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

Authors
  • Karttunen, J
  • Sanderson, S
  • Shastri, N
Type
Published Article
Journal
Proceedings of the National Academy of Sciences of the United States of America
Publication Date
Jul 01, 1992
Volume
89
Issue
13
Pages
6020–6024
Identifiers
PMID: 1378619
Source
Medline
License
Unknown

Abstract

The alpha/beta T-cell receptor 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 1:10(3)-10(4), 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 1:10(4) 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.

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