Reconstitution of T cell receptor signaling in ZAP-70-deficient cells by retroviral transduction of the ZAP-70 gene.
A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I-transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer 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 appropriately 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 cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.
Taylor
N
N
Division of Research Immunology and Bone Marrow Transplantation, Children's Hospital, Los Angeles, California 90027, USA.
Bacon
K B
KB
Smith
S
S
Jahn
T
T
Kadlecek
T A
TA
Uribe
L
L
Kohn
D B
DB
Gelfand
E W
EW
Weiss
A
A
Weinberg
K
K
D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D016511:Severe Combined Immunodeficiency / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D014161:Transduction, Genetic; D051746:ZAP-70 Protein-Tyrosine Kinase
D011948:Receptors, Antigen, T-Cell; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D002118:Calcium