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Type 1 diabetes is characterized by the autoimmune destruction of pancreatic β-cells. Recognition of major histocompatibility complex (MHC)-bound peptides is critical for both the initiation and progression of disease. In this study, MHC peptide complexes were purified from NIT-1 β-cells, interferon-γ (IFN-γ)-treated NIT-1 cells, splenic and thymic tissue of 12-week-old NOD mice, and peptides identified by mass spectrometry. In addition to global liquid chromatography–tandem mass spectrometry analysis, the targeted approach of multiple-reaction monitoring was used to quantitate the immunodominant Kd-restricted T-cell epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206–214. We identified >2,000 MHC-bound peptides; 1,100 of these presented by β-cells grown under normal conditions or after exposure to IFN-γ. These include sequences from a number of known autoantigens. Quantitation of IGRP206–214 revealed low-level presentation by Kd (∼25 complexes/cell) on NIT-1 cells after IFN-γ treatment compared with the simultaneous presentation of the endogenously processed Kd-restricted peptide Janus kinase-1355–363 (∼15,000 copies/cell). We have successfully sequenced peptides from NIT-1 β-cells under basal and inflammatory conditions. We have shown the feasibility of quantitating disease-associated peptides and provide the first direct demonstration of the disparity between presentation of a known autoantigenic epitope and a common endogenously presented peptide.
The cellular immune response depends upon T-cell recognition of peptides presented on the cell surface by molecules encoded by the major histocompatibility complex (MHC). Several thousand different MHC-bound peptides derived from the degradation of both intracellular and extracellular sources are displayed for scrutiny by T cells. In type 1 diabetes, the recognition of self-peptides leads to immune-mediated destruction of insulin-secreting β-cells and ultimately insulin deficiency. Presentation of peptides by class I MHC molecules on professional antigen-presenting cells and β-cells is critical for the development of disease. Nonobese diabetic (NOD) mice lacking class I MHC fail to develop diabetes, and studies aimed at specifically reducing class I expression on β-cells show an inverse correlation between the level of cell-surface expression and protection from disease (1,2). Moreover, increased expression of class I molecules on β-cells is observed in biopsies of patients with type 1 diabetes reflecting the inflammatory nature of the lesion (Original Nude Overlay Medium Vintage Norman Cocktail Beige Lace 1940's Dress Small AREnqB–5).
Identification of peptides presented by β-cells under basal and inflammatory conditions may provide insight into the mechanisms by which β-cells become targeted by autoreactive T cells. In particular, changes in the peptides presented by β-cells under inflammatory conditions may dictate the transition from benign to destructive insulitis in NOD mice. The generation of MHC-bound peptides is governed by a number of factors including protein accessibility, half-life, and protease resistance. Cytokine-induced expression of proteasome subunits and the action of signal peptidases together with cytoplasmic and endoplasmic reticulum–associated aminopeptidases contribute to the complexity and plasticity of peptide generation. Tissue-specific differences in antigen processing or differences in the milieu of inflammatory mediators at the site of antigen presentation may therefore facilitate the development of autoimmunity.
Mass spectrometry offers a powerful approach not only to identify new targets of immunity but also to provide accurate quantitative measurement of antigen presentation (6,7). Traditionally, T-cell epitopes have been defined using peptide libraries that span a given antigen and, less frequently, by Edman sequencing or tandem mass spectrometry. However, for many diseases the vast majority of peptide epitopes remain poorly defined, particularly in heterogeneous populations such as humans (7,8). One reason for this is the great complexity of the immunopeptidome and the relatively small proportion of antigen-specific peptides contained within it. Moreover, the immunopeptidome changes in response to inflammatory stimuli are chemically diverse and contain a significant proportion of peptides of atypical or heterogeneous length or bearing posttranslational modifications.
To examine the changes in peptide presentation by β-cells under inflammatory conditions, we have established a database of peptides presented by cultured β-cells in the presence or absence of interferon-γ (IFN-γ) by sequencing MHC-bound peptides using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Although sequences derived from known autoantigens could be identified among the MHC-bound peptides isolated from the surface of β-cells, these did not include previously identified T-cell epitopes. It has often been assumed that autoantigen-derived epitopes would be either unique to the tissue targeted by autoaggressive T cells or that they would be presented at relatively low levels that fail to induce tolerance. Since standard LC-MS/MS analysis did not reveal a number of known epitopes, we decided to use an approach that improves the selectivity and sensitivity of detection of known analytes. We therefore used the targeted mass spectrometry–based approach of multiple-reaction monitoring (MRM) to examine presentation of the dominant epitope from the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206–214 presented by β-cells. This approach has for the first time allowed direct quantitation of a known diabetes T-cell epitope and demonstrated the disparity in presentation of disease- and non–disease-related peptides. Moreover, we have provided a global dataset with which to probe tissues from NOD mice and examine the fluidity of antigen presentation during the development of disease.
RESEARCH DESIGN AND METHODS
NOD mice were purchased and housed in the Bio21 Institute animal facility. Studies were carried out in accordance with accepted standards of humane animal care and were approved by the animal ethics committee, University of Melbourne. Tissues were snap-frozen in liquid nitrogen and stored at −80°C.
The P815 (H-2d) mastocytoma and EL4 (H-2b) thymoma were maintained in RPMI 1640 (Invitrogen) medium supplemented with 10% FCS, 2 mmol/L glutamine, 50 IU/mL penicillin, and 50 µg/mL streptomycin. The NIT-1 insulinoma cell line (9) was maintained in low-glucose Dulbecco’s modified Eagle’s medium supplemented as described above. Expanded NIT-1 cells were washed in PBS, harvested by scraping, and snap-frozen. For cytokine treatment, cells were cultured with 100 IU/mL IFN-γ (eBioscience) for 24–72 h. For peptide pulsing, 5 × 107 cells were incubated with 1 μmol/L synthetic peptide in serum-free RPMI 1640 medium for 60 min at 37°C and washed with PBS.
Cells were incubated with fluorescein isothiocyanate–conjugated anti-Kd (SF1.1.10; BD Biosciences) antibody for 30 min on ice. Viable cells (determined by propidium iodide exclusion) were analyzed using a CyAn ADP flow cytometer (Beckman Coulter) in conjunction with FloJo software.
IGRP206–214 (VY*LKTNVFL; Kd restricted) and Janus kinase (JAK)-1355–363 (SY*FPEITHI; Kd restricted) absolute quantitation (AQUA) peptides were synthesized to incorporate an isotopically labeled amino acid (*) using standard solid-phase fluorenylmethyloxycarbonyl chemistry and purified to >95% by reversed-phase high-performance liquid chromatography (RP-HPLC). The identity of the peptides was confirmed by mass spectrometry and the concentration of peptides after dissolution in water determined by amino acid analysis as previously described (6).
Purification of MHC peptide complexes.
Cell pellets or tissues were ground in a Retsch Mixer Mill MM 400 under cryogenic conditions; resuspended in 0.5% IGEPAL, 50 mmol/L Tris (pH 8), 150 mmol/L NaCl, and protease inhibitors (Complete Protease Inhibitor Cocktail Tablet; Roche Molecular Biochemicals) at a density of 5 × 107 cells/mL; and incubated for 1 h at 4°C. Lysates were cleared by ultracentrifugation (200,000g) and MHC-peptide complexes immunoaffinity purified using solid-phase bound monoclonal antibodies SF1.1.10 (anti-Kd) and 28.8.6s (anti-DbKb) as previously described (10). Bound complexes were eluted with 10% acetic acid. The mixture of peptides and class I heavy-chain and β-2 microglobulin was fractionated on a 4.6-mm internal diameter × 50-mm long reversed-phase C18 HPLC column (Chromolith Speed Rod; Merck) using an ÄKTAmicro HPLC (GE Healthcare) as previously described (6). Fractions were collected, vacuum concentrated, and diluted in 0.1% formic acid.
Identification of MHC-bound peptides using LC-MS/MS.
Concentrated peptide fractions were analyzed by LC-MS/MS using an Eksigent NanoUltra HPLC and an AB SCIEX 5600 TripleTOF mass spectrometer as described in the Faux fur fur snow leopard fur Fake applique France fur Jacket with jacket by Women Exclusive jacket Tissavel fur jackets Luxury faux pzwqETT online. Data were analyzed with ProteinPilot software (Collar Mens Coat Suede Shearling Overcoat Medium DISTRESSED Fur 80s Jacket SHEEPSKIN Sherpa Long Brown Insulated Outerwear Winter Leather PBq54PY) and peptide identities determined subject to strict bioinformatic criteria that included the use of a decoy database to calculate the false discovery rate. A false discovery rate cutoff of 10% was applied, and the filtered dataset was further analyzed manually to exclude redundant peptides and known contaminants as described in the Faux fur fur snow leopard fur Fake applique France fur Jacket with jacket by Women Exclusive jacket Tissavel fur jackets Luxury faux pzwqETT online. Cellular localization of source proteins was analyzed using Software Tool for Researching Annotations of Proteins (12) and pathway analysis performed though the use of Ingenuity Pathway Analysis (IPA) (Ingenuity Systems; www.ingenuity.com).
Validation and quantitation of peptide epitopes by MRM
Selective validation of global LC-MS/MS analysis by MRM.
To determine the validity of the LC-MS/MS dataset generated from the TripleTOF 5600 experiments, 97 peptides from the Kd dataset and 96 peptides from the Db dataset were selected for MRM detection. For the Db set, two known subdominant peptides from IGRP were also included (IGRP225–233, LRLFGIDLL, and IGRPSweater Gray for Warm Cotton Hoodie for Woman Jacket woman Unisex Jacket hoodie Man Gray hood spring man Deep Gray Hoodie Big Long 241–249, KWCANPDWI). MRM transitions for these peptides were designed in silico using Skyline v1.1 (University of Washington, Seattle, WA). Peptide eluates were prepared from 1 × 108 IFN-γ–treated NIT-1 cells in duplicate and analyzed using a Tempo nanoLC-1Dplus in combination with a cHiPLC-nanoflex system (Eksigent) coupled to a 5500 QTRAP mass spectrometer (AB SCIEX) using experimental conditions described in the Faux fur fur snow leopard fur Fake applique France fur Jacket with jacket by Women Exclusive jacket Tissavel fur jackets Luxury faux pzwqETT online.
Quantitation of the immunodominant islet-specific epitope IGRP206–214 by MRM.
Transitions for MRM experiments were designed after inspection of experimental tandem mass spectrometry data. Three transitions were designed per peptide and validated through a series of experiments to investigate specificity, reproducibility, and background (Supplementary Figs. 1–4). Peptide fractions were concentrated and analyzed with a Tempo nanoLC-1Dplus system coupled to a 5500 QTRAP mass spectrometer as described in the Faux fur fur snow leopard fur Fake applique France fur Jacket with jacket by Women Exclusive jacket Tissavel fur jackets Luxury faux pzwqETT online. The amount of JAK-1355–363 and IGRP206–214 peptide present in each HPLC fraction was quantitated by examining the area under each MRM transition peak relative to an internal standard AQUA peptide as previously described (6). The addition of the AQUA peptide occurred immediately after immunoaffinity chromatography, since this is the first practical point for addition. This provides accurate quantitation of the peptide isolated from the immunoaffinity-purified MHC complexes and allows an estimation of the number of complexes per cell (6).
Dataset establishment and motif analysis.
For establishment of a database of peptides presented by β-cells, Kd and Db molecules were purified from cultured NIT-1 cells grown under basal conditions (3 × 109 cells) or after 48 h IFN-γ treatment (1.5 × 109 cells). For provision of a comparative dataset, peptides were also purified from pooled thymus and spleen from 12-week-old female NOD mice (n = 10). MHC-peptide complexes were isolated from detergent lysates using Kd- or Db-specific antibodies. Peptides were separated from class I heavy-chain and β-2 microglobulin by RP-HPLC and identified by LC-MS/MS. The complete dataset for IFN-γ–treated NIT-1 cells for both Kd (544 peptides from 484 source proteins) and Db (339 peptides from 297 source proteins) are shown in Supplementary Tables 1 and 2, respectively, and their presence in untreated NIT-1, spleen, or thymus indicated. The sequences of a further 221 peptides (51 Db, 170 Kd) identified in untreated NIT-1 cells (not in the thymus, spleen, or cytokine-treated cells by LC-MS/MS) are shown in Supplementary Table 3. These 221 peptides may be present in low abundance in the treated cells; however, manual inspection using extracted ion chromatograms generally did not provide definitive confirmation in the treated data. The predicted peptide-binding affinity was determined using the SYFPEITHI algorithm (13), and scores are recorded for Kd 9-mers and Db 9-mers and 10-mers. Of those peptides sequenced from the spleen, ∼80% were also detected in the thymus for both Kd and Db repertoires. The experimental data generated from IFN-γ–treated NIT-1 cells have been deposited with Tranche (https://proteomecommons.org/tranch/) and are Minimum Information About a Proteomics Experiment compliant (14).
No changes were noted in the amino acid preferences within peptides purified from cells (with or without IFN-γ) or tissues. The distribution of predicted peptide-binding scores remained unaltered between the untreated and IFN-γ–treated samples for Kd (Fig. 1) and Db (not shown). Motif analysis was therefore performed on the entire elution set obtained from NIT-1, NIT-1 plus IFN-γ, spleen, and thymus. A total of 1,146 and 910 peptide sequences for Kd and Db, respectively, were used to refine the motifs. Peptides bound to Kd were predominantly 9 amino acids in length (80%) with a small number of 10- or 11-mer peptides (Fig. 2A). Consistent with the published motif (15–17), position 2 was dominated by tyrosine and, in a small number of sequences, phenylalanine. The COOH-terminal position was occupied by Ile, Leu, or Val (Fig. 2B). Met and Phe were also tolerated at the COOH terminus; however, this preference was more apparent in peptides of 10 or 11 amino acids. Although the dominant anchor residues (P2 and PΩ) remained consistent, the binding motif for peptides of 9, 10, or 11 amino acids showed differential amino acid preferences in the COOH-terminal half of the peptide (Fig. 3). In particular, a strong preference for Pro at P7 and Gly at P8 in the 11-mer peptides was noted. Pro was also present at a higher frequency (>10%) at P6 in the 11-mer dataset. Ser and Thr remained moderate to strong anchors at all peptide lengths; however, the position of these residues shifted from P7 to P8 in 10-mer peptides and to P9 in the 11-mers.
While the majority of Db peptides were 9 amino acids in length (46%), more peptides of 10 and 11 amino acids in length (150 and 152 peptides, respectively) were isolated from Db than Kd (Fig. 2C). The number of peptides longer than 9 amino acids (∼53% of total sequences, 63% of these being 10- or 11-mer peptides) exceeds that previously reported for Db (18). The dominant anchors of Asn at P5 and Leu at the COOH terminus were present in 9-, 10-, and 11-mer peptide sets (Fig. 2D) as previously described (18). A strong preference for Ala at P2 was seen in the 9-mers, which became dominant in the 10- and 11-mer peptides (Fig. 3). The appearance of Pro at P6 was noted in 10- and 11-mer peptides, and Pro was also a moderate P7 preference in the 10-mer data set. Gly demonstrated a change in frequency, becoming a moderate anchor at P6 in 10-mer peptides and P8 in 11-mer peptides. Of final note, the preference for glutamic acid or aspartic acid shifted from P7 in the short peptide set to P8 in the 10-mers and P9 in the 11-mer dataset.
Peptides were derived largely from ubiquitous proteins, expressed in NIT-1, spleen, and thymus (Supplementary Tables 1 and 2). As expected, the majority of sequences were derived from either nuclear or cytoplasmic proteins (Fig. 4). No change in the localization of source proteins was noted following IFN-γ treatment; however, peptides from IFN-γ–inducible proteins were found in both datasets. Of particular note, peptides from the LMP2 and LMP7 subunits of the immunoproteasome- and the proteasome-associated protein ECM29 were found in IFN-γ–treated cells. This protein binds to the 26S proteasome and has been proposed to couple proteasomes to secretory compartments (19,20). IPA of source proteins within the larger Kd dataset showed a change in the top canonical pathways from caveolar-mediated endocytosis and insulin receptor signaling to ephrin receptor and AMP-activated protein kinase signaling. Phosphatidylinositol 3-kinase/AKT signaling remained a top canonical pathway in both IFNγ–treated and untreated datasets.
Consistent with previous reports, peptides from neuroendocrine or neuronal origin (e.g., α-internexin, neurofilament medium polypeptide, reticulon 4, γ-aminobutyric acid, and glutamate receptors) were identified in both datasets. A number of peptide sequences generated from proteins involved in secretory processes were also identified in untreated and treated NIT-1 cells. Within the IFN-γ–treated dataset, a single peptide derived from the known autoantigen IGRP (Db-restricted SGVLIIHHL) and a series of overlapping peptides from the potential autoantigen neuropeptide Y (Db and Kd restricted) were identified (Supplementary Tables 1 and 2). In addition to developing diabetes, NOD mice develop a lupus-like syndrome. Two peptides from proteins associated with Sjögren Syndrome and systemic lupus erythematosus were identified within the Db elution set, namely Sjögren Syndrome nuclear autoantigen 1 homolog (AALQNYNNEL) and calreticulin (EEESPGQAKDEL). A number of posttranslationally modified peptides were also identified, displaying pyroglutamate formation, deamidation, NH2-terminal acetylation, and glutathionylation. Such modifications occurred in both untreated and treated datasets.
The robustness of peptide identification was examined using a cohort of peptides identified in the discovery experiments in β-cell lines or in NOD mouse primary tissue. Duplicate samples of NIT-1 cells were solubilized and H-2Kd and Db peptides isolated. With use of in silico–generated MRMs, 77/97 H-2Kd– and 78/96 H-2Db–bound peptides were confirmed (representing a minimal 79% validation rate [for women dress print african ankara clothing for african dresses african Ankara women women mixed women fashion dresses clothing ankara for xOREwqnXC]). It should be noted that less material was used for the MRM analysis compared with the global LC-MS/MS discovery experiments (15-fold less). The high confirmation rate without the requirement for synthetic peptides provides a high-throughput means of validating initial discovery datasets.
Quantitation of IGRP206–214.
In these global LC-MS/MS experiments, we did not detect any of the immunodominant peptides previously reported in NOD mice. To determine whether this was due to the sensitivity of the approach, we used MRM for the specific detection of the immunodominant Kd-restricted IGRP206–214 peptide. MRM transitions were established for IGRP206–214 and the endogenously presented peptide JAK-1355–363 (Fig. 5A). JAK-1355–363 (derived from the interferon signaling molecule JAK-1) was selected, as it was readily identified in NIT-1, IFN-γ–treated NIT-1, thymus, and spleen samples and is a high-affinity Kd ligand. For confirmation of the specificity of these peptides for Kd, P815 (KdDd) and EL4 (KbDb) cells were incubated with a mix of IGRP206–214 and JAK-1355–363 AQUA peptides, class I complexes purified, and isolated peptides subjected to MRM analysis. Both IGRP206–214 and JAK-1355–363 were detected in the Kd eluate of peptide-pulsed P815 cells, as shown in Fig. 5B. Although P815 cells endogenously present JAK-1for Hoodie Sweater man Jacket Unisex Jacket Gray woman spring hood Gray Gray hoodie Long Woman Deep Man for Cotton Hoodie Big Warm 355–363, the use of the AQUA version of the peptide to pulse these cells allows a distinction to be made between endogenous presented peptide and exogenous loading of the mass-shifted AQUA peptide by liquid chromatography–mass spectrometry. Neither peptide was detected in fractions affinity purified from peptide-pulsed EL4 cells (Kb or Db) or the Dd fractions of P815 cells (Fig. 5C).
We next used this approach to detect peptides presented by NIT-1 cells. NIT-1 cells were treated with IFN-γ and MHC-peptide complexes isolated at 0, 24, 48, and 72 h posttreatment. The increase in Kd expression following cytokine treatment is shown in Fig. 6A. Both JAK-1355–363 and IGRP206–214 were detected from NIT-1 cells treated with IFN-γ over 24–72 h (Fig. 6B). A striking difference was observed between the number of MHC/peptide complexes for the two peptides isolated from the same sample. JAK-1355–363 was quantified at ∼15,000 copies per cell after 72 h of IFN-γ treatment; however, IGRP206–214 did not exceed >25 copies per cell. In the absence of IFN-γ treatment, JAK-1355–363 but not IGRP206–214 could be detected. To determine whether the inability to detect IGRP206–214 in untreated cells was due to the low level of class I expression, a second larger sample of untreated cells was prepared (3 × 109 cells). JAK-1355–363 was detected at ∼2,000 copies per cell (Fig. 6C), consistent with the 0-h time point in the IFN-γ dataset. IGRP206–214 was also detected in this sample at ∼1 copy/cell.
We have established a database of MHC class I peptides endogenously processed and presented by the NIT-1 β-cell line and in tissues of NOD mice. This is the largest dataset of H-2Kd and Db peptides reported, allowing refinement of the binding motifs for these molecules. Because these peptide sequences are derived from high confidence mass spectrometry identifications of naturally processed and presented peptide ligands, they do not share the inherent bias in amino acid motif definition seen in pool Edman sequencing (21) because of the dominance of highly abundant peptides. In general, the peptide motifs observed in this study were in good agreement with published motifs (15–18) and were further developed to include amino acids present with dominant, strong, and moderate frequencies. Moreover, discrete motifs were also apparent for peptides longer than the canonical 8–9 amino acids. Such long peptides have typically been difficult or impossible to predict because of the lack of a significant number of bona fide longer ligands. In particular, bioinformatics tools have not performed well for Db in comparison with other alleles (18), attributed to the low number of long natural ligands used to train predictive algorithms. In our study, we identified >400 naturally processed peptides of 10 or 11 amino acids in length, with the selection of such long peptides more frequent in the Db compared with the Kd repertoire. For both class I molecules, Gly and Pro became more common in peptides of 10 and 11 amino acids, reflecting a requirement for flexibility and kinking or bulging in the peptide backbone of longer peptides (22). For longer peptides bound to H-2Db, Ala became a dominant motif at P2. Identification of these long, naturally processed peptides and refinement of the current motifs should enhance the success of bioinformatic predictions, providing new opportunities to predict autoantigen-derived epitopes in the NOD mouse.
Treatment of NIT-1 cells with IFN-γ did not significantly change the length of peptides presented by either Kd or Db or the distribution of predicted binding affinities. This suggests that cytokine treatment does not bias toward high-affinity ligands. Recent studies examining the peptide repertoire of a number of human alleles have documented the presence of NH2-terminally extended peptides within the endogenous peptide pool (23). Consistent with this observation, we have also identified a small number of nested NH2-terminally extended peptides. Trimming of the NH2 terminus by the aminopeptidase ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing) is required for most class I peptides (24). The presence of NH2size cardigan black asymmetrical Transparent linen S XATOqt-terminally extended peptides is of interest given the reported immunogenicity of such sequences produced in the absence of ERAAP through formation of novel MHC-peptide complexes (25). Although the precise COOH terminus of MHC class I peptides is thought to be generated primarily by the proteasome in the cytoplasm, we also found peptides containing COOH-terminal extensions within the datasets for both Kd and Db. The identification of these peptides suggests that additional trimming of the COOH terminus may occur after translocation into the endoplasmic reticulum and MHC complex formation.
The majority of peptides sequenced were common to NIT-1, spleen, and thymus. Many of the β-cell–specific peptides were of neuronal origin, consistent with previous data on elution of class II peptides in NIT-1 cells overexpressing the class II transactivator (26). We identified a number of sequences from known autoantigens such as insulin, chromogranin A, and islet amyloid polypeptide. We also identified peptides from other secretory proteins including neuropeptide Y, secretogranin, and proSAAS. These included nested sets of peptides ranging in length from 9 to 23 amino acids, and a number of these were bound to both Kd and Db (e.g., secretogranin-1, SGKEVKGEEKGENQNSKFEVRLL; secretogranin2, YLNQEQAEQGREHL; neuropeptide Y, RYYSALRHYINLITRQRYG; and chromogranin A, LEGEDDPDRSM). The presence of unusually long peptides bound to class I molecules isolated from β-cells highlights a potential nonconventional antigen-presentation role in NOD mice. Whether these represent cleavage intermediates that are further processed following cytokine treatment and whether peptides derived from these sequences are recognized by autoreactive T cells warrants further investigation given the number of autoantigenic epitopes already identified from secretory granules. Recently, islet amyloid polypeptide was identified as the target of the BDC T-cell clone 5.2.9 (27). The CD4+ restricted epitope (KCNTATCATQRLANFLVRSS) was mapped using synthetic peptides spanning the entire molecule. We have identified a peptide that overlaps the COOH terminus of this sequence in untreated NIT-1 cells, bound by Kd (LVRSSNNLGP [Supplementary Table 3]). The propensity of certain immunogenic regions of targeted proteins to yield a number of class I and class II restricted epitopes is well documented. We also sequenced a Db-restricted peptide derived from IGRP in IFN-γ–treated NIT-1 cells (IGRP7–15 SGVLIIHHL). This peptide has a moderate binding score of 17 using the SYFPEITHI algorithm. The immunogenicity of this epitope has previously been tested using synthetic peptides (28). IGRP7–15 failed to elicit a response from islet-infiltrating T cells isolated from NOD mice of varying ages. Although our data demonstrate that IGRP7–15 is naturally processed and presented by NIT-1 β-cells, it is likely that the levels of presentation are sufficient to induce tolerance.
The ability to detect a tolerogenic peptide from IGRP by LC-MS/MS and not the dominant IGRP206–214 epitope led us to address the issue of epitope abundance using the sensitive approach of MRM. Using MRM, we were able to detect both the dominant IGRP206–214 peptide and the subdominant epitope IGRP225–233 (for women dress print african ankara clothing for african dresses african Ankara women women mixed women fashion dresses clothing ankara for xOREwqnXC). Moreover, this targeted method allows quantitation of peptide epitopes through the inclusion of an isotopically labeled standard. We found that IGRP206–214 was presented at very low levels on NIT-1 cells grown under basal conditions (∼1 copy/cell). Conversely, JAK-1355–363 was presented on the same cells at ∼2,000 copies/cell. Treatment of cells with IFN-γ increased the level of presentation of both IGRP206–214 and JAK-1355–363. Although the total number of IGRP206–214 peptide MHC complexes remained extremely low (25 copies/cell) compared with JAK-1355–363 (15,000 copies/cell), the fold increase in presentation of the two epitopes was greater for IGRP206–214, and did not merely mirror the fold increase in class I expression (fivefold increase in Kd at 72 h). This underscores the complexities of epitope generation and for the first time highlights the disparity between presentation levels of a known autoantigen and a normal endogenous ligand.
We have used a discovery-based approach to characterize the repertoire of peptides presented by pancreatic β-cells in the presence and absence of an inflammatory signal. Global LC-MS/MS analysis has successfully identified peptides from known autoantigens; however, many of these may represent sequences that are tolerogenic and, indeed, a large number of these peptides were present in the thymus. The quantitation of IGRP206–214 provides the first demonstration of low-level presentation of an immunodominant autoantigenic determinant in absolute terms. Moreover, the disparity in levels of presentation of IGRP7–15 and the immunodominant IGRP206–214 highlights differences in epitope liberation even within the same antigen. Such quantitative information can be used to guide discovery-based approaches; finding the threshold at which autoantigenic peptides are likely to be present will allow sequencing of peptides that may normally be masked by more abundant species. We propose that the work flows of LC-MS/MS and MRM analysis can be used in tandem to mine deeper into the immunopeptidome and that comparative analysis of datasets sequenced from cell lines and tissues in the presence and absence of inflammation can be used to guide selection of potential autoantigens. This may be particularly relevant to granule-associated proteins, the presentation of which clearly changed upon cytokine stimulation in this study. One of the most important applications of MRM analysis of MHC-peptide epitopes will lie in the ability to examine hundreds of peptides in a single analysis. This will not only allow changes in the presentation of known autoantigenic peptides to be determined but will also enable changes in the presentation of peptides generated from a single antigen to be assessed. This will have important implications for understanding epitope liberation and the contribution of epitope abundance to immunodominance hierarchies.
The authors acknowledge funding from Diabetes Australia Research Trust and infrastructure funding from the Australian Research Council. A.W.P. is supported by a National Health and Medical Research Council Australia Senior Research Fellowship.
No potential conflicts of interest relevant to this study were reported.
N.L.D. performed the majority of experiments, designed the study, and wrote the manuscript. C.T.T. designed and executed the MRM experiments. D.G.G., N.P.C., and P.T.I. provided technical help and expert guidance in mass spectrometry. A.W.P. designed the study, supervised experimental work, and wrote the manuscript. N.L.D. and A.W.P. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1333/-/DC1.
- Received September 22, 2011.
- Accepted May 17, 2012.
- © 2012 by the American Diabetes Association.
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.
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