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A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts.

Bonci F, Zabogli E, Conti F, Merico A, Freer G, Bendinelli M, Pistello M - BMC Biotechnol. (2009)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Retrovirus Center and Virology Section, Department of Experimental Pathology, University of Pisa, Via San Zeno, 35, 56127 Pisa, Italy. f.bonci@kedrion.com

ABSTRACT

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.

Conclusion: 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.

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Env-specific CTL activity in acutely FIV infected cats. The PBMCs were obtained at the indicated times post-infection and used as effector cells without ex vivo restimulation. The dotted line indicates the cut-off value. Cat GP died 40 days post inoculation due to unexplained renal failure.
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Figure 4: Env-specific CTL activity in acutely FIV infected cats. The PBMCs were obtained at the indicated times post-infection and used as effector cells without ex vivo restimulation. The dotted line indicates the cut-off value. Cat GP died 40 days post inoculation due to unexplained renal failure.

Mentions: Collectively, the above data had demonstrated that the Env+GFP+ fibroblasts expressed the target and reporter genes in stable and correct fashion. To test whether they were also suitable targets for CTL assays, we employed them in the F-CTL assay using, as effector cells, PBMCs collected from cats during acute FIV infection. Fibroblasts were harvested from SPF cats GN, GO, GP and GQ, processed as above, and frozen. The animals were then inoculated with a dose of FIV-Pet which produced the expected levels of viremia as determined starting 2 weeks post-infection. CTL activity was examined 2, 4, 8 and 12 weeks after infection. The test was carried out both with fresh PBMCs and with PBMCs that, prior to the F-CTL assay, were restimulated ex vivo with autologous Env+GFP+ fibroblasts as described above. Target cells were autologous Env+GFP+ fibroblasts and, as controls, autologous GFP+ fibroblasts and heterologous Env+GFP+ fibroblasts; however, significant cell lysis was observed only with the former cells. As depicted by Figure 4, which shows the percent specific lysis values observed with the unstimulated PBMCs at effector:target (E:T) ratios of 1:10 and 1:50, in one cat CTL activity could already be detected 2 weeks post-infection. In the other animals, the test was first found positive at 4 weeks post-infection, usually at the E:T ratio 50:1 only. At later times of infection, this picture did not change significantly, except for the fact that E:T ratio 10:1 yielded a measurable level of lysis more frequently than at earlier times. Percent lysis obtained with ex vivo restimulated PBMCs were only marginally higher than obtained with fresh PBMCs (data not shown), suggesting that in acutely infected cats Env-specific CTL activity cannot be significantly expanded ex vivo. Of note, cat GQ who exhibited the most prompt and robust CTL response developed levels of plasma viremia, proviral DNA in the PBMC, and a reduction of circulating CD4+ T-lymphocyte counts similar to the other animals (data not shown).


A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts.

Bonci F, Zabogli E, Conti F, Merico A, Freer G, Bendinelli M, Pistello M - BMC Biotechnol. (2009)

Env-specific CTL activity in acutely FIV infected cats. The PBMCs were obtained at the indicated times post-infection and used as effector cells without ex vivo restimulation. The dotted line indicates the cut-off value. Cat GP died 40 days post inoculation due to unexplained renal failure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662824&req=5

Figure 4: Env-specific CTL activity in acutely FIV infected cats. The PBMCs were obtained at the indicated times post-infection and used as effector cells without ex vivo restimulation. The dotted line indicates the cut-off value. Cat GP died 40 days post inoculation due to unexplained renal failure.
Mentions: Collectively, the above data had demonstrated that the Env+GFP+ fibroblasts expressed the target and reporter genes in stable and correct fashion. To test whether they were also suitable targets for CTL assays, we employed them in the F-CTL assay using, as effector cells, PBMCs collected from cats during acute FIV infection. Fibroblasts were harvested from SPF cats GN, GO, GP and GQ, processed as above, and frozen. The animals were then inoculated with a dose of FIV-Pet which produced the expected levels of viremia as determined starting 2 weeks post-infection. CTL activity was examined 2, 4, 8 and 12 weeks after infection. The test was carried out both with fresh PBMCs and with PBMCs that, prior to the F-CTL assay, were restimulated ex vivo with autologous Env+GFP+ fibroblasts as described above. Target cells were autologous Env+GFP+ fibroblasts and, as controls, autologous GFP+ fibroblasts and heterologous Env+GFP+ fibroblasts; however, significant cell lysis was observed only with the former cells. As depicted by Figure 4, which shows the percent specific lysis values observed with the unstimulated PBMCs at effector:target (E:T) ratios of 1:10 and 1:50, in one cat CTL activity could already be detected 2 weeks post-infection. In the other animals, the test was first found positive at 4 weeks post-infection, usually at the E:T ratio 50:1 only. At later times of infection, this picture did not change significantly, except for the fact that E:T ratio 10:1 yielded a measurable level of lysis more frequently than at earlier times. Percent lysis obtained with ex vivo restimulated PBMCs were only marginally higher than obtained with fresh PBMCs (data not shown), suggesting that in acutely infected cats Env-specific CTL activity cannot be significantly expanded ex vivo. Of note, cat GQ who exhibited the most prompt and robust CTL response developed levels of plasma viremia, proviral DNA in the PBMC, and a reduction of circulating CD4+ T-lymphocyte counts similar to the other animals (data not shown).

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Retrovirus Center and Virology Section, Department of Experimental Pathology, University of Pisa, Via San Zeno, 35, 56127 Pisa, Italy. f.bonci@kedrion.com

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus