Cytosolic free calcium concentration in the mitogenic stimulation of T lymphocytes by anti-CD3 monoclonal antibodies

Abstract The effects of anti-CD3 monoclonal antibodies on cytosolic free Ca 2+ concentration, [Ca 2+ ] i , were investigated in freshly isolated lymphocytes, T cell lines, T clones and the leukemic T cell line Jurkat with three different methodologies, i.e. classical cuvette experiments, cytofluorimetry and videoimaging. With any technique, concentrations of anti-CD3 antibodies optimal for stimulation of DNA synthesis were completely ineffective at inducing early increases of [Ca 2+ ] i in freshly isolated lymphocytes. At supraoptimal mitogenic concentrations: (i) anti-CD3 mAb induced negligible increases of [Ca 2+ ] i when tested in suspensions of freshly isolated lymphocytes, but the response increased progressively during in vitro culturing with IL2; (ii) most, but not all, T clones, when tested in suspension, were responsive to these concentrations of anti-CD3 antibodies in terms of [Ca 2+ ] i ; (iii) using the videoimaging technique at the single cell level, it was demonstrated that the anti-CD3 antibodies induced large increases of [Ca 2+ ] i in lymphocytes only under conditions which allowed adherence of the antibodies (and of the cells) to the glass surface. In all T cell types investigated, the [Ca 2+ ] i increases were most often composed by multiple, asynchronous oscillations. The buffering of [Ca 2+ ] i increases, obtained by loading the cells with membrane permeant esters of Quin-2 and Fura-2, inhibited anti-CD3 mAb induced DNA synthesis, but this appeared entirely attributable to a toxic side effect of the ester hydrolysis. The relevance of these data is discussed in terms of their methodological and functional implications for the understanding of the role of Ca 2+ in mitogenic stimulation of T cells.