Hypersensitivity to Diphtheria Toxin by Mouse Cells Expressing both Diphtheria Toxin Receptor and CD9 Antigen

DTS-II is a highly diphtheria toxin (DT)-sensitive cell line previously isolated by transfection of wild-type DT-resistant mouse L-M(TK-) cells with the cDNA encoding a monkey Vero cell DT receptor. DTS-II cells are as toxin-sensitive as Vero cells, have ≈3-fold more receptors than Vero cells, and have ≈10-fold lower affinity for DT than Vero cells. We now cotransfected DTS-II cells with a plasmid containing the Vero cell cDNA coding for CD9 antigen (pCD9) and with a plasmid containing the gene for hygromycin resistance (pHyg). The stably transfected hygromycin-resistant colonies were screened for DT hypersensitivity employing a replica plate system. A DT-hypersensitive colony was isolated and purified. The purified DT-hypersensitive cells, DTS-III, (i) are ≈10-fold more toxin-sensitive than DTS-II and Vero cells and (ii) bear ≈106DT receptors per cell (i.e., ≈20-fold and ≈60-fold more receptors than DTS-II and Vero cells, respectively), but their receptor affinity is still ≈10-fold lower than that of Vero cells. Cross-linking experiments employing125I-labeled DT demonstrated that DTS-II and DTS-III cells have essentially the same profile of DT-binding cell-surface protein(s), suggesting that CD9 antigen, although expressed on the cell surface of DTS-III cells, may not be in close proximity to the DT-binding domain of the receptor. CD9 may affect DT receptor expression by increasing receptor density at the cell surface. By employing DTS-III cells it should be possible to purify and characterize the DT cell-surface receptor protein(s).