Mapping MHC-II binding peptides derived from an antigenic protein for potential CD4+ T-cell epitopes has been challenging due to a lack of experimental approaches that are both quantitative and rapid. The rate-limiting steps in current approaches include the construction of single MHC allele expressing cell lines and/or the purification of the MHC-II allelic proteins for peptide elution (i.e., mass spectrometry) or in vitro peptide binding (i.e., ELISA) assays. These labor-intensive steps typically take up to 4 months or more. In this protocol, we describe a system that uses yeast cells to display "empty" (i.e., without covalently linked peptides) MHC-II heterodimers that are capable of binding exogenously adde... More
Mapping MHC-II binding peptides derived from an antigenic protein for potential CD4+ T-cell epitopes has been challenging due to a lack of experimental approaches that are both quantitative and rapid. The rate-limiting steps in current approaches include the construction of single MHC allele expressing cell lines and/or the purification of the MHC-II allelic proteins for peptide elution (i.e., mass spectrometry) or in vitro peptide binding (i.e., ELISA) assays. These labor-intensive steps typically take up to 4 months or more. In this protocol, we describe a system that uses yeast cells to display "empty" (i.e., without covalently linked peptides) MHC-II heterodimers that are capable of binding exogenously added peptides of interest. This yeast-MHC-II system eliminates the time-consuming soluble MHC-II purification steps, allowing rapid identification of peptide ligands from protein antigens (RIPPA). The amount of peptide loading to MHC-II or the extent of competition between indicator and competitor peptides at the surface of yeast cells can be quantitatively determined using flow cytometric analysis. Importantly, the protocol only takes ∼1 month from the construction of plasmids and the yeast display of "empty" MHC-II to the quantitative determination of MHC-II binding peptides from a given antigen. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Yeast display of "empty" MHC-II Support Protocol: Construction of yeast shuttle vector expressing "empty" MHC-II Basic Protocol 2: Peptide competition on the surface of yeast cells Alternate Protocol: RIPPA in a 96-well format.
