Citrullination of glucokinase linked to autoimmune diabetes

Inflammation, including reactive oxygen species and inflammatory cytokines in tissue microenvironments amplify the appearance of various post-translational modifications (PTMs) of self-proteins. Previously, a number of PTMs have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes (T1D). Herein, we have identified the citrullination of glucokinase (GK) as a result of inflammation, triggering autoimmunity and affecting its biological functions. Glucokinase is predominantly expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells, where it acts as a glucose sensor to initiate glycolysis and insulin signaling. Herein, we demonstrate that glucokinase is citrullinated in inflamed non-obese diabetic (NOD) islets as well as in human GK. Autoantibodies against both native and citrullinated GK arise in both spontaneous human T1D and murine models. Likewise, autoreactive CD4+ T cells to both native and citrullinated glucokinase epitopes are present in the circulation of T1D patients. Finally, citrullination alters GK biologic activity and suppresses glucose-stimulated insulin secretion. Our studies define glucokinase as a T1D biomarker, providing new insights into altering glucose metabolism, creating neoautoantigens, and better define the broad impact of PTMs on the tissue pathology of T1D.


INTRODUCTION
Glucokinase (GK, hexokinase IV; EC 2.7.1.2) is one of four isoenzymes of the hexokinase family. Glucose homeostasis in humans is highly regulated by the activity of glucokinase, catalyzing glucose phosphorylation, the first and rate-limiting step of glycolysis in the liver and pancreas 1 . More than 600 mutations of the human glucokinase gene have been described. The majority of these mutations result in reduced glucokinase enzyme activity in pancreatic b-cells and in hepatocytes 2 . As previously described, patients with maturity onset diabetes of the young (MODY) is linked to specific mutations of the glucokinase gene, classified as MODY2 3,4 .
Thus, glucokinase in particular has been considered as a potential therapeutic target for patients with various forms of diabetes. Since 2008, clinical trials of glucokinase activators have been investigated in patients with type 2 diabetes (T2D), including piragliatin, MK-0941, AZD1656 5 and dorzagliatin 6 . In contrast to T2D and monogenic diabetes, much less is known regarding the role of glucokinase in autoimmune type 1 diabetes (T1D).
T1D is characterized as an insulin-dependent glucose metabolic disorder arising from inflammation of the pancreatic islets and amplified by autoimmune B and T lymphocyte responses. Besides the role of infiltrating leukocytes into the pancreatic microenvironment, local inflammatory cytokines and reactive oxygen also amplify post-translational protein modifications (PTMs), including deamidation, oxidation, carbonylation and citrullination, that are capable of compromising immune tolerance 7 . Citrullination is an inflammation-dependent process and has been identified in the variety of inflamed tissues and studied extensively as biomarkers of rheumatoid arthritis (RA) 8 . Anti-cyclic citrullinated peptide antibodies (anti-CCP) arise early in RA, correlate with disease severity, and are now routinely used for the diagnosis of RA 9 . T cell recognition of citrullinated epitopes from joint-associated proteins is Moreover, the observations support a potential therapeutic strategy of inhibiting PAD enzymes for T1D.

Glucokinase citrullination in inflamed islets from NOD mice and inflammatory stressedbeta cells.
We first performed immunohistochemistry staining and confocal microscope to examine if protein citrullination is increased in islets from the spontaneous NOD murine model of T1D.
NOD mice develop hyperglycemia, insulitis, and lymphocyte infiltration of the pancreatic islets as one model of human T1D. Protein citrullination in islets is present already in 3.5 week old NOD mice and increases in staining intensity with increasing age of the mice (Fig. 1a).
Citrulline staining coincided with insulitis, as confirmed by hematoxylin-eosin (H&E) staining and anti-CD3 staining from 16 week old NOD mice (Fig. 1b). Protein citrullination is not detected in liver and pancreas extracts from age matched C57Bl/6 mice or in NOD liver, but is significantly present in NOD pancreas (Fig. 1c). PAD treated human GK served as a citrulline staining control.
Glucokinase is the primary glucose sensor since the small fluctuations of its enzyme activity alter the threshold of glucose-stimulated insulin secretion in pancreatic b-cells 1,23 . In liver, the major role of glucokinase is to regulate the glycogen synthesis. As previously reported 24 and confirmed here, GK levels are expressed in significantly higher levels relative to total protein in liver compared to pancreas from both C57Bl/6 and NOD mice (Fig. 1d). We next immunoprecipitated GK with specific antibody to confirm GK citrullination in NOD pancreas.
As expected, we captured significantly more glucokinase from NOD liver extracts compared to pancreas from 16 week old NOD mice (Fig. 1e). In contrast, the citrullinated glucokinase signal is significantly increased in pancreas compared to liver from NOD mice (Fig. 1e). These data suggest that little overall GK citrullination occurs in the liver, while significant pancreatic GK citrullination arises in the same NOD mouse as disease progresses.
To assess whether inflammation triggers GK citrullination in insulin secreting b-cells, a cocktail of proinflammatory cytokines (rmIFNγ and rhIL-1β) were used to model the insulitis using rat insulinoma cell line, INS-1 cells which displays the key characteristics of pancreatic β cells. The level of protein citrullination was increased in cytokine treated INS-1 cells over time (12,24 or 48 hours) by immunofluorescence and by flow cytometry (supplementary Fig. 1a, 1b, respectively). As expected, GK + /citrullination + populations of INS-1 cells was also increased after cytokine treatment compared to untreated cells in a time dependent manner (supplementary Fig.1 c). Finally, cytokine triggered glucokinase citrullination in INS-1 cells was confirmed by immunoprecipitation with anti-glucokinase and immunoblot with anti-peptidyl-citrulline (supplementary Fig.1d). Collectively, these results illustrate overall pancreatic citrullination and that citrullination of GK can be specifically amplified by inflammatory cytokines in tissue and cell based systems.

Autoantibodies against glucokinase and citrullinated glucokinase arise in both murine and human models of T1D.
We next determined if citrullinated GK may be a neoantigen in triggering autoimmunity in T1D. NOD mouse serum recognized both rhGK and PAD-treated rhGK proteins by immunoblot, while serum from control B10.BR mice did not (Fig. 2a). We next examined IgG autoantibodies to both GK and citrullinated GK by solid phase immunoassay (ELISA) in serum of NOD mice. Anti-GK and anti-citrullinated-GK IgG titers were significantly higher in both pre-diabetic NOD mice (n=78) and diabetic NOD mice (n=16; blood glucose content greater than 250 mg/dl) compared to control, age matched B10.BR mice (n=52) (Fig. 2b). Subdividing the samples by age groups show that anti-GK and anti-citrullinated-GK IgG arises early in NOD mice, already at 4-6 weeks of age, and before the onset of hyperglycemia. The autoantibody titers against GK and citrullinated GK continue to increase up to 20 week old NOD mice (Supplementary Fig. 2a & b).
To assess B cell reactivity against citrullinated GK and its native counterpart in patients with T1D, serum from patients with T1D (n=55) and healthy control subjects (n=18) were examined by ELISA. As shown in Fig. 2c, patients with T1D had significantly higher anti-GK and anticitrullinated-GK IgG titer compared to healthy subjects. Other established autoantibodies were also screened within the same set of human serum -including anti-insulin, anti-GAD65, anti-IA2 and anti-ZnT8. Interestingly, anti-GK and anti-citrullinated-GK antibodies correlated positively with anti-ZnT8. There is a biological and metabolic link between zinc ion transport and insulin secretion because mature insulin is stored in the form of Zn-containing hexamers. However, anti-citrullinated-GK antibodies correlated negatively with anti-insulin (Supplementary Table 1).
Prior studies documented that citrullination can enhance peptide binding to DR0401, generating GAD65 neo-epitopes 26 . Therefore, we sought to identify glucokinase sequences that can be bound and presented by DR0401 by scoring all possible nine amino acid motifs within GK using a previously published algorithm 13,27 . For these predictions, Arg residues that were shown by the mass spectrometry data (Fig. 5b) set to be accessible for modification by PAD enzyme were replaced by citrulline. A total of 38 peptides were synthesized and their binding to recombinant DR0401 protein was assessed using a competition assay 13,26 . Nine candidate peptides that bound to DR0401 with appreciable affinity (Supplementary Table 2). We then produced DR0401 tetramers for all of those peptides and used these to investigate the ability of each peptide to elicit CD4 + T cell responses in vitro. PBMCs from subjects with T1D were stimulated with pools of glucokinase peptides for two weeks, and subsequently stained with the corresponding individual tetramers, revealing five peptides that elicited detectable populations of tetramer-positive T cells in multiple subjects ( Supplementary Fig. 3). The sequences of these immunogenic peptides and their predicted motifs are summarized in Table 1. Tetramer-positive T cell clones were isolated for all of those peptides, further confirming T cell recognition of these candidate epitopes ( Supplementary Fig. 4).
Among the five immunogenic peptides, three had an Arg residue within the predicted minimal motif (GK 266, GK 270, and GK346) but only one of these, GK residue 358, was shown to be citrullinated. To investigate whether citrullination influenced the binding of these glucokinase epitopes, we compared the HLA binding of citrulline and Arg containing versions of these peptides (Supplementary Table 3). The results indicated that citrullination did not enhance or alter binding for any of the peptides, which implies that the citrulline/Arg are not T cell contact residues.
To enumerate glucokinase-reactive T cells in human subjects, we applied a direct tetramer enrichment approach 28 to measure their frequency in the peripheral blood of subjects with T1D and HLA matched controls. We labeled individual specificities with PE, PE-CF594, or PE-Cy5 labeled tetramers, staining two separate aliquots of cells to assess the five glucokinase epitopes simultaneously (representative results shown in Supplementary Fig. 5). Some glucokinasereactive CD4 + T cells were present in controls. However, the combined frequency of glucokinase-reactive CD4 + T cells in subjects with T1D was significantly higher than in controls (p=0.0007) (Fig. 3a). Utilizing CD45RA and CCR7 as markers to distinguish antigenexperienced (CD45RA -) versus naïve T cells (CD45RA + CCR7 + ), healthy subjects tended to have a higher proportion of glucokinase-reactive T cells that were naïve than subjects with T1D (p=0.06) (Fig. 3b). Thus, subjects with established T1D had significantly greater numbers of glucokinase-reactive CD4 + T cells than controls and their glucokinase-reactive T cells tended to show increased signs of antigen exposure. Considering individual specificities, only GK 346 specific T cells were significantly more frequent in subjects with T1D than in controls (p=0.0004), but GK 199 and GK 270 also trended toward trended toward having higher frequencies in subjects with T1D (p=0.08 and p=0.09, respectively) (Fig. 3c). We expressed T cell specificity data into a heat map to visualize patterns of glucokinase-specific T cells in different individuals (Fig. 3d). From this analysis it was evident that some subjects had high frequencies for multiple epitopes (e.g., T1D #2 and T1D #5) whereas others had very few glucokinase-specific T cells (e.g., T1D #6). However, T1D #2, #5 and #6 all have detectable anti-glucokinase IgG (indicated at the bottom of Fig. 3d). We next asked whether the frequency of glucokinase-reactive T cells might be correlated with characteristics such as disease duration, age at diagnosis biological sex, or levels of glucokinase specific antibodies. Considering both T1D patients and controls, we observed a significant positive correlation between the combined frequency of glucokinase-reactive T cells and levels of anti-glucokinase antibodies (Fig. 3e). No other associations reached significance.

The effect of citrullination on the biological function of glucokinase in pancreatic beta cells.
Pro-inflammatory cytokines are known to attenuate glucose stimulated insulin secretion (GSIS), mediated by glucokinase, as studied elsewhere 29 Interestingly, YW3-56 was able to correct IFNg +IL-1b suppressed GSIS at 9mM glucose if INS-1E cells were cultured with YW3-56 throughout all the experiment setting including 48 hrs of cytokine treatment and 1 hr of glucose stimulation (data not shown). Using pyruvate instead of glucose, beta cells will bypass glucokinase to stimulate insulin secretion 30 . Pyruvate stimulated insulin secretion was also found to decrease under IFNg +IL-1b exposure (the right panel of Fig. 4b). Moreover, YW3-56 was also able to correct cytokine-mediated suppression of insulin secretion upon pyruvate stimulation. However, insulin secretion upon to depolarization with KCL was not affected by cytokines in the presence or absence of YW3-56 (the middle panel of Fig. 4b). In toto, our data suggest that IFNg +IL-1b-triggered citrullination affects not only glycolysis (mediated by glucokinase) but also downstream metabolism from glycolysis in the insulin biosynthesis pathway. However, there appears to be no effect of citrullination on the depolarization step of insulin secretion in beta cells.
Recombinant human pancreatic glucokinase (rhGK) was used to map potential citrullination residues and ask if citrullination alters glucokinase catalytic activity. Full length of recombinant human pancreatic GK (rhGK) was incubated with PAD in the presence of Ca ++ . Citrullinated rhGK migrates slightly slower on SDS-PAGE due to the loss of positive charge as shown in the left panel of Fig. 5a. Citrullination of GK was also confirmed by immunoblot (the right panel of Fig. 5a). By using mass spectrometry, there were 16 citrullination modification residues, found in PAD-treated rhGK (Fig. 5b). Thus, 50% of Arg residues (16 Arg out of total 32 Arg residues) were available to be citrullinated in vitro by PAD. One representative mass spectra demonstrated the citrullination sites are at Arg 428 and Arg 429 in PAD-treated rhGK peptides (a.a. 423-447) (supplementary Fig. 6a). As shown in Fig. 5c, citrullination of rhGK by PAD in vitro was found to reduce the catalytic activity of the enzyme by 20%. Next, we subjected the native rhGK and PAD-treated rhGK to steady-state kinetic analyses at 37°C with various concentration of substrate (glucose). PAD-treated rhGK demonstrated similar Vmax compared to native rhGK (119.9±7.7 µmol/mg/min of PAD-treated rhGK versus 115.1±10.5 µmol/mg/min of native rhGK). However, the Km of PAD-treated rhGK was two-fold increased (16.2±2.3 mM) compared to native rhGK (8.48±2.1 mM) (Fig. 5d). These data reveal that glucokinase is a substrate of PAD and suggests citrullination can modulate its glucose threshold for insulin secretion in beta cells, mediated by reduced substrate binding affinity of glucokinase enzyme.

DISCUSSION
In the current study, our results reveal that citrullination alters the enzyme kinetics of glucokinase and reduces glucose stimulated insulin secretion (GSIS), the major biological function of pancreatic beta cells. We also demonstrate immune recognition of glucokinase in subjects with T1D, in that both antibodies against and autoreactive T cells that recognize glucokinase epitopes are present in the periphery.
The role of protein citrullination by PAD2/PAD4 in promoting loss of self-tolerance is well studied in RA. This represents a potentially effective therapeutic axis, in that several potent PAD4 specific inhibitors have been developed and were shown to disrupt mouse and human NET formation (NETosis) 22,32,33,34 leading to improved cancer prognosis. NETosis has also been shown to be a major source of autoantigens in RA 33,35 and its activity is also believed to form neo-epitopes in other autoimmune disease settings 16,26,36 . For the first time, we show that one PAD2/PAD4 inhibitor, YW3-56, can protect from inflammation-induced citrullination and partially restore insulin secretion in beta cells upon stimulation with glucose or pyruvate.
Besides YW3-56, a specific PAD2 inhibitor, CAY10723, was also found to be able to restore cytokine-suppressed insulin secretion in INS-1E cells (data not shown). However, neither YW3-56 nor CAY10723 can completely restore cytokine-suppressed insulin secretion response to glucose or pyruvate. These observations suggest that inflammation-induced citrullination may not be the only mechanism for defective insulin biosynthesis in T1D.
As mentioned above, ATP-dependent glucokinase converts glucose to glucose-6-phosphate, the critical step in glucose metabolism. Alternative splicing of the glucokinase gene results in three tissue-specific isoforms, isoform 1 (pancreas) and isoforms 2 and 3 (liver). The only difference among three glucokinase isoforms is in the amino acid sequence of exon 1in the first fifteen amino acids of human pancreatic glucokinase 37 . Unlike other hexokinases, glucokinase is not regulated by feedback inhibition by glucose-6-phosphate. Therefore, glucokinase can constitutively trigger insulin secretion in pancreatic b-cells under high glucose conditions. To date, few studies demonstrate any role of PTMs in altering glucokinase activity.
Polyubiquitination of human glucokinase, both pancreatic isoform 1 and hepatic isoform 2 increases catalytic activity up to 1.4 fold 38 . SUMOylation (small ubiquitin-like modifiers) of glucokinase was found in MIN6 and INS-1 cells and results in increased pancreatic glucokinase stability and activity 39 . In the current study, our data suggest that citrullination impairs the major function of glucokinase in pancreatic beta cells, glucose sensor function, due to the suppression of substrate binding affinity of recombinant human pancreatic glucokinase (Fig. 5d).
Pancreatic beta cell-specific glucokinase knockout mice die within the first week of birth of severe hyperglycemia 40 . Besides NOD mice, the streptozotocin (STZ)-induced model of murine diabetes also resembles human T1D, recapitulating the phenomena of insulitis and insulin deficiency. Relevant to this study, STZ-induced diabetic mice exhibit decreased glucokinase expression with hyperglycemia 41 . PKM2 (one of four pyruvate kinase isozyme L, R, M1 and M2) activation protects against diabetes by increasing glucose metabolic flux and inducing mitochondria biogenesis 42 . Of note, the catalytic activity of PK is increased 2 to 3-fold after in vitro citrullination by PAD 43 . Our pilot studies identified citrullinated-and acetylated-PKM2 in IFN-g and TNF-a-treated human islets but no such PTMs found in PKM2 from untreated control islets (data not shown). Collectively, our data suggest that citrullinated GK and perhaps other pathway proteins contributes to the defects of insulin secretion in beta cells.
In the current study, we found that the total citrullination levels were higher in 16 week old NOD inflamed pancreas compared to age-matched B6 pancreas. We found that both anti-GK and anti-citrullinated GK IgG are significantly higher in NOD serum compared to age-matched B10.BR serum even before the diabetic onset, as early as 4-6 weeks of age. Similarly,, both anti-GK and anti-citrullinated GK IgG titers were significantly higher in patients with T1D compared to healthy subjects. Interestingly, both diagnostic anti-insulin and anti-ZnT8 antibodies correlated with anti-citrullinated GK in patients with T1D, raising the possibility of linked development. However, the broad disease duration (0.1-55 years) in our T1D serum samples limits our ability to determine whether anti-GK and/or anti-citrullinated GK could serve as a novel diagnostic antibody for T1D (Fig. 2c).
The loss of immune tolerance to PTMs within the stressed beta cells triggers an autoreactive T cell response and contributes to the destruction of insulin-producing beta cells in autoimmune diabetes. Emerging data demonstrate that autoreactive T cells against variety of PTMsneoepitopes are found in patients with T1D including oxidation, deamidation, phosphorylation and citrullination 14 . For example, CD4 T cells from patients with T1D were identified to specifically recognize the oxidized insulin neoepitopes, near disulfide bond region in A chain, in the context of HLA-DR4 44 . Previous studies demonstrated that the citrullination and transglutamination can enhance GAD65 peptide binding to HLA-DRB1*04:01 (DR0401) 26 . To date, GAD65, GRP78, islet-specific glucose 6 phosphatase catalytic subunit-related protein (IGRP) and islet amyloid polypeptide (IAPP) are identified as the citrullinated-neoepitopes recognized by autoreactive T cells in the development of T1D 7 . Herein, we investigated the relevance of T cell responses to glucokinase in human disease by identifying citrullinated and unmodified peptides that are bound and presented by DR0401, a part of the high-risk T1Dassociated DR4/DQ8 haplotype 25 , and recognized by human CD4 + T cells. We then utilized the corresponding HLA class II tetramers to determine whether T cells that recognize glucokinase are present within the peripheral blood of subjects with T1D. We identified a total of five immunogenic DR0401-restricted GK epitopes, one of which was citrullinated at Arg 358 residue of GK. Notably, among the various GK epitopes identified through our study, the citrullinated GK 436 epitope emerged as the specificity with the highest median frequency and that best differentiated controls and subjects with T1D. Consistent with this data, we observed that Arg 358 residue of GK is highly susceptible to citrullination in PAD-treated recombinant human pancreatic GK protein (Fig. 5b). Moreover, Arg 358 residue of GK was identified to be citrullinated in cytokine treated-but not in untreated INS-1E beta cells ( Supplementary Fig. 6 Neoepitopes derived from citrullination modification of islet proteins, citrullinated GK and/or other unknown glucose-insulin metabolic proteins, breaks immune tolerance to trigger T and B cell autoimmunity. Perhaps more importantly, citrullination of GK impairs islet responses to glucose and overall glucose homeostasis. The pathway of cytokine stress and citrullination is an amplifying feedback loop of T1D pathology that includes both autoimmunity and altered beta cell metabolism. The pathway can potentially be interrupted by therapeutic manipulation of the tissue microenvironment preventing the development of citrullination (PAD inhibitors) or by the inhibition of proinflammatory cytokines in the pancreas.

Mice.
Female NOD/ShiLt, C57BL/6, and B10.BR mice were purchased at 3 wks of age from Jackson Laboratories (Bar Harbor, Maine). All mice were housed in microisolator cages with free access to food and water and maintained at the Yale Animal Resource Center at Yale University. All animal studies were performed in accordance with the guidelines of the Yale University Institutional Animal Care and Use Committee. Glucose levels were measured in blood withdrawn from the retroorbital sinus of anesthetized mice using Lite glucometer and test strips (Abbott Laboratories, Abbott Park, IL, USA). Mice with blood glucose values greater than 250 mg/dL were considered diabetic. Serial serum samples were acquired from NOD and age matched control animals. Pancreas and liver tissue extract were prepared in RIPA buffer containing a mixture of protease inhibitors (Complete protease inhibitor, Roche Diagnostics).

Protein citrullination detection.
Presence of citrullinated protein was detected by using anticitrulline (ab6464; Abcam) for immunohistochemistry as described elsewhere 45 , by using antipeptidyl-citrulline, clone F95 (Millipore) for flow cytometry and confocal microscopy and by using anti-modified citrulline detection kit (clone C4; Millipore) for immunoblot according to manufacturer's protocol. incorporating Percolator for peptide validation. Same parameters of variable modifications and fixed modification were set as mentioned above. Two missed cleavages were allowed, peptide tolerance was set at 5 ppm and MS/MS tolerance at 20 mmu. The MS/MS spectra were carefully checked manually for the presence of citrullinated residues.
ELISA and immunoblot assay. Immunoreactivity of human and mouse serum to rhGK and PAD-treated-rhGK was performed by ELISA. Briefly, 0.5 µg of rhGK or PAD-treated-rhGK in 0.05 M carbonate-bicarbonate buffer (pH = 9.6; Sigma) was coated onto ELISA plates (Thermo Fisher Scientific) overnight at 4°C and blocked with 1%BSA in PBST containing 1% Tween-20.
Sera were diluted as 1:100 in diluting buffer (0.03% BSA in PBST) and incubated 2 h at room temperature. Species-specific goat anti-IgG alkaline phosphatase was used as a secondary reagent (Southern Biotech). Color was developed via the addition of pNPP substrate (Sigma) and absorbance was read at 405nm (Synergy HT Multi-Mode Reader, BioTek Instruments).
Individual signals were normalized to no-antigen control wells. Rabbit polyclonal antibody against glucokinase (Proteintech) served as positive control in ELISA. All readings were normalized to non-specific serum binding to no-antigen control wells. Autoantibodies against to rhGK or PAD-treated-rhGK was designating as positive with an OD > 2 standard deviation (SD) above B10.BR serum or human healthy serum.
For immunoblotting, 1 µg of rhGK or PAD-treated-rhGK was subjected to electrophoresis, blotted onto a nitrocellulose membrane, and probed with serum (1:100) from B10.BR or NOD mice and incubated with the alkaline phosphatase-conjugated anti-mouse IgG, then visualized by NBT/BCIP substrate (Thermo Fisher Scientific).
Catalytic activity of recombinant human pancreatic glucokinase. After in vitro citrullination by PAD as described above, recombinant human pancreatic glucokinase (rhGK; LS-G5486, LSBio) was put on the ice to stop the reaction for 5 min. Then glucokinase activity was measured spectrophotometrically (A340nm) at 37°C by a glucose 6-phosphate dehydrogenase (G6PDH) coupled assay as described previously 48  Peptide prediction. The probability that citrullinated and unmodified glucokinase peptides would be bound and presented by DR0401 was evaluated based on a previously published prediction matrix 13,26 . Briefly, coefficients corresponding to each anchor residue for all possible core 9-mers within the protein were multiplied, yielding a predicted relative binding affinity score. Sequences were chosen to include at least 2 flanking residues on each side of the predicted minimal 9-mer motif.
Peptide binding measurements. Peptides with predicted binding to DR0401 (Supplemental Table 3) were synthesized (Sigma) and their binding to DR0401 was assessed through a competition assay, as previously described 49 . Briefly, increasing concentrations of each citrullinated glucokinase peptide were incubated in competition with a biotinylated reference influenza hemagglutinin peptide (HA306-318) at 0.02 μM in wells coated with DR0401 protein.
Curves were simulated using Prism software (Version 5.03, GraphPad Software Inc.) and IC50 values calculated as the concentration needed to displace 50% of the reference peptide.

In vitro tetramer assays and T cell clone isolation. Peripheral blood mononuclear cells
(PBMCs) were isolated by Ficoll underlay, resuspended in T cell media (RPMI, 10% pooled human serum, 1% Penicillin-Streptomycin, 1% L-glutamine) at 4×10 6  T cell clone maintenance and characterization. Clones specific for citrullinated Glucokinase peptides were maintained in supplemented RPMI and re-stimulated using PHA (2 µg/mL; Remel) every two weeks. Specificity was confirmed by re-staining expanded clones with tetramer.
Statistical analysis. Statistics were performed using a Student's unpaired two-tailed t test unless indicated. A value of p < 0.05 was regarded as significant.
helpful advice and edited the manuscript. M.J.M. directed the project, edited the manuscript and was responsible for coordination and strategy.    frequencies of glucokinase-reactive T cells (GK 192,199,231,266,270,and 346 combined) were significantly higher (p=0.0007, Mann Whitney test) in subjects with T1D (black circles) than in HLA matched controls (white circles). b, Glucokinase specific CD4 + T cells tended to be more naïve (CD45RA+CR7+) in healthy controls than in subjects with T1D had a significantly higher (p=0.064, Mann Whitney test). c, Individual frequencies were significantly higher in subjects with T1D (black circles) than in healthy controls (white circles) for GK 346 (p=0.0004, ANOVA followed by Sidak's multiple comparisons test). Individual frequencies trended toward being higher for GK 199 and GK 270 (p=0.08 and p=0.09 respectively) but did not reach statistical significance. d, A heatmap analysis of individual glucokinase T cell frequencies in subjects with T1D, indicating patterns of reactivity that differed between subjects. In the heatmap each column represents one subject and each row reflects one glucokinase specificity, with each square color coded to indicate the observed T cell frequency in cells per million. The antibody readout is indicated at the bottom individually. e, There was a significant positive correlation between the combined frequency of glucokinase-reactive T cells and levels of antiglucokinase antibodies (p=0.030, simple linear regression).