Low calcium concentrations support killing by some but not all cytolytic T lymphocytes, and reveal inhibition of a postconjugation step by calcium antagonists

Previous findings support the prediction that drugs which antagonize the action of calcium should inhibit cytolytic T lymphocyte (CTL)-mediated killing without inhibiting the formation of Ag-specific CTL-target cell conjugates. This would contrast with other CTL-inhibiting drugs, nearly all of which inhibit conjugate formation. Testing this prediction, we found that two calcium channel blockers (verapamil and ruthenium red) inhibit killing only when the extracellular calcium concentration is low (100 microM), and, as predicted, do not inhibit conjugate formation. Surprisingly, the esterase inhibitor N alpha-p-tosyl-L-lysine choloromethylketone also inhibited killing without inhibiting conjugate formation. Unexpectedly, we found that the amount of calcium required by CTL varies by four-fold or more. CTL produced in vivo, or by a single Ag stimulation cycle in vitro, require more than 130 microM calcium for optimal killing, whereas 30 microM suffices for CTL primed in vivo plus boosted in vitro. The rate of admission of calcium into the cytoplasm by physiologic channels did not appear to be the limiting factor for the former type of CTL. Recent findings indicate that allospecific CTL produced in vivo may lack cytoplasmic granules, and may kill by an unidentified mechanism distinct from the exocytosis of granules prominent in CTL lines or clones maintained in vitro. The differences in calcium requirements reported here may reflect differences in mechanisms of killing.