title: Direct mapping of MHC class II epitopes.
abstract: Despite technical improvements, the mapping of MHC class II epitopes within complex antigens by genetic or biochemical methods is still laborious and expensive. Here, we describe a simple and fast procedure to directly map T helper cell epitopes within known antigens by bacterial expression cloning. Short antigenic fragments, created by digestion of the coding sequence of the antigen with frequently cutting restriction enzymes, are randomly ligated to the coding sequence of GFP in a bacterial expression vector. Bacteria expressing antigen-GFP fusion proteins are then fed directly to MHC II+ antigen-presenting cells and probed with antigen-specific T cells. Bacterial colonies recognized by T cells are expanded, and the antigenic fragments identified by plasmid extraction and sequence analysis. This direct epitope identification (DEPI) approach offers several advantages. First, bacterial colonies expressing the antigen in frame with GFP are easily detectable by virtue of their green appearance and thereby reduce the screening effort significantly. Second, short antigenic peptides normally unstable in bacteria are highly expressed when fused to GFP. Third, the uniformly high level of expression of short antigenic peptides fused to GFP permits the identification of epitopes even within proteins which are difficult to express in bacteria. Furthermore, by fusing double-stranded oligonucleotides to the GFP gene, crucial amino acids within T cell epitopes may be defined. Thus, this method not only facilitates the identification of T cell epitopes, but also makes it possible to assess the role of individual amino acids for MHC binding or T cell recognition.
authors.affiliation: Clinical Cooperation Group, Children's Hospital, Hematology-Oncology, Technical University Munich, Kölner Platz 1, D-80804 Munich, Germany.
authors.forename: Slavoljub
authors.initials: S
authors.lastname: Milosevic
authors.forename: Uta
authors.initials: U
authors.lastname: Behrends
authors.forename: Heike
authors.initials: H
authors.lastname: Christoph
authors.forename: Josef
authors.initials: J
authors.lastname: Mautner
mesh_terms: D000595:Amino Acid Sequence; D000938:Antigen-Presenting Cells / Q000276:immunology; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018604:Epitope Mapping / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D018984:Epitopes, T-Lymphocyte; D000949:Histocompatibility Antigens Class II; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins