Binding of antigen-specific, H-2-restricted T cell hybridomas to antigen-pulsed adherent cell monolayers.
Two methods were investigated for studying the binding of radiolabeled hybridoma T cells to antigen (Ag) and H-2 products for which they bore receptors. In both cases hybridoma T cells were labeled with tritiated thymidine. In one method labeled cells were added to adherent splenic cells prepulsed with antigen, and the mixture was incubated overnight at 37 degrees C before nonadherent cells were gently washed away. The percent of adherent hybridoma T cells was then estimated by harvesting the adherent monolayers and measuring tritium counts bound. In a second method radiolabeled hybridoma T cells were added to adherent antigen-pulsed B cell lymphomas or hybridomas for between 15 min and 1 hr at 37 degrees C before removal of nonadherent cells and harvesting of the adherent monolayers. In both cases binding was both antigen- and I-region specific. In the second case binding was also rapid; significant binding could be measured after 15 min incubation. These techniques were used to study subclones of one of our T cell hybridomas that were thought by a functional assay (interleukin 2 release) to have lost receptors for Ag/H-2. It was found that subclones of the hybridoma that no longer secreted interleukin 2 in response to Ag/H-2, even though they continued to secrete interleukin 2 in response to concanavalin A, also no longer bound specifically to Ag-pulsed monolayers of the appropriate H-2 type. This confirmed the idea that these subclones had lost the ability to synthesize receptors for Ag/H-2. It is hoped that assays of this type will be useful in the future for the study of Ag/H-2 receptors on T cells.
D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D000939:Epitopes; D005802:Genes, MHC Class II; D006183:H-2 Antigens / Q000235:genetics / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008805:Mice, Inbred A; D008810:Mice, Inbred C57BL; D010047:Ovalbumin / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000032:analysis* / Q000235:genetics; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
D000939:Epitopes; D006183:H-2 Antigens; D011948:Receptors, Antigen, T-Cell; D010047:Ovalbumin