CD3, CD68, and glucagon stainingThe coimmunostaining for CD3, CD68, and glucagon was performed on the automated platform Discovery XT-VENTANA. After deparaffinization and antigen retrieval using CC1 solution, slides were incubated 1 hour at 37°C with a mix of anti-CD3 (A0452, Dako) and anti-CD68 (M0876, Dako) antibodies, before incubating anti-mouse (FP-SC 4110, Interchim) and anti-rabbit (FP-SB 5110, Interchim) secondary antibodies for 45 minutes at 37°C. Anti–glucagon-FITC antibody (BS-3796-A488, Interchim) was subsequently incubated for 45 minutes at 37°C. Slides were digitalized with the slide scanner NANOZOOMER 2.0RS/C10730-12 (Hamamatsu; objective ×20, resolution 0.46 μm/pixel). Image analysis was performed on the whole pancreas slide by using an algorithm from HALO platform combined with a tissue classifier (Indica Labs). Segmentation between exocrine/endocrine pancreas and other tissue types (connective tissues) were processed using Tissue Classifier based on color, texture, and contextual features. Islet regions were identified as a contiguous area of glucagon. CD3 and CD68 cells were detected according to thresholds of intensity within the endocrine and exocrine pancreas. The algorithm calculates the total number of cells within each pancreas part, the number of CD3+ cells, the number of CD68+ cells, and the areas of endocrine and exocrine parts of the pancreas. Quantification was performed under blinded conditions, with anonymized slides from control and T1D donors.

The HERV-W-Env IHC was performed on the automated platform Benchmark (Ventana, Roche) with the detection kit UltraView DAB (brown chromogen), without pretreatment. GN_mAb_Env03 monoclonal antibody was developed by GeNeuro and has already been validated in several publications (24, 25, 30). GN_mAb_Env03 monoclonal antibody was used at a concentration of 5 μg/ml for the 77 slides. Eight additional slides (4 T1D, 4 non-T1D) were used as controls, with mouse IgG2a isotype. Counterstaining was applied with hematoxylin II and bluing reagent. Slides were digitized with the slide scanner (Hamamatsu), objective ×20, and quantification was made using Indica Labs HALO platform. An algorithm was designed based on pattern recognition that discriminates pancreas tissue (analyzed areas) from fatty inclusions, vasculo-nervous structures, and surrounding connective tissue (excluded areas). Image analysis based on red, green, and blue (RGB) spectra was used to detect brown staining (DAB) within the positive areas (pancreas). The algorithm was designed to allow the detection of specific brown staining according to a threshold of intensity, and nonspecific edge staining of sections was not taken into account. It calculates pancreas area (mm²), staining area (mm²), and percentage (% stained area/pancreas area). Quantification was performed under blinded conditions, with anonymized slides from control and T1D donors.

Immunohistochemistry Sections (4μm) from formalin fixed paraffin embedded pancreas tissues were deparaffinized and rehydrated with serial passage through changes of xylene and graded ethanol. All slides were subjected to heat induced antigen retrieval in Target Retrieval Solution (Dako). The tissue sections were stained for insulin as a part of routine collection protocol for nPOD tissues (previously described (Campbell-Thompson et al., 2016; Campbell-Thompson et al., 2012b)) or double stained for insulin (polyclonal guinea pig anti-insulin,1:1000 dilution, Dako (Santa Clara, CA)) and glucagon (monoclonal mouse anti-glucagon, 1:5000 dilution, Abcam, Cambridge, MA) by immunohistochemistry (IHC), scanned using an Aperio CS Scanscope (Leica/Aperio, Vista, CA), and stored in the nPOD online digital pathology database (eSLIDE version 12, Leica/Aperio). For donors indicated in Table S1, scanned images of insulin-stained slides available from the block(s) nearest to the tissues used for total protein extraction (described above) were evaluated for fractional insulin area using Indica Labs, Inc image analysis software (Corrales, NM). For insulin and glucagon double stained slides, antigen-antibody binding was visualized using the EnVision G/2 Doublestain System (Dako). For control (n=5) and T1D subjects (n=11), glucagon + insulin positive islets, glucagon positive islets, insulin positive single cells, and clusters of two to five insulin positive cells were annotated by hand using the Aperio viewing platform and analyzed using Image Scope, Leica Biosystems analysis software (Version 12.1.0.5029, Buffalo Grove, IL).

Proinsulin, Insulin, C-peptide, Islet Amyloid Polypeptide and Glucagon by ELISA and Luminex Commercially available kits from ALPCO (Salem, NH) were utilized to measure total proinsulin, insulin and C-peptide from pancreas protein extracts as indicated in Table S1. The proinsulin, insulin and C-peptide assays are specific and do not cross react with each other. Glucagon was measured with a commercial ELISA assay provided by Mercodia (Winston Salem, NC). Islet amyloid polypeptide was measured by an assay from Millipore (Billerica, MA) using magnetic bead technology (Luminex). A total protein Bradford assay from Thermo-Fisher (Waltham, MA) was performed on each supernatant and used to normalize mass and extraction efficiency.

In situ hybridizationSections were dried for 1 hour at 60°C, pretreated and hybridized with an hs-ITPR2 probe (Homo sapiens inositol 145-trisphosphate receptor type 2 (ITPR2) mRNA) for 2 hours at 40°C. Probes were custom-designed and labeled for use with RNAscope 2.5HD (Advanced Cell Diagnostics, Newark, CA, USA). Some sections were stained with Hs-PPIB or dapB probes as positive and negative controls, respectively. Amplification steps were performed prior to the detection of signals with 3,3′-diaminobenzidine. Sections were counterstained and mounted with Permount (Fisher scientific, Waltham MA, USA).

ImmunofluorescencePancreatic slides were deparaffinized with Slidebrite (BioCare Medical, Concord CA) and dehydrated using graded ethanol concentrations. Slides were boiled in antigen retrieval citrate buffer pH6, blocked for 1hr with 2% normal goat serum, 2% bovine serum albumin, 0.5% Tween 20 in phosphate buffer saline followed by incubation with primary antibodies overnight at 4°C using different combinations. The staining series included antibodies to IP3R2 (Abcam, Cambridge, MA, USA 1:50), Translocase of outer mitochondrial membrane 20 (TOM20) (Santa Cruz biotechnologies, Dallas Texas, USA 1:50), VDAC-1 (Abcam, 1:100), mitofusin-2 (Abcam, 1:100), insulin (Dako, Carpinteria, CA, USA 1:150), glucagon (Abcam, 1:200). Secondary antibodies coupled to a fluorochrome (AF488, AF555, AF647, Life Technologies, Grand Island, NY, USA) were added for 30 min at RT according to the species of primary antibodies. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich) and preparations were mounted in Prolong Gold anti-fade reagent (Life Technologies). Slides were analyzed with a slide scanner AxioScan.Z1 (Carl Zeiss SAS, Marly le Roi, France) at x40 magnification.

In situ proximity ligation assayDuolink II in situ proximity ligation assay (PLA) (Olink Bioscience, Uppsala Sweden) enables detection, visualization, and quantification of protein interactions (<40 nm) as an individual dot by microscopy. Primary antibodies to assess ER-mitochondria interactions were against IP3R2 (1:100) and VDAC-1 (1:200) as previously described [7]. Digitized slides were analyzed at x20 magnification. When necessary, beta cells and alpha cells were identified using anti-insulin and anti-glucagon antibody staining on the same slide. Dots were quantified in each islet using the Zen program and Fiji-ImageJ software and expressed as percentage of dots per nucleus. Experiments were performed at least twice using 2 to 5 non-consecutive slides for each donor. For Min6-B1 cultures, ER-mitochondria-interactions were assessed using a fluorescent PLA assay, employing antibodies directed against VDAC-1 (Abcam, 1:100) and IP3R1 (Santa Cruz laboratories, Dallas TX, USA 1:500) as described previously [10]. Experiments with Min6-B1 cells were performed at least three times, with a minimum of five fields taken per condition.

pancreatic islets examined (methods as previously described by Campbell-Thompson et al. [11]) from nine blocks encompassing the pancreas head, body, and tail regions demonstrated CD3+ infiltration (insulitis)

Immunostaining and morphometric evaluation Paraffin sections were incubated overnight with primary antibody (Key Resources Table). For each antibody, sections were stained and imaged in parallel such that the staining intensity reflects the protein expression. For quantification, images were captured systematically covering the whole section in confocal mode on a Zeiss LSM 710 microscope. Every cluster of insulin-stained cells (3–7 cells) or islet (8 or more cells)/section was evaluated; sections were coded and read blindly. For each age, 3–4 animals were evaluated for each staining. For human samples, sections from one block from the body of the pancreas from donors as listed in Key Resources Table.

Immunofluorescence (IF) Image analysis was performed with Image Pro Premier software on randomly selected islets. For each islet, the difference of the mean intensity in the positive area (MIP), and the mean intensity in the negative area (MIN) (background) was measured (MIP-MIN). The differences between the groups and within the regions of each section were presented as the mean of (MIP-MIN) ± SD. All techniques, image acquisition and analysis have been validated by histology and microscopy specialists, at LJI which enabled us to rightfully quantify the IL-1β expression level.

2.4. AnalysisIn situ hybridization (ISH) Islets were quantified by Image-pro Premier software (Media Cybernetics, Rockville, MD). For each islet, the percentage of the positive area (or positive staining, defined by Image-pro Premier software) within the islet boundary was assessed and the differences between the groups and within the regions of each section were presented as the mean of analyzed islets (mean±SD).

Total terminal deoxynucleotide transferase–mediated dUTP nick end labelingApoptosis analysis was performed in a random sampling of T1D cases, as previously described (30), with modification; sections were predigested in 0.004% trypsin and identified with Cy5-labeled reagents. Total terminal deoxynucleotide transferase–mediated dUTP nick end labeling (TUNEL)–positive β cells were assessed in >95,000 islet cells per condition. In every sample, TUNEL-positive pancreatic ducts were imaged to ensure adequate TUNEL staining.

Proliferation analysisKi-67+ cells were measured within insulin-stained slides or ×20 magnification colorimetric images from optimal cutting temperature compound (OCT)–embedded pancreas sections prepared by the nPOD core laboratory, available through the nPOD Datashare. Colorimetric insulin and DAPI were identified using a sample-specific red-green-blue color range, established through sampling of multiple data points. Ki-67+ β-cell and Ki-67+ acinar proliferation was calculated as percent total cell population. Acinar cells were identified as nonislet cells containing DAPI.

Slides were imaged to quantify β-cell morphometry using Volocity 6.1.1 (PerkinElmer, Waltham, MA), as previously described (29). Images were acquired with Zeiss Axio Imager M1 (Carl Zeiss Microscopy, Thornwood, NY) with an automated X-Y stage and captured with Orca ER camera (Hamamatsu, Bridgewater, NJ), resulting in images of tens of thousands of individual nuclei per sample (summarized in Supplemental Table 3).

Whole sections of pancreas stained for insulin, Ki67, and alcian blue with hematoxylin counterstain were digitally scanned using Aperio ScanScope (Aperio Technologies, Vista, CA). Analysis was performed using Aperio ImageScope version 11.0.2.725. Three sections per case, 1 each from head, body, and tail, were analyzed, except in 1 T1D case [6031] and 7 control cases (6010, 6012, 6013, 6015, 6017, 6021, 6022) in which only head and tail sections were available. All sections were examined and quantified in a blinded manner.Interlobular ducts were defined as ductal structures embedded in mesenchyme and possessing a PDG compartment. PDGs were identified as coiled invaginations composed of columnar epithelium arising from interlobular ducts and lying within the mesenchyme surrounding those ducts. The number of interlobular duct epithelial cells and PDG cells was counted, as was the number of those cells with nuclei staining for Ki67 and cytoplasmic staining for insulin. Small ducts, defined as intralobular ducts not embedded in mesenchyme, were comparably analyzed.

Immunohistochemistry was performed using a standard immunoperoxidase approach, as previously described [21]. To examine multiple antigens within the same FFPE section, samples were probed in a sequential manner with up to three different antibodies (ESM Tables 3, 4). The mean fluorescence intensity (MFI) of stained antigens was measured using ImageJ Version 1.50b Java 1.8.0_77; https://imagej.nih.gov/ij/download.html. Some slides were processed with isotype control antisera to confirm the specificity of labelling (ESM Fig. 1). Frozen sections were stained using a standard immunofluorescence approach [22].

Using the Affymetrix Human Gene 2.0 ST array, CEL files were generated from both control and type 1 diabetic donors, as previously described [24]. Raw signal-intensity values from Affymetrix spike-in controls demonstrated that array hybridisation had been successful (i.e. bioB<bioC<bioD<Cre). Data quality was verified by measuring the positive vs negative area under the curve. Raw signal-intensity values from all arrays were robust multichip average background corrected, quantile normalised, median polish summarised and log2 transformed [25–27]. NetAffx-determined probe-set annotations for HLA genes (Affymetrix) were re-mapped according to RefSeq, release 73 (15 November 2015; see ftp://ftp.ncbi.nlm.nih.gov/refseq/release/release-catalog/archive/). For each HLA gene, where multiple mappings were possible (i.e. HLA-A, -B, -C and -F), probe sets were annotated according to eight major haplotypes incorporated into the human genome assembly, as previously described [28]. Because probe sets shared mappings, it was not possible to identify HLA subtypes uniquely using this gene chip; rather, transcript clusters were used to examine changes in global gene expression. Processing was carried out using the Partek Genomics Suite, version 6.5 (Partek, St Louis, MO, USA). The resulting normalised expression data for specific genes of interest were then subjected to analysis as described below.

Human pancreas sections were deparaffinized, followed by acidic-pH heat–mediated antigen retrieval. Cultures of single pancreatic islet cells were fixed with 4% paraformaldehyde. Samples were blocked and permeabilized in PBS with 0.3% Triton X-100 and 10% goat or donkey serum. Primary antibodies CHOP (1:100; sc-575, Santa Cruz Biotechnology), GAD6 mouse monoclonal antibody (mAb) against C-terminus of GAD65 (31) (1:1,000); N-GAD65 mouse mAb against the N-terminus of GAD65 (32) (1:300), giantin (1:1,000; ab24586, Abcam); insulin (1:10,000; 4011-01, Linco); insulin (1:2,000; ab14042, Abcam), and CD3 (1:30; M7254, Dako) were incubated overnight at 4°C in PBS with 0.3% Triton X-100 and 1% goat or donkey serum. Alexa Fluor conjugated secondary antibodies (Molecular Probes) were incubated at 1:200 dilution in PBS with 0.3% Triton X-100 for 30 min at room temperature.Image Capture, Analysis, and QuantificationSamples were imaged on a Zeiss LSM700 confocal microscope with 63×/1.40 NA Plan-Apochromat oil-immersion objective for single islet cells and 40×/1.30 NA Plan-Apochromat oil-immersion objective for pancreatic tissue sections. All images for quantification within a single experiment were captured with the same laser power and detector gain. The ratio of GAD65 mean fluorescence intensity (MFI) in the Golgi compartment and post-Golgi vesicles compared with the rest of the cytosol was calculated with a custom ImageJ macro. Individual β-cells in a given field of view were identified and outlined by hand. For each cell, the macro automatically defined a region of interest (ROI) outlining the Golgi compartment, identified by giantin costain or by characteristic morphology and brightness thresholding of GAD65 stain, and GAD65+ vesicles, identified by brightness thresholding of GAD65+ bright puncta. A second ROI defined the remainder of the cell, excluding the Golgi, GAD65+ vesicles, and nucleus. GAD65 Golgi accumulation was reported as the ratio of MFI for the two ROIs.

Morphometric analysis was carried out with a semiautomated process, as described in Supplemental Methods and Supplemental Figure 1. This process allows analysis of the whole section. Lymph nodes (found in five slides from control Ab− subjects, 1%), and regions with edge artifacts or nonspecific staining were manually excluded. For each slide, we quantified the total tissue area and separated it into exocrine and nonexocrine tissue (adipose and mesenchymal tissues, large ducts, and vessels) vs the insulin-stained area. The percentage of the total tissue area was converted into mass by multiplying it by the weight of the pancreas when available. When the weight of the pancreas was not available, only the ratio of the endocrine area to the total area was calculated. We used Photoshop CS4 (Adobe Systems) to perform thresholding on whole sections, and measurements were performed using ImageJ (http://rsbweb.nih.gov/ij/). Given that only the cytoplasm was stained for β-cells using the insulin Ab, we developed another Photoshop script to quantify the surface of the entire islet.

Insulitis Leukocyte PhenotypingParaffin sections from blocks having the maximum insulitis frequency for each donor were stained, and positive leukocytes/insulitic islets (six or more CD3+ cells) were counted using multi-immunofluorescence. Serial sections (4 μm) were dewaxed and rehydrated with Tris buffer. Heat-induced antigen retrieval was performed using Trilogy (Cell Marque, Rocklin, CA) at 95° for 20 min followed by rinsing in water for 20 min. The staining series was designed to phenotype leukocytes (total leukocytes [CD45], T [CD3] and B [CD20] lymphocytes, T-lymphocyte subsets [CD4 and CD8], and monocytes [dendritic cells (CD11c) and macrophages (CD68)]) (21) in conjunction with subtyping islets for insulin immunopositivity. The staining series was as follows: 1) CD45+glucagon+insulin, 2) CD20+CD3+glucagon, 3) CD8+CD4+glucagon, and 4) CD11c+CD68+insulin. Chromogranin A staining was also used to delineate endocrine cells. Antigens are listed in order of primary antibody incubation, and the corresponding secondary antibody and conjugated fluorochrome (AF488-AF555-AF647) (Supplementary Table 2). After blocking, sections were sequentially incubated with the primary antibody followed by the appropriate secondary antibody. For anti-CD4, a Cy3 Tyramide Signal Amplification Kit (PerkinElmer, Waltham, MA) was used according to the manufacturer's instructions. All sections were mounted with ProLong Gold Antifade mounting media containing DAPI (Life Technologies, Grand Island, NY). Positive controls included human spleen, tonsil, and donor intrapancreatic lymph nodes, and negative controls included omission of the primary antibody.The numbers of leukocytes/insulitic islets were determined using multichannel image acquisition software on a Zeiss Axiophot microscope (AxioVision; Carl Zeiss Inc., Thornwood, NY). Fluorescent channels were viewed in combination with DAPI to count the number of positive leukocytes/islet.

β-Cell and α-Cell Area and MassInsulin- and glucagon-immunopositive areas were determined using the IHC sections to estimate β-cell and α-cell areas, respectively, in relation to total tissue area using a single Aperio colocalization algorithm (22). An average of six sections was used per donor (two sections/head, body, and tail regions). The β-cell and α-cell areas were expressed as a ratio (percent) to the total sectional area, including acinar and interstitial regions, to permit the use of pancreata weights. The average β-cell and α-cell area per pancreas was calculated from regional area averages. The β-cell or α-cell mass (in milligrams) was calculated by multiplication of the respective average area and pancreas weight (in grams).

Insulitis Screening and Insulitic Islet Subtyping for Insulitis FrequenciesPancreata were processed to formalin-fixed paraffin blocks for each pancreas region (head, body, and tail) as previously described (20). For each donor, serial sections (average two blocks per region) were stained by hematoxylin-eosin and two double-immunohistochemistry (IHC) stains (Ki67 and insulin, CD3 and glucagon) (Supplementary Table 2) (21). When insulitic islets were found in a given donor, additional blocks were screened (as detailed below). Stained sections were scanned at ×20 magnification using an Aperio CS scanner (Leica/Aperio, Vista, CA), and all images were stored in an online pathology database (eSLIDE; Leica/Aperio).Screening for insulitic islets was performed on CD3+ glucagon–stained sections. An islet was defined as ≥10 α-cells. Insulitis was defined as an islet with six or more CD3+ cells immediately adjacent to or within the islet with three or more islets per pancreas section, according to recent criteria (4). Islets with insulitis were marked in an image layer using ImageScope software (Leica/Aperio). The two IHC serial images were aligned using the synchronization tool, and insulin+ islets were also marked on the image layer. All islets/sections from donors with insulitis were subsequently subtyped as follows: 1) insulin+ CD3−, 2) insulin+ CD3+, 3) insulin− CD3+, and 4) insulin− CD3− (see Table 1 for numbers of islets analyzed). Then, all islets were counted by subtype. The process was reversed for AAb+ donors (i.e., islet subtypes were counted using the Ki67-insulin image after markup for CD3+ insulitic islets and insulin− islets using the CD3-glucagon image). The number of pancreas sections subtyped for insulitis ranged from 2 to 16 sections/donor (8.1 ± 4.1 sections/donor, n = 162 sections) (Table 1). The lowest number of available sections was due to partial pancreas recovery (tail only in nPOD 6198).Insulitis frequency (percent) was calculated as the total number of insulitic islets (sum of insulin+ CD3+ and insulin− CD3+ islets) divided by the total number of islets (sum of four subtypes). The frequency of insulin+ insulitic islets in relation to the total number of insulin+ islets was determined by the ratio of (insulin+ CD3+ islets)/(sum of insulin+ CD3− and insulin+ CD3+ islets) with similar calculations for the frequency of insulin− insulitic islets (insulin− CD3+)/(sum of insulin− CD3+ and insulin− CD3−).

The pancreas was weighed intact and/or after dividing it into three regions (head, body and tail) by cutting the junction between head and body at the notch and dividing the remaining portion in half for body and tail regions.

To monitor the cellular distribution of cyclin-D isoforms, pancreatic sections were stained with either an anti-cyclin-D3 antibody plus HRP–goat anti-mouse IgG and Alexa Fluor 488 tyramide (Life Technologies, Eugene, OR, USA), or an anti-cyclin-D1 antibody plus HRP–goat anti-rabbit IgG and Alexa Fluor 488 tyramide. The tyramide amplification steps were used to enhance the fluorescence signal and were performed according to the manufacturer’s instructions (Life Technologies). Pancreatic sections were co-stained with an anti-glucagon antibody raised in either mouse or rabbit (each from Abcam) and with guinea pig anti-insulin (Dako, Ely, UK) plus relevant secondary antibodies labelled with Alexa Fluor 568 and Alexa Fluor 647 (Life Technologies), respectively. Images were captured under fluorescence illumination using a Leica AF6000 microscope (Leica, Milton Keynes, UK).

Insulitis was defined by a recent guideline which stipulates that the total number of leucocytes in close contact with the islet boundary (peri-insulitis) and within the intra-islet areas is equal to or greater than 15 [29].

Peri- and intra-islet CD45 cells were enumerated manually in all sections. For the diabetic group, observers were blinded to the case details, including autoantibody status. Islets with approximately ≥20 endocrine cells were analysed. Single glucagon and insulin cells scattered within the exocrine region were not enumerated, while sections from diabetic cases containing small islets (20 cells) but harbouring at least one insulin cell were recorded. The total numbers of insulin-positive and -negative islets in the pancreatic head, body and tail were also recorded in each section.

Sections (5 μm) were de-paraffinised, rehydrated and subjected to antigen retrieval with citrate buffer containing 0.05% Tween-20 (Sigma-Aldrich, St Louis, MO, USA). During immunohistochemistry, PBS, pH 7.4, was employed as a wash step. Sections were equilibrated in PBS and blocked with 5% normal goat serum (Sigma-Aldrich) for 1 h at 37°C. A mixture of guinea pig anti-insulin serum (A0564, dilution 1:600; Dako, Glostrup, Denmark) and rabbit anti-glucagon serum (A0565, dilution 1:200; Dako) in 5% normal goat serum (Sigma-Aldrich) was applied and incubated for 1 h at 37°C. Highly cross-adsorbed species-specific goat anti-guinea pig IgG-Alexa 568 (A11075; Invitrogen, Eugene, OR, USA) and goat anti-rabbit IgG-Alexa 488 (A11034, dilution 1:600 in 5% normal goat serum; Invitrogen) were then applied as a mixture and incubated as in the previous step. Sections were incubated with mouse anti-human CD45 (M0701; Dako; clones 2B11 + PD7/26, dilution 1:100 in PBS + 0.1% Tween-20) for 16 h at 4°C, washed and reacted with 3% H2O2 for 15 min. After washing, sections were incubated sequentially with donkey anti-mouse IgG-biotin (715-065-150; Jackson ImmunoResearch, West Grove, PA, USA, dilution 1:200 in PBS + 0.1% Tween-20) and streptavidin-horseradish peroxidase (016-030-084; Jackson ImmunoResearch, dilution 1:200 in PBS/0.1% Tween-20). They were finally exposed to a diaminobenzidine chromogenic mixture (Sigma-Aldrich) to visualise CD45 cells. Non-immune serum or IgG from the immunising species and omission of primary antibodies acted as negative controls.Sections were examined with a Nikon Eclipse E600 microscope under epifluorescence and bright field microscopy and digital images were recorded. All islets in a section with ≥20 endocrine cells were imaged for the presence of insulin, glucagon and CD45 cells and each of the three image sets from multiple acquisitions merged with Adobe Photoshop CS4 following conversion of CD45-positive cells to a greyscale fluorescence mode.

For morphometric comparison of human islets from 12-LO-positive and -negative cases, a composite image of 50 visual fields at ×200 magnification (area of 50 000 μm2) was captured using the mosaic function of AxioVision analysis software (v4.7; Zeiss). Five composite images of the pancreas were obtained per case, and at least 3 cases per group were studied. Areas positive for insulin or 12-LO were defined by setting a threshold signal for each antigen, and the size of the positive area was quantified automatically based on this threshold. Hematoxylin and eosin (HE)-stained images on the nPOD website from serial sections of the blocks we analyzed were used to determine the islet area for each donor. To quantitate the PP staining intensity, images of 30 islets from the uncinate and nonuncinate areas of pancreas sections immunostained for PP were analyzed for mean densitometric values of PP+ areas using AxioVision v4.7. To normalize staining, mean densitometric background staining with nonimmune serum was subtracted from values obtain for the PP sections. Observers blinded to the clinical information of donors performed all measurements

Paraffin embedded pancreatic sections from female cadaveric donors including non-diabetic controls and type 2 diabetes patients were obtained from the Juvenile Diabetes Research Foundation (JDRF)-sponsored Network for Pancreatic Organ Donors with Diabetes (nPOD) program (http://www.jdrfnpod.org/for-investigators/online-pathology-information/). Insulin positive (Ins+) β-cells were used for staining and quantification.

Concomitant fluorescence in situ hybridization (FISH) and immunofluorescenceParaffin sections were deparaffinized in a xylene series followed by heat mediated antigen retrieval. Slides were then dehydrated in an ethanol series (70%, 80% and 100%) and air dried. Frozen sections were fixed in ice-cold pure ethanol for 10 min and air-dried prior to in situ hybridization. Purified human islets were cultured on rat tail type I collagen coated coverslips. They were fixed in 1% paraformaldehyde for 15 min, then simultaneously permeabilized and blocked in 0.1% (w/v) saponin (Sigma, S4521), 3% BSA solution for 45 min, and subsequently dehydrated in ethanol series. After pre-treatments, tissue sections or cell preparations were incubated with X/Y chromosome FISH probes (Vysis CEP X Spectrum Orange™/Y Spectrum Green™ Direct labelled Fluorescent DNA probe, Abbot Molecular Inc., Des Plaines, IL) as described previously [10]. To study aneuploidy/polyploidy, the CEP 18 probe (Abbot Molecular Inc.) was used in combination with the X/Y probe. After post-hybridization washes, sections or cells were incubated with primary antibodies (and controls as appropriate) specific for insulin (DAKO, A0564), glucagon (DAKO, A0565), somatostatin (Santa Cruz, SC-13099), GATA4 (Santa Cruz, SC-1237), Cytokeratin-19 (Abcam, Ab9221), Ki67 (Abcam, Ab833), CD68 (DAKO, m0876), CD45 (DAKO, M0701), nestin (Millipore MAB5326), or CD34 (Invitrogen, 073403) for 2 hrs at 37°C. After probing with the corresponding fluorescent secondary antibody for 1 hr at room temperature, sections were washed three times in 1× PBS solution, dehydrated, and counterstained with DAPI permanent mounting media (VECTASHIELD®, Vector laboratories, CA).Image analysisFluorescent imaging Images were captured using an Olympus BX41 fluorescent microscope fitted with an AxioCam MRm digital camera (Zeiss, Germany). The X and Y chromosomes were analyzed using the ISIS fluorescent imaging software (Zeiss, Germany) designed for FISH.Confocal microscopy To confirm MMc frequencies, pancreatic tissue sections were screened using a Leica SP5 confocal imaging system at the Wolfson Bioimaging Facility, School of Biochemistry, University of Bristol, UK. Z-stack images of each tissue section were analyzed for FISH and immunofluorescence to allow visualization of X and Y chromosome signals in all planes of the nucleus.Counting strategyFISH signals were checked in individual filter channels to ensure signal fidelity and that the visualization of two red dots representing two copies of the X chromosome was not caused by immunofluorescence cross talk. Only cells that showed clear XY (red and green dot) or XX (two red dots) signals within single nuclei were counted. Any cells with overlapping nuclei were excluded from the analysis. X chromosome “splits” that occasionally appeared as two juxtapositional dots due to DNA breakage were not considered as XX signals. A FISH success rate (the frequency of nuclei with two clear signals visible) of greater than 60% was required before further analysis.All T1D and matched control tissues were stained for FISH and insulin. Overall, as many nuclei as possible were counted (>1000 at least), including at least 20 islets per section. MMc were counted in insulin positive and insulin negative cell fractions. For T1D case 1, 2, and 3, MMc were also examined in CD45+ population. An additional independent, blinded scorer reviewed each candidate female cell.

Human ADORA1-VAR. Human pancreas RNA samples with intact 18S and 28S rRNA were used for these studies. Primers were designed to examine if a human equivalent of the mouse Adora1-Var is expressed in the pancreas (Table 2, primer set 5). The forward primer spans exon 2 and 4 and only detects a splice variant lacking exon 3 of the ADORA1 gene ({"type":"entrez-nucleotide","attrs":{"text":"NM_000674.2","term_id":"115305570","term_text":"NM_000674.2"}}NM_000674.2). Primers targeting only exon 3 were designed to detect the full-length human ADORA1 gene (Table 2, primer set 4). For cloning into TOPO, primers spanning the start and stop codon of ADORA1-VAR were used (Table 2, primer set 3). Fusion proteins were synthesized by standard subcloning techniques using the PCR primers listed in Supplementary Table 1.

Paraffin-embedded pancreatic sections from diabetes-free controls and T1D patients were obtained from the Juvenile Diabetes Research Foundation (JDRF)-sponsored nPOD program (http://www.jdrfnpod.org/online-pathology.php). Sections were used for staining and quantification of expression levels of selected UPR markers.

Human Patient SamplesFrozen sections of human pancreata were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD), a collaborative T1D research project sponsored by the Juvenile Diabetes Research Foundation International (JDRF). We used sample/CaseID as indicated on the nPOD website. A total of four T1D and five T2D samples were selected for this study based on their blood glucose, body mass index (BMI) values, and history of diabetes. Samples from healthy patients (n=5) were used as a control. All patient samples used in the study were excised from the tail of the pancreas.Immunohistochemistry of Pancreatic Sections and CellsHuman and mouse pancreatic sections were co-immunostained with IC2, anti-insulin and anti-glucagon or anti-somatostatin antibodies. Frozen sections (5–7-µm thick) were fixed with 4% formaldehyde for 5 min, washed with PBS and blocked with 2% BSA in PBS for 1 hr at room temperature. IC2 (1 µg/ml) and guinea pig anti-insulin (Abcam, Cambridge, MA) and rabbit anti-glucagon antibodies (Abcam), diluted to 1:50 and 1:100, respectively, were mixed in 2% BSA, added to the sections and incubated at room temperature for 2 hr. Then, sections were washed with PBS and incubated in the mixture of goat anti-rat IgM-AF594 (1: 1000 dilution, Invitrogen, Carlsbad, CA), goat anti-guinea pig IgG (H+L)-FITC (1:200 dilution, Abcam) and goat anti-rabbit IgG (H+L) AF-680 (1:500 dilution, Invitrogen) for 2 hr. Next, the sections were washed with PBS, mounted in ProLong® Gold anti-fade reagent with DAPI (Invitrogen), and observed under fluorescence microscope. For pancreatic sections from human patients, the fluorescence intensity was scored on a scale of 1 to 5 by two blinded investigators. In all of these experiments, an irrelevant purified rat monoclonal IgM was used as control for IC2. Staining without primary antibodies was also used as a control.

Combined immunofluorescence For co-localisation studies, anti-insulin and anti-VP1 immunoreactivity were detected using an AlexaFluor 488-conjugated anti-guinea pig antibody and an AlexaFluor 568-conjugated anti-mouse antibody, respectively (Invitrogen, Paisley, UK). To determine whether the enteroviral VP1 protein co-localised with either PKR or Mcl-1 in beta cells, primary antibodies were incubated as described in ESM Table 3. The primary antibodies were detected with relevant goat secondary antibodies conjugated to AlexaFluor 488 or 568 (Invitrogen) or with goat anti-guinea pig DyLight 405 (Stratech, Newmarket, UK). Control sections were stained with relevant primary and secondary antisera to confirm that no cross-reactivity was detected. Sections were mounted in Vectashield hard-set mounting medium (Vector Laboratories, Peterborough, UK) under glass coverslips. Images were captured using a Nikon Eclipse 80i microscope (Nikon, Kingston upon Thames, UK) and overlaid using NIS-Elements BR 3.0 software (Nikon) to study the relative localisation of each antigen. Sections directly adjacent to those stained using the combined method were stained with an anti-glucagon (rabbit; Dako) or an anti-insulin (guinea pig; Dako) antibody using a standard immunoperoxidase technique to determine total islet numbers in the sections and to distinguish insulin-containing islets (ICIs) from insulin-deficient islets (IDIs).

Quantitative real-time RT–PCR in tissue, cells, and polysome fractionsTotal RNA was extracted using Trizol reagent and the Qiagen RNeasy mini kit or micro kit, as previously described (Yip et al., 2009). First-strand cDNA was generated using the iScript cDNA synthesis kit (Biorad). Quantitative PCR was performed to measure mouse Eif4g3, Eif4g1, Casp3, Deaf1, Ins2, Fgb, Ela, Ppy, Tyr, Ambp, Gapdh, and Actb mRNA levels, and human EIF4G3, EIF4G1, CASP3 and ACTB, and GAPDH mRNA levels. cDNA was preamplified using the Taqman PreAmp Mastermix (Applied Biosystems) prior to QPCR for Ins2 and for gene expression measured in polysome fractions and LNSC subsets. For all other experiments, cDNA was not pre-amplified. qPCR assays were performed using the 7900HT Fast Real Time PCR System (Applied Biosystems), Taqman Gene Expression Arrays (Applied Biosystems), and SsoFast Probes Supermix (Biorad). For human CASP3 measurements, Quantitect primers (Qiagen) and SsoFast EvaGreen Supermix (Biorad) were used. The comparative Ct method for relative quantification (ΔΔCt) was used, and expression was normalized with housekeeping gene expression.

Initial screening for insulin, MHC class I, and CD8. For characterization of the sections before tetramer staining, staining for insulin, CD8, and HLA-ABC (all at room temperature) was performed using the antibodies listed in Table 3. Fixation was with acetone and sections were blocked with goat serum in a standard immunofluorescent staining protocol. An islet was determined as hyperexpressing MHC class I based on a threshold of three unequivocally positive cells, which was the maximum number found in all control islets examined.Table 3.Antibodies used for characterization of the sections prior to tetramer stainingAntigenPrimary antibodyDetection antibodyInsulinPolyclonal guinea pig anti-insulin (Dako; 1/140 dilution, 1-h incubation)Polyclonal goat anti–guinea pig IgG, highly cross-adsorbed, Alexa Fluor 488 (Invitrogen; 1/1000 dilution, 30-min incubation)HLA-ABCMouse monoclonal (clone W6/32) IgG2a against a monomorphic epitope on the 45 kD polypeptide products of the HLA-A, B and C loci (Dako; 1/100 dilution, 1 h incubation)Polyclonal goat anti–mouse IgG2a, isotype-specific, Alexa Fluor 594 (Invitrogen; 1/1000 dilution, 30-min incubation)CD8Mouse monoclonal (clone HIT8a) IgG1 against the CD8 alpha subunit (BD; 1/100 dilution, 1-h incubation)F(ab’)2 fragment of goat anti-mouse IgG, Alexa Fluor 594 (Invitrogen; 1/1000 dilution, 30-min incubation)

nPOD central AAb core laboratoryThe nPOD central AAb core laboratory has a long history of excellence in the type 1 diabetes AAb field 10, 13, 15, 21, 22, participating routinely in the Diabetes AAb Standardization Program (DASP), now renamed IASP. The nPOD central AAb core began testing for ZnT8A in nPOD cases using RBA prior to the introduction of the ZnT8A ELISA. This core also tests for IAA as there is currently no reliable ELISA for this analyte. Every case with available serum that is referred to nPOD is tested via RBA for GADA, IA‐2A, ZnT8A and IAA for either confirmation of the ELISA screening results, or for determination of final AAb status. In cases of discrepancy, the RBA supersedes the ELISA as the result reported on the nPOD website (www.jdrfnpod.org).

Morphometric analysis Beta cell area (defined as the area of cells stained by insulin) was quantified using computer-assisted morphometric analysis of slides digitally stored in the Aperio System. We obtained the ratio between the insulin stained areas and the total pancreatic section area, i.e. the relative volume of beta cells in patients with diabetes and in normal controls. For each diabetic pancreas we then determined the beta cell index defined as the relative volume divided by the mean relative volume of beta cells of controls multiplied by 100. The beta cell index thus expressed the beta cell area of each diabetic pancreas as a percentage of normal control pancreases. Double immunofluorescent staining for insulin and glucagon was used to determine the percentage of islets devoid of beta cells in pancreases with residual beta cells (i.e. the percentage of insulin-deficient islets). In each section we determined the percentage of insulin-deficient islets (i.e. islets composed of non-beta cell vs the total number of islets). Only islets completely devoid of beta cells were considered insulin-deficient islets.

For each childhood-onset case, the pancreas was processed in three regions (head, body and tail) and fixed overnight in 10% (vol./vol.) neutral buffered formalin (ThermoFisher, Waltham, MA, USA) prior to processing to paraffin blocks. Tissue sections (5 μm) were obtained stained with haematoxylin and eosin, immunohistochemistry and immunofluorescence. Pancreatic tissue sections were stained: (1) with antibodies to insulin or glucagon, and a panleucocyte marker CD45 and/or a T cell marker (CD3; and in a subset of diabetic pancreases with CD4, CD8, CD20 and CD68 antibodies); and (2) with a cocktail of antibodies to endocrine non-beta cell hormones composed of glucagon, pancreatic polypeptide and somatostatin. In diabetic pancreases with insulitis (as defined below), the infiltrating T cells were further characterised with antibodies to CD4 and CD8 (as well antibodies to CD20 and CD68 to detect B lymphocytes and macrophages). Antibody binding was detected with appropriate secondary antibodies to guinea pig, rabbit and mouse immunoglobulins conjugated with alkaline phosphatase and peroxidase (MACH2 Polymer Systems; Biocare, Concord, CA, USA), followed by chromagen development with fast red (Vector, Burlingame, CA, USA) or diaminobenzidine (Vector). A scanner (CS ScanScope; Aperio, Vista, CA, USA) was used to produce whole slide images for both haematoxylin and eosin and immunohistochemistry-stained slides. In a subset of diabetic pancreases (pancreases containing residual beta cells), the pancreas was also double-stained with antibodies to insulin and class I HLA molecules. Immunofluorescence was performed by incubating the tissue sections with antibody to insulin, glucagon and survivin (Abcam, Cambridge, MA, USA), and with appropriate anti-rabbit and anti-guinea pig immunoglobulins conjugated with aminomethylcoumarin acetate, Cy3 or Cy5. The sections were photographed at ×20 magnification using a microscope B651 (Olympus America, Center Valley, PA, USA) connected with a digital imaging system (Image pro plus, version 6.2; Media Cybernetics, Bethesda, MD, USA) with a camera (Pro 150ES; Pixera, San Jose, CA, USA). Additional images were recorded on an epifluorescence microscope (Microphot FXA; Nikon Instruments, Melville, NY, USA) with a monochrome digital camera (Roper Micromax; PerkinElmer, Waltham, MA, USA) and appropriate software (Intelligent Imaging Innovations, Denver, CO, USA). The photos are displayed in pseudocolour. Immunoperoxidase staining of survivin was performed by incubating the sections with antibody to survivin and visualising with Cytomation Envision+System-HRP (DAB; Dako, Carpenteria, CA, USA). Class I HLA immunostaining was performed with mouse monoclonal anti-human HLA-ABC, Clone W6/32 (Dako), directed against a monomorphic epitope on the 45 kDa polypeptide products of the HLA-A, -B and -C loci. Insulin was detected with polyclonal guinea pig anti-swine insulin (Dako). All secondary goat antibodies (Life Technologies, Carlsbad, CA, USA) were highly cross-adsorbed against the host species of the other primary antibodies and conjugated with Alexa 488 (green) or 594 (red). Anti-fade reagent (Prolong Gold) with DAPI (Life Technologies) was applied upon staining and sections were imaged using an epifluorescence microscope (Eclipse 80i; Nikon) equipped with a mercury arc lamp (X-Cite, Mississauga, ON, Canada) and a digital camera (DXM1200C; Nikon). An air objective (×20; Nikon) was used with a 0.75 N.A. Positive and negative controls for HLA-ABC staining included human spleen and isotope-matched primaries, respectively. Cross-reactivity with the insulin primary was also experimentally excluded. In additional experiments, HLA immunofluorescence staining was performed with rabbit polyclonal antibody (Abcam). An irrelevant rabbit polyclonal antibody served as a negative control.

SGCG

SGCA

TPM2

TPM2

TNNT3

MYH7

SLC1A1

FBXO11

developmental delay

FBXO11

RNF13

NEB

EXTL3

MED12

HNF4α

UBE2A

UBE2A

PLAA

EXTL3

PPM1D

EMC1

EXTL3

UBE2A

EXTL3

EXTL3

EXTL3

LINC00520