Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens

J. Karttunen, S. Sanderson, N. Shastri

Research output: Contribution to journalArticle

Abstract

The α/β T-cell receptor recognizes a complex ligand formed by the association of antigenic peptides with molecules of the major histocompatibility complex (MHC). The inherent limitations of the conventional T-cell activation assays used to detect these peptide/MHC ligands have, until now, hampered the development of expression cloning systems for T-cell antigens. To overcome these limitations, we have recently introduced a method for detecting ligand-induced activation of individual T cells. This assay, which makes use of a lacZ reporter construct, differs from conventional ligand-induced activation assays in that it allows the detection of single, activated T cells in large pools of resting cells. We applied the lacZ assay to the problem of screening expression libraries, which requires the ability to detect ligand-bearing antigen-presenting cells when they are present at very low frequency. We show here that ligand-expressing antigen- presenting cells can be detected at frequencies of 1:103-104, a level of sensitivity compatible with the screening of cDNA libraries. Furthermore, by using as antigen-presenting cells COS-7 cells stably transfected with the murine Kb class I MHC molecule, we demonstrate that transiently expressed ovalbumin is efficiently processed and presented to an ovalbumin/Kb-specific T-cell hybridoma. lacZ expression is induced in a detectable number of cocultured T cells, even when the ovalbumin cDNA consists of only 1:104 of the total DNA used to transfect the COS cells. These results suggest that unknown T-cell antigens may be identified by screening cDNA libraries in MHC- expressing COS cells using lacZ-inducible T cells as indicators of peptide antigen expression.

Original languageEnglish (US)
Pages (from-to)6020-6024
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume89
Issue number13
DOIs
StatePublished - 1992

Keywords

  • antigen presentation
  • major histocompatibility complex
  • ovalbumin
  • β-galactosidase

ASJC Scopus subject areas

  • General

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