Differential of I-A b Immunopeptidome of Thymus, Splenic B cells, and Splenic DCs from 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, comprehensive immunopeptidome analysis of the thymus has not been reported for either MHC I or MHC II. To map the immunopeptidome in the thymus of C57BL/6 mice presented by IAb, we used immunoaffinity coupled to mass spectrometry. We identified >1000 peptides using data dependent acquisition strategy. One challenge in thymic peptidome analysis is the possibility of co-eluting non-specifically bound peptides present as a result of the low abundance of MHC II antigen presenting cells (<10% of total thymic cells). We used several approaches to reduce non-specific background peptides, including analyzing elutions from isotype control antibody conjugated beads, considering only peptides present in nested sets and using predicted binding motif analysis to identify core epitopes. We compared the immunopeptidome of thymus to those of the more easily characterized splenic B cells and splenic DCs. Some of the core epitopes were unique to thymus, splenic B cells or splenic DCs, although most were shared. We observed a broader length distribution of thymic peptidome as compared to splenic B cell and DC peptidomes. To help understand these differences, we selected for detailed analysis 57 peptides representative of thymic derived, splenic B derived or shared peptidomes and studied their binding affinity to IAb. The results suggested that the pool of peptides presented by IAb in thymus has lower MHC affinity than does the pool presented by splenic B cells. Thus our data support the idea of differential antigen processing and presentation in central versus peripheral tissues.