Abstract
Antigen presentation by MHC-II proteins in the thymus is central to selection of CD4 T cells, but analysis of the full repertoire of presented peptides responsible for positive and negative selection is complicated by the low abundance of antigen presenting cells. A key challenge in analysis of limiting abundance immunopeptidomes by mass spectrometry is distinguishing true MHC-binding peptides from co-eluting non-specifically bound peptides present in the mixture eluted from immunoaffinity-purified MHC molecules. Herein we tested several approaches to minimize the impact of non-specific background peptides, including analyzing eluates from isotype-control antibody-conjugated beads, considering only peptides present in nested sets, and using predicted binding motif analysis to identify core epitopes. We evaluated these methods using well-understood human cell line samples, and then applied them to analysis of the I-Ab presented immunopeptidome of the thymus of C57BL/6 mice, comparing this to the more easily characterized splenic B cell and dendritic cell populations. We identified a total of 3473 unique peptides eluted from the various tissues, using a data dependent acquisition strategy with a false-discovery rate of <1%. The immunopeptidomes presented in thymus as compared to splenic B cells and DCs identified shared and tissue-specific epitopes. A broader length distribution was observed for peptides presented in the thymus as compared to splenic B cells or DCs. Detailed analysis of 61 differentially presented peptides indicated a wider distribution of I-Ab binding affinities in thymus as compared to splenic B cells. These results suggest different constraints on antigen processing and presentation pathways in central versus peripheral tissues.
Highlights
MHC-II antigen presentation pathways have been explored mostly in professional antigen-presenting cells (APC) such as B cells and dendritic cells, because of their important role in generating and regulating immune responses
This was somewhat surprising given the idea that efficient T cell negative selection against all possible self-antigens depends on antigen presentation in the thymus of peptides derived from tissue-specific gene products, many of which would not be expected to be expressed in the spleen
We explored several methods to mitigate the effects of the low abundance of MHC-II-expressing cells in the thymus, including characterization of peptides captured by non-specific immunoaffinity and control resins, enrichment of membrane and vesicular compartments prior to isolation, and consideration of MHC-II length preferences and antigen presentation pathways leading to nested sets of peptides sharing a core epitope
Summary
MHC-II antigen presentation pathways have been explored mostly in professional antigen-presenting cells (APC) such as B cells and dendritic cells, because of their important role in generating and regulating immune responses. AIRE expression by medullary thymic medullary epithelial cells provides a regulated mechanism for stochastic expression of genes usually expressed in a tissue-specific fashion and is required for efficient T cell negative selection [3, 4]. Pioneering differential peptidome analysis by Marrack et al, showed that the major peptides presented by MHC-II in the thymus were associated with MHC-II in the spleen [8]. This was somewhat surprising given the idea that efficient T cell negative selection against all possible self-antigens depends on antigen presentation in the thymus of peptides derived from tissue-specific gene products, many of which would not be expected to be expressed in the spleen. Much of the technology development for immunopeptidome analysis has grown out of analysis of cell lines and highly enriched samples, and strategies for dealing with analysis of limiting abundance biological samples are being developed [13,14,15,16,17]
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