HYPERSENSITIVITY TO DIPHTHERIA-TOXIN BY MOUSE CELLS EXPRESSING BOTH DIPHTHERIA-TOXIN RECEPTOR AND CD9 ANTIGEN

DT(S)-II is a highly diphtheria toxin (DT)-sensitive cell fine previously isolated by transfection of wild-type DT-resistant mouse L-M(TK-) cells with the cDNA encoding a monkey Vero cell DT receptor. DT(S)-II cells are as toxin-sensitive as Vere cells, have almost-equal-to 3-fold more receptors than Vero cells, and have almost-equal-to 10-fold lower affinity for DT than Vero cells. We now cotransfected DT(S)-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, DT(S)-III, (i) are almost-equal-to 10-fold more toxin-sensitive than DT(S)-II and Vero cells and (ii) bear almost-equal-to 10(6) DT receptors per cell (i.e., almost-equal-to 20-fold and almost-equal-to 60-fold more receptors than DT(S)-II and Vero cells, respectively), but their receptor affinity is still almost-equal-to 10-fold lower than that of Vero cells. Cross-linking experiments employing I-125-labeled DT demonstrated that DT(S)-II and DT(S)-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 DT(S)-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 DT(S)-III cells it should be possible to purify and characterize the DT cell-surface receptor protein(s).