Comprehensive Immunopeptidome Analysis of I-Ab-bound peptides from Thymus and Splenic B cells in C57BL/6 mice

Abstract

Abstract Antigen presentation in the thymus is central to selection of CD4 and CD8 T cells. Despite postulated roles for differential central and peripheral antigen presentation pathways for MHC-I and MHC-II, comprehensive immunopeptidome analysis of the thymus has not been reported. Advances in mass spectrometry (MS) technology now allow analysis of the MHC-presented peptidomes of tissue samples containing small numbers of antigen-presenting cells. To map the immunopeptidome in the thymus of C57/BL6 mice presented by I-Ab, we used immunoaffinity coupled to MS. We identified >1000 peptides using a conventional data-dependent acquisition strategy (DDA). We compared the immunopeptidome of thymus to that of the splenic B cells. Some of the core epitopes were unique to thymus, and some were unique to splenic B cells, although most were shared. We observed differences in the general characteristics of the peptidomes presented by thymus and splenic B cells, including length distribution and hydrophobicity. To help understand the impact of the hydrophobicity differences, we selected 57 peptides representative of thymic-derived, splenic B-derived, or shared peptidomes, and studied their binding affinity to IAb. Results suggest that the pool of peptides presented by I-Ab in thymus has lower MHC binding than does the pool presented by splenic B cells. We are exploring a data-independent acquisition strategy (DIA) for quantitative comparison of peptides identified in thymus and splenic B cells to further characterize antigen presentation differences in thymus and periphery.