Immunopeptidome Landscape During Brucella melitensis Infection in Mice.

Mouse bone marrow-derived dendritic cells (BMDCs) were infected in vitro with the recombinant Brucella melitensis M5+gfp strain. The immunopeptidome of Brucella melitensis M5+gfp, which presented peptides bound to MHC class II molecules on their surface, was isolated and characterized. BMDCs infected with Brucella melitensis M5+gfp were subjected to hypotonic lysis. The associated immunopeptidome was then isolated and characterized using co-immunoprecipitation (Co-IP) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 289 MHC-II-bound Brucella melitensis M5+gfp peptide sequences were identified, mapping to 183 distinct proteins. We successfully define the Brucella melitensis M5+gfp immunopeptidome presented by MHC-II on infected BMDCs. The source proteins of these peptides exhibited significant abundance and functional, structural, and pathway diversity. This study demonstrates that during antigen presentation by antigen-presenting cells (APCs), Brucella melitensis peptides presented by MHC-II originate from a broad repertoire of proteins, not limited to surface antigens. This complex immunopeptidome, shaped by active selection mechanisms, provides diverse targets for host immune recognition. These findings establish a foundation for further investigation into the transfer of comprehensive immune information between immune cells and the elicitation of immune responses. This work also paves the way for identifying specific T-cell receptors involved in recognition and immune activation, thereby facilitating the analysis of adaptive immunity's molecular basis. Furthermore, this study provides an innovative approach for immunopeptidome analysis, providing a crucial theoretical foundation for developing novel Brucella melitensis subunit vaccines.