gene (encoding PPI) and the HLA class I genes HLA- A*0201 and A*2402 derived from the signal peptide region In Vitro Transcription, Translation, and Translocation (4,6). These signal peptide–derived epitopes are recognized and Analysis of SPP Processing by patient CTLs and presented by b-cells bearing the rele- Plasmid pcDNA3/PPI was linearized with EcoRI and tran- vant HLA class I molecule. Here we examine whether PPI scribed in vitro with T7 RNA polymerase at 42°C using signal peptide is a more general source of epitopes by study- 500 mmol/L m7G(59)ppp(59)G CAP analog (New England ing additional HLA class I molecules associated with type 1 Biolabs, Ipswich, MA) (30). mRNAs were translated in vitro diabetes. We show that generation of epitopes from the PPI in 25 mL rabbit reticulocyte lysate (Promega, Madison, WI) signal peptide is driven by the intramembrane protease containing [35S]-methionine and [35S]-cysteine (PerkinElmer, SPP and that loading into nascent HLA class I molecules Waltham, MA) and, where indicated, two equivalents of requires trimming by ERAP1 and is either direct or follows nuclease-treated dog pancreas rough microsomes (31). cytoplasmic translocation and TAP, with the selected path- (Z-LL)2-ketone (Calbiochem SPP inhibitor, 5 mmol/L; Merck, way being determined by the HLA allele.
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