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Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells.
- Source :
-
Journal of Virology . 11/15/2014, Vol. 88 Issue 22, p12992-13004. 13p. - Publication Year :
- 2014
-
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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 0022538X
- Volume :
- 88
- Issue :
- 22
- Database :
- Academic Search Index
- Journal :
- Journal of Virology
- Publication Type :
- Academic Journal
- Accession number :
- 103592805
- Full Text :
- https://doi.org/10.1128/JVI.01914-14