Identification of T-Cell epitopes: Rapid isolation of class i-presented peptides from viable cells by mild acid elution

Walter J. Storkus, Herbert J. Zeh, Russell D. Salter, Michael T. Lotze

Research output: Contribution to journalArticle

203 Scopus citations

Abstract

A novel method was developed to isolate immunogenic peptides (CD8+T-cell epitopes) from class I complexes expressed at the cell surface of viable cells. Cells treated at pH 3.3 with citrate-phosphate buffer for periods as short as 15 s remained viable and became phenotypically class I deficient. Qualitative loss of class I determinants was verified both serologically and by the incapacity of acid-treated cells to be lysed by class I-restricted cytolytic T lymphocytes (CTLs) in contrast to non-acid-treated controls. Flow cytometric analysis of acid-treated cells suggests that class I heavy chains remain associated with the cell membrane, while the class I light chain (β2-microglobulin) is absent. Since the physical dissociation of β2-microglobulin from class I heavy chain is correlated with the release of previously class I-bound peptides, we examined acid-eluted cell-free supernatants for the presence of immunogenic peptides. Peptides were acid eluted from an influenza A strain-infected, HLA-A2+cell line and were subsequently fractionated by reverse-phase high performance liquid chromatography (RP-HPLC). These fractionated peptides were examined for their capacity to sensitize an HLA-A2+B cell line to lysis mediated by an influenza A matrix peptide- (Flu Ml 57–68) specific, HLA-A2-restricted CTL line. A single peak of biologic activity was identified in HPLC fractions 47 and 48 derived from influenza-infected cells. These fractions contained a peptide of Mr 968 with a sequence similar to the Flu Ml 58–66 sequence GILGFVFTL. The application of this technique to other T-cell-based systems may aid in the definition of peptide epitopes relevant to viral, autoimmune, or neoplastic disorders.

Original languageEnglish (US)
Pages (from-to)94-103
Number of pages10
JournalJournal of Immunotherapy
Volume14
Issue number2
DOIs
StatePublished - Aug 1993
Externally publishedYes

Keywords

  • Amino acid sequence
  • Class I complexes
  • Cytolytic T lymphocytes
  • Isolation
  • Peptide
  • T-cell epitopes

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Pharmacology
  • Cancer Research

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