title: Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells.
abstract: Identification of CD8(+) cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4(+) SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)-mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4(+) T cells into strategies designed to enhance T cell immunity.
  
  
  OBJECTIVE
  HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4(+) T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Jane C
authors.identifier: http://orcid.org/0000-0002-5786-194X
authors.initials: JC
authors.lastname: Yaciuk
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Skaley
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Bardet
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Fredda
authors.initials: F
authors.lastname: Schafer
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Danijela
authors.initials: D
authors.lastname: Mojsilovic
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Cate
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Stewart
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Curtis
authors.initials: C
authors.lastname: McMurtrey
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Jackson
authors.affiliation: Pure Protein, LLC, Oklahoma City, Oklahoma, USA.
authors.forename: Rico
authors.initials: R
authors.lastname: Buchli
authors.affiliation: Laboratori de Retrovirologia, Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Autonomous University of Barcelona, Badalona, Spain.
authors.forename: Alex
authors.initials: A
authors.lastname: Olvera
authors.affiliation: Laboratori de Retrovirologia, Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Autonomous University of Barcelona, Badalona, Spain.
authors.forename: Samandhy
authors.initials: S
authors.lastname: Cedeño
authors.affiliation: IDIBAPS, Hospital Clinic, University of Barcelona, Barcelona, Spain.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Plana
authors.affiliation: Laboratori de Retrovirologia, Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Autonomous University of Barcelona, Badalona, Spain University of Vic and Central Catalonia, Vic, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Mothe
authors.affiliation: Laboratori de Retrovirologia, Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Autonomous University of Barcelona, Badalona, Spain University of Vic and Central Catalonia, Vic, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.
authors.forename: Christian
authors.initials: C
authors.lastname: Brander
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: John T
authors.initials: JT
authors.lastname: West
authors.affiliation: Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA William-Hildebrand@ouhsc.edu.
authors.forename: William H
authors.initials: WH
authors.lastname: Hildebrand
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology*; D002853:Chromatography, Liquid; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D015497:HIV-1 / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis* / Q000276:immunology; D014764:Viral Proteins / Q000032:analysis* / Q000276:immunology
chemical_list: D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D010455:Peptides; D014764:Viral Proteins