Immunopeptidome Landscape During Brucella melitensis Infection in Mice

Mouse bone marrow-derived dendritic cells (BMDCs) were infected in vitro with the recombinant <i>Brucella melitensis M5^+gfp</i> strain. The immunopeptidome of <i>Brucella melitensis M5^+gfp</i>, which presented peptides bound to MHC class II molecules on their surface, was isolated and characterized. BMDCs infected with <i>Brucella melitensis M5^+gfp</i> 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 <i>Brucella melitensis M5^+gfp</i> peptide sequences were identified, mapping to 183 distinct proteins. We successfully define the <i>Brucella melitensis M5^+gfp</i> 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), <i>Brucella melitensis</i> 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 <i>Brucella melitensis</i> subunit vaccines.