Signal transduction properties of the T cell activation monoclonal antibody panel: ubiquitous increase in cellular substrate tyrosine phosphorylation in the absence of detectable calcium mobilization

A panel of coded monoclonal antibodies (mAbs) submitted to the T cell section of the Vth International Workshop and Conference on Human Leukocyte Differentiation Antigens was examined for the ability to induce cellular activation. Intracellular calcium levels were examined by loading resting T cells with the Ca++-specific dye Indo-1, incubating the cells with Ab, cross-linking with goat anti-mouse Ab and analyzing by flow cytometry. Only 11 out of 68 Abs induced a detectable rise in Ca++; two of these Abs induced a substantial rise in Ca++. In resting T cells, 63 of 68 Abs induced increased tyrosine phosphorylation of substrates compared to negative control. Approximately half of the Abs that induced tyrosine phosphorylation induced substrates different from those seen following treatment of cells with anti-CD3 Ab. All mAbs that induced a rise in Ca++ also induced increased tyrosine phosphorylation. These experiments show a distinct difference in the ability of cross-linked Ab to induce changes in tyrosine phosphorylation and intracellular free Ca+. Furthermore, these results indicate that transient increases in cellular substrate phosphorylation may have questionable biologic significance in T cells.