THE INTRACELLULAR CA2+ CONCENTRATION OPTIMAL FOR T-CELL ACTIVATION IS QUITE DIFFERENT AFTER IONOMYCIN OR CD3 STIMULATION

The relationship between the initial increase of intracellular Ca2+ concentration ([Ca2+](i)) (measured at the single-cell level with an imaging system) and the ensuing proliferation was examined in a human T cell clone stimulated by a phorbol ester in combination with ionomycin, thapsigargin or an anti-CD3 mAb (monoclonal antibody against the CD3 molecule, UCHT1). From the responses to various ionomycin concentrations, one can define a range of [Ca2+](i) values (400-900 nM) which appears optimal for T cell proliferation; lower [Ca2+](i) values are suboptimal, higher values are cytotoxic. It was then examined if the [Ca2+]i requirements were similar following anti-CD3 stimulation. [Ca2+](i) oscillations elicited by a concentration of UCHT1 (1/1,000) optimal for mitogenicity fall precisely within the 400-900 nM range. However, very low concentrations of UCHT1 (1/100,000) which evoke barely detectable [Ca2+](i) responses still cause the cells to proliferate. The possibility that the lower [Ca2+](i) requirements observed following anti-CD3 stimulation was due to [Ca2+](i) oscillations was tested under conditions which prevented the appearance of these oscillations. It turns out that an oscillatory Ca2+ signal is not more mitogenic than a sustained augmentation of [Ca2+](i). Finally, it was examined if overstimulation via CD3 could have toxic consequences similar to those elicited after ionomycin overstimulation. Large transient [Ca2+](i) responses can be observed following anti-CD3 stimulation in appropriate conditions, and namely in T cells pretreated with interleukin-2. These [Ca2+](i) augmentations are not cytotoxic. A role for the plasmalemmal Ca2+ pump in the prevention of cytotoxicity can be demonstrated. In conclusion, the correspondence between the [Ca2+](i) response and cell proliferation is entirely different following stimulation by ionomycin and by anti-CD3. In addition, cell proliferation evoked by very low UCHT1 concentration might reveal the existence of a yet unidentified activation pathway.