Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells

Jane C. Yaciuk, Matthew Skaley, Wilfried Bardet, Fredda Schafer, Danijela Mojsilovic, Steven Cate, Christopher J. Stewart, Curtis McMurtrey, Kenneth W. Jackson, Rico Buchli, Alex Olvera, Samandhy Cedeño, Montserrat Plana, Beatriz Mothe, Christian Brander, John T. West, William H. Hildebrand

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29 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)12992-13004
JournalJournal of Virology
Volume88
Issue number22
DOIs
Publication statusPublished - 1 Jan 2014

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    Yaciuk, J. C., Skaley, M., Bardet, W., Schafer, F., Mojsilovic, D., Cate, S., Stewart, C. J., McMurtrey, C., Jackson, K. W., Buchli, R., Olvera, A., Cedeño, S., Plana, M., Mothe, B., Brander, C., West, J. T., & Hildebrand, W. H. (2014). Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells. Journal of Virology, 88(22), 12992-13004. https://doi.org/10.1128/JVI.01914-14