Graphical Abstract graphic file with name fx1.jpg [41]Open in a new tab Highlights * • Obesity reshapes the landscape of VAT-derived MIP * • The obese VAT-exclusive MIP reflects an obesity-associated signature * • An obese VAT-exclusive peptide LDHA[237-244] can stimulate CD8^+ T cell responses * • LDHA[237-244]-reactive CD8^+ T cells were present in obese mice but not lean mice __________________________________________________________________ Diabetology; Immunology; Proteomics Introduction The prevalence of obesity and its associated metabolic abnormalities, including insulin resistance, type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD), and non-alcoholic fatty liver disease (NAFLD), has attained epidemic proportions, severely affecting health and global mortality rates. Obesity-induced visceral adipose tissue (VAT) chronic inflammation is now considered to portray a pivotal role in the development of metabolic diseases. Increasing data indicate alterations in multiple immune cells in obese VAT, including alterations in macrophages, mast cells, eosinophils, as well as in adaptive immune cells ([42]Sun et al., 2012). T lymphocytes have been regarded as unexpected contributors to obesity-induced VAT inflammation and insulin resistance ([43]Wu et al., 2007). The inflammatory reaction within obese VAT has been characterized by a striking influx of CD8^+ T cells ([44]Rausch et al., 2008). Similarly, the ratio of CD8^+ to CD4^+ T cells increased in obese VAT weeks before macrophage infiltration ([45]Feuerer et al., 2009, [46]Winer et al., 2009). The infiltration of significant number of CD8^+ T cells preceded macrophage recruitment in obese VAT of mice after short-term feeding of a high-fat diet (HFD). Depletion of CD8^+ T cells lowered macrophage infiltration and adipose tissue inflammation, and ameliorated systemic insulin resistance, whereas adoptive transfer of CD8^+ T cells into CD8-deficient mice reverted these effects and increased the numbers of pro-inflammatory macrophages in VAT ([47]Nishimura et al., 2009). These evidences indicate that CD8^+ T cells may portray a significant role in the initiation of obesity-induced VAT inflammation. Obese VAT-infiltrated T cells have a restricted TCR-Vβ repertoire, suggesting that expansion of these T cells in progressive obesity is possibly driven by VAT-specific antigens as a result of obesity ([48]McDonnell et al., 2018, [49]Nishimura et al., 2009, [50]Yang et al., 2010). It has been reported that obese adipose tissue possesses the ability to activate CD8^+ T cells, whereas lean fat does not ([51]Nishimura et al., 2009). However, the antigenic mechanisms underlying the activation, proliferation, and pro-inflammatory responses of CD8^+ T cells in obese VAT remain unknown. Numerous peptides presented on the cell surface of major histocompatibility complex (MHC) class I molecules are collectively referred to as the MHC class I-associated immunopeptidome (MIP) interacting with adaptive CD8^+ T cells ([52]Granados et al., 2015). Recent studies have adopted large-scale mass spectrometry (MS) as the sole direct approach for analyzing the global composition of MIP. The molecular composition of MIP is complex and varies from one cell/tissue type to another; it is intertwined with protein metabolism and is ultimately shaped by two processes: protein translation and degradation ([53]Adamopoulou et al., 2013, [54]de Verteuil et al., 2010). Under certain pathological conditions, selected intrinsic and extrinsic cellular factors, including neoplastic transformation, infection, as well as metabolic perturbations, can restructure self-MIPs, possibly resulting in the generation of immunogenic peptides ([55]de Verteuil et al., 2012). Malignant transformation has a profound impact on the MIPs. Numerous tumor-associated MIPs have been described to encompass immunogenic peptides that are recognized by CD8^+ T cells ([56]Bassani-Sternberg et al., 2016, [57]Boon et al., 2006, [58]Dutoit et al., 2012, [59]Kowalewski et al., 2015, [60]Loffler et al., 2018). Alteration of cellular metabolism via the inhibition of the mammalian target of rapamycin results in dynamic alterations in the cell surface MIP landscape as well as generation of immunogenic peptides ([61]Caron et al., 2011). Additionally, inflammatory cytokines induce the alteration of human β cell MIPs and yield conventional and neo-antigenic peptides recognized by CD8^+ T cells in type 1 diabetes and healthy donors ([62]Gonzalez-Duque et al., 2018). However, it remains to be determined whether obesity reshapes VAT-derived MIP and generates immunogenic peptides for driving CD8^+ T cell responses. We performed large-scale high-resolution MS to analyze VAT-derived MIPs isolated from lean and obese mice fed a normal chow diet (NCD) and HFD, respectively, and observed that HFD-induced obesity led to significant alterations in the VAT MIP landscape. Moreover, abnormal adipocyte metabolism under obese conditions generated obese VAT-exclusive immunogenic peptides that elicited the pro-inflammatory responses of CD8^+ T cells, representing a mechanism underlying the obesity-induced VAT inflammation. Results Characterization of MHC Class I-Associated Immunopeptidome Derived from the Visceral Adipose Tissue of NCD-Fed (Lean) and HFD-Fed (Obese) Mice First, the HFD-induced obese mouse model was well established. HFD-fed obese mice exhibited significantly increased weight and fat mass, along with impaired glucose tolerance and insulin sensitivity ([63]Figures S1A–S1E) at 8 weeks post HFD administration. The epididymal fat pads (VAT samples) freshly harvested from HFD-fed obese mice and NCD-fed lean mice, respectively, were lysed to immunopurify H2-Kb-peptide complexes. Although the mRNA expression level of H2-Kb was lower in obese VAT from HFD-fed mice compared with that from NCD-fed mice, there was no significant difference in H2-Kb protein expression levels in VAT between lean and obese mice ([64]Figures S1F and S1G). The quantity of H2-Kb protein purified from obese VAT was marginally lower than that obtained from lean VAT ([65]Figure S1G). H2-Kb-bound peptides were acid-eluted from the H2-Kb molecules and analyzed using reverse-phase HPLC-tandem mass spectrometry (LC-MS/MS) ([66]Figure 1A). By combining the Mascot and Sequest search results from three technical repetitions, we identified 913 unique peptides from NCD VAT and 404 unique peptides from HFD VAT, which met the following criteria: FDR<5%, either Ionscore >20 in Mascot search or q values < 0.05 in Sequest search, and 8–12 amino acids in length. When applying a more stringent filter using the predicted H2-Kb binding affinity with IC[50] <500 nM (netMHCpan4.0), we eventually discovered 324 ([67]Table S1. The identified unique high confidence H2-Kb-bound peptides from visceral adipose tissues of NCD-fed mice, related to [68]Figure 1) and 171 ([69]Table S2. The identified unique high confidence H2-Kb-bound peptides from visceral adipose tissues of HFD-fed mice, related to [70]Figure 1) unique high confidence H2-Kb-bound peptides from NCD and HFD VAT, respectively. The two MIP datasets indicated the characteristic length distribution for H2-Kb-restricted peptides, predominantly 8 amino acids in length, with a small number of nonameric peptides and very few of 10- to 11-mer peptides. Notably, the proportion of octapeptides in HFD VAT MIP was higher than that in NCD VAT MIP ([71]Figure 1B). Most of these MIPs (>80%) were predicted to elicit strong H2-Kb binding affinity with IC[50] < 100 nM ([72]Figure 1C). Similarly, both octapeptides and nonapeptides from the two MIPs exhibited the typical anchor motifs (P5 and P8/9) for H2-Kb molecules binding ([73]Figure 1D). The results confirm the predominant presence of tyrosine and phenylalanine in position P5 and that of leucine, valine, and methionine in the C-terminal positions of peptides. No significant alterations were noted in the amino acid preferences within the MIPs purified from NCD- and