title: High efficiency ex vivo cloning of antigen-specific human effector T cells.
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.
authors.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.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Neller
authors.affiliation: Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors.forename: Michael H-L
authors.initials: MH
authors.lastname: Lai
authors.affiliation: Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Lanagan
authors.affiliation: Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors.forename: Linda E
authors.initials: LE
authors.lastname: O'Connor
authors.affiliation: Oncogenomics Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors.forename: Antonia L
authors.initials: AL
authors.lastname: Pritchard
authors.affiliation: Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Martinez
authors.affiliation: Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Schmidt
mesh_terms: D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000378:metabolism*; D000956:Antigens, Viral / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D004854:Herpesvirus 4, Human / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D008969:Molecular Sequence Data; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D000956:Antigens, Viral; D007371:Interferon-gamma