Page 1 of 44 Diabetes Peptides derived from insulin granule proteins are targeted by CD8+ T cells across MHC Class I restrictions in humans and NOD mice. Marie Eliane Azoury1*, Mahmoud Tarayrah1*, Georgia Afonso1, Aurore Pais1, Maikel L. Colli2, Claire Maillard1, Cassandra Lavaud1, Laure Alexandre-Heymann1,3, Sergio Gonzalez- Duque1,4, Yann Verdier4, Joelle Vinh4, Sheena Pinto5, Soren Buus6, Danièle Dubois- Laforgue1,3, Etienne Larger1,3, Jean-Paul Beressi7, Graziella Bruno8, Decio L. Eizirik2, Sylvaine You1, Roberto Mallone1,3. 1Université de Paris, Institut Cochin, CNRS, INSERM, 75014 Paris, France. 2Université Libre de Bruxelles, Center for Diabetes Research and Welbio, Medical Faculty, Université Libre de Bruxelles, 1070 Brussels, Belgium. 3Assistance Publique Hôpitaux de Paris, Hôpitaux Universitaires de Paris Centre-Université de Paris, Cochin Hospital, Service de Diabétologie et Immunologie Clinique, 75014 Paris, France. 4ESPCI Paris, PSL University, Spectrométrie de Masse Biologique et Protéomique, CNRS USR3149, 75005 Paris, France. 5DKFZ, Division of Developmental Immunology, 69120 Heidelberg, Germany. 6University of Copenhagen, Department of Immunology and Microbiology, Laboratory of Experimental Immunology, DK-2200 N Copenhagen, Denmark. 7Centre Hospitalier de Versailles André Mignot, Service de Diabétologie, 78150 Le Chesnay, France. 8University of Turin, Department of Medical Sciences, 10126 Turin, Italy. *These authors contributed equally to this work. Running title: T-cell antigens from insulin granule proteins. Corresponding author: Roberto Mallone, [email protected] Manuscript length: 4,480 words, 1 table, 5 figures. Online Appendix: 3 tables, 3 figures. Tweet (204 characters): Proteins of the insulin granules in beta cells are key autoimmune targets in #type 1 diabetes. The list of antigens available to understand disease mechanisms and monitor its progression is growing longer! @InstitutCochin, @Inserm, @Innodiagroup 1 Diabetes Publish Ahead of Print, published online September 14, 2020 Diabetes Page 2 of 44 ABSTRACT The antigenic peptides processed by β cells and presented through surface HLA Class I molecules are poorly characterized. Each HLA variant, e.g. the most common HLA-A2 and HLA-A3, carries some peptide-binding specificity. Hence, features that, despite these specificities, remain shared across variants may reveal factors favoring β-cell immunogenicity. Building on our previous description of the HLA-A2/A3 peptidome of β cells, we analyzed the HLA-A3-restricted peptides targeted by circulating CD8+ T cells. Several peptides were recognized by CD8+ T cells within a narrow frequency (1-50/106), which was similar in donors with and without type 1 diabetes and harbored variable effector/memory fractions. These epitopes could be classified as conventional peptides or neo-epitopes, generated either via peptide cis-splicing or mRNA splicing, e.g. secretogranin-5 (SCG5)-009. As reported for HLA-A2-restricted peptides, several epitopes originated from β- cell granule proteins, e.g. SCG3, SCG5 and urocortin-3. Similarly, H-2Kd-restricted CD8+ T cells recognizing the murine orthologues of SCG5, urocortin-3, and proconvertase-2 infiltrated the islets of NOD mice and transferred diabetes into NOD/scid recipients. The finding of granule proteins targeted in both humans and NOD mice supports their disease relevance and identifies the insulin granule as a rich source of epitopes, possibly reflecting its impaired processing in type 1 diabetes. 2 Page 3 of 44 Diabetes INTRODUCTION Autoimmune CD8+ T cells are held as the final effectors of β-cell destruction in type 1 diabetes. Indirect support for this tenet comes from the observations that the pancreatic immune infiltrates of type 1 diabetic patients are dominated by CD8+ T cells (1); that at least some of these CD8+ T cells recognize islet epitopes (1-3); and that islet-reactive cytotoxic CD8+ T-cell clones can lyse β cells in vitro (4-7). However, clones with similar cytotoxic activity can be derived from healthy donors (2). More generally, recent reports from our laboratory (2, 3) and others (8) documented that the frequency of islet-reactive CD8+ T cells in peripheral blood is similar between donors with and without type 1 diabetes, while these T cells are enriched in the pancreas of diseased individuals (2, 3). We therefore proposed a novel paradigm of “benign” islet autoimmunity, which is present in all individuals and imprinted in the thymus, due to a marginal impact on clonal deletion of the antigens presented by thymic medullary epithelial cells (mTECs) (2, 9, 10). The progression of this benign autoimmune state toward type 1 diabetes may rely on different, non-exclusive mechanisms. First, regulation may be at play, either T-cell-extrinsic (e.g. suppression by regulatory T cells, Tregs) or intrinsic (e.g. anergy, exhaustion). Indeed, conventional T cells from T1D patients are more resistant to Treg suppression (11, 12) and exhaustion (8, 13), and assays relying on T-cell activation consistently detected higher frequencies of some islet-reactive fractions in T1D patients (2, 4, 14-17). Second, a higher vulnerability of target β cells may favor type 1 diabetes progression (18). The lines of evidence for this ‘β-cell-centric’ hypothesis include observations that: i) some T1D susceptibility gene variants modulate islet inflammation (19-21), which makes β cells more ‘visible’ to autoimmune CD8+ T cells by upregulating HLA Class I (HLA-I) expression and peptide-HLA-I (pHLA-I) presentation (3); ii) the benefit of effective immunotherapies is limited in time, which may hint at β-cell-intrinsic components not impacted by treatment (22); iii) contrary to what found in the blood, islet-reactive CD8+ T cells are enriched in the type 1 3 Diabetes Page 4 of 44 diabetic pancreas (2, 3), pointing to local factors that promote their disease-specific homing to the target organ; and iv) diseased β cells display endoplasmic reticulum (ER) stress (23) and impaired proinsulin processing (24, 25), which may promote their immunogenicity by favoring HLA-I presentation of potentially modified proinsulin peptides. Overall, a unifying picture is emerging that prompts to consider type 1 diabetes as a disease of both the immune system and the β cell (18, 26). The identification of the peptides naturally processed by β cells that trigger CD8+ T-cell recognition through presentation by surface HLA-I molecules may provide novel and relevant information on how β cells exert an active role in disease pathogenesis. We recently provided a first description of the HLA-I peptidome of human β cells under basal and inflammatory conditions, and detailed novel antigenic peptides recognized by circulating CD8+ T cells in the frame of the most prevalent HLA- A*02:01 (HLA-A2) variant (3). As observed for known islet epitopes (2), the frequency of these CD8+ T cells was similar in the blood, but higher in the pancreas, of type 1 diabetic patients compared with healthy donors (3). Several other HLA-I-eluted peptides were predicted to bind the other common HLA-A*03:01 variant (HLA-A3 from hereon) (3), which presents peptides carrying different amino acid (aa) motifs compared to HLA-A2. Whether CD8+ T cells recognize these peptides was not assessed. We here studied this recognition in order to define whether HLA-A2 and HLA-A3, despite their different peptide binding preferences, present peptides with any shared feature. We report that peptides derived from insulin granule proteins are common for both variants, and are also targeted by CD8+ T cells that infiltrate the islets of prediabetic non-obese diabetic (NOD) mice and transfer disease into NOD/scid recipients. 4 Page 5 of 44 Diabetes RESEARCH DESIGN AND METHODS HLA-A3-restricted candidate epitopes identified by HLA-I peptidomics and in-silico mining of mRNA splice variants The first set of candidate HLA-A3-restricted epitopes was previously identified by HLA-I peptidomics (3) (Table 1, top). Potential HLA-A3 binding was assigned based on predicted affinity (NetMHC 4.0) and stability (NetMHCstab 1.0) scores. Candidates derived from mRNA splice variants were identified by a parallel in-silico mining of previous RNA-seq datasets from primary human islets exposed or not to interleukin (IL)-1 and interferon (IFN)- and from human mTECs, as described in (3) and summarized in Figure S1. Relevant mRNA splice variants were first selected based on a median reads per kilobase per million mapped reads (RPKM) >5 in islets, based on the median RPKM of known islet antigens (3, 27). Second, mRNA isoforms poorly expressed in mTECs, which might favor T-cell escape from clonal deletion, were selected based on an RPKM <0.1 in mTECs or with a fold decrease >100 versus islets (3). Third, only mRNA isoforms with >10-fold higher expression in islets versus other tissues were retained (3). We then analyzed the predicted aa neo- sequences encoded by these mRNA variants, taking the longest and/or most prevalent islet mRNA isoform as reference. This neo-sequence filter yielded 88/166 mRNA variants (53%) and 340 peptide neo-sequences identified as a potential source of neo-epitopes. Thirty-seven peptides predicted to bind HLA-A3 were thus identified. We further restricted this list by focusing on 9- to 10-aa-long peptides (29/37, 78%) carrying a neo-sequence stretch ≥3 aa, with 24/29 (83%) candidates finally retained (Table 1, bottom). Peptides and HLA-A3 binding assays Peptides (>90% pure, Synpeptides) were tested for binding to HLA-A3 by flow cytometry using biotin-tagged HLA-A3 monomers (immunAware), per the manufacturer’s protocol. Briefly, biotinylated monomers (final concentration 1.2 nM) were folded as described (3) and 5 Diabetes Page 6 of 44 captured on 6-8 µm streptavidin-coated beads (Spherotech). Beads were subsequently incubated with an anti-β2-microglobulin BBM.1 monoclonal antibody (mAb; Santa Cruz), followed by an AlexaFluor (AF)488-labeled goat polyclonal anti-mouse IgG (RRID:AB_2728715). The HLA-A3-binding peptide Flu NP265-273 (ILRGSVAHK), and a non-binding peptide CHGA382-390 (HPVGEADYF) were included as positive and negative controls, respectively.