title: A broadly applicable approach to T cell epitope identification: application to improving tumor associated epitopes and identifying epitopes in complex pathogens.
abstract: Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. michael_valentino@urmc.rochester.edu
authors.forename: Michael D
authors.initials: MD
authors.lastname: Valentino
authors.forename: C Siddiq
authors.initials: CS
authors.lastname: Abdul-Alim
authors.forename: Zachary J
authors.initials: ZJ
authors.lastname: Maben
authors.forename: Denise
authors.initials: D
authors.lastname: Skrombolas
authors.forename: Lucinda L
authors.initials: LL
authors.lastname: Hensley
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Kawula
authors.forename: Michelle
authors.initials: M
authors.lastname: Dziejman
authors.forename: Edith M
authors.initials: EM
authors.lastname: Lord
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Frelinger
authors.forename: John G
authors.initials: JG
authors.lastname: Frelinger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005604:Francisella tularensis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000276:immunology / Q000378:metabolism; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D009369:Neoplasms / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D019151:Peptide Library; D017430:Prostate-Specific Antigen / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D014406:Tularemia / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D000939:Epitopes; D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D019151:Peptide Library; D017430:Prostate-Specific Antigen