tetano
Editor, Senior Moderator
Methods Mol Biol. 2019;2024:213-234. doi: 10.1007/978-1-4939-9597-4_13.
[h=1]Construction and Screening of an Antigen-Derived Peptide Library Displayed on Yeast Cell Surface for CD4+ T Cell Epitope Identification.[/h] Wen F[SUP]1,[/SUP][SUP]2[/SUP], Smith MR[SUP]2[/SUP], Zhao H[SUP]3,[/SUP][SUP]4,[/SUP][SUP]5,[/SUP][SUP]6[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] Antigenic peptides (termed T cell epitopes) are assembled with major histocompatibility complex (MHC) molecules and presented on the surface of antigen-presenting cells (APCs) for T cell recognition. T cells engage these peptide-MHCs using T cell receptors (TCRs). Because T cell epitopes determine the specificity of a T cell immune response, their prediction and identification are important steps in developing peptide-based vaccines and immunotherapies. In recent years, a number of computational methods have been developed to predict T cell epitopes by evaluating peptide-MHC binding; however, the success of these methods has been limited for MHC class II (MHCII) due to the structural complexity of MHCII antigen presentation. Moreover, while peptide-MHC binding is a prerequisite for a T cell epitope, it alone is not sufficient. Therefore, T cell epitope identification requires further functional verification of the MHC-binding peptide using professional APCs, which are difficult to isolate, expand, and maintain. To address these issues, we have developed a facile, accurate, and high-throughput method for T cell epitope mapping by screening antigen-derived peptide libraries in complex with MHC protein displayed on yeast cell surface. Here, we use hemagglutinin and influenza A virus X31/A/Aichi/68 as examples to describe the key steps in identification of CD4+ T cell epitopes from a single antigenic protein and the entire genome of a pathogen, respectively. Methods for single-chain peptide MHC vector design, yeast surface display, peptide library generation in Escherichia coli, and functional screening in Saccharomyces cerevisiae are discussed.
[h=4]KEYWORDS:[/h] CD4+ T cell epitope mapping; Flow cytometry; High-throughput screening; Human leukocyte antigen DR1 (HLA-DR1); Influenza A virus; MHC-binding peptides; Major histocompatibility complex; Peptide library; Yeast display
PMID: 31364052 DOI: 10.1007/978-1-4939-9597-4_13
[h=1]Construction and Screening of an Antigen-Derived Peptide Library Displayed on Yeast Cell Surface for CD4+ T Cell Epitope Identification.[/h] Wen F[SUP]1,[/SUP][SUP]2[/SUP], Smith MR[SUP]2[/SUP], Zhao H[SUP]3,[/SUP][SUP]4,[/SUP][SUP]5,[/SUP][SUP]6[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] Antigenic peptides (termed T cell epitopes) are assembled with major histocompatibility complex (MHC) molecules and presented on the surface of antigen-presenting cells (APCs) for T cell recognition. T cells engage these peptide-MHCs using T cell receptors (TCRs). Because T cell epitopes determine the specificity of a T cell immune response, their prediction and identification are important steps in developing peptide-based vaccines and immunotherapies. In recent years, a number of computational methods have been developed to predict T cell epitopes by evaluating peptide-MHC binding; however, the success of these methods has been limited for MHC class II (MHCII) due to the structural complexity of MHCII antigen presentation. Moreover, while peptide-MHC binding is a prerequisite for a T cell epitope, it alone is not sufficient. Therefore, T cell epitope identification requires further functional verification of the MHC-binding peptide using professional APCs, which are difficult to isolate, expand, and maintain. To address these issues, we have developed a facile, accurate, and high-throughput method for T cell epitope mapping by screening antigen-derived peptide libraries in complex with MHC protein displayed on yeast cell surface. Here, we use hemagglutinin and influenza A virus X31/A/Aichi/68 as examples to describe the key steps in identification of CD4+ T cell epitopes from a single antigenic protein and the entire genome of a pathogen, respectively. Methods for single-chain peptide MHC vector design, yeast surface display, peptide library generation in Escherichia coli, and functional screening in Saccharomyces cerevisiae are discussed.
[h=4]KEYWORDS:[/h] CD4+ T cell epitope mapping; Flow cytometry; High-throughput screening; Human leukocyte antigen DR1 (HLA-DR1); Influenza A virus; MHC-binding peptides; Major histocompatibility complex; Peptide library; Yeast display
PMID: 31364052 DOI: 10.1007/978-1-4939-9597-4_13