title: Development and implementation of a direct detection, quantitation and validation system for class I MHC self-peptide epitopes.
abstract: Gene and protein expression studies demonstrate that viral-infected and malignant cells undergo a complex series of transcriptional and translational changes. As class I MHC molecules reflect the proteome (and changes therein) by presenting intracellular peptide epitopes, the development of a direct discovery and validation technology for the identification of these epitopes is needed. We developed our technology using HIV-1-infected cells as a model. A combination of hollow fiber class I HLA protein production and mass spectrometric epitope analysis indicated a 3-fold increase in the host-peptide VLMTEDIKL(720-728), [eIF4G((720))] presented by the HLA-A*0201 of HIV-1-infected cells. This peptide is derived from the host-protein translation of eukaryotic initiation factor 4-gamma (eIF4G) that plays a pivotal role in cellular protein synthesis. Direct confirmation of expression of this self-encoded antigen was performed through development of a T cell receptor mimic (TCRm) monoclonal antibody (mAb). The resulting 4F7 TCRm demonstrated specific recognition of the eIF4G((720))-A*0201 complex. Staining of normal PBMCs with 4F7 showed only low levels of endogenous eIF4G((720)) presentation by HLA-A*0201, while 4F7 staining of HIV-1-infected PBMCs revealed an approximately 3-fold increase in eIF4G((720))-A*0201. The MHC-peptide complex was initially detectable by 4F7 at 3 days post-infection, with a steady increase through day 8. We therefore demonstrate the successful development and implementation of an integrated discovery and validation technology system for direct identification and confirmation of class I MHC-peptide epitopes on cells.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA. jon.weidanz@ttuhsc.edu
authors.forename: Jon A
authors.initials: JA
authors.lastname: Weidanz
authors.forename: Paolo
authors.initials: P
authors.lastname: Piazza
authors.forename: Heather
authors.initials: H
authors.lastname: Hickman-Miller
authors.forename: David
authors.initials: D
authors.lastname: Woodburn
authors.forename: Tiffany
authors.initials: T
authors.lastname: Nguyen
authors.forename: Angela
authors.initials: A
authors.lastname: Wahl
authors.forename: Francisca
authors.initials: F
authors.lastname: Neethling
authors.forename: Maurizio
authors.initials: M
authors.lastname: Chiriva-Internati
authors.forename: Charles R
authors.initials: CR
authors.lastname: Rinaldo
authors.forename: William H
authors.initials: WH
authors.lastname: Hildebrand
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000276:immunology; D001324:Autoantigens / Q000032:analysis* / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000821:virology; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000032:analysis* / Q000276:immunology; D039603:Eukaryotic Initiation Factor-4G / Q000276:immunology; D005434:Flow Cytometry; D015497:HIV-1 / Q000276:immunology; D015234:HLA-A Antigens / Q000276:immunology; D015789:HLA-A2 Antigen; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology; D010455:Peptides / Q000032:analysis* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology / Q000821:virology
chemical_list: D000911:Antibodies, Monoclonal; D001324:Autoantigens; C113920:EIF4G1 protein, human; D018984:Epitopes, T-Lymphocyte; D039603:Eukaryotic Initiation Factor-4G; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D015395:Histocompatibility Antigens Class I; D010446:Peptide Fragments; D010455:Peptides