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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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Flow cytometry plot of the CTL assay and comparative efficiency at restimulating CTL activity in vitro of Env transduced and Env peptides loaded immortalized fibroblasts. A: CTL activity following lymphocyte restimulation with Env-transduced immortalized fibroblasts as measured by F-CTL. Right and left panels show the results obtained with cats BR and BS, respectively. The gray line indicates the target population incubated alone, the thick line indicates residual target cells after incubation with restimulated lymphocytes. B: Comparative efficiency of Env transduced and Env peptides-loaded immortalized fibroblasts at restimulating CTL activity. Prior to the assay, the effector cells (PBMCs of chronically FIV infected cats boosted with p-Env-GMCSF 18 weeks earlier) were restimulated with Env+GFP+ fibroblasts (solid columns) or immortalized fibroblasts pulsed for 1 hour with pooled peptides covering the entire Env (empty columns).
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Figure 3: Flow cytometry plot of the CTL assay and comparative efficiency at restimulating CTL activity in vitro of Env transduced and Env peptides loaded immortalized fibroblasts. A: CTL activity following lymphocyte restimulation with Env-transduced immortalized fibroblasts as measured by F-CTL. Right and left panels show the results obtained with cats BR and BS, respectively. The gray line indicates the target population incubated alone, the thick line indicates residual target cells after incubation with restimulated lymphocytes. B: Comparative efficiency of Env transduced and Env peptides-loaded immortalized fibroblasts at restimulating CTL activity. Prior to the assay, the effector cells (PBMCs of chronically FIV infected cats boosted with p-Env-GMCSF 18 weeks earlier) were restimulated with Env+GFP+ fibroblasts (solid columns) or immortalized fibroblasts pulsed for 1 hour with pooled peptides covering the entire Env (empty columns).

Mentions: In this experiment we investigated if and how effectively the Env+GFP+ fibroblasts could be used as a stimulus to expand Env-specific CTL activity ex vivo by comparing them with immortalized fibroblasts loaded with a pool of peptides covering the whole FIV Env. This was done by using the peripheral blood mononuclear cells (PBMCs) of BR, BS and CF, 3 chronically FIV infected cats known to possess substantial Env-specific CTL activity as a result of boosting with a DNA immunogen (see below). These PBMCs were co-cultured for 5 days with autologous immortalized fibroblasts that had been transduced with vEnv-GFP or had been pulsed for 1 hour with the pooled peptides or, as controls, with autologous GFP+ fibroblasts or fibroblasts that had not been further manipulated. The PMBCs thus incubated were then tested for CTL activity against Env+GFP+ fibroblasts. Lysis by PBMCs restimulated with autologous GFP+ fibroblasts or immortalized fibroblasts was at background levels (data not shown). The Env+GFP+ fibroblasts caused an expansion of CTL activity readily measurable (Figure 3A) and substantially higher than the peptide-loaded counterparts (Figure 3B). As expected, either stimulus had no effect on the PBMCs of an uninfected cat used as control.


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)

Flow cytometry plot of the CTL assay and comparative efficiency at restimulating CTL activity in vitro of Env transduced and Env peptides loaded immortalized fibroblasts. A: CTL activity following lymphocyte restimulation with Env-transduced immortalized fibroblasts as measured by F-CTL. Right and left panels show the results obtained with cats BR and BS, respectively. The gray line indicates the target population incubated alone, the thick line indicates residual target cells after incubation with restimulated lymphocytes. B: Comparative efficiency of Env transduced and Env peptides-loaded immortalized fibroblasts at restimulating CTL activity. Prior to the assay, the effector cells (PBMCs of chronically FIV infected cats boosted with p-Env-GMCSF 18 weeks earlier) were restimulated with Env+GFP+ fibroblasts (solid columns) or immortalized fibroblasts pulsed for 1 hour with pooled peptides covering the entire Env (empty columns).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Flow cytometry plot of the CTL assay and comparative efficiency at restimulating CTL activity in vitro of Env transduced and Env peptides loaded immortalized fibroblasts. A: CTL activity following lymphocyte restimulation with Env-transduced immortalized fibroblasts as measured by F-CTL. Right and left panels show the results obtained with cats BR and BS, respectively. The gray line indicates the target population incubated alone, the thick line indicates residual target cells after incubation with restimulated lymphocytes. B: Comparative efficiency of Env transduced and Env peptides-loaded immortalized fibroblasts at restimulating CTL activity. Prior to the assay, the effector cells (PBMCs of chronically FIV infected cats boosted with p-Env-GMCSF 18 weeks earlier) were restimulated with Env+GFP+ fibroblasts (solid columns) or immortalized fibroblasts pulsed for 1 hour with pooled peptides covering the entire Env (empty columns).
Mentions: In this experiment we investigated if and how effectively the Env+GFP+ fibroblasts could be used as a stimulus to expand Env-specific CTL activity ex vivo by comparing them with immortalized fibroblasts loaded with a pool of peptides covering the whole FIV Env. This was done by using the peripheral blood mononuclear cells (PBMCs) of BR, BS and CF, 3 chronically FIV infected cats known to possess substantial Env-specific CTL activity as a result of boosting with a DNA immunogen (see below). These PBMCs were co-cultured for 5 days with autologous immortalized fibroblasts that had been transduced with vEnv-GFP or had been pulsed for 1 hour with the pooled peptides or, as controls, with autologous GFP+ fibroblasts or fibroblasts that had not been further manipulated. The PMBCs thus incubated were then tested for CTL activity against Env+GFP+ fibroblasts. Lysis by PBMCs restimulated with autologous GFP+ fibroblasts or immortalized fibroblasts was at background levels (data not shown). The Env+GFP+ fibroblasts caused an expansion of CTL activity readily measurable (Figure 3A) and substantially higher than the peptide-loaded counterparts (Figure 3B). As expected, either stimulus had no effect on the PBMCs of an uninfected cat used as control.

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