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, Cell Metabolism, vol.28, p.15, 2018.

. Engelbreth-holm, They were maintained in DMEM/F12 medium supplemented with 2% bovine serum albumin, 6.7 ng/ml sodium selenite, 10 mM nicotinamide, 50 mM b-mercaptoethanol and penicillin/streptomycin. IFN-g (R&D) was added to the cell culture at 80%-90% confluence at a final concentration of 500 U/ml for 16-18 h. IFN-g, TNF-a and IL-1b (all from R&D) were added at a final concentration of 2,000 U/ml, 1,100 U/ml, and 1,000 U/ml, respectively. Primary Human Tissues and PBMCs For HLA-I peptidomics experiments, Swarm murine sarcoma

H. , ) were obtained from a brain-dead non-diabetic organ donor (age 49 years, male, BMI 37 kg/m 2 ; protocol approved by the Agence de la Biome´decine) with standard procedures and maintained in CMRL 1066 medium (Sigma) supplemented with 10% fetal bovine serum. For RNA-seq analyses, primary human islets from 5 brain-dead non-diabetic organ donors

, Italy) were exposed or not to IFN-g (1,000 U/ml) and IL-1b (50 U/ml) for 48 h. For HLA-I expression analyses (Figure 2G), primary human islets were from 3 non-diabetic organ donors (mean age 54.7 ± 14.3 years, 1 female, 2 males, BMI 29 ± 5 kg/m 2 ; protocol approved by the Ethics Committee of the University of, p.367, 2002.

I. Ile-de-france, Informed consent was obtained from all subjects, or next-of-kin for islet donors. For in-situ MMr staining, pancreas and PLN sections were provided by nPOD

, The W6/32 mAb recognizes a conformational epitope formed by the interaction of the HLA-I heavy chain and b2-microglobulin and was used for purifying pHLA-I complexes and for flow cytometry in conjunction with an FITC-conjugated goat anti-mouse Ig Ab (BD). The HC10 mAb recognizes a linear epitope on the HLA-I heavy chain and was used for western blotting along with an anti-a-tubulin mAb for loading control (Figure 1B). Since a-tubulin expression is higher in ECN90 b cells than in primary islets, an anti-b-actin mAb was used for loading control to compare HLA-I expression and upregulation between the two cell types (Figure 2G), W6/32 and HC10 anti-HLA-I mAbs were purified on a protein A Prosep Ultraplus column (Millipore) from hybridoma supernatants

, Lysates were cleared by centrifugation and pHLA-I complexes immunoaffinity-purified with the W6/32 mAb covalently bound to Protein A Sepharose CL-4B beads (GE Healthcare) by dimethyl pimelimidate cross-linking. Beads were subsequently loaded on GELoader Tips (20 ml; ThermoFisher) and washed before elution of pHLA-I complexes with 10% acetic acid. Aliquots were collected at each washing and elution step for analysis by 12% SDS-PAGE and western blot using the HC10 mAb to verify the yield and purity of the eluted HLA-I. Eluted peptides and the associated HLA-I heavy chain and b2-microglobulin obtained from 20x10 6 cells were concentrated to 20 ml by vacuum centrifugation, acidified with 10 ml of 1% formic acid (Normapur) and loaded on C18 stage tips (ThermoFisher) prewashed with 100% methanol and equilibrated with 2% acetonitrile (ACN) in 0.1% formic acid in LC-MS grade water. After loading, the C18 stage tips were washed with 2% ACN/0.1% formic acid and peptides separated from the HLA-I heavy chain and b2-microglobulin species by eluting them with 50% ACN/0.1% formic acid. The ACN was evaporated by vacuum centrifugation and the peptides resuspended up to 6 ml of volume in a solution of 2% ACN/0.1% formic acid and spiked with 10 fmol/ml of a cytomegalovirus pp65 495-503 peptide (NLVPMVATV) as internal control. For LC-MS analysis, 5 ml of this peptide solution were used, The HLA-I peptidome of the ECN90 b-cell line was obtained from 5 biological replicates. A single biological replicate was available for primary human islets. Frozen cell pellets ($20x10 6 /condition for ECN90 cells; $25,000 islet equivalents/condition for primary islets, corresponding to $19x10 6 b cells) were resuspended in a buffer containing 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 0

. Dionex, Peptides were eluted with a linear gradient of 2%-50% buffer B (80% ACN, 0.05% formic acid) at a flow rate of 220 nl/min over 60 min at 35 C. Data were acquired using a data-dependent acquisition ''Top 20'' method. We acquired one full-scan MS spectrum at a resolution of 60,000 at 200 m/z with an automatic gain control (AGC) target value of 3x10 6 ions, followed by 10 MS/MS spectra in higher energy collisional dissociation mode on the 10 most intense ions at a resolution of 15,000 at 200 m/z with an AGC target value of 1x10 5 with a maximum injection time of 120 ms and a dynamic exclusion of 20 s. Unassigned precursor ion charge states and charge states >4 were excluded. The peptide match option was set to 'preferred'. The resulting spectra were analyzed by MaxQuant, electrospray ionization Q Exactive HF mass spectrometer (ThermoFisher) with a glass emitter (New Objective), 2012.

. Berkers, 2015) from major known and candidate b-cell protein Ags (Figure S1 and Data S1); and c) e3, Cell Metabolism, vol.28, pp.1-15, 2018.

, PTM candidates were not further analyzed. The other candidates underwent additional filtering steps to focus subsequent studies on HLA-A2restricted peptides. Predicted HLA-A2 binders were first selected in silico using NetMHC 4.0 and NetMHCstab 1.0 (except for in silico mRNA splice candidates, which had already been filtered for predicted HLA-A2 binding in the same way), then in vitro for experimental HLA-A2 binding and, PTM variants) and 2 in vitro mRNA splice candidates

, This expression is stabilized in the presence of HLA-A*02:01-binding peptides, resulting in a higher surface expression that can be monitored by flow cytometry. T2 cells were washed in RPMI medium and plated in round-bottom 96-well plates at 0.2x10 6 cells/200 ml in the presence of 5 mg/ml b2-microglobulin. Peptides prepared in DMSO were sequentially diluted 4-fold in RPMI and added to final concentrations of 102.4 to 0.1 mM for 24 h at 37 C, 5% CO 2. The HLA-A*02:01-binding peptide Flu MP 58-66 (GILGFVFTL), and a non-binding peptide CHGA 382-390 (HPVGEADYF) were included as positive and negative controls, respectively. After incubation, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and stained with the viability marker Live/Dead Red and mouse anti-HLA-A2 mAb BB7.2, followed by an Alexa Fluor 488-labeled goat-anti-mouse IgG Ab (Interchim), HLA-A2 Binding Assays Peptide binding to HLA-A*02:01 was measured using the transporter associated with Ag processing (TAP)-deficient T2 cell line

. Culina, The concentration of each fluorescent MMr was corrected for the variable staining index of each streptavidin, in order to obtain a distinct double-MMr + population for each fluorochrome pair. The identification of the same MMr + population by each pair validated the panel. PBMCs were isolated by density gradient centrifugation using 50 ml Leucosep tubes (Greiner/Dominique Dutscher), washed twice in RPMI medium supplemented with AB human serum (Sigma), counted on a ThermoFisher Countess II automated counter and frozen in pre-chilled 10% DMSO solution in AIM-V medium (ThermoFisher) using CoolCell containers (Biocision) stored overnight at À80 C prior to transfer into liquid nitrogen. At thawing, PBMCs were immediately diluted in pre-warmed AIM-V medium. Following centrifugation and one additional wash in AIM-V, PBMCs were counted and rested in the presence of 50 nM dasatinib for 30 min at 37 C before magnetic depletion of CD8-cells (StemCell Technologies). Staining was performed for 20 min at 20 C in 20 ml PBS-dasatinib for 10 7 cells with the combinatorial double-coded MMr panels (Culina et al., 2018) detailed in Figure 3, followed, without washing, by mAb and Live/Dead Aqua staining at 4 C for 20 min. After one wash, cells were acquired using a FACSAria III cytometer configured as detailed in Table S6. Candidate epitopes binding to HLA-A2 (Figure S2) that did not yield any appreciable MMr staining provided negative controls for each panel. Data were analyzed with FlowJo software as described in Figure 3. Cells were sequentially gated on small lymphocytes, singlets, live cells, Each pHLA-A2 complex was used at a final concentration of 8-27 nM and conjugated with fluorochrome-labeled streptavidin at a 1:4 ratio, pp.3-4, 2018.

, MMr + cells (PE + PE-CF594 + ) were visualized by gating on events that were PE + PE-CF594 +

A. Culina, After a final wash, sections were counterstained with hematoxylin, dehydrated via sequential passages in 95%-100% ethanol and xylene, mounted, and analyzed using a Nikon Eclipse Ni microscope with NIS-Elements D software v4.40. In-situ immunofluorescence staining was performed similarly, but non-specific reactions were blocked with 5% goat serum for 2 h at room temperature before serial incubations with rabbit anti-PE Ab (1:250, 1.5 h at room temperature) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (ThermoFisher; 1:500, 1 h at room temperature). After a further wash, sections were incubated for 1 h at room temperature with rat anti-CD8 mAb (Abcam; 1:100) together with mouse anti-CD45RO mAb, Events negative for all MMr fluorochromes (PE À PE-CF594 À APC À BV650 À BV711 À BV786 À ) were represented in the same PE/PE-CF-594 dot plot to set the double-MMr + gate, as shown in Figures 4A-4F. CD45RA and CCR7 staining was subsequently visualized by gating on these MMr + cells, 2018.