A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts
<p>Abstract</p> <p>Background</p> <p>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 pres...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2009-03-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/9/18 |
| _version_ | 1818163665696718848 |
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| author | Bendinelli Mauro Freer Giulia Merico Antonio Conti Francesca Zabogli Elisa Bonci Francesca Pistello Mauro |
| author_facet | Bendinelli Mauro Freer Giulia Merico Antonio Conti Francesca Zabogli Elisa Bonci Francesca Pistello Mauro |
| author_sort | Bendinelli Mauro |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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 hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with <sup>51</sup>chromium 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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p> |
| first_indexed | 2024-12-11T16:53:10Z |
| format | Article |
| id | doaj.art-202c0d3daddb4085aca23724de822b63 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-11T16:53:10Z |
| publishDate | 2009-03-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-202c0d3daddb4085aca23724de822b632022-12-22T00:58:03ZengBMCBMC Biotechnology1472-67502009-03-01911810.1186/1472-6750-9-18A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hostsBendinelli MauroFreer GiuliaMerico AntonioConti FrancescaZabogli ElisaBonci FrancescaPistello Mauro<p>Abstract</p> <p>Background</p> <p>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 hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with <sup>51</sup>chromium 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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p>http://www.biomedcentral.com/1472-6750/9/18 |
| spellingShingle | Bendinelli Mauro Freer Giulia Merico Antonio Conti Francesca Zabogli Elisa Bonci Francesca Pistello Mauro A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts BMC Biotechnology |
| title | A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts |
| title_full | A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts |
| title_fullStr | A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts |
| title_full_unstemmed | A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts |
| title_short | A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts |
| title_sort | novel method for producing target cells and assessing cytotoxic t lymphocyte activity in outbred hosts |
| url | http://www.biomedcentral.com/1472-6750/9/18 |
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