A new approach to the cloning of genes encoding T-cell epitopes.
The molecular structure of antigens recognized exclusively by T cells, such as minor histocompatibility antigens and some antigens that provoke autoimmune responses, has proved difficult to determine. Recently, several antigens induced on tumor cells by mutagen treatment have been cloned by transfection of genomic DNA libraries into P1.HTR cells, screening for antigen expression using T-cell clones, and subsequent recovery of the integrated DNA by cosmid rescue. We have modified this technique and have stably transfected P1.HTR cell lines with polyoma T antigen, which allows episomal replication of the shuttle vector, pCDM8. Using pCDM8-CAT constructs, we have determined the frequency of transfection and plasmid copies taken up per cell under optimal transfection conditions. Using a pCDM8 construct which expresses the tumor-specific antigen, P91A (pCDM8-tum-), that is recognized by a T-cell clone, we have found that cells transfected with this antigen can be recognized by the T-cell clone when they are present at only 1%-3% of a mixed population. Progeny of a single cell transfected with pCDM8-tum-: pCDM8-CAT at proportions of 1:10, 1:25, and 1:50 are recognized by the T-cell clone. Furthermore, Hirt extracted plasmid DNA from transfectants expressing the tum- antigen can be amplified in bacteria, transfected back into P1.HTR recipients, and recognized by the T-cell clone. This approach should enable reasonably rapid screening of cDNA libraries for even relatively low abundance messages encoding, for example, minor histocompatibility and alloantigens, and allow their subsequent cloning.
Scott
D M
DM
Transplantation Biology Section, Clinical Research Centre, Harrow, Middlesex, UK.
Dyson
P J
PJ
Simpson
E
E
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology; D000954:Antigens, Surface / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004261:DNA Replication; D000939:Epitopes; D015723:Gene Library; D005822:Genetic Vectors; D008213:Lymphocyte Activation; D051379:Mice; D008811:Mice, Inbred DBA; D010957:Plasmids; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D000954:Antigens, Surface; D000939:Epitopes