Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens.
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.
Karttunen
J
J
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Sanderson
S
S
Shastri
N
N
D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology*; D001681:Biological Assay; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D004306:Dose-Response Relationship, Immunologic; D006183:H-2 Antigens / Q000276:immunology; D006825:Hybridomas; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000378:metabolism; D008024:Ligands; D008213:Lymphocyte Activation; D051379:Mice; D010047:Ovalbumin / Q000276:immunology; D013194:Staining and Labeling; D013601:T-Lymphocytes / Q000276:immunology*; D001616:beta-Galactosidase / Q000378:metabolism
D006183:H-2 Antigens; D007376:Interleukin-2; D008024:Ligands; D010047:Ovalbumin; D004247:DNA; D001616:beta-Galactosidase