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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 chronically FIV infected cats before and after boosting with a DNA immunogen. The effector PBMCs were obtained at the indicated times and used at E:T ratio 10:1 after ex vivo restimulation with Env+GFP+ fibroblasts. The dotted line indicates the cut-off value.
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Figure 5: Env-specific CTL activity in chronically FIV infected cats before and after boosting with a DNA immunogen. The effector PBMCs were obtained at the indicated times and used at E:T ratio 10:1 after ex vivo restimulation with Env+GFP+ fibroblasts. The dotted line indicates the cut-off value.

Mentions: In these experiments, Env+GFP+ fibroblasts and F-CTL assay were used to measure anti-Env CTL in the PBMCs of three SPF cats infected with FIV-Pet 5 years earlier. As determined by periodical monitoring, these animals were in a steady state phase of infection since they had stable or slightly fluctuating moderate levels of viremia, high FIV antibodies titers, and moderately reduced circulating CD4+ T-lymphocytes counts (data not shown). A fourth cat (BO), who had been inoculated exactly as the others but had remained consistently FIV negative, was used as noninfected control. Effector PBMCs were either used fresh or restimulated ex vivo by co-cultivation with autologous Env+GFP+ fibroblasts; however, in no case they exhibited measurable CTL activity (time -8 weeks in Figure 5). An attempt was therefore done to boost anti-Env immune responses of the cats by DNA immunization. Starting 2 weeks after the above CTL assay, the 4 animals were given 3 doses of a plasmid encoding the Env of FIV-Pet and feline granulocyte-macrophage-colony stimulating factor (p-Env-GMCSF), 3 weeks apart, and after 2 further weeks again tested for CTL activity. As shown by Figure 5, the uninfected control cat showed no evidence that this immunization had elicited significant CTL activity. In contrast, all 3 FIV chronically infected animals exhibited robust CTL activity, ranging between 16% and 35% at E:T ratio 10:1. When the test was repeated 18 and 20 weeks after completion of DNA immunization, CTL activity was found to have substantially declined; however, it was generally in the measurable range (Figure 5) and, at least at week 18, it could be substantially expanded ex vivo (Figure 3). Incidentally, this immunization was also followed by a reduction of FIV RNA and proviral DNA loads and by an increment of circulating CD4 T+ lymphocytes, thus showing that it had exerted a beneficial effect on infection course (Pistello et al., manuscript in preparation).


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 chronically FIV infected cats before and after boosting with a DNA immunogen. The effector PBMCs were obtained at the indicated times and used at E:T ratio 10:1 after ex vivo restimulation with Env+GFP+ fibroblasts. The dotted line indicates the cut-off value.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Env-specific CTL activity in chronically FIV infected cats before and after boosting with a DNA immunogen. The effector PBMCs were obtained at the indicated times and used at E:T ratio 10:1 after ex vivo restimulation with Env+GFP+ fibroblasts. The dotted line indicates the cut-off value.
Mentions: In these experiments, Env+GFP+ fibroblasts and F-CTL assay were used to measure anti-Env CTL in the PBMCs of three SPF cats infected with FIV-Pet 5 years earlier. As determined by periodical monitoring, these animals were in a steady state phase of infection since they had stable or slightly fluctuating moderate levels of viremia, high FIV antibodies titers, and moderately reduced circulating CD4+ T-lymphocytes counts (data not shown). A fourth cat (BO), who had been inoculated exactly as the others but had remained consistently FIV negative, was used as noninfected control. Effector PBMCs were either used fresh or restimulated ex vivo by co-cultivation with autologous Env+GFP+ fibroblasts; however, in no case they exhibited measurable CTL activity (time -8 weeks in Figure 5). An attempt was therefore done to boost anti-Env immune responses of the cats by DNA immunization. Starting 2 weeks after the above CTL assay, the 4 animals were given 3 doses of a plasmid encoding the Env of FIV-Pet and feline granulocyte-macrophage-colony stimulating factor (p-Env-GMCSF), 3 weeks apart, and after 2 further weeks again tested for CTL activity. As shown by Figure 5, the uninfected control cat showed no evidence that this immunization had elicited significant CTL activity. In contrast, all 3 FIV chronically infected animals exhibited robust CTL activity, ranging between 16% and 35% at E:T ratio 10:1. When the test was repeated 18 and 20 weeks after completion of DNA immunization, CTL activity was found to have substantially declined; however, it was generally in the measurable range (Figure 5) and, at least at week 18, it could be substantially expanded ex vivo (Figure 3). Incidentally, this immunization was also followed by a reduction of FIV RNA and proviral DNA loads and by an increment of circulating CD4 T+ lymphocytes, thus showing that it had exerted a beneficial effect on infection course (Pistello et al., manuscript in preparation).

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