RECONSTITUTION OF T-CELL RECEPTOR SIGNALING IN ZAP-70-DEFICIENT CELLSBY RETROVIRAL TRANSDUCTION OF THE ZAP-70 GENE

A variant of severe combined immunodeficiency syndrome (SCID) with a s elective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4(+) cells has recently been show n to be the result of mutations in the ZAP-70 gene. T cell receptor (T CR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I-transf ormed CD4(+) T cell Lines were established from ZAP-70-deficient patie nts and normal controls. ZAP-70 was expressed and appropriately phosph orylated in normal T cell lines after TCR engagement, but was not dete cted in T cell lines from ZAP-70-deficient patients. To determine whet her signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer produ cer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropri ately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced c ells, but not ZAP-70-deficient cells, were able to mobilize calcium af ter receptor ligation, indicating that proximal TCR signaling was reco nstituted. These results indicate that this form of SCID may be correc ted by gene therapy.