Respiratory tract gene transfer. Transplantation of genetically modified T-lymphocytes directly to the respiratory epithelial surface.
To evaluate the strategy for potentially treating respiratory disorders with genetically modified T-lymphocytes, the interleukin-2 (IL-2)-dependent murine T-cell line, CTLL2, was genetically altered with the Escherichia coli beta-galactosidase (beta-gal) gene (lacZ) in vitro with a retroviral vector and the modified T-cells were transplanted directly to the respiratory epithelial surface of syngeneic C57Bl/6 mice. Southern and Northern analyses confirmed that the neomycin-selected modified T-cells contained and expressed the lacZ gene. The fate of the modified T-cells (CTLL2/lacZ) was followed by flow cytometry with T-cell surface marker Thy1.2 and fluorescent beta-gal analysis. One day after transplantation (7.5 x 10(5) CTLL2/lacZ T-cells/g of body weight), 95 +/- 3% of the Thy1.2+ T-cells recovered from respiratory epithelial lining fluid (ELF) were beta-gal+. Importantly, the modified T-cells remained in the lung for some time; at 3 days, Thy1.2+ beta-gal+ T-cells represented 63 +/- 12% of ELF Thy1.2+ T-cells and 59 +/- 6% of Thy1.2+ T-cells recovered from the whole lung. At 7 days, 33 +/- 8% of the Thy 1.2+ cells in ELF and 75 +/- 6% of the Thy1.2+ cells in whole lung were Thy1.2+ beta-gal+. In contrast, the proportion of the Thy1.2+ beta-gal+ T-cells in the spleen, the major extrapulmonary lymphatic organ, never rose above 3 +/- 1% of the total Thy1.2+ cells. The number of Thy1.2+ beta-gal+ T-cells in the lung could be modified by the systemic administration of IL-2, with whole lung Thy1.2+ beta-gal+ T-cells increasing 4.6-fold 3 days after transplantation, compared with non-IL-2-treated animals. These studies suggest that direct transplantation of genetically modified T-cells into the lung is feasible and represents a viable strategy for lung-specific gene transfer.
Fukayama
M
M
Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Kanno
T
T
Brody
S L
SL
Kirby
M
M
Crystal
R G
RG
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D004848:Epithelium; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005798:Genes, Bacterial; D005814:Genes, Viral; D005818:Genetic Engineering; D005822:Genetic Vectors; D007376:Interleukin-2 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D012313:RNA / Q000032:analysis; D012137:Respiratory System; D012190:Retroviridae / Q000235:genetics; D013601:T-Lymphocytes / Q000637:transplantation; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D007376:Interleukin-2; D012313:RNA; D001616:beta-Galactosidase