CD2-mediated NFAT transcriptional activity and IL-2 transcription are dependent on ZAP70/Syk and LAT expression.
(A) Wild-type Jurkat cells (J77) and the ZAP70/Syk-deficient clone P116 were transiently transfected with 10 μg of a reporter plasmid p3xNFAT-luc, carrying the luciferase gene driven by 3 tandem repeats of the distal NFAT sequences derived from the IL-2 promoter, and 1 μg pRL-TK vector, which provides constitutive expression ofRenilla luciferase, as described in “Materials and methods.” Twenty-four hours after transfection, 106cells/test were left unstimulated or stimulated with plate-bound OKT3, T112+T113, each either alone or in combination with 10 ng/mL PMA or 1.5 μmol/L ionomycin (I), or PMA plus ionomycin for 6 hours. Cells were then washed and lysed, and the soluble extract was assayed for luciferase activity. Results are reported as activity of firefly luciferase normalized to that of Renillaluciferase (to correct for transfection efficiency) in the lysate, and expressed as percentage of response obtained with PMA plus ionomycin (P+I). Results are representative of 3 independent experiments. (B) A comparable experiment to that in panel A was performed using the LAT-deficient cells ANJ3 and the ANJ3 cells reconstituted with wild-type LAT (ANJ3-LATwt). (C) CD2-induced IL-2 transcription is dependent on ZAP70/Syk expression. Wild-type Jurkat and the ZAP70/Syk-deficient cells, P116 (1 × 106/test) were either left unstimulated or stimulated with plate-bound OKT3, 1 μL of T112 plus 1 μL of T113, either alone or in combination with PMA, or PMA plus ionomycin for 4 hours. Total RNA was then extracted and subjected to RT-PCR for IL-2 as described in “Materials and methods.” RT-PCR for glyceraldehyde-3-phosphate dehydrogenase was performed as control (not shown).
