Pleiotropic consequences of metabolic stress on the major histocompatibility complex class II molecule antigen processing and presentation machinery

Summary

Hyperglycemia and hyperlipidemia are often observed in patients with Type II Diabetes (T2D) and related mouse models. One dysmetabolic biochemical consequence is the non-enzymatic reaction between sugars, lipids and proteins, favoring protein glycation, glycoxidation and lipoxidation. Here, we identified oxidative alterations in key components of the major histocompatibility complex (MHC) class-II molecule antigen processing and presentation machinery in vivo, in conditions of hyperglycemia-induced metabolic stress. These modifications were linked to epitope-specific changes in endosomal processing efficiency, MHC-class-II-peptide binding and DM editing activity. Moreover, we observed some quantitative and qualitative changes in the MHC-class-II immunopeptidome of Ob/Ob and mice on a high fat diet as compared to controls, including changes in the presentation of an apolipoprotein B100 peptide previously associated with T2D and metabolic-syndrome-related clinical complications. These findings highlight a link between glycation reactions and altered MHC-class-II antigen presentation that may contribute to T2D complications.

Graphical Abstract

eTOC

Hyperglycemia and hyperlipidemia during diabetes and metabolic syndrome induce protein oxidative post-translational modifications (PTMs) that affect the MHC-class-II processing and presentation machinery. Clement et al. reveal that these PTMs are linked to epitope-specific changes in the MHC-class-II peptidome, with increased presentation of an apolipoprotein B100 peptide, contributing to diabetes-related complications.

Introduction

Proteomic modifications linked to non-enzymatic glycation and glycoxidation are common in all tissues under hyperglycemic conditions, as present in metabolic syndrome, type 2 diabetes (T2D), mice bearing a homozygous mutation in leptin (Ob/Ob mice) and mice subjected to a high fat diet (HFD) (Brownlee, 1995, 2000) (Cannizzo et al., 2012). Glycation refers to the non-enzymatic, covalent binding of carbohydrate moieties to reactive protein residues, as a consequence of the nucleophilic reaction between the carbonyl group of a reducing sugar and either the ε- amino group of lysine residues or the α-amino group at the N-terminus of proteins (Miksik and Deyl, 1997). Glycoxidation resembles glycation in mechanism, but it involves a previously oxidized monosaccharide (i.e., an α-oxaldehyde, such as glyoxal, methylglyoxal, or 3-deoxyglucosone) rather than a reducing sugar (Miksik and Deyl, 1997).

Since both glycation and glycoxidation are non-enzymatic reactions (Maillard reactions), the protein-sugar bond formation rate strictly depends on the sugar concentration. Protein glycation is minimal in physiological conditions, originating from the daily fluctuations in glycemia, and proceeds at increased rate in the context of hyperglycemia. Indeed, the circulating amounts of glycated or glycoxidated proteins (e.g., glycated hemoglobin or albumin) are commonly employed as a reliable indicator of glycemic status.

Over time, glycated proteins undergo a series of more stable rearrangements, cumulatively known as initial Amadori products. Over one hundred different Amadori products have been described; α-pyranose, α-furanose, β-pyranose and β-furanose groups linked to the amino group of lysines and arginines, or more rarely, the thiol group of cysteines or the protein N-terminus, are among the best characterized (Miksik and Deyl, 1997; Rosca et al., 2005). Albeit these initial non-enzymatic reactions are reversible, following a series of intramolecular rearrangements protein glycation and glycoxidation become irreversible, generating the so-called advanced glycation end-products (AGEs) (Arena et al., 2014; Lapolla et al., 2013; Scharf et al., 2013).

The increase in glycated proteins experienced by diabetic individuals has important clinical implications, as it has been implicated in a variety of complications including nephropathy, neuropathy, retinopathy and coronary artery disease (Mullarkey et al., 1990; Yamagishi et al., 2017). Immunologically, extracellular AGEs mediate robust pro-inflammatory effects by binding to and activating the receptor for advanced-glycation end products (RAGE), as well as Toll-like receptor 4 (TLR4) on monocytes, dendritic cells and macrophages (Adamopoulos et al., 2016; Poulsen et al., 2013). Additionally, proteins modified by AGE and other oxidative post-translational modifications (PTMs) upon processing, can generate antigenic neo-epitopes that promote autoimmunity. For instance, carbonylated transthyretin has been associated with increased T cell reactivity in juvenile idiopathic arthritis (Clement et al., 2016b). Protein citrullination is routinely employed as a diagnostic test for rheumatoid arthritis and – together with protein carbamylation – has been mechanistically linked to autoantibody production and pathogenic T cell responses (Mydel et al., 2010; Wiik et al., 2010). Myelin basic protein (MBP) phosphorylation and myelin oligodendrocyte glycoprotein (MOG) malondialdehyde modifications have been linked to autoimmunity in multiple sclerosis, and acetylation of the MBP peptide 1–11 is required to stimulate T cells in experimental autoimmune encephalomyelitis (van Stipdonk et al., 1998; Warnecke et al., 2017; Zamvil et al., 1986). Similarly, peroxidation, phosphorylation and deamidation of several proteins have been implicated in the pathogenesis of systemic lupus erythematosus (Liu et al., 2012; Utz et al., 1997), and carbonylation of insulin A chain and chromogranin A, which greatly increases their antigenicity, has been documented in patients with autoimmune diabetes (Haskins and Cooke, 2011).

As an additional mechanism linking protein PTMs to altered immunity, herein we demonstrated that several components of the MHC class II antigen processing and presentation machinery are glycated in metabolic conditions associated with increased oxidative and metabolic stress, resulting in qualitative and quantitative changes in the MHC-class-II immunopeptidome, including increased presentation of Apolipoprotein B (APOB) peptides that contribute to the pathogenesis and complications of metabolic disorders in humans.

Results

The dendritic cell proteome is carbonylated and AGE-modified in Ob/Ob mice

We initially set out to investigate the impact of T2D and metabolic syndrome on the proteome of antigen presenting cells (APCs). To this aim, we isolated dendritic cells (DCs) from the lymph nodes of Ob/Ob mice and syngeneic, age-matched control C57BL/6 (B6) mice, followed by the immunoblotting-assisted quantification of AGEs and carbonyl modifications. This approach revealed a significant increase in the amount of AGE-modified proteins (p=0.0015) and carboxymethyl-lysine (CML) (p=0.0185) in the proteome of DCs from Ob/Ob vs B6 mice (Fig. 1a, 1b). To further map these oxidized proteins at the molecular and cellular level, we performed label-free mass spectrometry (Suppl. Table S1), on three biologically independent DCs lysates harvested from B6 and Ob/Ob mice (Suppl. Table S1). Analysis of the cellular proteome PTMs ranked formyl-lysine and CML as the most abundant AGEs found in Ob/Ob DCs (Figure 1c and Suppl. Table S1). Additional glycoxidation-specific PTMs, mapped only to the Ob/Ob proteome, were glyceryl lysine and 3-deoxyglucosone. An additional proteomic analysis, performed on gradient purified late endosomes also confirmed the increased number of glycated proteins in the Ob/Ob vs B6 organelles (Suppl. Table S1).

Figure 1. Increased number of oxidative PTMs (AGE, HNE and carbonyl groups) are present on the Ob/Ob DCs-proteome as compared to B6 mice.

a) Representative immunoblots to detect AGEs and protein carbonylation in CD11c DCs lysates from B6 and Ob/Ob mice (one blot out of three blots is shown). Bar graphs report the mean and S.E. of densitometric analysis of three independent membranes. b) Data were analyzed by one-way ANOVA and Dunnett’s multiple comparisons test, significance: **P < 0.005. c) Major AGEs and carbonyl-PTM found in the combined analysis of three biological samples of DCs purified from Ob/Ob and B6 mice. Bar graph reports the total spectral count of oxidative PTMs, quantified in the Ob/Ob and B6 DCs proteome from four biologically independent proteomic experiments. d) IPA generated view of the major protein networks involved in carbohydrate metabolism and protein catabolism, generated by the analysis of the Ob/Ob PTM-modified DCs proteome (Suppl. Table S1). Proteins highlighted in brown indicate presence of AGEs or carbonyl groups. e) IPA generated view of the endosomal antigen processing pathway generated by the analysis of the PTM-modified Ob/Ob DCs proteome (Suppl. Table S1). Proteins highlighted in red indicate presence of AGEs or carbonyl groups. f) IPA-generated analysis of proteins with AGEs and carbonyl PTMs retrieved the major biochemical and cellular pathways affected by glycation. The probability of having a relationship between each IPA indexed biological function and the experimentally determined protein was calculated by a right-tailed Fisher’s exact test with the Benjamini-Hochberg Correction.

Ingenuity pathway analysis (IPA) analysis, performed on the PTM-modified proteins, identified metabolic pathways as well as phagocytosis, antigen processing and presentation and phagosome maturation amongst the top pathways including a higher number of proteins modified by AGEs or carbonylation in primary DCs from Ob/Ob vs. B6 mice (Fig. 1d, 1e, 1f). The site-specific amide-AGE modifications that we detected also mapped to proteins involved in response to DCs signaling, immune cell trafficking, cytoskeleton signaling and cell movement, (Fig. 1f). Overall, these findings indicated that the DCs proteome of Ob/Ob mice has an increased number of carbonyls PTMs and AGEs as compared to DCs of B6 mice.

Oxidative alterations of the DC proteome affect antigen presentation to T cells in Ob/Ob mice

To investigate whether the PTMs observed on the antigen processing and presentation machinery were associated with alteration in antigen presentation to T cells we analyzed the immune response to two model antigens: OVA_323–329 and HEL_74–96. Specifically, we immunized B6 and Ob/Ob mice with OVA and HEL peptides by subcutaneous inoculation in complete Freund adjuvant (CFA), and collected draining lymph nodes 2 weeks later, followed by restimulation of total nodal cells with the relevant peptide and assessment of CD4⁺ T cell proliferation. We observed no significant differences in CD4⁺ T cell responses to OVA_323–329 between immunized control and Ob/Ob mice (Fig. 2a). However, the proliferative response of nodal CD4⁺ T cells to HEL_74–96 was limited in immunized Ob/Ob mice as compared to their control immunized counterparts (Fig. 2b), indicative of a reduction in presentation of the HEL_74–96 epitope.

Figure 2. Glycation and Carbonylation of the Ob/Ob Dendritic Cell Proteome Affect T cell presentation.

a, b) B6 and Ob/Ob mice were immunized with 100 μg of OVA or HEL proteins. Two weeks later draining lymph nodes were harvested, total cells were labeled with Violet-Blue and incubated with 20 μg of OVA for 4 days. CD4 T cell proliferation to OVA and HEL was assessed as percentage of Violet-Blue proliferating CD4⁺ T cells. Experiments were performed in biological triplicates for both OVA (a) and HEL (b). Statistical analysis was performed using a paired two-tailed t-test. c and d) Flow cytometry analysis of B6 or Ob/Ob DCs cultured for different time points with 50 μg of Eα-RFP. Protein phagocytosis was quantitatively evaluated by RFP fluorescence whereas peptide processing and I-A^b loading was quantified using the conformational antibody Y-Ae specific for the I-A^b-Eα−52–68 peptide complex. Experiments were performed in biological quadruplicate and mean fluorescence index (MFI) of Eα-RFP and Y-Ae staining reported as the average plus standard deviation. Statistical analysis was performed using a paired two-tailed t-test e) Endosomes were gradient-purified from B6 and Ob/Ob DCs and incubated with OVA or HEL proteins for 6 hours at 37°C. e f) Number and sequences of OVA peptides, quantified by MS/MS, processed by purified B6 and Ob/Ob endosomes (detailed sequence information is presented in Suppl. Table S2). g) MS1 quantification of the OVA I-A^b-binding epitopes as processed by B6 and Ob/Ob endosomes (Suppl. Table S2). h, i) Number and sequences of HEL peptides, quantified by MS/MS, processed by purified B6 and Ob/Ob endosomes. j) MS1 quantification of the HEL- I-A^b-binding epitopes as processed by B6 and Ob/Ob endosomes (Suppl. Table S2). The OVA and HEL processing pattern, as determined by hot-spot analysis, is reported in Supplement Figures S1 and S2.

As endocytosis, and antigen processing, were among the pathways mostly affected by oxidative modifications, we next analyzed the efficiency of MHC-class-II presentation and T cell priming using a system that allows tracking of antigen phagocytosis, processing and MHC-class-II presentation. DCs purified from the spleen of Ob/Ob or B6 mice were incubated with recombinant Eα-RFP, which comprises the Eα_52–68 epitope restricted by mouse histocompatibility 2, class II antigen A, beta 1 (H2-Ab1; I-A^b) followed by the concomitant assessment of protein uptake (based on RFP fluorescence) and presentation (based on the Y-Ae antibody, specific for I-A^b complexed with Eα_52–68) by flow cytometry. In this setting, we detected no differences in the capacity of DCs from Ob/Ob vs. control mice to take up Eα-RFP after 30 min of exposure (16% vs. 15% RFP⁺ DCs, Fig. 2c, 2d). However, over time DCs from control mice became more enriched in Y-Ae⁺ cells than their control counterparts (16% vs. 10% at 2 hours (T2), 32% vs 19% at 6 hours (T6), and 32% vs 16% at 12 hours (T12) (Fig. 2c, 2d), indicative of an increased presentation of the Eα_52–68 epitope.

Altogether, these data demonstrate that the overall efficiency of antigen presentation is impaired in Ob/Ob mice only for selected MHC-Class II-restricted antigens including Eα- and HEL-derived epitopes.

The effects of oxidative alterations on MHC-Class-II antigen processing are protein and epitope-specific

To investigate in detail the defect in antigen presentation manifested by DCs from Ob/Ob mice, we purified the late endosomal compartment of nodal DCs from Ob/Ob or B6 mice, incubated it with 2 μg recombinant OVA or HEL, and quantified OVA- and HEL-derived peptides by MS/MS. A total of 630 OVA-derived unique peptides were generated by the late endosomes of DCs from Ob/Ob mice as compared to 509 generated by the late endosomes of B6 mice (Fig. 2e, 2f, Suppl. Table 2). Precursor MS1-area quantification confirmed that nodal DCs from Ob/Ob mice were quantitatively more efficient at processing the I-A^b OVA_323–339 peptides then their control counterparts (Fig. 2g). Conversely, DCs late endosomes from Ob/Ob mice generated a reduced number of HEL unique peptides (87) as compared to their control counterparts (161 unique peptides), including 71 overlapping sequences (Fig. 2h, 2i). The overall decreased efficiency of HEL processing by Ob/Ob endosomes was confirmed by MS1 quantification (Suppl. Table 2). Importantly, the I-A^b-restricted HEL_81–96 peptides could only be detected in the late endosomal compartment of DCs from control (but not Ob/Ob) mice (Fig. 2j). The impaired generation of the HEL-I-A^b epitopes, but not the OVA-I-A^b epitopes, by Ob/Ob DC endosomes could explain the differential CD4⁺ T cell proliferative response observed upon immunization with HEL and OVA (Fig 2a, 2b).

To further map how OVA and HEL were differently processed by the C57BL/6 and Ob/Ob late endosomes we performed hot spot analysis, which allows visualization of differences in protein processing patterns (Suppl. Fig. S1). Hot spot analysis of HEL processing indicated that, albeit a lower number of peptides was generated by the late endosomal compartment of DCs from Ob/Ob as compared to control mice, the pattern of HEL processing at the N-terminus did not exhibit major alterations (Suppl. Fig. S1). Conversely, peptides corresponding to the C-terminal domain of HEL (encompassing aa 81 to 96, where the I-A^b immunodominant epitopes localize)(Gammon et al., 1987) could not be mapped in the late endosomal compartment of DCs from Ob/Ob mice (Suppl. Fig. S1). These data further confirm that the decreased proliferative response to HEL, following immunization, is related to impair HEL endosomal processing.

On the other hand hot spot analysis confirmed that Ob/Ob DC endosomes processed a higher number of OVA peptides than their control counterpart, including the immunodominant OVA_323–339 epitope, and that there were no differences in the OVA processing pattern between C57BL/6 and Ob/Ob endosomes (Suppl. Fig. S2).

Taken together, these findings indicate that the endosomal compartment of DCs from Ob/Ob mice exhibits alterations in the overall pattern of antigen processing that, at least in the case of specific antigens, may compromise the generation of MHC-Class-II immunodominant epitopes.

Oxidative alterations affect H2-DM CLIP-peptide exchange

Next, we set to analyze the efficiency of peptide loading on MHC-class-II molecules in the endosomal compartment of DCs from Ob/Ob vs. control mice. We were prompted to delve into this aspect by the increased amounts of MHC-class-II I-A^b molecules complexed with class II invariant chain-associated peptide (CLIP) that we observed on splenic APCs from Ob/Ob mice, as well as from control mice subjected to a high-fat, high-fructose (HFHF) diet (Fig. 3a, Suppl. Fig. S3), and by proteomic data indicating that CLIP Met90, Met92, Met98 and Met101 were oxidized, to different degrees, only in DCs from Ob/Ob mice (Fig. 3b, 3c, 3d). Albeit M oxidation is known to occur during sample preparation the higher amount of oxidized CLIP peptides in Ob/Ob mice prompted us to believe that endogenous oxidative stress could also pay a role in M oxidation. To estimate the impact of such oxidative alterations on the affinity of CLIP for I-A^b, we chemically synthesized CLIP variants with (1) oxidized Met90, Met92, Met98 and Met101, or (2) oxidized Met101 (which was present on >30% of sequenced CLIP peptides, Fig. 3b, 3c, 3d), and assessed their relative binding to I-A^b in vitro by a competitive peptide-exchange assay. We observed an oxidation degree-dependent decrease in CLIP affinity for I-A^b, resulting in a compromised ability of I-A^b to exchange CLIP for a test peptide (Fig. 3e), consistent with the location of Met90 and Met98 in the I-A^b P1 and P9 pockets, as observed in the crystal structure of the I-A^b-CLIP complex (Zhu et al., 2003).

Figure 3. Glycation and Carbonylation of the Antigen Processing and Presentation Machinery Affects I-A^b-CLIP binding affinity.

a) Surface I-A^b CLIP analyzed by flow cytometry. Graphs show quantification of the geometric mean fluorescence intensity obtained for I-A^b-CLIP on the surface of nodal (left) and splenic (right) B cells from B6, Ob/Ob or mice maintained on a high-fat high-fructose diet for 12 weeks (HFHF). Values were corrected for any differences in I-A^b expression and data is normalized to the values obtained for control B6 mice (Suppl. Figure S3). Horizontal lines indicate the mean of values obtained for each individual data point. Each dot corresponds to a biological replicate. Significance was calculated using an unpaired t test. b and c) sequence, PTM and percentage of oxidized CLIP peptides found in the endosomes of Ob/Ob mice. d) MS/MS fragmentation of non-oxidized and oxidized CLIP at positions m98 and m101. e) Binding affinities of I-A^b to native CLIP peptide and oxidized CLIP peptides were calculated using fluorescence-based peptide exchange. The oxidized CLIP peptides showed reduction in binding to I-Ab (>150μM) as compared to the native CLIP peptide (0.5μM). The p values were calculated using unpaired t test.

One potential mechanism to explain the increased abundance of I-A^b/CLIP complexes on the surface of splenic APCs from Ob/Ob mice involve I-A^b glycation, as our proteomic studies mapped several AGE-modified amino acids to I-A^b (Suppl. Table S1). To investigate the relevance of these alterations, we mapped AGE-modified residues onto the I-A^b crystal structure (Zhu et al., 2003) and identified multiple amino acids at the I-A^b surface, including the antigen-binding pocket and sites of H2-DM interaction (Fig. 4a, 4b, 4c, Suppl. Table S3). In particular, oxidative alterations have been documented at βHis81, which stabilizes peptide binding by forming hydrogen bonds surrounding the peptide P1 residue (Painter et al., 2011; Stern et al., 1994; Zhu et al., 2003). Oxidation was also mapped at βPro56, which is part of the PD-VS polymorphism associated with type 1 diabetes in humans and NOD mice (Morel et al., 1988; Singer et al., 1998) and is responsible for peptide specificity in the minor P10 pocket (Zavala-Ruiz et al., 2004) (Fig. 4a, 4b, 4c). Other alterations were observed at αMet44, which is adjacent to the key I-A^b residue αTrp45 that flips out of I-A^b structure during peptide exchange to form the primary interaction site with H2-DM, as well as at βTrp189 and βArg190, which are adjacent to a key residue (βGlu187) in the formation of a salt bridge with H2-DM βArg110 as part of the H2-DM and MHC-Class-II lower Ig domain interaction (Painter et al., 2011; Painter and Stern, 2011; Pos et al., 2012; Yin and Stern, 2014) (Fig. 4b, 4c). We used an in vitro modification system to evaluate whether MHC-class-II glycation could be responsible for to alterations in peptide loading. Thus, we incubated recombinant class II human major histocompatibility protein HLA-DRB1*01:01, hereafter abbreviated as DR1, with 5 or 20 mM glucose for 3 days at 37 °C, in the presence of recombinant human CLIP. Thereafter, the DR1 and CLIP monomeric fractions were purified by gel filtration in presence of the corresponding glucose concentration, which revealed no signs of unfolding or aggregation (Suppl. Fig. S4a). We evaluated the effects of these modifications on DM-catalyzed peptide exchange, using a fluorescence polarization assay (Yin and Stern, 2014). Specifically, DR1 optionally exposed to 5 or 20 mM glucose for 3 days was mixed with a fluorescent viral peptide – i.e., hemagglutinin (HA) – in the absence or presence of DM, and HA binding to DR1 was followed over time by fluorescence polarization. In the absence of DM, when the assay was performed in the presence of the same glucose concentrations employed for DR1 glycation, peptide exchange rate was not affected by glycation (Fig. 4d, Suppl. Fig. S4b). Conversely, the ability of DM to promote peptide exchange was reduced when DR1 had previously been exposed to 5 or 20 mM glucose, but only if glucose was included in the polarization assay (Fig. 4e Suppl. Fig. 4c). These findings indicated that rapidly reversible oxidative DR1 alterations (most likely Schiff bases or Amadori products) interfered with DM activity. Since the conditions we initially employed for DR1 glycation did not introduce irreversible oxidative changes as observed in vivo, we incubated recombinant DR1 with 500 mM glucose for 15 days, as per a protocol previously shown to cause extensive AGE accumulation (Sadowska-Bartosz and Bartosz, 2015). Quantitative LFQ mass spectrometry identified many amido-glycated arginine and lysine residues at sites similar to those observed on I-A^b from the DC compartment of Ob/Ob mice (Suppl. Table S4 Suppl. Fig S4d Suppl. Fig S4e). We mapped the sites of oxidative modifications onto the DR1 crystal structure (Painter et al., 2011) and, as previously observed for I-A^b, several of these sites were located at the interface through which DR1 interacts with DM (Suppl. Fig. S4e). We assessed the DM-independent and -dependent peptide loading activity of the modified protein. While in the absence of DM the pre-exposure of DR1 to glucose had no effect on peptide loading (Fig. 4f, Suppl. Fig. S4f), DM-dependent peptide exchange was considerably reduced when DR1 had been subjected to glycation, even in the absence of glucose from the polarization assay (Fig. 4f, Suppl. Fig. S4g). Such a defect could not be corrected by increasing DM concentrations, indicating that DR1 residues that are critical for DM-mediated peptide exchange were stably modified by non-enzymatic glycation. To assess the effects of DR1 glycation in vitro over a shorter time scale, we incubated DR1 with the highly reactive glucose metabolite methylglyoxal (MGO), found in high concentration in subject with hyperglycemia and T2D. Following MGO incubation DR1 PTMs modifications were similar to the ones observed following glucose incubation (Suppl. Fig. S4d and Suppl. Fig. 4e), and in an earlier study of DR1 chemical modification (Carven and Stern, 2005). Again a reduction in DM-dependent peptide loading activity was observed (Fig. 4g, Suppl. Fig. S4h), with the magnitude of the effect depending on the MGO concentration (Suppl. Fig. S4i). Overall, these results indicate that DM-catalyzed peptide exchange on DR1 is reduced by both reversible and irreversible glucose-dependent modifications.

Figure 4. Glycation and Carbonylation of the Antigen Processing and Presentation Machinery Affects MHC-class-II-DM-CLIP- peptide exchange.

a, b) Mapping of AGEs PTMs identified by MS/MS analysis on the 1MUJ.pdb crystal structure of I-A^b bound to the CLIP peptide. Site specific AGEs were identified both in the peptide binding site (a, b) and in the DM interaction site (b) (in magenta-the glycation sites; in yellow-the oxidation and carbonylation sites; in green-the HNE and HNE derivatives of lipoxidation sites). PTMs identification was performed using PEAKS 8.0 in combination with Scaffold PTM 3.1.0. c) The major AGEs and carbonyl PTMs for each site on I-A^b are shown as % after dividing the total MS1-derived areas for the modified peptides with the total sum of MS1-derived area of all modified and non-modified peptides (Suppl. Table S3) The data were derived combining seven independent proteomics and immunopeptidomics experiments for each Ob/Ob and B6 primary DC samples. The type and the total AGE-unique sites associated “A scores” and the corresponding localization probability on each alpha and beta chains of MHC-class-II (I-A^b and DR1) are presented in Suppl. Table S3, S4 and Suppl. Figure S4. Binding of labeled HA peptide was measured by fluorescence polarization over time: d) The initial peptide binding rates for DR1 incubated with 0 mM, 5mM or 20 mM glucose in absence or presence of DM for 3 independent experiments each done in triplicate. e) Same as in panel (d) but modified DR1 was purified and assayed in absence of glucose. f) Same as in panels (d) and (e), but DR1 was incubated in the absence (gray traces) or presence of 500 mM glucose (green traces) for 15 days at 37 °C and purified and assayed in absence of glucose. g) Same as in panel (d), (e), and (f), but DR1 was incubated in the absence (gray traces) or presence of 1 mM methylglyoxal (red traces) for 24 hours. Three independent experiments were performed, and the indicated p value corresponds to significance of difference of fit values. Asterisks indicate significance of difference between samples for each DM concentration, were ** correspond to p<0.005, and *** to p<0.0005. p values were calculated using a two-tailed unpaired t test.

Changes in the MHC-class-II immunopeptidome affect T cell responses in obese mice

To determine whether the glycation-driven changes in the interaction between MHC-class-II molecules and DM are associated with alteration in the immunopeptidome, we eluted immunoaffinity-purified I-A^b from conventional DCs isolated from Ob/Ob or control mice (and analyzed MHC-class-II-associated peptides by LC/MS/MS by a data-dependent acquisition (DDA) approach (Suppl. Table S5). Although the total amount of I-A^b was not different between Ob/Ob and control mice (6.9 ± 2.2 vs. 9.6 ± 3.4 μg, respectively, n=3, p=0.31), the number of unique peptides (Fig. 5a) and distinct core epitopes (Fig. 5b) was higher for Ob/Ob as compared to control mice, indicating that a more diverse repertoire of peptides was presented. To help understand the greater diversity of epitopes presented by DCs from Ob/Ob as compared to control mice, we searched for core epitopes unique to Ob/Ob mice or control mice, or present in the both peptidomes. Of 227 distinct core epitopes detected in each of the 3 Ob/Ob replicates tested, 13 core sequences were not detected in any of the control samples. By contrast, only 3 core epitopes were present in each of the control samples but not detected in any of the Ob/Ob samples (Fig. 5c). Similar results were observed by relaxing the identification criteria and considering core epitopes present in fewer samples: 38 additional core epitopes were present in 2 of the 3 Ob/Ob replicate samples but were absent from any control mice sample, whereas only 10 additional core epitopes were detected in 2 of 3 control mice samples but were absent from any Ob/Ob sample. Overall, by DDA analysis the Ob/Ob peptidome appears to contain around 1/5 of the core epitopes that are absent, or present at much lower frequency in the control mice peptidome. Peptide-binding motifs (Fig. 5d, 5e) as well as peptide length distributions were similar in DCs from Ob/Ob and control mice (Fig. 5f). The distribution of peptide abundances, as measured by total ion current intensity (TIC, Suppl. Table S5) also differed between the two conditions, with low-abundance MHC-class-II-binding peptides being enriched on the surface of DCs from Ob/Ob vs. control B6 mice (Fig.5g).

Figure 5. Metabolic stress qualitatively and quantitatively changes the I-A^b immunopeptidome.

a) Number of unique peptides identified in biological triplicates following I-A^b immune-affinity purification and equal amount of I-A^b elution from C57BL/6 (B6) or obese (Ob/Ob) DCs. p values were calculated by two tailed unpaired t test. b) As panel (a) but showing number of core epitopes, as such peptides in the same nested set are counted only once. c) Unique core epitopes identified in Ob/Ob as compared with B6 samples. Bar shading indicates number of replicate samples for which the core epitope was identified. For example, the black bar labeled “3X” indicates core epitopes identified in each one of the biological replicates, unique to either the B6 or the Ob/Ob peptidome. The light gray bar labeled “2X” indicates core epitopes identified in 2 out of 3 of the biological replicates, unique to either the B6 or the Ob/Ob peptidome. The white bar labeled “1X” indicates core epitopes identified in 1 out of 3 biological replicates, unique to either the B6 or the Ob/Ob peptidome. d, e) I-A^b eluted peptides from B6 and Ob/Ob DCs were analyzed using GibbsCluster-2.0 Server to identify binding motif and displayed using Seq2Logo. No differences were seen in the binding motives for B6 versus Ob/Ob mice. f) Length distribution of I-A^b peptides eluted from B6 and Ob/Ob DCs were similar, with median length of ~16aa. g) Violin plot showing the distribution of fractional abundance for the 500 most intense B6 and Ob/Ob peptides. The Ob/Ob immunopeptidome seems to be more diverse than the control immunopeptidome, with increases representation of low-abundance peptides. h) Volcano plot indicates the quantitative differences for the core epitopes, I-A^b-eluted from B6 and Ob/Ob DCs and analyzed by DIA. The peptides showing differences with p<0.05 (B6>Ob/Ob in blue, Ob/Ob>B6 in red, only present in Ob/Ob in green) were analyzed for the I-A^b predicted affinity using NetMHCIIpan 3.2 Server. P values were calculated by unpaired two-tailed t test. i) The peptides present in Ob/Ob>B6 and Ob/Ob only are weaker predicted binders than peptides present in control>Ob/Ob or only Ob/Ob subsets. P values were calculated by two-tailed Mann Whitney t test.

However, to more accurately quantify possible differences between control C57BL/6 and Ob/Ob peptidomes, we analyzed additional biologically-independent triplicate samples for each condition using a data-independent acquisition (DIA) approach. Specifically, we analyzed DIA data by employing a reference spectral library consisting of all peptides identified by database matching in the pool of spectra from combined DDA dataset, which ultimately enables a direct, label-free quantitation of relative abundance (Jensen et al., 2018). Using DIA analysis the number of C57BL/6 or Ob/Ob I-A^b-eluted differentially expressed peptides, greatly decreased indicating how the analysis of all MS precursor ions (within a defined m/z window), instead of the 10 more abundant peaks, provide less missing values and a more reliable quantitative measurement of the MHC-class-II immunopeptidome (Collins et al., 2017). In other words, by employing DIA we were able to detect low abundance epitopes that were undetected by DDA analysis (Supplement Table S5).

Importantly, even after DIA analysis several core epitopes were still found to be in greater amounts in DC samples from control vs. Ob/Ob samples (p<0.05), and vice versa (Fig. 5h, Suppl. Table S5). Fourteen core epitopes were found to be in greater amounts in DC samples of control mice as compared to DC samples of Ob/Ob mice, 19 core epitopes were found to be in greater amounts in DC samples of Ob/Ob mice as compared to DC samples of control mice, whereas 3 core epitopes were identified in DC samples of Ob/Ob mice only and none of DC samples of control mice. Predicted affinity for I-A^b (based on NetMHCIIpan3.2) was significantly higher for peptides containing core epitopes that were enriched in control (over Ob/Ob) samples, as compared to peptides containing core epitopes enriched in Ob/Ob (over control) samples, including the three core epitopes detected by DIA analysis only in the MHC-Class-II DCs immunopeptidome from Ob/Ob mice (Fig. 5i). Accordingly, the peptides enriched in Ob/Ob (over control) samples and unique to Ob/Ob samples were predicted to be much weaker binders than their control counterparts. These findings are consistent with a defect in H2-DM-mediated peptide editing in Ob/Ob mice, resulting in a broader spectrum of peptides with increased representation of low-abundance and low-affinity peptides. Overall, the quantitative analysis of I-A^b-eluted DC immunopeptidomes from Ob/Ob and control mice reveal important differences in peptide presentation and epitope selection (Suppl. Table S5).

Metabolic stress potentiates T cell response to apolipoprotein B100

To investigate the immunological relevance of differential MHC-class-II antigen presentation, from Ob/Ob and control mice we focused apolipoprotein B 100 (APOB), since DCs from Ob/Ob mice presented an average 50% increase in the APOB core epitope as compared to DCs eluted from C57BL/6 mice (Fig. 6a, Suppl. Table S6) and an average 40% increase of APOB peptides with oxidative PTMs (Suppl. Table S6). In addition, the absolute quantitation of the APOB core epitope YSGSVANEAN by MS-based parallel reaction monitoring (PRM) confirmed that metabolic stress enhances YSGSVANEAN presentation on I-A^b molecules from Ob/Ob vs. control C57BL/6 mice. Alongside, we analyzed the I-A^b peptidome of DCs from C57BL/6 mice kept for three months on a HFHF diet. I-A^b-eluted peptides from C57BL/6 mice, Ob/Ob and HFHF mice were spiked with two heavy isotope-labeled standards (the core epitope YSGSV(+6)ANEAN and the L(+7)SQEYSGSVANEAN peptide, which was previously identified to be more abundant in the Ob/Ob DC immunopeptidome by DIA analysis, Suppl. Table S6). Targeted PRM analysis established the transition parameters and retention times for the two individual heavy peptide standards (Suppl. Table S7 b–c). The endogenous core epitope YSGSVANEAN and LSQEYSGSVANEAN peptide were identified and quantified in the spiked peptidomes samples by monitoring the co-elution with heavy standards, as shown in one representative experiment from Ob/Ob sample (Figure 6b, 6c). The absolute abundance of the APOB core peptide confirmed an increased presentation of APOB by DCs from mice under metabolic stress (Figure 6d).

Figure 6. Metabolic stress qualitatively and quantitatively changes T cell response to APOB.

a) Label-free quantitation (LFQ) of the APOB-I-A^b core epitope YSGSVANEAN (Suppl. Table S6). Average intensities from three independent biological samples are presented, normalized to the total I-A^b-eluted peptidome. b) MS-based parallel reaction monitoring (PRM), for absolute peptide quantitation, and representative ion extracted chromatogram of the core epitope YSGSVANEAN is shown (one of two quantification is reported). The spiked heavy peptide YSGSV(+6)ANEAN is shown in blue while the corresponding endogenous light peptide is shown in red, co-eluting with the heavy standard. c) Example of PRM analysis of the Ob/Ob peptidome ion extracted chromatogram with the identified peak corresponding to the endogenous YSGSVANEAN peptide (one of two quantification is reported). d) The absolute abundance (fmole) of YSGSVANEAN, quantified in I-A^b immunopeptidome samples from DCs of mice feed a high fat high sucrose diet (HFHF), Ob/Ob and B6 as determined by PRM analysis. Quantification was performed by comparing the MS1 area of each samples with the MS1 area, integrated for 10 fmole, of the co-eluting heavy standards. Average of two biological replicates is show. e) Nodal T cell proliferation of B6 and Ob/Ob mice immunized with the APOB core epitope YSGSVANEAN. Lymph nodes were collected 2 weeks following immunization and incubated with titrated amounts of the APOB peptide for 4 days. T cell division were quantified by incorporation of EdU using Click-iT EdU proliferation assay. Each concentration point is reported as biological quintuplicates. P-values were calculated by multiple t-tests comparison analysis and the statistical significance determined using the Holm-Sidak method, with alpha = 0.05. f) T cell proliferation in abdominal fat collected from B6 and Ob/Ob mice immunized with the APOB peptide. Abdominal fat was collected 2 weeks following immunization and single cell suspension labeled with CFSE and incubated with titrated amounts of the APOB peptide for 4 days. CD4 T cell proliferation to APOB peptide was assessed as percentage of CFSE low/proliferating CD4⁺ T cells. Average of three biological replicates is shown. p values were calculated by unpaired two-tailed t-test. Additional proliferation experiments are reported in Fig. S5 g) IL-6, IL-10, TNFα, IFNγ and IL-17A were quantified in the culture supernatant of proliferating nodal T cell as in (e), cultured for 4 days in presence or absence of 20 μg/ml of APOB peptide. p values were calculated by unpaired two tailed t-tests comparison analysis. h) Congo red staining of the aorta harvested from B6 and Ob/Ob mice. Representative H&E staining of aortic tissue harvested from B6 and Ob/Ob mice (one staining out of 12 biological replicates is shown). i) Representative Ki-67 staining of aortic tissue harvested from B6 and Ob/Ob mice (one staining out of 12 biological replicates is shown). j, k) Quantification of j) immune infiltrates and k) Ki67+ cells in aortic tissue harvested from B6 and Ob/Ob mice. p-values were calculated by unpaired two tailed t-tests analysis. l) B6 and Ob/Ob mice were immunized with 100 μg of APOB peptide. Two weeks later the aorta was harvested, digested and single cell suspensions were labeled with CFSE and incubated with or without 20 μg of APOB peptide for 4 days. CD4⁺ T cell proliferation to APOB peptide was assessed as percentage of CFSE low/proliferating CD4⁺ T cells. Average of three biological replicates is shown. m) Bar graph report the percentage of CFSE low/proliferating CD4+ T cells from biological triplicates. p values were calculated by unpaired two-tailed t-test.

Accordingly, Ob/Ob mice, immunized with the APOB core peptide, exhibited a higher proliferative response than their control counterparts (Fig. 6e). Similarly, a trend towards higher amounts of CD4⁺ T cells responding to the APOB core peptide was observed in visceral fat of Ob/Ob vs. control mice (Fig. 6f). Such an increased proliferative response was also associated with augmented production (either at baseline or upon stimulation) of pro-inflammatory cytokines, including tumor necrosis factor (TNF), interferon gamma (IFNG), interleukin 10 (IL-10) and interleukin 17 alpha (IL-17A), but not IL-6 (Fig. 6g).

Since APOB has previously been associated with inflammatory responses and atheroma plaque formation in large arteries, which is one of the complications of T2D and metabolic syndrome (Shaw et al., 2017), we set to investigate signs of exacerbated inflammation in the aorta of control and Ob/Ob mice upon immunization with the APOB core peptide. We found an increased Congo Red-positive area, indicative of lipid accumulation, in immunized Ob/Ob mice as compared to their immunized control counterparts (Fig. 6h). Moreover, the aortic wall of immunized Ob/Ob mice exhibited increased infiltrating lymphocytes and Ki67⁺ proliferating cells as compared to control mice (Fig. 6h, 6i).

Finally, to determine whether APOB-specific CD4⁺ T cells infiltrated the aortic wall of control and Ob/Ob mice immunized with the APOB core peptide, we prepared single-cell suspensions from collagenase-digested aortic tissue harvested 2 weeks after immunization and investigated T cell proliferative responses upon peptide re-stimulation. We observed a pronounced proliferative response in CD4⁺ T cells from Ob/Ob mice (over WT mice) immunized with the APOB core peptide (Fig. 6j, 6k, 6l, 6m).

Altogether, these findings indicate that CD4⁺ T cells from the aortic wall of Ob/Ob mice are hyperresponsive to the APOB core peptide, most likely as a consequence of increased MHC-class-II presentation of the APOB epitopes as well as increased amount of oxidative PTM-modification on APOB imposed by the Ob/Ob genotype. Conversely, nodal CD4⁺ T cells from Ob/Ob mice immunized with the APOB core epitope were slightly hyporesponsive to splenic DC stimulation as compared to their counterparts from immunized WT mice, irrespective of DC source (Suppl. Fig. S5), and this did not depend on altered type I (cDC1) vs. type II (cDC2) splenic DC composition (Suppl. Fig. S5).

Discussion

Systemic metabolic dysregulation associated with hyperlipidemia and hyperglycemia is a key pathological component of T2D and metabolic syndrome. Elevated amounts of circulating glucose and lipids generate reactive molecular species that favor protein oxidation and glycation (Brownlee, 2000; Greifenhagen et al., 2016; Schmidt et al., 2015; Spiller et al., 2017). Hyperlipidemia and hyperglycemia also promote the generation of mitochondrial and cytosolic reactive oxygen species (ROS) and reactive nitrogen species (RNS), which further contribute to non-enzymatic protein oxidation (Araki and Nishikawa, 2010; Bansal et al., 2012; Bonnefont-Rousselot et al., 2003; Mullarkey et al., 1990; Wong et al., 2002). Moreover, reactive lipids and sugars can also be ingested with high-fat or high-sugar diets (Poulsen et al., 2013). Thus altogether, tissues from patients with T2D or metabolic syndrome are exposed to dysmetabolic conditions that can alter proteins primary structure through rapid, non-enzymatic glycation and oxidation (Ahmad et al., 2014; Andriani et al., 2016; Bavkar et al., 2019; Bo et al., 2016; Brownlee, 1994, 1995).

The pathogenicity of AGE-bearing proteins is related to these structural alterations, which promote proteins unfolding, degradation, and/or aggregation (Cannizzo et al., 2012). Such functional degeneration is actively prevented by multiple mechanisms of protein quality control, including AGE removal by dedicated enzymes such as glyoxalase I (GLO1) (Brouwers et al., 2011; Shinohara et al., 1998), and by proteasomal and autophagic degradation of irreversible protein aggregates (Cannizzo et al., 2012; Rybstein et al., 2018; Uchiki et al., 2012). However, these mechanisms can be overwhelmed in the context of prolonged oxidative stress, ultimately enabling the accumulation of dysfunctional proteins. Indeed the impact of AGEs on various degenerative disorders including T2D has been extensively described (Dobi et al., 2019; Hanssen et al., 2014; Ma et al., 2009; Ma et al., 2017; Sima et al., 2010; Wei et al., 2013; Yao and Brownlee, 2010; Zakliczynski et al., 2009) and mechanistically validated in several disease models (Li et al., 1996; Maruf et al., 2015; Matsui et al., 2010; Soro-Paavonen and Forbes, 2006).

A developing area of research focuses on the precise mechanisms though which oxidative alterations modify protein functionality. Glycation has been shown to reduce the stability of apolipoprotein A1 (APOA1) and thus decrease cholesterol transport by high density lipoproteins (HDLs) (Nobecourt et al., 2010; Nobecourt et al., 2008; Passarelli et al., 2005), to affect the ability of hemoglobin to release oxygen (Saleh, 2015; Shimada et al., 2005), and to decrease the enzymatic activity of various enzymes (Backos et al., 2013; Fujita et al., 1998; Liu et al., 2009; Yan and Harding, 1997). Moreover, glycation reportedly induces apoptotic cell death by influencing cellular chaperons and signal transduction pathways including Janus kinase 1 and NF-kB (Jiang et al., 2015). Finally, oxidative protein alterations in immune cells have been shown to compromise the catalytic activity of the proteasome (Uchiki et al., 2012), immunoglobulin functions (Lapolla et al., 2013), as well as inflammasome-dependent cytokine production and phagocytic activity (Liu et al., 1999; Son et al., 2017).

Along these lines of investigation, we delved into possible functional alterations imposed on the MHC-class-II antigen processing and presentation machinery by oxidative changes linked to T2D. We found that several proteins involved in antigen uptake, endosomal processing, and MHC-class-II peptide loading were affected by oxidative changes in the DCs harvested from Ob/Ob mice. Specifically, the late endosomes of Ob/Ob DCs, continuously exposed to oxidative stress conditions, exhibited alterations in the pattern of antigen processing, favoring formation of an OVA- derived epitope while inhibiting formation of HEL- and IE-α- derived epitopes.The immunological consequences of the differential endosomal processing were reflected in the outcome of T cell priming, following immunization with each antigen. Indeed, whereas similar OVA-specific T cell responses were observed in control B6 and Ob/Ob mice, decreased T cell expansion was observed following HEL, and IE-α presentation.

Another important consequence of the redox inbalance in Ob/Ob mice and mice subjected to a HFHF diet was the glycation of MHC-class-II molecules. Oxidative PTMs were mapped at peptide-interacting sites (βPro56, βHis81, βAsn82) or sites directly involved or adjacent to residues required for DM binding (αMet44, βTrp189, βArg190). CLIP oxidation was mapped at Met90, Met92, Met98 and Met101.

To fully explore the consequences of these PTMs on MHC-class-II-restricted immune responses we analyzed the I-Ab immunopeptidome. Since both B6 and Ob/Ob mice express I-A^b as the only MHC-class-II molecule, the peptide repertoire should be similar. However, using both DDA and DIA approaches to analyze the eluted peptidome, we found selected peptides that were eluted only from B6 or Ob/Ob mice, as well as several peptide, that were represented in a quantitatively different manner between the two immunopeptidomes. Additionally, as expected, several eluted epitopes from Ob/Ob mice showed direct oxidative alterations.

To further delve into the pathophysiological relevance of these findings, we analyzed one peptide, (APOB) that was enriched in the MHC-class-II immunopeptidome of Ob/Ob DCs and presented increased oxidative PTMs in the same mice. This epitope had previously been shown to be involved in the atherosclerotic complications of T2D (Shaw et al., 2017). We found that CD4⁺ T cells from the aortic wall of Ob/Ob mice exhibited increased responsiveness to the APOB peptide in immunization experiments, and that an increased number of APOB - specific T cells infiltrate the aortic tissue.

Overall our data indicate that DCs generated in a dysmetabolic environment, such as present in Ob/Ob and HFHF mice, may utilize “dysregulated” cellular pathways that can ultimately affect their ability to efficiently process and present antigens, including changes in their efficiency for antigen phagocytosis, cellular trafficking, CD4 priming and activation. Herein we specifically focused on MHC-class-II antigen presentation and determined that oxidative alterations may contribute to generation of a CD4⁺ T cell compartment with increased reactivity to several otherwise non-immunogenic peptides, ultimately fostering disease progression by virtue of accrued inflammation at target tissues.

Limitations of the study

Our study was conducted in Ob/Ob mice and mice kept on a high fat high fructose diet. Albeit these models are considered a phenocopy of many of the proteomic modifications observed in T2D patients, human disease is obviously more variable. Genetic variability, presence of co-morbidities and therapeutic protocols that control glycaemia are all important variables that could change, qualitatively and quantitatively, protein oxidative PTMs. We have shown that oxidative modifications to MHC class II proteins induced by elevated glucose directly affect their ability to bind peptides and interact with the peptide exchange catalyst HLA-DM, but the effects of elevated glucose on MHC-II peptidomes that we observed in vivo could also result from indirect effects involving other components of the antigen processing and presentation machinery.

STAR METHODS

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to Laura Santambrogio, Weill Cornell Medical College e-mail: las4011@med.cornell.edu

Materials Availability

This study did not generate new unique reagents

Data and Code Availability

Proteomic data that support the findings of this study have been deposited in the PRIDE database Project Name: Characterization of I-A^b MHCII immunopeptidome eluted from control and obese (Ob/Ob) mice. Project accession: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers: PXD023581, 10.6019/PXD023581, PXD018783, 10.6019/PXD018783, PXD024239 and 10.6019/PXD024239.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals

C57BL/6, Stock No: 000664 and Ob/Ob C57BL/6.Cg-Lepob/J, Stock No: 000632 were all purchased from the Jackson laboratory. All experiments were performed using 12–14 weeks old mice with a 50% combination of male and female mice. All experiments were performed under the WCM IACUC Protocol #: 2019-0024.

HFHF Diet

Seven to eight weeks old B6 mice were housed in the WCM/MSKCC animal facility with 12‐h light and dark cycles. The animal room was maintained at a temperature range of 23 ± 1 °C. All mice were randomly divided into two groups including a control group, in which the mice were fed the normal Laboratory Rodent Diet (Purina, St Louis, MO, USA) and HFHF (high fat and high fructose) group fed a high‐fat diet (60kcal % fat) containing 245 gm of lard, 200 gm Casein, lactic, 3 gm cysteine, 125 gm lodex10, 72.80 gm Sucrose, 50 gm Solka Floc, 25 gm soybean oil, 50 gm S10026B, 2 gm Choline itartrate and 1gm V10001c (cat# D12492, Research Diets Inc) and high fructose diet containing 64kcal% (cat # D10012G and D13061501, Research Diets Inc.,) in the ratio of 1:1 for every day up to 3month.

Mice Immunization

Female and male (50% of both sex) B6 and Ob/Ob mice were immunized with a total of 100 μg of the core epitope APOB (YSGSVANEAN) peptide in CFA. Mice were injected at two sites, nape of the neck and base of the tail. Two weeks following immunization mice were anesthetized with isoflurane and inguinal and axillary lymph node harvested for T cell proliferation assays.

Abdominal Fat Harvesting

B6, Ob/Ob and HFHF mice were anesthetized using isoflurane followed by cervical dislocation. Mice were then positioned on the surgical board and the peritoneum was cut transversely, directly below the diaphragm to expose the abdominal organs. White adipose tissue was carefully removed and transferred to test tube containing DMEM complete medium for further processing.

Aorta Harvesting

B6, Ob/Ob and HFHF mice were anesthetized using isoflurane followed by cervical dislocation. Mice were positioned on a surgical board in the supine position and appendages secured to the board using surgical tape. A 10cc syringe, filled with 10 mL ice cold 1X PBS, and attached to a 25 gauge needle was gently inserted into the left ventricle of the heart. At the same time the right atrium was cut to allow tissue perfusion for 8–10 minutes. Upon completion of perfusion, the thoracic cavity was exposed, rinse with 1x PBS and all fluid removed by absorption with a sterile gauze. Using sterile micro scissors and micro forceps, the aorta was separated from the spine dorsally and from the esophagus ventrally. The isolated aorta was immediately transferred to test tube containing DMEM complete medium for further processing or placed in OCT for histological analysis.

Ex vivo Primary Cell Cultures

Mouse Dendritic Cells preparation:

Ob/Ob mice and B6 controls were injected subcutaneously with 40×10⁶ B16-FLt3-L-producing melanoma cells to in vivo expand the number of DCs in secondary lymphatic organs (Clement et al., 2016a). Twelve to 14 days after injection spleens and lymph nodes (cervical, axillary, inguinal and mesenteric) were harvested and DCs were purified by density-gradient centrifugation using 30% Bovine Albumin solution (Sigma-Aldrich). Three mls of sterile BSA (30%) was mixed 1:1 (v/v) with RPMI1640 complete media on top of which an extra layer of 3 mL sterile BSA (30%) was added to complete the gradient premixing solution in a centrifuge polycarbonate tube that fits inside the Beckman SW Ti41 rotor. One-two mL of cell suspension was added on top of the premixed gradient solution and the mixture was centrifuged at 8500 rpm, 1 hour at 4°C using the Optima XPN-100k UltraCentrifuge (Bekman Coulter). The white ring of cells isolated after centrifugation routinely contains 70 to 80% CD11c+ DCs (Clement et al., 2016a), with no significant differences observed in the numbers of cells isolated from control or Ob/Ob mice.

METHOD DETAILS

SDS-PAGE and Immunoblot Analysis of AGE, and CML-modified proteins.

DCs were lysed in RIPA buffer (catalog# R0278, SIGMA) supplemented with 1 × protease inhibitor cocktail (catalog# 04693116001, Roche) for 30 minutes on ice. Lysates were spun twice at 20,000 g to collect the post-nuclear supernatant. Protein concentration was determined using Bradford micro BCA (ThermoFisher Scientific, catalog# 23235) protein assays and prediluted lysates in a microplate assay format following the manufacturer instructions. Equal amount of proteins were suspended in 2% SDS, 50 mM DTT, 50 mM Tris pH 7.4 at RT before loading on a 4–15% SDS-PAGE (Mini-PROTEAN® TGX^™ Gels, BioRad, CA). Blotted membranes were blocked in 5% nonfat milk in TBST for 1 h, at RT, and probed with the polyclonal goat-anti AGE (catalog # AB9890, from EMD-Millipore) at 1:200–1:500 dilution, overnight at 4 °C. Donkey anti goat secondary antibody (IRdye 800CW, catalog # 925–32214) (1:5000–1:3000) was added for 2 h at room temperature. Blots were imaged using an Odyssey Infrared Imaging System (LI-COR Biosciences, Cambridge, UK). The ImageJ software (https://imagej.nih.gov/ij) was used to perform the densitometry analysis of all membranes stained with Ponceau S. All Immunoblots were performed in a minimum of triplicate experiments. Detection of carbonylated proteins. Cell lysates (10 μg total proteins) were derivatized using the Oxyblot Protein Oxidation Detection Kit (catalog # S7150 Millipore, USA), separated on 4–15% SDS-PAGE as described above. Transferred membranes were incubated with a rabbit polyclonal anti-DNPH antibody followed by a goat anti-rabbit IgG–HRP antibody (OxiSelect^™ Protein Carbonyl Immunoblot Kit (catalog # STA-308) from Cell Bioloabs, Inc). Blots were imaged using an Odyssey Infrared Imaging System (LI-COR Biosciences, Cambridge, UK). The ImageJ software (https://imagej.nih.gov/ij) was used to perform the densitometry analysis of all membranes stained with Ponceau S. All Immunolots were performed in a minimum of triplicate experiments.

Antigen processing assay in primary DCs

Primary splenic DCs from B6 and Ob/Ob mice, purified on a BSA gradient as described above, were pulsed at 2×10^6 cells in presence of 50 μg of fluorescent Eα-RFP protein for 0.5 hour (T0) (Cannizzo et al., 2012). Cells were then washed twice in PBS and chased for different time points (T2 (2 hours), T6 (6 hours) and T12 (12 hours). After collection DCs were analyzed by Flow Cytometry to detect processing of Eα-RFP as well as MHC-class-II loading I-A^b/Eα−52–68 using the conformational Ab YA-e (Cannizzo et al., 2012).

Cell Purification from Abdominal Fat and Aorta

Abdominal fat and Aorta were harvested, as described above, from B6 or Ob/Ob mice. Tissues were dissociated with the Tissue Dissociation Kits (#130-105-808 and # 130-101-540 respectively, Miltenyi Biotec), as per manufacturer instructions. Single cells were labeled with the CFSE Cell Division Tracker Kit according to the manufacturer suggestions (#423801, Biolegend). Cells were then washed in PBS and incubated with or without the APOB core peptide (2 or 20 ug/mL) for 4 days. Finally, samples were stained with the Zombi NIR Fixable Viability Kit (#423106, Biolegend) and CD4-PerCP (#561090, Clone RM4–5, BD) antibodies, and acquired on a MACSQuant Analyzer 10 (Miltenyi). Data were analyzed with FlowJo ver10.5.3, with CD4⁺ T cell proliferation being assessed as percentage of CFSE^low events over total CD4⁺ T cells.

Lymph node T cell proliferation and CD4 T cell proliferation

Inguinal and axillary lymph nodes from B6 and Ob/Ob mice immunized with the core epitope APOB (YSGSVANEAN), as described above, were collected in sterile Hank’s solution and a single-cell suspension was prepared through a 100-μm cell strainer (Cat.# 22363549 Fisher Brand). Cells were washed twice in complete DMEM (cat# 45000–324, VWR) medium containing [+] 4.5 g/L glucose, [+] L-glutamine, sodium pyruvate, HEPES buffer, 10% Fetal bovine serum, antibiotic (Streptomycin and Penicillin) antimycotic (Ambotericin B) and seeded at 5 × 10⁵ cells/100 μl/well in MICROTEST U-Bottom 96-well polystyrene sterile plates (cat.#353077 Fisher Scientific). Cells were incubated with or without increasing concentrations of the APOB core peptide epitope (5, 20, 40, and 80 μg/ml) in quadruple replicates. Cells were incubated for 3 days at 37°C, supplemented with 5% CO2. Cell proliferation was measured on the fourth day using Click-iT EdU Microplate Assay (Invitrogen^™, cat nr. C10214) following the manufacturer’s instructions. Data were recorded in a microplate reader with the following setting for fluorescence: (excitation/emission 565/585 nm). Data were expressed as mean ± SD of five independent biological replicates. Statistical significance was determined using the Holm-Sidak method, with alpha = 0.05. In other experiments CD4+ T cells were purified from C57BL/6 and Ob/Ob mice (immunized as reported above) using CD4 (L3T4) MicroBeads (Miltenyi Biotec, 130-117-043), and stained with CFSE (Biolegend, 423801). At the same time DCs were isolated from splenocytes with the Pan DC isolation kit (Miltenyi Biotec, 130-100-875). Two x10⁵ cells CD4⁺ T cells and 5 ×10⁴ DCs were co-cultured for 4 days, and stained with 10 ng/mL DAPI (Sigma-Aldrich, D9542) and CD4-APCcy7 (Biolegend, 100526). Samples were acquired using a MACSQuant Analyzer 10 (Miltenyi Biotec) and data analysis was performed with FlowJo (BD).

Cytokines Quantification

Culture supernatant was collected four days later and IFN-γ, IL-1b, IL-10, IL-12p70, Il-17a, IL-6, and TNF-a measured using a ProcartaPlex multiplex immunoassays (Thermo Fisher Scientific) according to the manufacturer’s instructions. The concentration of each cytokine was calculated according to the assay standard curve. Each condition of stimulation was assessed in triplicates.

Flow Cytometry

DCs were incubated for 30 minutes on ice with saturating amounts of Y-Ae-FITC anti-mouse I-A^b/Eα−52–68 (eBioscience, Cat # 11-5741-82) in staining buffer (PBS, 0.1% BSA, 0.01% NaN₃). Following washing in staining buffer, samples were analyzed with the FACScan flow cytometer (Becton Dickinson, N.J, USA).

For measurement of MHC-class-II and I-A^b-CLIP, spleen and nodal cells were surface stained with the following antibodies prior to data acquisition by flow cytometry: CD19-PE (clone 1D3, BD Biosciences #557399), MHCII-AF594 (clone 212.A1), I-A^b-CLIP-AF488 (clone 15G4(Liljedahl et al., 1998), and CD3-AF700 (clone 500A2, eBioscience #56-0033-82). Live cells were also stained with DAPI for dead cell exclusion. For determination of H2-O and H2-M amount, spleen and nodal cells were surface stained with MHCII-AF594 (clone 212.A1 (Landias et al., 1986)), CD3-AF700 (clone 500A2, eBioscience #56-0033-82), and eFluor 506 fixable viability dye (eBioscience # 65-0866-14) for dead cell exclusion prior to fixation and permeabilization (BD Fixation/Permeabilization Solution Kit # 554714) and staining with H2-M-AF488 (clone 23CA (Fallas et al., 2007)) and H2-Ob-AF647 (clone Mags.Ob1 (Fallas et al., 2007)). Data was acquired using a BD LSRII cytometer and analyzed with FlowJo software (BD).

DCs subset analysis

Spleens from B6 or Ob/Ob mice were incubated with collagenase D (200 ug/mL) for 30 minutes to harvest splenocytes. After red blood cell lysis, 2× 10⁶ cells live splenocytes were incubated with a CD16/32 blocker (Biolegend, 101320), and stained with XCR1-Brilliant Violet 510 (Biolegend, 148218), CD172a-Alexa Fluor 488 (SIRPα, Biolegend, 144023), MHC-Class II-PerCP/Cyanine5.5 (I-A/I-E, Biolegend, 107625), CD11c-PE/Cyanine7 (Biolegend, 117318), F4/80-Alexa Fluor® 647 (Biolegend, 123122) for 30 min at 4 C under protection from light. After 2 washes in PBS, samples were stained with 10 microg/mL DAPI, and acquired using a MACSQuant Analyzer 10 (Miltenyi Biotec). Data analysis was performed with FlowJo (BD).

Proteomic Analysis of redox induced posttranslational modifications (PTMs) in mouse DCs

Sample preparation:

Equal protein amounts (50 μg) from total DCs lysates (B6 and Ob/Ob), were reduced in 20 mM TCEP.HCl (Thermo Scientific), 50 mM ammonium bicarbonate buffer, at pH 8.5 containing 8 M urea for 35 min at room temperature. The reduced proteins were alkylated with 100 mM iodoacetamide solution, for 50 min at room temperature in the dark. Three different enzymes were used for “in solution” digestion in 50 mM ammonium bicarbonate buffer, pH 8.5, for 18 h, at 37 °C: endoproteinase Lys-C (1:50 enzyme: protein ratio); trypsin (1:20 enzyme: protein ratio) and Glu-C (1:10 enzyme: protein ratio) (sequencing grade Promega, Madison, WI, USA. Peptides mixture, extracted from all enzymatic digestions, were desalted on C18 Prep clean columns before high resolution liquid chromatography tandem mass spectrometry (LC-MS/MS).

nanoLC MS/MS:

Technical duplicates for each of the biological triplicate samples were analyzed on a Q Exactive HF quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Easy nLC 1000 UHPLC (Thermo Fisher Scientific) through a nanoelectrospray ion source. After equilibration with 5% acetonitrile, 0.1% formic acid, peptides were separated by a 120 min linear gradients from 4% to 30% acetonitrile, 0.1% formic acid at 400nL/min (Optima^™ LC/MS, Fisher Scientific, Pittsburgh, PA). The mass spectrometer was operated in a positive ion mode and in data–dependent acquisition (DDA) mode. Full MS scans were obtained within a 300 to 1600 m/z range with a mass resolution of 120,000 at m/z 200, and a target value of 1.00E+06 with a maximum injection time of 50 ms. HCD collision was performed on the 15 most prominent peaks, and tandem mass spectra were acquired at a mass resolution of 30,000 at m/z 200 and a target value of 1.00E+05. Isolation of precursors was performed with a window of 1.2 Th. The dynamic exclusion time was 20s. The normalized collision energy was 32%. We excluded precursor ions with single, unassigned, or eight and higher charge states from fragmentation selection.

Protein identification:

The raw files from each technical and biological replicate were filtered, de novo sequenced and assigned a protein ID using Peaks 8.5/X software (Bioinformatics Solutions, Waterloo, Canada), by searching against the mouse (Mus musculus) Swiss-Prot database (17,046 entries). The following search parameters were applied for LFQ analysis: trypsin, Lys-C and GluC restriction enzymes and one allowed missed cleaved at one or both peptide end. The parent mass tolerance was set to 15 ppm using monoisotopic mass, and fragment ion mass tolerance was set at 0.05 Da. Carbamidomethyl cysteine (+57.0215 on C) was specified in PEAKS as a fixed modification. Methionine, lysine, proline, arginine, cysteine and asparagine oxidations (+15.99 on CKMNPR), deamidation of asparagine and glutamine (NQ-0.98) and pyro-Glu from glutamine (Q-18.01 N-term) were set as variable modifications.

Post-translational analysis of amide-AGE peptides:

The analysis of amide-AGE focused on the following PTMs on lysine and arginine: Carboxymethylation (CML, CMA) (+58.005; K, R); Carboxyethylation (CEL) (+72.021; K, R); Fructosylation (N-hexosyl lysine) (+162.053; K) Pyrraline (+108.021; K); GLAP (+109.029; K); Glycerinylation (+88.016; K); Acetylation (+42.011; K); Formylation (+27.995; K); Glarg (+39.995; R); MG-H (+54.011; R); G-H1 (+39.990; R); Argpyrimidine (+79.966; R) Tetrahydropyrimidine (3-deoxyglucosone) (+144.042; R); Dihydroxyimidazolidine (CEAMGDHI) (+72.020; R); N-Pentosyl Lysine (+132.0575; K); N-tetrosyl Lysine (+102.0317; K) (as reported in other proteomic AGE analyses. All AGE PTMs were added to the list of other variable PTM described above and included in the workflow for the protein identification using the de novo algorithm provided by PEAKS 8.0 software.

Post-translational analysis of carbonylated peptides:

The following posttranslational modifications were specified in PEAKS 8.0 as variable modifications for carbonylation: Lys->Allysine (−1.03; K); Lys->AminoadipicAcid (+14.96; K); Amino (Y) (+15.01, Y); Trp->Oxolactone (W) (+13.98, W); Pro->Pyrrolidone (P) (−27.99; P); Pro->Pyrrolidinone (P) (−30.01; P); Arg->GluSA (−43.05; R); Dehydrated 4-hydroxynonenal (HNE-Delta:H(2)O(H)) (+138.21, C,H,K); reduced 4-Hydroxynonenal (HNE+Delta:H(2) (H)) (+158.13, C,H,K); HNE (+156.12, C,H,K); 4-Oxononenal (ONE) (4-ONE) (+154.10, C,H,K); dehydrated 4-Oxononenal Michael adduct (4-ONE+Delta:H(−2)O(−1)) (+136.09; C,H,K).

PTMs validation and normalized spectral counts:

Data were validated using the false discovery rate (FDR) method built in PEAKS 8.5/X and protein identifications were accepted if they could be identified with a confidence score (−10lgP)>20 for peptides and (−10lgP)>20 for proteins; a minimum of 1 peptide per protein after data were filtered for less than 1.0% FDR for peptides and less than 1.5 % FDR for proteins identifications. In addition, selected peptides and proteins with 15< (−10lgP) <20 were included in the data set after manual inspection of their MS/MS spectra. An independent validation of the MS/MS-based peptides and protein identification was performed with Scaffold (version Scaffold_4.6.2, Proteome Software Inc.) using the compatible “mzid” files of all samples exported from PEAKS 8.0. The Scaffold built in option “MuDPIT” was used to combine multiple files from biological and/or technical replicates of C57BL6 and Ob/Ob mice. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 1 identified peptide. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Scaffold PTM 3.1.0 and later versions (Proteome Software Inc, Portland Oregon, USA) was used to annotate PTM sites derived from MS/MS sequencing using Scaffold (version 4.7.2 and later versions). Scaffold PTM (Proteome Software, Portland, Oregon, USA) was used to annotate PTM sites derived from MS/MS sequencing results obtained using Scaffold (version Scaffold_4.10.0). Using the site localization algorithm (Beausoleil et al., 2006) Scaffold PTM re-analyzed MS/MS spectra identified as modified peptides and calculated the Ascore values and site localization probabilities to assess the level of confidence in each PTM localization. Scaffold PTM then combined localization probabilities for all peptides containing each identified PTM site to obtain the most accurate estimated probability that a PTM is present at that site. The AGE-amide modified peptides, identified by both PEAKS 8.5/X and Scaffold PTM software, were also manually inspected as previously described (Butterfield et al., 2014; Colombo et al., 2019; Jahouh et al., 2012; Lapolla et al., 2013) using the following criteria: 1) missed cleavage at the modified residue; 2) the accurate mass increase corresponding to AGE modification; 3) presence of unmodified peptide with matching fragments for each modified peptide; 4) presence of fragment ions retaining modification; 5) if modification is at the N terminus, then presence of b-ions retaining modification and unmodified y-ions; 6) if modification is at the middle position, then presence of b- or y-ions retaining modification; 7) presence of at least a few consecutive b- or y-ions; and 8) presence of complementary b- or y-ions. The total number of unique AGE and carbonylation sites were calculated for each patient, treated as an independent biological replicate following MuDPIT of the technical duplicates.

Quantitation of AGE-PTM peptides:

Quantitation of selected AGE-PTM was performed using the PEAKS 8.5 and PEAKS X (Bioinformatics Solutions Inc) using the built-in option for label-free quantitation of PTM based on the MS1 extracted peak areas for each oxPTM site of interest, including the AGE-PTM sites. When available, in each sample, the % abundance PTM for each modified site was calculated by dividing the peak area of modified peptides by the total peak areas corresponding to the (modified + unmodified) peptides followed by multiplication with 100. A similar quantitative analysis was performed in Scaffold PTM where the total modified spectral counts were divided with the total spectral counts for each type of PTM and finally expressed as %PTM for each identified and validated amino acid site.

Gene ontology (GO), molecular and cellular pathways enrichment analysis

Networks functional analyses were generated by employing the ingenuity pathway analysis (IPA; Ingenuity Systems, Redwood City, CA, USA) using the list of identified proteins in the C57BL6 and Ob/Ob mice. For network generation, datasets containing gene identifiers (gene symbols) were uploaded into the IPA application together with their rescaled log2 transformation of protein’s area ratios. The networks were then algorithmically generated based on their connectivity index using the built-in IPA algorithm. The significance was set to a P-value of <0.05.

Late endosomes preparation

Flt3L-induced splenic murine DCs (1–3 × 10⁸), prepared as described above, were pelleted, washed in PBS, and resuspended in PBS containing 0.25 M sucrose and 20 mM HEPES (pH 7.4). Late endosomes were isolated as reported previously (Clement et al., 2016a). Briefly, the DCs were homogenized in a Dounce homogenizer and spun at 150 × g for 10 min. The supernatant was loaded on a 27% Percoll gradient laid over a 2.5 M sucrose cushion and centrifuged for 1 h at 34,000 × g in a SW Ti41 rotor. The band above the sucrose cushion corresponds to the total lysosomal fraction. The band at the interface, enriched in late and early endosomes was further separated on a 10% Percoll gradient by centrifugation at 34,000 × g for 1 h to obtain late endosomes.

In vitro antigen processing of HEL and OVA by late endosomes

The amounts of substrate proteins and late endosomes were titrated to determine the optimal experimental conditions for analyzing the processed peptidome as described previously (Clement et al., 2016a). Native OVA and HEL (3 μg)(Sigma-Aldrich) were incubated with late endosomes purified from control B6 or Ob/Ob mice (1 μg total endosomal protein) in 50 mM sodium phosphate buffer pH 5.5 (with 50mM NaCl and 2mM EDTA) for 6–12 hours at 37°C. The proteolysis reaction was quenched with 0.5% acetonitrile and 5% formic acid. Processed peptides were filtered through a 10-kDa MWCO (molecular weight cut-off) and submitted for nanoLC/MS/MS analysis. The peptidome analysis was performed as described below (analysis of I-A^b eluted peptides).

Isolation of I-A^b peptide complexes from mouse DCs and peptide elution.

MHC-class-II complexes from DCs of B6 and Ob/Ob mice were isolated using immunoaffinity chromatography. Three independent samples of B6 and Ob/Ob were isolated in parallel. Cell pellets were resuspended in 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, containing protease inhibitors and 5% β-octylglucoside, freeze-thawed for 5–6 times, homogenized, and the solubilized whole cell fraction was recovered by centrifugation at 100,000×g for 1 h at 4 °C. The supernatant was used for the isolation of the MHC-class-II-peptide complexes using an immunoaffinity column of M5/114 monoclonal antibody immobilized onto CNBr activated sepharose. The column was equilibrated with buffer (50 mM Tris-HCl, 150 mM NaCl pH 8.0, containing protease inhibitors) for 2 h. The lysates were pre-cleared with the isotype control antibody slowly for 1 h at 4 °C to prevent nonspecific binding of the proteins to the beads. After pre-clearing the lysate was incubated with M5/114 conjugated beads and allowed to mix slowly for 1 h at 4 °C. The column was washed with several buffers in succession as follows: (1) 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, containing protease inhibitors and 5% β-octylglucoside (5 times the bead volume); (2) 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, containing protease inhibitors and 1% β-octylglucoside (10 times the bead volume); (3) 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, containing protease inhibitors (30 times the bead volume); (4) 50 mM Tris-HCl, 300 mM NaCl, pH 8.0, containing protease inhibitors (10 times the bead volume); (5) 1X PBS (30 times the bead volume); and (6) HPLC water (100 times the bead volume). MHC-class-II-peptide concentration was measured by ELISA to calculate the total amounts of I-A^b in each preparation. Briefly, the monoclonal antibody 17/227 (200ng/well), diluted in bicarbonate/carbonate buffer pH 9.0, was used to coat the wells of high binding 96 well plates (Immulon 4 HBX-ND541225). The plates were incubated at 4 °C for O/N or at 37 °C for 2hrs. The wells were blocked using 3% BSA in 1X PBS for 1hr at 37 °C. The monoclonal anti-MHC-Class-II antibody (M5/114) was used as the primary antibody for the detection. HRP-conjugated goat anti-rat IgG (KPL:14-16-12) was used as the secondary antibody followed by the ABTS substrate solution (Roche-11 684 302 001) for the colorimetric detection. Incubations were done at 1hr at 37 °C and the washes between every incubation was performed using 1X PBST buffer (1X PBS, 0.05% triton X-100 three times. The dilutions of protein and antibody was done in dilution buffer 0.3% BSA, 0.1% Triton-X100 in 1X PBS. Recombinant I-A^b-peptide complex was used as standard protein (1 ng to 2ug) to calculate the amounts of I-A^b in each preparation.

Peptides were further separated using a Vydac C4 macrospin column (The Nest Group, USA). I-A^b peptides were eluted using 30% acetonitrile in 0.1% TFA. Eluted peptides were lyophilized using a Speed-Vac.

Analysis of I-A^b eluted peptides.

Peptide extracts were reconstituted in 25 μl 5% acetonitrile containing 0.1% (v/v) trifluoroacetic acid and separated on a nano-ACQUITY (Waters Corporation, Milford, MA) UPLC with technical triplicate injections. In brief, a 3.0 μl injection was loaded in 5% acetonitrile containing 0.1% formic acid at 4.0 μl/min for 4.0 min onto a 100 μm I.D. fused-silica precolumn packed with 2 cm of 5 μm (200Å) Magic C18AQ (Bruker-Michrom, Auburn, CA) and eluted using a gradient at 300 nL/min onto a 75 μm I.D. analytical column packed with 25 cm of 3 μm (100Å) Magic C18AQ particles to a gravity-pulled tip. The solvents were A) water (0.1% formic acid); and B) acetonitrile (0.1% formic acid). A linear gradient was developed from 5% solvent A to 35% solvent B in 90 min. Ions were introduced by positive electrospray ionization via liquid junction into a Q Exactive hybrid mass spectrometer (Thermo Fisher Scientific). Mass spectra were acquired over m/z 300–1750 at 70,000 resolution (m/z-200), and data-dependent acquisition (DDA) selected the top 10 most abundant precursor ions in each scan for tandem mass spectrometry by HCD fragmentation using an isolation width of 1.6 Da, collision energy of 27, and a resolution of 17,500. To determine fractional intensities, the intensity for each of the top 500 peptides in each sample was divided by the total intensity for all 500 peptides.

DIA Analysis of the I-A^b-peptidomes eluted from B6 and Ob/Ob DCs.

DIA data were analyzed using Scaffold DIA (1.2.1) (Proteome Software Inc., Portland) with the raw data files converted to mzML format using ProteoWizard (3.0.11748). To generate the spectral libraries, the acquired DDA raw files were searched with PEAKS 8.5 and then filtered with Scaffold software (version 4.6.2) using the same setting described above for the protein identification in the proteomic samples, except for the enzyme restriction which was set up as “no enzyme” option to fit the endogenously processed peptides. Then, the spectral library was exported as “DDA pDC ctr obob ptm.blib” file using the built-in available option from the Scaffold software. The analytic samples were aligned based on retention times and individually searched against “DDA pDC ctr obob ptm.blib spectral library” with a peptide mass tolerance of 10 to 15 ppm and a fragment mass tolerance of 15 to 50.0 ppm. Variable modifications were imported from the DDA based spectral library which are specified in Supplementary table S1. The “no enzyme” option was used with variable allowed 8–12 missed cleavage site(s). Only peptides with charges in the range [2–8] and length in the range [5–25] were exported for further quantitation. Peptides identified in each sample were filtered by Percolator (3.01. nightly-13–655e4c7-dirty) to achieve a maximum FDR between 0.01–0.05. Individual search results were combined, and peptide identifications were assigned posterior error probabilities and re-filtered to FDR thresholds of 0.01–0.05 by Percolator (3.01. nightly-13–655e4c7-dirty). Peptide quantification was performed by Encyclopedia (0.7.2). For each peptide, the 5 highest quality fragment ions were selected for quantitation. The intensities for the proteins were calculated and normalized by summation of the peptide intensities using the Scaffold DIA’s built-in normalization algorithm. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD023581, 10.6019/PXD023581, PXD018783 and 10.6019/PXD018783.

Absolute peptide quantification using stable isotope-labeled peptides

Two peptides, 1) core epitope YSGSVANEAN and 2) LSQEYSGSVANEAN, which was identified to be an abundant peptide, containing the core epitope (underlined) in the peptidome of Ob/Ob DCs, as mapped by DIA analysis (Suppl. Table S6) were selected for synthesis and absolute quantification studies. Peptides with 13C and 15N labeled amino acids (e.g. V+6Da:13C(5)15N(1) in YSGSV(+6)ANEAN and L+7Da:13C(6)15N(1) in L(+7)SQEYSGSVANEAN were synthesized by Pierce Custom Peptides (Thermo Fisher Scientific, Rockford, IL, USA) and spiked at 10fmole/peptide/injection into immunopeptidomes samples from B6 (normal diet), Ob/OB (normal diet) and B6 (High Fat High Fructose diet) to quantify the chemically-identical but unlabeled (light) endogenous peptides present in the I-A^b-eluted peptidomes. I-A^b-eluted peptides were resuspended in 10 μL of 3% ACN/0.1% formic acid, spiked with 10 fmol of each of the heavy synthetic peptide, and injected in the Thermo Scientific^™ Orbitrap Fusion^™ Tribrid^™ mass spectrometer with PRM method for peptide MS1 and MS/MS analysis. The UltiMate 3000 UHPLC system (Thermo Scientific) and EASY-Spray PepMap RSLC C18 50 cm × 75 μm ID column (Thermo Fisher Scientific) coupled with Orbitrap Fusion (Thermo) were used to separate fractioned peptides with a 5–30% acetonitrile gradient in 0.1% formic acid over 70 min at a flow rate of 250 nL/min. After each gradient, the column was washed with 90% buffer B for 5 min and reequilibrated with 98% buffer A (0.1% formic acid, 100% HPLC-grade water) for 20min. MS data were acquired by combining to scan events corresponding to full scan and a Parallel Reaction Monitoring (PRM) method targeting the ten HLA peptides. A target value for the full scan MS spectra was 1 × 106 ions in the 300–1500 m/z range with a maximum injection time of 60 ms and resolution of 120,000 at 200 m/z with data collected in profile mode. The PRM method was employed at a resolution of 30,000 at 200 m/z, a target AGC value 1 × 106, and maximum fill times of 110 ms. The precursors ions of each targeted peptide were isolated using a 1.7 m/z unit window and fragmented by higher-energy C-trap dissociation with a normalized collision energy of 27 eV. Data analysis was performed using Skyline (version: 20.2.0.286) and the area under the curve of MS1 and selected intense fragment ions summed to determine the quantity of the respective endogenous and reference peptides.

HLA-DR1 and HLA-DM expression and purification.

HLA-DR1 (DRA*01:01/DRB1*01:01) extracellular domains were expressed in Drosophila S2 cells and purified by immunoaffinity chromatography with LB3.1 antibody followed by Superdex200 (GE Healthcare) size exclusion chromatography as described (Sloan et al., 1995). HLA-DM (DMA*01:01/DMB*01:01) extracellular domains were expressed similarly but were purified using a C-terminal FLAG-tag, as described (Busch et al., 1998).

Expression and purification of I-A^b-3R complex

The soluble I-A^b-3R, a mouse MHC-class-II protein contains α and β subunit. The peptide 3R (FEAFMARAKAAV) was engineered at its C-terminus on the beta subunit and expressed from a baculovirus plasmid p3288 I-A^b-BirA. The I-A^b-3R protein complex is about 62kDa molecular weight. The I-A^b-3R was expressed in Hi5 cells in a shake flask to a density of 2 million cells/ml in a mixture of 70% EX-CELL 405 serum free medium for insect cells (Sigma, cat. #14405C) and 30% complete graces medium (Thermo Fisher Scientific, cat. #11605–094) in presence of antimycotic. The cells were further infected using I-A^b-3R virus and incubated for 5 days in a 27 °C at 100 rpm. Post-incubation, the supernatant was collected and filtered through a 0.45μm filter. Protease inhibitors (0.02% NaN₃, 0.7μg/ml pepstatin, 1μg/ml leupeptin and 0.25mM PMSF) were added to supernatant and purified using M5/114 antibody conjugated Sepharose CL-4B column. The eluted purified protein was further purified using Superdex 200 gel filtration column.

Peptide synthesis and labeling

The HA_306–318-derived peptide Ac-PRYVKQNTLRLAT and CLIP peptide Ac-VSKMRMATPLLMQ were synthesized (21st Century Biochemicals, Marlboro, MA) and labeled with Alexa Fluor 488 tetrafluorophenyl ester (Invitrogen, Eugene, OR) through primary amine of K5 (HA) and K3 (CLIP). For this purpose, the peptide (2 mg) was dissolved in 400 μl of sodium bicarbonate (150 mM pH 9.8) and mixed with Alexa488-tetrafluorophenyl ester (1mg) (Molecular Probes). After one-hour incubation at room temperature, labeled peptide was purified by reverse HPLC (Agilent) using a C18 column (Jupiter 300A 00G-4053-E0) and a gradient of acetonitrile in 0.02% trifluoracetic acid.

I-A^b-CLIP peptide exchange assay

A fluorescence polarization (FP) assay was used to measure the IC50 of CLIP (KPVSQMRMATPLLMRPM), M-CLIP 1 (KPVSQM[Oxy]RM[Oxy]ATP[Hyp]LLM[Oxy]RPM[Oxy]) and M-CLIP 2 peptide (KPVSQMRMATPLLM[Oxy]RPM) using N-terminally-acetylated CLIP peptide labeled with Alexa Fluor 488 tetrafluorophenyl ester (Invitrogen, Carlsbad, CA) via the primary amine at K3 as probe peptide as previously described. The binding reactions were carried in buffer conditions of 100 mM sodium citrate, 50 mM sodium chloride, 0.1% octyl β-D-glucopyranoside, 5 mM ethylenediaminetetraacetic acid, 0.1% sodium azide, 0.2 mM iodoacetic acid, 1 mM dithiothreitol). The I-A^b-3R complex has a thrombin linker to cleave off the 3R peptide from I-A^b protein. Thrombin was added during all the reactions at a concentration of 1U/ug and inactivated after 3hrs of reaction using 0.1mM phenylmethanesulfonyl fluoride. The I-A^b-3R concentration used was selected by titrating I-A^b-3R against fixed labeled peptide concentration (25 nM) and choosing the concentration of I-A^b-3R that showed ~50% maximum binding. For calculating IC50 values, 100 nM I-A^b-3R was incubated with 25 nM Alexa488-labeled CLIP probe peptide, in combination with a serial dilution of test peptides, beginning at 10 μM followed by 5-fold dilutions in presence of 0.5μM HLA-DM. The reaction mixture was incubated at 37 °C. The capacity of each test peptide to compete for binding of probe peptide was measured by FP after 72 h at 37 °C. The assay was read using a Victor X5 Multilabel plate reader (PerkinElmer, Shelton, CT). FP values were converted to fraction bound by calculating [(FP_sample - FP_free)/(FP_no_comp - FP_free)], where FP_sample represents the FP value in the presence of test peptide; FP_free represents the value for free Alexa488-conjugated CLIP peptide; and FP_no_comp represents values in the absence of competitor peptide. We plotted fraction bound versus concentration of test peptide and fit the curve to the equation y = 1/(1 +[pep]/IC50), where [pep] is the concentration of test peptide, y is the fraction of probe peptide bound at that concentration of test peptide, and IC50 is the 50% inhibitory concentration of the test peptide.

HLA-DR1 glycation.

For evaluating post-translational modifications induced by glucose, DR1 was incubated at 37 °C with CLIP peptide (Ac-VSKMRMATPLLMQ) for 3 days in the absence or presence of 5 mM or 20 mM glucose, or for 15 days in the presence or absence of 500 mM glucose, in PBS with 0.1% octyl β-D-glucopyranoside. After incubation, the monomeric fraction of DR1-Clip was purified away from detergent, free peptide, glucose, and aggregated protein that formed during the incubation using Superdex 200 (GE Healthcare) size exclusion chromatography in PBS. Aggregation did not differ appreciably for samples incubated in the presence or absence of glucose and represented less than 15% of the total protein. For the samples incubated for 3 days with glucose, we used PBS with matching concentration of glucose present in the DR1-Clip sample. For evaluating post-translational modifications induced by methylglyoxal, DR1-Clip complexes were first purified using Superdex200 in PBS, and then incubated for 24 hours in the absence or presence of methyl glyoxal at the indicated concentrations (0.2, 1, 5 mM) in PBS with 0.1% octyl β-D-glucopyranoside. After incubation the complexes were purified from residual methylglyoxal and detergent using Amicon Ultra centrifical ultrafiltration devices with 10kD cutoff.

DR1 binding with labeled-HA.

Peptide binding was monitored using a fluorescence polarization assay (Yin and Stern, 2014), Purified monomeric DR1Clip was added to a black, polystyrene half well area of a 96 well plate (Corning) with or without DM in binding buffer (100 mM sodium citrate, 50 mM sodium chloride, 0.1% octyl β-D-glucopyranoside, 5 mM ethylenediaminetetraacetic acid, 0.1% sodium azide, 0.1 μg/ml phenylmethanesulfonyl fluoride, 0.2 mM iodoacetic acid, 1 mM dithiothreitol). For the samples incubated for 3 days with glucose, we added matching concentration of glucose with the DR1Clip sample. The plate was sealed and incubated at 37 °C for 3 minutes. Labeled-HA was diluted in binding buffer in a black tube and incubated at 37 °C for 3 minutes. The warmed labeled-HA was added to the warmed DR1Clip and DM to have final 100 nM DR1Clip, 0 or 1 μM DM, and 25 nM labeled-HA in a final volume of 50 μl. The plate was transferred to a Victor X5 Multilabel plate reader (PerkinElmer, Shelton, CT) to measure fluorescence polarization. The DR1Clip complexes incubated with methylglyoxal (0, 0.2, 1, 5 mM) for 24 hours were tested similarly for the DR1 binding with labeled-HA in absence or presence of 1 μM DM. With the mp values, a plot of labeled-HA binding to DR1 vs time was obtained, and a line was fitted to the initial portion of the binding reaction. The slope of the line represents the initial peptide binding rate.

Unsupervised clustering and alignment of MHC-class-II I-A^b eluted peptidome sequences from control and Ob/Ob mice.

Gibbs cluster analysis (www.cbs.dtu.dk) was used to assess whether the I-A^b-binding motif was present in the eluted peptidomes from control and Ob/Ob mice. Since the I-A^b binding motif was reported previously by others (Andreatta et al., 2017; Wan et al., 2020; Zhu et al., 2003) we constrained the P1 position to hydrophobic amino acids and we used the “clustering” algorithm for the sequence weighing. This type of analysis retrieved at least 50% of the eluted peptidome displaying the expected I-A^b binding motif. Sequences lacking the hydrophobic amino acids in P1 were excluded in the final analysis output. IC₅₀ values and core epitopes were predicted using the NetMHCIIpan 3.2 algorithm.

QUANTIFICATION AND STATISTICAL ANALYSYS

Statistical analysis was performed using Windows GraphPad Prism 7.0 (GraphPad Software, La Jolla, California, USA). Numerical results are reported as mean +/− SE or +/−SDV as indicated. Data are derived from a minimum of three independent experiments unless stated otherwise. Statistical significance of the difference between experimental groups, in instances of multiple means comparisons, was determined using one-way ANOVA, followed by the Bonferroni post hoc test. In addition, two-tailed unpaired 1-way ANOVA and two-tailed t-test were performed for immunoblot, ELISA and LFQ analyses. A “p” value less than 0.05 was considered significant. For multiple t-tests comparison analysis, such as quantitative ELISA for cytokines measurements, the statistical significance was determined using the Holm-Sidak method, with alpha = 0.05. Each row was analyzed individually, without assuming a consistent SD. An independent validation was performed using the multiple t-tests comparison and two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 1%. The adjusted p values were reported for each data set.

Immunoblots

All immunoblots were run in a minimum of biological triplicates and densitometric analysis, performed on each membrane. Data are reported as bar graphs (mean and SE) of three or more independent membranes. Data were analyzed by one-way ANOVA and Dunnett’s multiple comparisons test,

T cell proliferation

OVA, HEL, and APOB proliferation assays. All assays were run in biological triplicates (OVA, HEL) or quintuplicates (APOB). Statistical differences in proliferation values were calculated using an unpaired two-tailed t-test.

CD4⁺ T cells proliferation in adipose and aortic tissues

Proliferating CD4⁺ T cells were analyzed in biological triplicates. Statistical differences in proliferation values were calculated using an unpaired two-tailed t-test.

Ki67⁺ cells in liver tissue

Proliferating Ki67⁺ cells were analyzed in biological triplicates. Statistical differences in proliferation values were calculated using an unpaired two-tailed t-test.

CLIP staining

Cells were stained in biological quadruplicates. Statistical differences in mean fluorescence index values were calculated using an unpaired two-tailed t-test.

Cytokines

Cytokines were quantified in biological triplicates. Statistical differences in cytokines values were calculated using an unpaired two-tailed t-test and multiple t-tests comparison analysis and the statistical significance was determined using the Holm-Sidak method, with alpha = 0.05. An independent validation was performed using the multiple t-tests comparison and two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 1%. The adjusted p values were reported for each data set.

IPA analysis

The probability of having a relationship between each IPA indexed biological function and the experimentally determined protein was calculated by right-tailed Fisher’s exact test with the Benjamini-Hochberg Correction. The statistical significance was set to a p-value of <0.05.

HLA-DR1/DM peptide editing

Statistical differences in binding values were calculated using an unpaired two-tailed t-test.

Peptidome analysis

Statistical differences among the eluted peptides (total number, I-A^b predicted affinity) were calculated by two tailed unpaired t test.

Supplementary Material

1

Supplement Table S1: Proteomic Analysis of DCs from B6 and Ob/Ob mice: Related to Figure 1

2

Supplement Table S2: OVA and HEL peptides generated by B6 and Ob/Ob Endosomal Processing: Related to Figure 2

3

Supplement Table S3: Oxidative-PTMs mapped on I-Ab: Related to Figure 4

4

Supplement Table S4: Oxidative-PTMs mapped on HLA-DR1: Related to Figure 5 and Figure S4

5

Supplement Table S5: I-Ab-eluted Immunopeptidome from B6 and Ob/Ob mice: Related to Figure 5

6

Supplement Table S6: Apo-B I-Ab-eluted Immunopeptidome: Related to Figure 5 and 6

7

Supplement Table S7: PRM Absolute Quantification of ApoB100 Peptides: Related to Figure 6

Key Resources Table

Reagent	Source	Identifier
Bovine Albumin Solution	Sigma-Aldrich	Catalog # A9576
RIPA buffer	Sigma-Aldrich	Catalog # R0278
Protease inhibitor cocktail	Roche	Catalog # 04693116001
4–15% SDS-PAGE (Mini-PROTEAN® TGX™) Gels	BioRad, CA	Catalog # 4561084
Oxyblot Protein Oxidation Detection Kit	Millipore	Catalog # S715
OxiSelect™ Protein Carbonyl Immunoblot Kit	Cell Biolabs, Inc.	Catalog # STA-308
Braford protein assay kit	BioRad, CA	Catalog # 500006
Micro BCA protein assay kit	ThermoFisher Scientific	Catalog # 23235
Cell Proliferation ELISA BrdU (colorimetric) assay	Roche	Catalog # 11647229001
Cell Proliferation Click-iT EdU microplate assay	Invitrogen	Catalog # C10214
Braford protein assay kit	BioRad, CA	Catalog # 500006
Tissue Dissociation Kit Adipose Tissue	Miltenyi Biotec	Catalog #130-105-808
Tissue Dissociation Kit Aorta	Miltenyi Biotec	Catalog # 130-101-540
CFSE Cell Division Tracker Kit	Biolegend	Catalog #423801
Zombi NIR Fixable Viability Kit	Biolegend	Catalog # 423106
DMEM	VWR	Catalog # 45000–324
Streptomycin, Penicillin and l-glutamine	Gibco	Catalog # 10376–016
Antimycotic (Ambotericin B)	Gibco	Catalog # 15290–026
Sodium Pyruvate	Gibco	Catalog # 11360–070
HEPES Buffer	Fisher Scientific	Catalog # BP299–100
Fetal Bovine Serun	Sigma-Aldrich	F0926
CFSE	Biolegend	Catalog # 423801
DAPI	Millipore Sigma	Catalog # D9542
eFluor 506 fixable viability dye	eBioscience	Catalog # 65-0866-14
Fixation/Permeabilization Solution Kit	BD Pharmingen	Catalog # 554714
Collagenase D	Roche	Catalog # 11088858001
XCR1-Brilliant Violet 510	Biolegend	Catalog # 148218
Endoproteinase Lys-C	Promega	Cat.# VA1170
Trypsin	Promega	Catalog # V5111
Glu-C	Promega	Catalog # V1651
Acetonitrile Optima™ LC/MS	Fisher Scientific	Catalog # A955-4
Formic Acid Optima™ LC/MS	Fisher Scientific	Catalog # A11710X1-AMP
Percoll	Millipore Sigma	Catalog # P1644-100ML
OVA (albumin from chicken egg white)	Millipore Sigma	Catalog # A5503-1G
HEL (lysozyme from chicken egg white)	Millipore Sigma	Catalog # L6876-1G
NaCl	Millipore Sigma	Catalog # S7653-1KG
EDTA	Millipore Sigma	Catalog # EDS-500G
Tris-Hcl	Fisher Scientific	Catalog # BP153-500
β-octylglucoside	Sigma-Aldrich	Catalog # O8001
CNBr activated sepharose	GE Healthcare	Catalog # 17-0981-01
ABTS substrate solution	Roche	Catalog # 11-684 302 001
Triton X 100	Fisher Scientific	Catalog # BP151-500
Trifluoroacetic acid	Sigma-Aldrich	Catalog # T6508
APOB Peptides	ThermoFisher Scientific	Custom
HPLC-grade water	ThermoFisher Scientific	Catalog # TS-51140
Pepstatin	Millipore Sigma	Catalog # 10253286001
Leupeptin	Millipore Sigma 21st Century Biochemicals,	Catalog # L2884-1 MG
HA CLIP Peptides	Marlboro, MA	Custom
Alexa Fluor 488 tetrafluorophenyl ester	ThermoFisher Scientific	Catalog # A37570
Sodium citrate	Millipore Sigma	Catalog # W302600-1KG-K
Sodium chloride	Millipore Sigma	Catalog # S7653-250G
Phenylmethanesulfonyl fluoride	Millipore Sigma	Catalog # P7626-1G
Methylglyoxal	Millipore Sigma	Catalog # M0252-25ML
Octyl β-D-glucopyranoside	Millipore Sigma	Catalog # O8001
Sodium azide	Millipore Sigma	Catalog # S2002-5G
Dithiothreitol (DTT)	Millipore Sigma	Catalog # 10197777001
Iodoacetamide	Millipore Sigma	Catalog # I1149-5G
TCEP, Hydrochloride, Reagent Grade	Millipore Sigma	Catalog # 580567
Antibodies
Polyclonal goat-anti AGE	Millipore	Catalog # AB9890
Donkey anti goat secondary antibody	Irdye 800CW	Catalog # 925-32214
CD4-PerCP (	BD Pharmingen	Catalog #561090
CD4 (L3T4) MicroBeads	Miltenyi Biotec	Catalog # 130-117-043
Pan DC isolation kit	Miltenyi Biotec	Catalog # 130-100-875
CD4-APCcy7	Biolegend	Catalog # 100526
Y-Ae-FITC anti-mouse I-Ab/Eα-52-68	eBioscience	Catalog # 11-5741-82
CD19-PE	BD Pharmingen	Catalog # 557399
MHC-class II M5/114	ThermoFisher Scientific	Catalog # 17-5321-82
CD3-AF700	BD Pharmingen	Catalog # 56-0033-82
I-Ab-CLIP	Santa Cruz	Catalog # sc-53946
H2-M	Denzin Lab	Clone YoDMA.1
H2-Ob	Denzin Lab	Clone Ob.1
CD16/32 blocker	Biolegend	Catalog # 101320
CD172a-Alexa Fluor 488	Biolegend	Catalog # 144023
MHC-Class II-PerCP/Cyanine5.5	Biolegend	Catalog # 107625
CD11c-PE/Cyanine7	Biolegend	Catalog # 117318
F4/80-Alexa Fluor® 647	Biolegend	Catalog # 123122
Software
Scaffold Q+S (version 4.6.2,)	Scaffold Q+S (proteomesoftware.com)
Scaffold PTM 3.1.0 and later versions	Scaffold PTM (proteomesoftware.com)
PEAKS 8.5 and PEAKS X	www.bioinfor.com/peaks-software/
Scaffold DIA (1.2.1)	Scaffold DIA (proteomesoftware.com)
Gibbs cluster analysis	www.cbs.dtu.dk
Windows GraphPad Prism 7.0–9.0	www.graphpad.com/scientific-software/prism/
Deposited Data
Supplement Table S1 & S3	PXD024239, 10.6019/PXD024239
Supplement Table S5	PXD023581, 10.6019/PXD023581, PXD018783, 10.6019/PXD018783
ProteomeXchange Consortium via the PRIDE partner repository (http://www.proteomexchange.org/)

Highlights.

• Oxidative post-translational modifications (PTMs) are observed in T2D subjects

• PTMs were mapped to the MHC-class-II antigen processing and presentation machinery

• PTMs affected epitope-specific antigen processing and MHC-class-II presentation

• Increased presentation of an APO-B peptide associated with clinical complications of T2D