In this study, we have developed a workflow to improve the experimental identification of allele-specific MHC class II-associated peptides by biotinylating the α chain of HLA-DR in live cells. This was accomplished using the HLA-DR-null HL-60 cell line, cotransfecting with BirA and Avi-tagged MHC expression constructs. Supply of exogenous biotin ensured the specific biotinylation of MHC, which was then enriched from lysates using either streptavidin or HLA-DR-specific antibody. Peptides were eluted from these complexes and identified by mass spec. This procedure successfully captured all HLA-DR expressed by the cells and peptides identified by the traditional immunoaffinity approach were compared with the biotinylation approach.

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To cite this poster: Ho, B., Good, P., Nyayapathy, S., et al. Improved identification of allele-specific MHC class II immunopeptidome. Poster presented at: The Annual Meeting of the American Association of Immunologists; May 3-7, 2024. Chicago, IL.