Improved identification of allele-specific MHC class II immunopeptidome

Abstract

Abstract In humans, there are three main MHC class II alleles that present peptides: HLA-DR, HLA-DQ and HLA-DP. While peptides presented by HLA-DR have been well characterized due to the availability of a high-quality antibody to this allele, peptides presented by HLA-DQ and HLA-DP are less well characterized. In this study, we have developed a workflow to improve the experimental identification of allele-specific MHC class II-associated peptides by biotinylating the alpha 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. Detailed characterization of peptides which bind to MHC class II haplotypes has the potential to aid in the development of therapies for autoimmune disorders in two ways. Better understanding of the antigens driving disease could lead to better therapies, and further, ensuring that biologic drugs used to treat these conditions do not themselves elicit immune responses through class II presentation will improve existing treatments.