A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts.
BACKGROUND
Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major histocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with 51chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry.


RESULTS
The method consists of culturing skin fibroblasts, immortalizing them with a replication defective clone of simian virus 40, and finally transducing them with a bicistronic vector encoding the target antigen and the reporter green fluorescent protein. When used in a flow cytometry-based assay, the target cells obtained with this method proved valuable for assessing the viral envelope protein specific cytotoxic T lymphocyte activity in domestic cats acutely or chronically infected with feline immunodeficiency virus, a lentivirus similar to human immunodeficiency virus and used as animal model for AIDS studies.


CONCLUSIONS
Given the versatility of the bicistronic vector used, its ability to deliver multiple and large transgenes in target cells, and its extremely wide cell specificity when pseudotyped with the vesicular stomatitis virus envelope protein, the method is potentially exploitable in many animal species.
Bonci
Francesca
F
Retrovirus Center and Virology Section, Department of Experimental Pathology, University of Pisa, Via San Zeno, 35, 56127 Pisa, Italy. f.bonci@kedrion.com
Zabogli
Elisa
E
Conti
Francesca
F
Merico
Antonio
A
Freer
Giulia
G
Bendinelli
Mauro
M
Pistello
Mauro
M
D000818:Animals; D002415:Cats; D002461:Cell Line, Transformed; D003601:Cytotoxicity Tests, Immunologic; D016181:Feline Acquired Immunodeficiency Syndrome / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D015686:Gene Products, env / Q000276:immunology; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000276:immunology; D016089:Immunodeficiency Virus, Feline / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000821:virology; D014161:Transduction, Genetic
D015686:Gene Products, env; D049452:Green Fluorescent Proteins