Dataset Information

Characterization of I-Ab MHCII immunopeptidome eluted from control and obese (Ob/Ob) mice using a combination of data-dependent (DDA) and data-independent acquisition (DIA) nanoLC mass spectrometry

ABSTRACT: In Dendritic cells (DC), the MHC II eluted immunopeptidome reflects the antigenic composition of the microenvironment. Proteins are transported and processed into peptides in endosomal MHC II compartments through autophagy or phagocytosis; extracellular peptides can also directly bind MHC II proteins at the cell surface. Altogether, these mechanisms allow DC to sample both the intra and extracellular environment. With an increase in mass spectrometry sensitivity and accuracy, we can now finally tackle important questions on the nature and plasticity of the MHC-II immunopeptidome in health and disease. Presented epitopes, neoepitopes, and PTM-modified epitopes can be quantitatively and qualitatively analyzed to provide a comprehensive picture of DC role in immunosurveillance. To determine whether the redox metabolic conditions induce an altered spectrum of presented peptides, we eluted immunoaffinity-purified I-Ab from conventional dendritic cells isolated from control B6 or obese Ob/Ob mice, and analyzed MHC-II-associated peptides by LC/MS/MS using combined data-dependent (DDA) and data-independent acquisition (DIA) approaches. We analyzed the 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, thus allowing a direct label-free quantitation of relative abundances between the two sample categories. The quantitative analysis of the I-Ab-eluted immunopeptidomes pinpoint important differences in peptide presentation and epitope selection in obese mice.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Dendritic Cell

DISEASE(S): Disease Of Metabolism

SUBMITTER: Cristina Clement

LAB HEAD: Laura Santambrogio and Lawrence J.Stern

PROVIDER: PXD023581 | Pride | 2021-03-21

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	CristinaClement_Fusion_Spleen_pDC_Control_BL6_DDA_Rep1.mgf	Mgf
	CristinaClement_Fusion_Spleen_pDC_Control_BL6_DDA_Rep1.mzxml	Mzxml
	CristinaClement_Fusion_Spleen_pDC_Control_BL6_DDA_Rep1.raw	Raw
	CristinaClement_Fusion_Spleen_pDC_Control_BL6_DDA_Rep2.mgf	Mgf
	CristinaClement_Fusion_Spleen_pDC_Control_BL6_DDA_Rep2.mzxml	Mzxml

Items per page:

1 - 5 of 37

Publications

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

Clement Cristina C CC Nanaware Padma P PP Yamazaki Takahiro T Negroni Maria Pia MP Ramesh Karthik K Morozova Kateryna K Thangaswamy Sangeetha S Graves Austin A Kim Hei Jung HJ Li Tsai Wanxia TW Vigano' Marco M Soni Rajesh K RK Gadina Massimo M Tse Harley Y HY Galluzzi Lorenzo L Roche Paul A PA Denzin Lisa K LK Stern Lawrence J LJ Santambrogio Laura L

Immunity 20210315 4

Hyperglycemia and hyperlipidemia are often observed in individuals 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 under conditions of hyperglycem ...[more]

PMID: 33725478

Similar Datasets

Project description: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 (so-called Ob/Ob mice, a common model of obesity and T2D) and mice subjected to a high fat diet (HFD). It has been already shown that the circulating levels of glycated or glycoxidated proteins (e.g., glycated hemoglobin or albumin) are commonly employed as a reliable indicator of overall glycemic status. Albeit these initial non-enzymatic reactions are reversible, following a series of chemical rearrangements protein glycation and glycoxidation become irreversible, generating the so-called advanced glycation end-products (AGEs). AGEs are commonly detected in tissue proteins with an extended half-life, including dermal collagens as well as extracellular matrix proteins. In these proteins, Nϵ-carboxymethyllysine (CML), pentosidins, and glucosepane represent the most prevalent AGEs. As an additional, novel mechanism linking protein PTMs to altered immunity, the research presented herein highlights that several components of the MHC class II antigen processing and presentation machinery are glycated in metabolic conditions associated with increased oxidative stress, leading to qualitative and quantitative changes in the MHC class II immunopeptidome. To investigate the impact of T2D and metabolic syndrome on the proteome of antigen presenting cells (APCs) we isolated dendritic cells (DCs, which are key for the initiation of antigen-specific immunity) from the lymph nodes of Ob/Ob mice and syngeneic, age-matched control C57BL/6 (B6) mice and mapped the oxidized proteins at the molecular and cellular level by employing label-free mass spectrometry using at least three biologically independent whole lysates of DCs. Analysis of the type of post-translational modifications (PTMs) accumulated in the proteome from the combined three biological Ob/Ob samples ranked the formyl-lysine and site-specific carboxymethylation on lysine (CML) as the most abundant AGEs found in the glycated proteome; with CML having about two-fold increase in their total PTM-modified spectral counts in the Ob/Ob vs control dendritic cell proteome. Additional glycoxidation-specific PTMs, that mapped only on the Ob/Ob proteome, albeit at a smaller amount than CML and formyl lysine, were glyceryl lysine and 3-deoxyglucosone. Finally, a separate proteomic analysis, performed on gradient purified late endosomes also mapped an increased number of PTM-modified proteins in the Ob/Ob organelles. Ingenuity pathway analysis (IPA) analysis 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. control mice. The site-specific amide-AGE modifications that we detected also mapped to proteins involved in response to DC signaling, immune cell trafficking, actin and cytoskeleton signaling, cell movement, and carbohydrate, nucleic acids and protein metabolism. Overall, these findings indicate that the DC proteome of Ob/Ob mice has an increased number of carbonyl PTMs and AGEs as compared to DC of C57BL/6 mice. The results presented herein are one of the first to map the redox mediated PTM in the primary mouse DC in healthy vs T2DM type syndrome physiological conditions.

Project description:Purpose: Production of broad-spectrum antibodies against T cell-dependent antigens requires marginal zone (MZ) B cells to present antigenic peptides complexed to MHC II. Here we describe the trogocytic acquisition of these complexes from dendritic cells (DCs). Complement C3 (C3) binds to MHC II on murine and human DCs. Recognition of C3 by MZ B cell complement receptor 2 triggers trogocytosis of DC plasma membrane and its receptors, which become exposed on the MZ B cells. Excessive trogocytosis is deleterious but prevented by membrane ubiquitin ligase MARCH1, which limits the number of MHC II–C3 complexes displayed on the DC surface. MHC II molecules are thus receptors for constitutively activated and potentially harmful C3, an activity that enables acquisition of DC-like functions by trogocytic MZ B cells. Methods: mRNA profiles of splenic cDC1s, B cells and CD11cintCD24+CD8int cells of wild type (WT) and March1-/- mice mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. All 100-base-pair single-end reads were mapped to the reference mouse genome (GRCm38/mm10) using STAR aligner, and gene-wise counts were obtained using Subread package. Differential expression analysis of RNA sequencingdata was carried out using the edgeR and limma-voom packages in Bioconductor. Results: Using an optimized data analysis workflow, we mapped about 14 million sequence reads per sample to the mouse genome (build mm10) and identified 16,182 genes in the different cell types from WT and March1−/− mice.Transcriptomic profiling revealed over 6500 genes differentially expressed between cDC1s and B cells. Principle component analyses revealed very few differences between WT and March1−/− cDC1s, and between WT and March1−/− B cells. Notably, the transcriptome of CD11cintCD24+CD8int cells was similar to those of WT or March1−/− B cells, indicating they belonged to the B cell lineage. Conclusions: In our study we have described that MHC II ubiquitination by MARCH1 plays two important roles: first, to enhance the removal of surface MHC II–C3 complexes on all APCs; and second, to limit MZ B cell trogocytosis and elimination of cDCs. It is conceivable these two functions, more than the regulation of MHC II antigen presentation, have been the major drivers for the conservation of MHC II ubiquitination through evolution.

Project description:In Dendritic cells (DC), the MHC II eluted immunopeptidome reflects the antigenic composition of the microenvironment. Proteins are transported and processed into peptides in endosomal MHC II compartments through autophagy or phagocytosis; extracellular peptides can also directly bind MHC II proteins at the cell surface. Altogether, these mechanisms allow DC to sample both the intra and extracellular environment. To understand the contribution of the lymph proteome to the MHC-II immunopeptidome we eluted I-Ab complexes from DCs harvested from the deep cervical or mesenteric nodes and investigated whether the I-Ab presented peptidome reflects the anatomical distribution of the proteomes carried by the lymph collected from the same anatomical districts. Heatmap representation and cluster analysis indicated differences among the two immunopeptidome. Similarly, regression analysis showed a much higher regression coefficient among MHC II immunopeptidomes eluted from the same anatomical district (r=0.699) as compared to the one eluted from the two different anatomical districts (deep cervical, average r = 0.877 and mesenteric, average r = 0.937). Overall, all analyzed peptides displayed the expected I-Ab binding motives, binding affinity and expected range of peptide length. A combination of data dependent (DDA) and data independent (DIA) analysis indicated that around 36% of the eluted peptides were shared by both the cervical and mesenteric lymph nodes. The rest of the eluted peptides were distinct to each lymph node reflecting the qualitatively different proteome associated with each of the two anatomical districts: peptides unique to the cervical lymph nodes displayed many proteins known to be enriched in brain tissue, whereas those unique to mesenteric lymph nodes were enriched in mesenteric organ proteins. As such, the quantitative analysis of the I-Ab-eluted immunopeptidomes pinpoint important differences in peptide presentation and epitope selection in distinct anatomical districts.