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High efficiency ex vivo cloning of antigen-specific human effector T cells.

Neller MA, Lai MH, Lanagan CM, O'Connor LE, Pritchard AL, Martinez NR, Schmidt CW - PLoS ONE (2014)

Bottom Line: Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen.We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells.Cloning efficiency, clonality, and retention/loss of function are described.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; School of Medicine, The University of Queensland Mayne Medical School, Brisbane, Queensland, Australia.

ABSTRACT
While cloned T cells are valuable tools for the exploration of immune responses against viruses and tumours, current cloning methods do not allow inferences to be made about the function and phenotype of a clone's in vivo precursor, nor can precise cloning efficiencies be calculated. Additionally, there is currently no general method for cloning antigen-specific effector T cells directly from peripheral blood mononuclear cells, without the need for prior expansion in vitro. Here we describe an efficient method for cloning effector T cells ex vivo. Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen. In combination with multiple phenotypic markers, single effector T cells are sorted using a flow cytometer directly into multi-well plates, and cloned using standard, non antigen-specific expansion methods. We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells. Cloning efficiency, clonality, and retention/loss of function are described. Ex vivo effector cell cloning provides a rapid and effective method of deriving antigen-specific T cells clones with traceable in vivo precursor function and phenotype.

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Related in: MedlinePlus

Retention of IFN-γ production by T cell clones.ELISA was used to measure IFN-γ in supernatants of T cell clones (▴CD4+; • CD8+) stimulated with (A) autologous or allogeneic lymphoblastoid cell lines (LCL), (B) autologous LCL either unloaded or pre-incubated with recombinant pp65 for 1-2 h, or (C, D) autologous or allogeneic melanoma cells (as indicated in graph). The stimulator: T cell responder ratios were (A–C) 50,000∶1,000 or (D, patient D14) 100,000∶10,000. Dotted line indicates>100 pg/ml in excess of control, the cut-off for positivity used in Table 1, which summarises the data from this figure.
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pone-0110741-g004: Retention of IFN-γ production by T cell clones.ELISA was used to measure IFN-γ in supernatants of T cell clones (▴CD4+; • CD8+) stimulated with (A) autologous or allogeneic lymphoblastoid cell lines (LCL), (B) autologous LCL either unloaded or pre-incubated with recombinant pp65 for 1-2 h, or (C, D) autologous or allogeneic melanoma cells (as indicated in graph). The stimulator: T cell responder ratios were (A–C) 50,000∶1,000 or (D, patient D14) 100,000∶10,000. Dotted line indicates>100 pg/ml in excess of control, the cut-off for positivity used in Table 1, which summarises the data from this figure.

Mentions: CD4+ and CD8+ clones were stimulated with autologous or allogeneic melanoma cells, LCL or LCL loaded (or untreated) with CMV pp65 protein, as appropriate, and then culture supernatants were analysed for IFN-ã by ELISA (Fig. 4 A–D). The proportion of clones that retained IFN-γ production varied from 8% (CD8+ clones derived from LCL stimulation) to 77% (CD8+ clones from melanoma patient D14; Table 1). As with cloning efficiency, the ability of clones to secrete IFN-γ was not related to higher precursor frequency – indeed, the opposite may be true, as the high proportion of cells producing IFN-γ ex vivo in response to CMV and EBV translated into lower proportions of functional CD8+ T cells (Table 1).


High efficiency ex vivo cloning of antigen-specific human effector T cells.

Neller MA, Lai MH, Lanagan CM, O'Connor LE, Pritchard AL, Martinez NR, Schmidt CW - PLoS ONE (2014)

Retention of IFN-γ production by T cell clones.ELISA was used to measure IFN-γ in supernatants of T cell clones (▴CD4+; • CD8+) stimulated with (A) autologous or allogeneic lymphoblastoid cell lines (LCL), (B) autologous LCL either unloaded or pre-incubated with recombinant pp65 for 1-2 h, or (C, D) autologous or allogeneic melanoma cells (as indicated in graph). The stimulator: T cell responder ratios were (A–C) 50,000∶1,000 or (D, patient D14) 100,000∶10,000. Dotted line indicates>100 pg/ml in excess of control, the cut-off for positivity used in Table 1, which summarises the data from this figure.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219695&req=5

pone-0110741-g004: Retention of IFN-γ production by T cell clones.ELISA was used to measure IFN-γ in supernatants of T cell clones (▴CD4+; • CD8+) stimulated with (A) autologous or allogeneic lymphoblastoid cell lines (LCL), (B) autologous LCL either unloaded or pre-incubated with recombinant pp65 for 1-2 h, or (C, D) autologous or allogeneic melanoma cells (as indicated in graph). The stimulator: T cell responder ratios were (A–C) 50,000∶1,000 or (D, patient D14) 100,000∶10,000. Dotted line indicates>100 pg/ml in excess of control, the cut-off for positivity used in Table 1, which summarises the data from this figure.
Mentions: CD4+ and CD8+ clones were stimulated with autologous or allogeneic melanoma cells, LCL or LCL loaded (or untreated) with CMV pp65 protein, as appropriate, and then culture supernatants were analysed for IFN-ã by ELISA (Fig. 4 A–D). The proportion of clones that retained IFN-γ production varied from 8% (CD8+ clones derived from LCL stimulation) to 77% (CD8+ clones from melanoma patient D14; Table 1). As with cloning efficiency, the ability of clones to secrete IFN-γ was not related to higher precursor frequency – indeed, the opposite may be true, as the high proportion of cells producing IFN-γ ex vivo in response to CMV and EBV translated into lower proportions of functional CD8+ T cells (Table 1).

Bottom Line: Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen.We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells.Cloning efficiency, clonality, and retention/loss of function are described.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; School of Medicine, The University of Queensland Mayne Medical School, Brisbane, Queensland, Australia.

ABSTRACT
While cloned T cells are valuable tools for the exploration of immune responses against viruses and tumours, current cloning methods do not allow inferences to be made about the function and phenotype of a clone's in vivo precursor, nor can precise cloning efficiencies be calculated. Additionally, there is currently no general method for cloning antigen-specific effector T cells directly from peripheral blood mononuclear cells, without the need for prior expansion in vitro. Here we describe an efficient method for cloning effector T cells ex vivo. Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen. In combination with multiple phenotypic markers, single effector T cells are sorted using a flow cytometer directly into multi-well plates, and cloned using standard, non antigen-specific expansion methods. We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells. Cloning efficiency, clonality, and retention/loss of function are described. Ex vivo effector cell cloning provides a rapid and effective method of deriving antigen-specific T cells clones with traceable in vivo precursor function and phenotype.

Show MeSH
Related in: MedlinePlus