An improved retroviral gene transfer technique demonstrates inhibition of CD4-CD8- thymocyte development by kinase-inactive ZAP-70.
ZAP-70 is a Syk family tyrosine kinase that plays an essential role in initiating TCR signals. Deficiency in ZAP-70 causes a defect in the development at CD4+CD8+ thymocytes due to defective TCR-mediated positive and negative selection. Using a newly devised retrovirus gene transfer and an efficient green fluorescence protein detection technique in fetal thymus organ cultures, the present study shows that forced expression in developing thymocytes of a catalytically inactive mutant of ZAP-70, but not wild-type ZAP-70, inhibits T cell development at the earlier CD4-CD8- stage. The ZAP-70 mutant blocked the generation of CD4+CD8+ thymocytes even in the absence of endogenous ZAP-70. Thus, the present results demonstrate a novel technique for gene transfer into developing T cells and suggest that ZAP-70/Syk family tyrosine kinases are involved in the signals inducing the generation of CD4+CD8+ thymocytes.
Sugawara
T
T
Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba, Japan.
Di Bartolo
V
V
Miyazaki
T
T
Nakauchi
H
H
Acuto
O
O
Takahama
Y
Y
D016475:3T3 Cells; D000818:Animals; D015704:CD4 Antigens / Q000235:genetics*; D016827:CD8 Antigens / Q000235:genetics*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D004789:Enzyme Activation / Q000235:genetics; D005333:Fetus; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000276:immunology; D049452:Green Fluorescent Proteins; D006131:Growth Inhibitors / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D016296:Mutagenesis; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology; D012190:Retroviridae / Q000235:genetics* / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D051746:ZAP-70 Protein-Tyrosine Kinase
D015704:CD4 Antigens; D016827:CD8 Antigens; D006131:Growth Inhibitors; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497262:Zap70 protein, mouse