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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

Overview of T cell clone generation.The procedure for cell stimulation, enrichment, single cell sorting, clone maintenance and characterisation is outlined. After PBMC were stimulated with antigen for 14 h, the IFN-γ capture assay was used to label functional cells. Cell phenotype and viability were revealed by the addition of fluorescent mAb against surface markers and propidium iodide. A MoFlo cell sorter enriched for viable, CD14– CD16– CD19– cells that were either CD4+ IFN-γ+ or CD8+ IFN-γ+. A FACS Vantage cell sorter then confirmed the phenotype of purified cell populations, and deposited functional cells into 96 well plates at one cell per well. Plates contained medium with feeder cells and phytohaemagglutinin-L, to non-specifically stimulate T cell clones. The growth and function of clones were assessed following expansion for three weeks, with weekly medium changes.
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pone-0110741-g001: Overview of T cell clone generation.The procedure for cell stimulation, enrichment, single cell sorting, clone maintenance and characterisation is outlined. After PBMC were stimulated with antigen for 14 h, the IFN-γ capture assay was used to label functional cells. Cell phenotype and viability were revealed by the addition of fluorescent mAb against surface markers and propidium iodide. A MoFlo cell sorter enriched for viable, CD14– CD16– CD19– cells that were either CD4+ IFN-γ+ or CD8+ IFN-γ+. A FACS Vantage cell sorter then confirmed the phenotype of purified cell populations, and deposited functional cells into 96 well plates at one cell per well. Plates contained medium with feeder cells and phytohaemagglutinin-L, to non-specifically stimulate T cell clones. The growth and function of clones were assessed following expansion for three weeks, with weekly medium changes.

Mentions: Cell suspensions were filtered through sterile 37 µm nylon mesh immediately prior to purification sorting of CD4+ IFN-γ+ and CD8+ IFN-γ+ populations using a MoFlo cell sorter running Summit software (Beckman Coulter, Fullerton, CA, USA). Sorting gates were determined by the bimodal expression of phenotypic markers (CD4, CD8, CD14, CD16, CD19) and IFN-γ, and in most cases were confirmed using negative controls. Subsequently a FACSVantage SE cell sorter running CellQuest and ClonCyt software (BD Biosciences) and equipped with a single cell deposition unit was used to sort single CD4+ or CD8+, IFN-γ+, CD14- CD16- CD19- cells into wells of U-bottom 96-well plates containing clone medium and feeder cells consisting of 2×104 irradiated allogeneic LCL (a mixture of three different lines) and 1×105 irradiated allogeneic PBMC per well. The overall cloning procedure is summarised in Fig. 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)

Overview of T cell clone generation.The procedure for cell stimulation, enrichment, single cell sorting, clone maintenance and characterisation is outlined. After PBMC were stimulated with antigen for 14 h, the IFN-γ capture assay was used to label functional cells. Cell phenotype and viability were revealed by the addition of fluorescent mAb against surface markers and propidium iodide. A MoFlo cell sorter enriched for viable, CD14– CD16– CD19– cells that were either CD4+ IFN-γ+ or CD8+ IFN-γ+. A FACS Vantage cell sorter then confirmed the phenotype of purified cell populations, and deposited functional cells into 96 well plates at one cell per well. Plates contained medium with feeder cells and phytohaemagglutinin-L, to non-specifically stimulate T cell clones. The growth and function of clones were assessed following expansion for three weeks, with weekly medium changes.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110741-g001: Overview of T cell clone generation.The procedure for cell stimulation, enrichment, single cell sorting, clone maintenance and characterisation is outlined. After PBMC were stimulated with antigen for 14 h, the IFN-γ capture assay was used to label functional cells. Cell phenotype and viability were revealed by the addition of fluorescent mAb against surface markers and propidium iodide. A MoFlo cell sorter enriched for viable, CD14– CD16– CD19– cells that were either CD4+ IFN-γ+ or CD8+ IFN-γ+. A FACS Vantage cell sorter then confirmed the phenotype of purified cell populations, and deposited functional cells into 96 well plates at one cell per well. Plates contained medium with feeder cells and phytohaemagglutinin-L, to non-specifically stimulate T cell clones. The growth and function of clones were assessed following expansion for three weeks, with weekly medium changes.
Mentions: Cell suspensions were filtered through sterile 37 µm nylon mesh immediately prior to purification sorting of CD4+ IFN-γ+ and CD8+ IFN-γ+ populations using a MoFlo cell sorter running Summit software (Beckman Coulter, Fullerton, CA, USA). Sorting gates were determined by the bimodal expression of phenotypic markers (CD4, CD8, CD14, CD16, CD19) and IFN-γ, and in most cases were confirmed using negative controls. Subsequently a FACSVantage SE cell sorter running CellQuest and ClonCyt software (BD Biosciences) and equipped with a single cell deposition unit was used to sort single CD4+ or CD8+, IFN-γ+, CD14- CD16- CD19- cells into wells of U-bottom 96-well plates containing clone medium and feeder cells consisting of 2×104 irradiated allogeneic LCL (a mixture of three different lines) and 1×105 irradiated allogeneic PBMC per well. The overall cloning procedure is summarised in Fig. 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