FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2885248212> ?p ?o ?g. }

• W2885248212 endingPage "960.e6" @default.
• W2885248212 startingPage "946" @default.
• W2885248212 abstract "•Peptide HLA class I presentation by β cells is increased by inflammatory cytokines•Peptide sources feature several insulin granule proteins, e.g., SCG5, PCSK2, UCN3•SCG5-009 mRNA splice products and IAPP fusion peptides are also presented•In type 1 diabetes, peptide-reactive CD8+ T cells are enriched in the pancreas Although CD8+ T-cell-mediated autoimmune β cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by β cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known β cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by β cells and for the development of T cell biomarkers and tolerogenic vaccination strategies. Although CD8+ T-cell-mediated autoimmune β cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by β cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known β cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by β cells and for the development of T cell biomarkers and tolerogenic vaccination strategies. Autoimmune CD8+ T cells dominate the pancreatic immune infiltrates of human type 1 diabetes (T1D) (Coppieters et al., 2012Coppieters K.T. Dotta F. Amirian N. Campbell P.D. Kay T.W. Atkinson M.A. Roep B.O. von Herrath M.G. Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients.J. Exp. Med. 2012; 209: 51-60Crossref PubMed Scopus (463) Google Scholar) and lyse β cells by recognizing surface peptide-human leukocyte antigen class I (pHLA-I) complexes. Identifying these peptides is therefore critical for developing tolerogenic vaccination strategies and immune staging tools targeting islet-reactive CD8+ T cells. Most islet antigens (Ags), namely insulin (INS) and its precursor preproinsulin (PPI), glutamic acid decarboxylase (GAD65/GAD2), islet Ag (IA)-2 (PTPRN) (Mallone et al., 2007Mallone R. Martinuzzi E. Blancou P. Novelli G. Afonso G. Dolz M. Bruno G. Chaillous L. Chatenoud L. Bach J.M. et al.CD8+ T-cell responses identify beta-cell autoimmunity in human type 1 diabetes.Diabetes. 2007; 56: 613-621Crossref PubMed Scopus (157) Google Scholar, Martinuzzi et al., 2008Martinuzzi E. Novelli G. Scotto M. Blancou P. Bach J.M. Chaillous L. Bruno G. Chatenoud L. van E.P. Mallone R. The frequency and immunodominance of islet-specific CD8+ T-cell responses change after type 1 diabetes diagnosis and treatment.Diabetes. 2008; 57: 1312-1320Crossref PubMed Scopus (77) Google Scholar), and zinc transporter 8 (ZnT8/SLC30A8) (Scotto et al., 2012Scotto M. Afonso G. Larger E. Raverdy C. Lemonnier F.A. Carel J.C. Dubois-Laforgue D. Baz B. Levy D. Gautier J.F. et al.Zinc transporter (ZnT)8(186-194) is an immunodominant CD8+ T cell epitope in HLA-A2+ type 1 diabetic patients.Diabetologia. 2012; 55: 2026-2031Crossref PubMed Scopus (48) Google Scholar), have been identified based on their targeting by autoantibodies (aAbs). Other Ags such as islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) (Mallone et al., 2007Mallone R. Martinuzzi E. Blancou P. Novelli G. Afonso G. Dolz M. Bruno G. Chaillous L. Chatenoud L. Bach J.M. et al.CD8+ T-cell responses identify beta-cell autoimmunity in human type 1 diabetes.Diabetes. 2007; 56: 613-621Crossref PubMed Scopus (157) Google Scholar), chromogranin A (CHGA) (Li et al., 2015Li Y. Zhou L. Li Y. Zhang J. Guo B. Meng G. Chen X. Zheng Q. Zhang L. Zhang M. et al.Identification of autoreactive CD8+ T cell responses targeting chromogranin A in humanized NOD mice and type 1 diabetes patients.Clin. Immunol. 2015; 159: 63-71Crossref PubMed Scopus (22) Google Scholar), and islet amyloid polypeptide (IAPP) (Standifer et al., 2006Standifer N.E. Ouyang Q. Panagiotopoulos C. Verchere C.B. Tan R. Greenbaum C.J. Pihoker C. Nepom G.T. Identification of novel HLA-A∗0201-restricted epitopes in recent-onset type 1 diabetic subjects and antibody-positive relatives.Diabetes. 2006; 55: 3061-3067Crossref PubMed Scopus (73) Google Scholar) have been identified based on studies in the non-obese diabetic mouse and/or its islet-enriched expression. A systematic discovery effort is missing, and the available catalog may be biased by the lack of information about the peptides that are naturally processed and presented by β cells. Mutated sequences in tumor proteins become preferential CD8+ T cell targets (Gubin et al., 2014Gubin M.M. Zhang X. Schuster H. Caron E. Ward J.P. Noguchi T. Ivanova Y. Hundal J. Arthur C.D. Krebber W.J. et al.Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.Nature. 2014; 515: 577-581Crossref PubMed Scopus (1433) Google Scholar), possibly because they are regarded as non-self and therefore not efficiently tolerized. Similarly, other processes in β cells may facilitate tolerance escape: post-translational modifications (PTMs) (McGinty et al., 2014McGinty J.W. Chow I.T. Greenbaum C. Odegard J. Kwok W.W. James E.A. Recognition of post-translationally modified glutamic acid decarboxylase 65 epitopes in subjects with type 1 diabetes.Diabetes. 2014; 63: 3033-3040Crossref PubMed Scopus (109) Google Scholar, Rondas et al., 2015Rondas D. Crevecoeur I. D’Hertog W. Bomfim Ferreira G. Staes A. Garg A.D. Eizirik D.L. Agostinis P. Gevaert K. Overbergh L. et al.Citrullinated glucose-regulated protein 78 is an autoantigen in type 1 diabetes.Diabetes. 2015; 64: 573-586Crossref PubMed Scopus (124) Google Scholar), transpeptidation, i.e., the splicing and fusion of non-contiguous peptide fragments from the same protein or from different ones (Babon et al., 2016Babon J.A. DeNicola M.E. Blodgett D.M. Crevecoeur I. Buttrick T.S. Maehr R. Bottino R. Naji A. Kaddis J. Elyaman W. et al.Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes.Nat. Med. 2016; 22: 1482-1487Crossref PubMed Scopus (187) Google Scholar, Delong et al., 2016Delong T. Wiles T.A. Baker R.L. Bradley B. Barbour G. Reisdorph R. Armstrong M. Powell R.L. Reisdorph N. Kumar N. et al.Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion.Science. 2016; 351: 711-714Crossref PubMed Scopus (326) Google Scholar), and the use of alternative transcription start sites (Kracht et al., 2017Kracht M.J. van Lummel M. Nikolic T. Joosten A.M. Laban S. van der Slik A.R. van Veelen P.A. Carlotti F. de Koning E.J. Hoeben R.C. et al.Autoimmunity against a defective ribosomal insulin gene product in type 1 diabetes.Nat. Med. 2017; 23: 501-507Crossref PubMed Scopus (139) Google Scholar). These studies have mostly focused on CD4+ T cells, which are stimulated by pHLA class II complexes presented by professional Ag-presenting cells that take up β cell material. These indirect Ag-processing pathways do not reflect those that are specific to β cells. Indeed, several arguments suggest an active role of β cells in their own demise (Eizirik et al., 2009Eizirik D.L. Colli M.L. Ortis F. The role of inflammation in insulitis and beta-cell loss in type 1 diabetes.Nat. Rev. Endocrinol. 2009; 5: 219-226Crossref PubMed Scopus (736) Google Scholar). First, we recently showed that some T1D susceptibility gene variants modulate islet inflammation (Marroqui et al., 2014Marroqui L. Santin I. Dos Santos R.S. Marselli L. Marchetti P. Eizirik D.L. BACH2, a candidate risk gene for type 1 diabetes, regulates apoptosis in pancreatic beta-cells via JNK1 modulation and crosstalk with the candidate gene PTPN2.Diabetes. 2014; 63: 2516-2527Crossref PubMed Scopus (71) Google Scholar, Marroqui et al., 2015Marroqui L. Dos Santos R.S. Floyel T. Grieco F.A. Santin I. Op de Beeck A. Marselli L. Marchetti P. Pociot F. Eizirik D.L. TYK2, a candidate gene for type 1 diabetes, modulates apoptosis and the innate immune response in human pancreatic beta-cells.Diabetes. 2015; 64: 3808-3817Crossref PubMed Scopus (71) Google Scholar, Moore et al., 2009Moore F. Colli M.L. Cnop M. Esteve M.I. Cardozo A.K. Cunha D.A. Bugliani M. Marchetti P. Eizirik D.L. PTPN2, a candidate gene for type 1 diabetes, modulates interferon-gamma-induced pancreatic beta-cell apoptosis.Diabetes. 2009; 58: 1283-1291Crossref PubMed Scopus (128) Google Scholar), suggesting that the β cell response to inflammation is genetically modulated. This response triggers cytokine/chemokine release, endoplasmic reticulum (ER) stress, and HLA-I upregulation (Eizirik et al., 2009Eizirik D.L. Colli M.L. Ortis F. The role of inflammation in insulitis and beta-cell loss in type 1 diabetes.Nat. Rev. Endocrinol. 2009; 5: 219-226Crossref PubMed Scopus (736) Google Scholar, Marroqui et al., 2017Marroqui L. Dos Santos R.S. Op de Beeck A. Coomans de Brachene A. Marselli L. Marchetti P. Eizirik D.L. Interferon-alpha mediates human beta cell HLA class I overexpression, endoplasmic reticulum stress and apoptosis, three hallmarks of early human type 1 diabetes.Diabetologia. 2017; 60: 656-667Crossref PubMed Scopus (96) Google Scholar), which facilitate a productive autoimmune response. The alternative mRNA splicing signature induced by β cell inflammation (Eizirik et al., 2012Eizirik D.L. Sammeth M. Bouckenooghe T. Bottu G. Sisino G. Igoillo-Esteve M. Ortis F. Santin I. Colli M.L. Barthson J. et al.The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines.PLoS Genet. 2012; 8: e1002552Crossref PubMed Scopus (323) Google Scholar, Ortis et al., 2010Ortis F. Naamane N. Flamez D. Ladriere L. Moore F. Cunha D.A. Colli M.L. Thykjaer T. Thorsen K. Orntoft T.F. et al.Cytokines interleukin-1beta and tumor necrosis factor-alpha regulate different transcriptional and alternative splicing networks in primary beta-cells.Diabetes. 2010; 59: 358-374Crossref PubMed Scopus (123) Google Scholar) has received less attention but may similarly generate neo-sequences not translated in the thymus and regarded as non-self. Second, we recently reported a circulating islet-reactive CD8+ T cell repertoire that is predominantly naive and largely overlapping between T1D and healthy subjects (Culina et al., 2018Culina S. Lalanne A.I. Afonso G. Cerosaletti K. Pinto S. Sebastiani G. Kuranda K. Nigi L. Eugster A. Osterbye T. et al.Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.Sci. Immunol. 2018; 3: eaao4013Crossref PubMed Scopus (116) Google Scholar). These findings reveal a general leakiness of central tolerance irrespective of T1D status, begging the question of what determines T1D progression versus the maintenance of a “benign” state of autoimmunity. One hypothesis is that the target β cell and its response to inflammation may be critical in the progression toward T1D in the face of similar autoimmune T cell repertoires across individuals. In this context, it is crucial to understand the “image” that human β cells deliver to CD8+ T cells through pHLA-I complexes. To this end, we implemented a strategy combining HLA-I peptidomics on β cells and RNA sequencing (RNA-seq) analysis of the splice isoforms transcribed by primary islets exposed or not to inflammatory cytokines and by thymic medullary epithelial cells (mTECs), focusing primarily on the most prevalent HLA-A2 variant. Our first epitope discovery pipeline employed HLA-I peptidomics experiments on the ECN90 β cell line (Culina et al., 2018Culina S. Lalanne A.I. Afonso G. Cerosaletti K. Pinto S. Sebastiani G. Kuranda K. Nigi L. Eugster A. Osterbye T. et al.Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.Sci. Immunol. 2018; 3: eaao4013Crossref PubMed Scopus (116) Google Scholar), which carries the HLA-I haplotype A∗02:01/A∗03:01/B∗40:01/B∗49:01/C∗03:04/C∗07:01 (hereafter A2/A3/B40/B49/C3/C7). ECN90 β cells were cultured overnight with or without interferon-γ (IFN-γ), alone or in combination with tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), and lysed to immunopurify pHLA-I complexes. HLA-I-bound peptides were then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although ECN90 β cells expressed surface HLA-I under basal conditions, this expression was significantly upregulated upon cytokine treatment (Figures 1A and 1B ), without significant cell death (Culina et al., 2018Culina S. Lalanne A.I. Afonso G. Cerosaletti K. Pinto S. Sebastiani G. Kuranda K. Nigi L. Eugster A. Osterbye T. et al.Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.Sci. Immunol. 2018; 3: eaao4013Crossref PubMed Scopus (116) Google Scholar). The 2,997 eluted peptides were mostly (93%) 8- to 12-mers (Figure 1C), i.e., the length required for HLA-I binding. The amino acid (aa) identity at pHLA-I anchor positions (p2 and p9) also revealed the preferences expected based on the aforementioned HLA-I haplotype (Figures 1D and 1E). In line with the observed HLA-I upregulation, the number of eluted peptides was significantly higher in the presence of cytokines and higher in β cells exposed to IFN-γ, TNF-α, and IL-1β compared with IFN-γ alone (Figure 1F). These peptide datasets were subsequently analyzed using a stepwise bioinformatics pipeline (Figure 1G). First, only peptides that were reproducibly detected in at least two of five biological replicates (85%; all percentages are given in relation to the number of peptides retained by the previous filter) and that displayed the expected 8- to 12-aa length (93%) were selected. β cell-enriched peptides (both conventional and with PTMs, excluding those derived from peptide or mRNA splicing; red pipeline in Figure 1G) were subsequently filtered based on non-ubiquitous (16%) and enriched β cell expression (34%) of their source proteins. For other non-conventional peptides (i.e., PTM or transcriptional variants), no expression filter was applied, as these species could be β cell specific despite a ubiquitous expression of the source protein or mRNA. PTM peptides (methionine, tryptophan, histidine, and cysteine oxidation, tryptophan conversion to kynurenine) derived from ubiquitous proteins accounted for 8% of the whole dataset (blue pipeline in Figure 1G). Compounds potentially corresponding to peptide splice variants (0.5%; brown pipeline) were identified using an in-house script (Figure S1 and Data S1) based on reported peptide splicing preferences (Berkers et al., 2015Berkers C.R. de Jong A. Schuurman K.G. Linnemann C. Meiring H.D. Janssen L. Neefjes J.J. Schumacher T.N. Rodenko B. Ovaa H. Definition of proteasomal peptide splicing rules for high-efficiency spliced peptide presentation by MHC Class I molecules.J. Immunol. 2015; 195: 4085-4095Crossref PubMed Scopus (47) Google Scholar) applied to known and putative Ags. For peptides derived from mRNA splice variants (dotted and green pipeline in Figure 1G), the peptidomics dataset was interrogated against RNA-seq datasets from primary human islets exposed or not to cytokines and from human mTECs (Data S2). First, higher gene expression can favor peptide processing and presentation. Hence, mRNA splice variants were selected based on a median reads per kilobase per million mapped reads (RPKM) > 5 in islets (27%), based on the median RPKM of known islet Ags (Eizirik et al., 2012Eizirik D.L. Sammeth M. Bouckenooghe T. Bottu G. Sisino G. Igoillo-Esteve M. Ortis F. Santin I. Colli M.L. Barthson J. et al.The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines.PLoS Genet. 2012; 8: e1002552Crossref PubMed Scopus (323) Google Scholar). Second, mRNA isoforms poorly expressed in mTECs might favor T cell escape from clonal deletion. Thus, mRNA variants with an RPKM < 0.1 in mTECs or with a fold decrease >100 versus islets were selected (6%). Third, we selected mRNA isoforms with >10-fold higher expression in islets versus other tissues. We then analyzed the predicted aa neo-sequences encoded by these mRNA variants, yielding 88/166 mRNA variants (53%) and 336 peptide neo-sequences that were used to interrogate the HLA-I peptidomics dataset, with two hits found. Finally, each dataset was filtered to retain only those peptides found enriched in HLA-I- versus mock-purified samples, leading to the overall exclusion of 48% hits. We focused on predicted HLA-A2-restricted peptides for subsequent HLA-A2 binding and CD8+ T cell recognition studies. Collectively, these results show that inflammatory cytokines increase pHLA-I presentation and that these peptides display the aa signatures required for HLA-I binding. The filtered HLA-I peptidomics dataset obtained is described in Figure 2 and detailed in Table S1. While 42 of 98 (43%) eluted peptides were shared among basal and cytokine-treated conditions, 45 of 98 (46%) peptides were only detected upon cytokine exposure (Figure 2A). Also, quantitatively, most peptides (62/98; 63%) were exclusively or more presented in cytokine-treated ECN90 β cells (Table S1). Among the 40 source proteins of HLA-I-eluted peptides (Figure 2B), the most represented ones were the known islet Ags CHGA (n = 15 peptides) and PPI (n = 12, plus one derived from an INS-006 mRNA splice variants). Besides the other known Ag IA-2 (PTPRN; n = 3), the five top-scoring proteins included two putative Ags: kinesin family member 1A (KIF1A; n = 9) and secretogranin V (SCG5/7B2; n = 3, plus one derived from a SCG5-009 mRNA splice variant). Other proteins included known Ags, i.e., GAD2 (GAD65), SLC30A8 (ZnT8), and IAPP (splice peptide), and several putative ones. Notably, all the HLA-A2-restricted PPI peptides identified, namely PPI2–10, PPI6–14, PPI15–24, PPI29–38 (INSB5–14), and PPI34–42 (INSB10–18) (Figure 2C), are already described as major CD8+ T cell epitopes, thus validating our discovery strategy. Source proteins were enriched for insulin granule products (14/42, 33%; Figure 2D), namely CHGA, INS, SCG5, PTPRN, ATP-binding cassette subfamily C member 8 (ABCC8), proprotein convertase 1 (PCSK1/PC1), urocortin-3 (UCN3), chromogranin B (CHGB), carboxypeptidase E (CPE), proprotein convertase 2 (PCSK2/PC2), secretogranin III (SCG3), SLC30A8, and IAPP and neuropeptide Y (NPY) generating splice peptides. The predicted HLA-I restrictions of these peptides (Figure 2E) comprised all the alleles expressed by ECN90 β cells, while 10% of restrictions could not be assigned. For peptides derived from β cell-enriched proteins, 11 of 98 (11%) carried PTMs, with most of them (8/11; 73%) representing variants of unmodified peptides identified in this same dataset. Most of these modifications (7/11; 64%) were M(+15.99) methionine, C(+47.98) cysteine, and W(+15.99) tryptophan oxidations, with W(+3.99) tryptophan to kynurenine transitions also detected. The 99 PTM peptides derived from ubiquitous source proteins are listed in Table S2. To validate the results obtained using the ECN90 β cell line, we similarly analyzed an HLA-A2+ primary human islet preparation that did not share other HLA-I alleles with ECN90 cells. The major source proteins of the HLA-I-bound peptides identified were largely overlapping with those found in ECN90 cells (Figure 2F), with INS (n = 12 peptides), CHGA (n = 4), KIF1A (n = 3), and SCG5 (n = 3) ranking highest for both cells and CHGB (n = 3), PCSK2 (n = 1), and an identical IAPP splice peptide also detected in both. When analyzing the identity of individual peptides (including length and PTM variants) (Table S3), 16 of 33 (48%) were shared between ECN90 and primary islet cells. This common repertoire increased to 12 out of 13 peptides (92%) when considering only predicted HLA-A2 binders, supporting the validity of the ECN90 β cell model. Of note, shared peptides included all the PPI species already described as CD8+ T cell epitopes, SCG5186–196 along with a shorter SCG5186–195 variant with higher HLA-A2 affinity, and the IAPP15–17/IAPP5–10 splice peptide VAL/KLQVFL. Although this product could also reflect PTPRN596–598/IAPP5–10 trans splicing, IAPP15–17/IAPP5–10 is more likely because the intra-protein vicinity of these fragments is more favorable for transpeptidation. The new hits identified were mostly predicted to bind to the HLA-I molecules not shared with ECN90 cells, barring an HLA-A2-restricted CHGB440–448 peptide retained for further validation. Contrary to ECN90 cells, most peptides were detected at similar levels under basal and cytokine-treated conditions (Table S3). This mirrored a higher basal HLA-I expression in primary islets, possibly reflecting peri-mortem and tissue isolation stress conditions, which was less upregulated by cytokine treatment (Figure 1G). Moreover, detection sensitivity may have been limited by the concomitant isolation of non-β cell peptides, i.e., pancreatic polypeptide- and glucagon-derived sequences likely eluted from δ and α cells (n = 4 and n = 5, respectively; not shown because they were excluded by the β cell enrichment filter). The fragmentation profile of the identified peptides was confirmed by comparing their MS/MS spectra with those of the corresponding synthetic peptides. Finally, the predicted HLA-A2 binding was experimentally verified (Figures S2A–S2H), leading to the final selection of 18 of 19 (95%) HLA-I-eluted peptides for CD8+ T cell studies (including CHGB440–448 eluted from primary islets). Collectively, these data show that β cells process and present several known HLA-A2-restricted PPI epitopes and additional candidate ones, which are enriched for secretory granule products. The RNA-seq dataset used for assigning m/z species was further mined in silico, independently of the HLA-I peptidomics pipeline, to identify other potential HLA-A2-restricted peptides (Figure 1G, dotted pipeline). Selection was based on a predicted HLA-A2 binding, a 9- to 10-aa length, and a neo-sequence stretch ≥3 aa, with 39 candidates retained (Figure S2I). These were splice variants of known β cell Ags (GAD2-003, IAPP-002, IAPP-004, PTPRN-021, SLC30A8-002) and of candidate ones. Most of the source mRNA splice variants (35/39, 90%) were similarly expressed in untreated and cytokine-treated islets. HLA-A2 binding was experimentally confirmed for 34 of 39 candidates (87%; Figure S2I), which were retained for further validation along with the 18 HLA-A2 binders identified by HLA-I peptidomics. We previously documented that the vast majority of individuals, both T1D and healthy, harbor similar frequencies of circulating, predominantly naive HLA-A2-restricted CD8+ T cells reactive to known PPI, GAD65, IA-2, IGRP, and ZnT8 epitopes (Culina et al., 2018Culina S. Lalanne A.I. Afonso G. Cerosaletti K. Pinto S. Sebastiani G. Kuranda K. Nigi L. Eugster A. Osterbye T. et al.Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.Sci. Immunol. 2018; 3: eaao4013Crossref PubMed Scopus (116) Google Scholar). Similarly, we reasoned that the presence of a cognate naive CD8+ T cell repertoire is the preliminary requirement for the immunogenicity of the HLA-A2-restricted candidate epitopes identified in the in vitro HLA-I peptidomics and in silico transcriptomics pipeline (n = 52, 18, and 34, respectively). We therefore started by screening these candidates for recognition by circulating CD8+ T cells in HLA-A2+ healthy donors (Table S4), using combinatorial double-coded HLA-A2 multimers (MMrs) loaded with the corresponding synthetic peptides (Culina et al., 2018Culina S. Lalanne A.I. Afonso G. Cerosaletti K. Pinto S. Sebastiani G. Kuranda K. Nigi L. Eugster A. Osterbye T. et al.Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.Sci. Immunol. 2018; 3: eaao4013Crossref PubMed Scopus (116) Google Scholar). We retained those candidates that harbored a cognate naive CD8+ T cell repertoire, based on (1) the frequency of this repertoire, which is typically in the range of 1–50/106 CD8+ T cells (Alanio et al., 2010Alanio C. Lemaitre F. Law H.K. Hasan M. Albert M.L. Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies.Blood. 2010; 115: 3718-3725Crossref PubMed Scopus (135) Google Scholar, Culina et al., 2018Culina S. Lalanne A.I. Afonso G. Cerosaletti K. Pinto S. Sebastiani G. Kuranda K. Nigi L. Eugster A. Osterbye T. et al.Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.Sci. Immunol. 2018; 3: eaao4013Crossref PubMed Scopus (116) Google Scholar), and (2) the pattern of HLA-A2 MMr staining, which is usually clustered rather than spread in the presence of a specific epitope-reactive population (James et al., 2018James E.A. Abreu J.R.F. McGinty J.W. Odegard J.M. Fillie Y.E. Hocter C.N. Culina S. Ladell K. Price D.A. Alkanani A. et al.Combinatorial detection of autoreactive CD8+ T cells with HLA-A2 multimers: a multi-centre study by the Immunology of Diabetes Society T Cell Workshop.Diabetologia. 2018; 61: 658-670Crossref PubMed Scopus (12) Google Scholar). The gating strategy is presented in Figure 3 and representative dot plots in Figures 4A–4F . Using these two criteria, several candidate epitopes displayed a cognate naive CD8+ T cell repertoire in the expected range in a sizable fraction (≥50%) of the healthy individuals analyzed. The frequency of CD8+ T cells recognizing the known β cell epitope PPI6–14 previously analyzed (Culina et al., 2018Culina S. Lalanne A.I. Afonso G. Cerosaletti K. Pinto S. Sebastiani G. Kuranda K. Nigi L. Eugster A. Osterbye T. et al.Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.Sci. Immunol. 2018; 3: eaao4013Crossref PubMed Scopus (116) Google Scholar) also fell in the same range, with some outliers noted. In total, 9 of 18 HLA-I-eluted peptides (50%; Figure 4G) were validated, namely CHGA344–352, insulin gene enhancer protein ISL1276–284, potassium channel subfamily K member 16 (KCNK16)129–137, KIF1A1347–1355, PCSK230–38, SCG5186–195, SCG5-009186–194, and UCN31–9. Despite recognition in only 1 of 6 donors analyzed, the peptide splice product IAPP15–17/IAPP5–10 was also retained, since it was identified in both ECN90 and primary islet cells. Using the same criteria, 11 of 34 candidates selected in silico were validated (32%; Figure 4H), namely cyclin I (CCNI)-00814–22, GAD2-003179–187, guanine nucleotide-binding protein G(s) subunit α isoforms short (GNAS)-03667–75, GNAS-036124–132, IAPP-00233–42, PTPRN-021392–402, PTPRN-021398–407, phogrin/receptor-type tyrosine-protein phosphatase N2 (PTPRN2)-00511–19, PTPRN2-00519–27, mitochondrial oligoribonuclease (REXO2)-0202–10, and SLC30A8-00216–25. As previously observed for other known β cell epitopes (Culina et al., 2018Culina S. Lalanne A.I. Afonso G. Cerosaletti K. Pinto S. Sebastiani G. Kuranda K. Nigi L. Eugster A. Osterbye T. et al.Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.Sci. Immunol. 2018; 3: eaao4013Crossref PubMed Scopus (116) Google Scholar), including the PPI6–14 here used as β cell positive control, only a minority (median 16.4%, interquartile range 8.5%–26.7%) of CD8+ T cells recognizing these candidate epitopes were Ag experienced (CD45RA+CCR7−, CD45RA−CCR7−, or CD45RA−CCR7+; Figures 4I and 4J). Conversely, the Flu MP58–66 peptide included as viral positive control displayed the expected predominantly Ag-experienced phenotype. The complete list of the 20 candidates validated for CD8+ T cell recognition is presented in Table 1. All the peptides validated came from source proteins whose gene expression was detected in islets, both under basal and cytokine-treated conditions. One notable exception was SCG5-009, whose expression was negligible under basal condition but strongly upregulated after cytokine treatment. Gene expression in mTECs was also negligible in all cases with the exception of CHGA, ISL1, and SCG5.Figure 4HLA-A2-Restricted β Cell Peptides Are Targeted by a Circulating Naive CD8+ T Cell Repertoire in Healthy DonorsShow full captionMMr+CD8+ cells reactive to HLA-A2-b" @default.
• W2885248212 created "2018-08-22" @default.
• W2885248212 creator A5000550995 @default.
• W2885248212 creator A5002497731 @default.
• W2885248212 creator A5004765717 @default.
• W2885248212 creator A5004800654 @default.
• W2885248212 creator A5007387317 @default.
• W2885248212 creator A5011634368 @default.
• W2885248212 creator A5013130720 @default.
• W2885248212 creator A5014161101 @default.
• W2885248212 creator A5014468236 @default.
• W2885248212 creator A5027032373 @default.
• W2885248212 creator A5034219724 @default.
• W2885248212 creator A5039277778 @default.
• W2885248212 creator A5040035539 @default.
• W2885248212 creator A5040317975 @default.
• W2885248212 creator A5044342048 @default.
• W2885248212 creator A5048913575 @default.
• W2885248212 creator A5049356241 @default.
• W2885248212 creator A5051191189 @default.
• W2885248212 creator A5051848877 @default.
• W2885248212 creator A5051941400 @default.
• W2885248212 creator A5053332237 @default.
• W2885248212 creator A5064248322 @default.
• W2885248212 creator A5069364785 @default.
• W2885248212 creator A5070276813 @default.
• W2885248212 creator A5077733570 @default.
• W2885248212 creator A5078636742 @default.
• W2885248212 creator A5081445488 @default.
• W2885248212 creator A5084381481 @default.
• W2885248212 creator A5086915876 @default.
• W2885248212 creator A5090781458 @default.
• W2885248212 date "2018-12-01" @default.
• W2885248212 modified "2023-10-18" @default.
• W2885248212 title "Conventional and Neo-antigenic Peptides Presented by β Cells Are Targeted by Circulating Naïve CD8+ T Cells in Type 1 Diabetic and Healthy Donors" @default.
• W2885248212 cites W1524604314 @default.
• W2885248212 cites W1554799697 @default.
• W2885248212 cites W1891992783 @default.
• W2885248212 cites W1990868550 @default.
• W2885248212 cites W2001058095 @default.
• W2885248212 cites W2004264117 @default.
• W2885248212 cites W2006740199 @default.
• W2885248212 cites W2013932739 @default.
• W2885248212 cites W2037347257 @default.
• W2885248212 cites W2044481899 @default.
• W2885248212 cites W2055492712 @default.
• W2885248212 cites W2065210931 @default.
• W2885248212 cites W2065776802 @default.
• W2885248212 cites W2066592806 @default.
• W2885248212 cites W2079018735 @default.
• W2885248212 cites W2089934054 @default.
• W2885248212 cites W2093090129 @default.
• W2885248212 cites W2097688270 @default.
• W2885248212 cites W2105353410 @default.
• W2885248212 cites W2115898840 @default.
• W2885248212 cites W2118705984 @default.
• W2885248212 cites W2129877119 @default.
• W2885248212 cites W2136199424 @default.
• W2885248212 cites W2141609931 @default.
• W2885248212 cites W2141927426 @default.
• W2885248212 cites W2157837394 @default.
• W2885248212 cites W2159694361 @default.
• W2885248212 cites W2160440883 @default.
• W2885248212 cites W2164707000 @default.
• W2885248212 cites W2218629053 @default.
• W2885248212 cites W2266478788 @default.
• W2885248212 cites W2536906012 @default.
• W2885248212 cites W2541443144 @default.
• W2885248212 cites W2570554925 @default.
• W2885248212 cites W2593862806 @default.
• W2885248212 cites W2772265469 @default.
• W2885248212 cites W2785684315 @default.
• W2885248212 cites W4240896746 @default.
• W2885248212 doi "https://doi.org/10.1016/j.cmet.2018.07.007" @default.
• W2885248212 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30078552" @default.
• W2885248212 hasPublicationYear "2018" @default.
• W2885248212 type Work @default.
• W2885248212 sameAs 2885248212 @default.
• W2885248212 citedByCount "144" @default.
• W2885248212 countsByYear W28852482122018 @default.
• W2885248212 countsByYear W28852482122019 @default.
• W2885248212 countsByYear W28852482122020 @default.
• W2885248212 countsByYear W28852482122021 @default.
• W2885248212 countsByYear W28852482122022 @default.
• W2885248212 countsByYear W28852482122023 @default.
• W2885248212 crossrefType "journal-article" @default.
• W2885248212 hasAuthorship W2885248212A5000550995 @default.
• W2885248212 hasAuthorship W2885248212A5002497731 @default.
• W2885248212 hasAuthorship W2885248212A5004765717 @default.
• W2885248212 hasAuthorship W2885248212A5004800654 @default.
• W2885248212 hasAuthorship W2885248212A5007387317 @default.
• W2885248212 hasAuthorship W2885248212A5011634368 @default.
• W2885248212 hasAuthorship W2885248212A5013130720 @default.
• W2885248212 hasAuthorship W2885248212A5014161101 @default.
• W2885248212 hasAuthorship W2885248212A5014468236 @default.
• W2885248212 hasAuthorship W2885248212A5027032373 @default.
• W2885248212 hasAuthorship W2885248212A5034219724 @default.
• W2885248212 hasAuthorship W2885248212A5039277778 @default.

• next