Approximately 32% of the population in the U.S. have metabolic syndrome, and about 10% have type 2 diabetes (T2D). In these hyperglycemic conditions proteomic modifications associated with glycation and glycoxidation are often observed. In this application we propose to analyze the role played by glycation and carbonylation on the MHC II antigen processing and presentation machinery and its implication for T cell immunity. Prompted by the observation that MHC II proteins, as well as processed peptides, are modified by advanced glycation endpoint (AGE) and carbonyl groups in patients with T2D, we aim to systematically dissect the role played by these moieties on antigen processing, MHC II-peptide binding, DM editing, and T cell presentation. In a step-by-step approach we will address how the antigen processing machinery is affected by glycation/carbonylation post-translational modifications (PTMs) and how this, in turn, affects T cell immunity. In this application we propose to: (i) map glycation and carbonylation on MHC II Molecules in T2D patients, Ob/Ob mice and relevant controls, using state-of- the-art tandem mass spectrometry, (ii) determine the effect of these PTMs on endosomal antigen processing using quantitative MS/MS and hot spot analysis, (iii) analyze the MHC-II immunopeptidomes in T2D patients, Ob/Ob mice and relevant controls to determine how the dysmetabolic environment can affect peptide selection, epitopes copy number and peptides PTM- modifications, (iv) analyze the MHC-II immunopeptidomes in Ob/Ob mice and relevant controls, prior to and after infection with S. Typhimurium, to determine how the dysmetabolic environment can affect MHC II restricted pathogen immunity, (v) characterize immune responses to novel PTM-modified autoantigens by CD4 T cell using tetramer staining following immunization with relevant epitopes, and T cell characterization (surface phenotype, proliferation and cytokine production following antigen stimulation). Ultimately our analysis will provide a mechanistic analysis of how T2D impacts MHC II- restricted immune responses and its consequences on immunity.
MHC II proteins bind peptides and present them to T cells as part of the system by which the immune system surveys the body for infection or malignancy. The major long term goal of our study is to determine how the set of MHC II-presented peptides changes during pathological conditions such as Diabetes and Metabolic Syndrome.
