Isolation of mutant T lymphocytes with defects in capacitative calcium entry

Calcium and calcium-binding proteins play important roles in the signaling cascade leading from the initial engagement of TCRs on T cells to the fully activated state. To undertake a molecular dissection of this cascade, we first isolated a Jurkat T cell line derivative containing the NF-AT promoter element driving transcription of the diphtheria toxin A chain gene (dipA), resulting in rapid cell death. Selecting viable cells that fail to activate NF-AT-dependent transcription, we isolated two independent cell lines possessing defects in capacitative Ca²⁺ entry. NF-AT-dependent transcription can be restored in these cells by expression of a constitutively active calcineurin, but not by overexpression of the Ca²⁺ regulatory protein CAML, which can normally replace the Ca²⁺ signal. The defect in these cell lines probably lies between CAML and calcineurin in the T cell activation cascade.