High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells

Autor: Catherine M. Lanagan, Linda E. O′Connor, Antonia L. Pritchard, Michelle A Neller, Michael H.-L. Lai, Nathan R. Martinez, Christopher W. Schmidt
Jazyk: angličtina
Rok vydání: 2014
Předmět:
CD4-Positive T-Lymphocytes
Herpesvirus 4
Human
Cytomegalovirus
CD8-Positive T-Lymphocytes
Memory T cells
Spectrum Analysis Techniques
Cellular types
Medicine and Health Sciences
Cytotoxic T cell
Immune Response
Antigens
Viral
Cells
Cultured
Multidisciplinary
Effector
Infectious Disease Immunology
Flow Cytometry
Vaccination and Immunization
Spectrophotometry
Medicine
White blood cells
Cytophotometry
Clone (B-cell biology)
Research Article
Tumor Immunology
Cell biology
Blood cells
Science
Immune Cells
Immunology
Molecular Sequence Data
T cells
Enzyme-Linked Immunosorbent Assay
Streptamer
Molecular cloning
Biology
Research and Analysis Methods
Interferon-gamma
Antigen
Antigens
Neoplasm
Humans
Amino Acid Sequence
Cell Proliferation
Cloning
Biology and life sciences
Molecular biology
Animal cells
Leukocytes
Mononuclear
Clinical Immunology
Ex vivo
T-Lymphocytes
Cytotoxic
Zdroj: PLoS ONE
PLoS ONE, Vol 9, Iss 11, p e110741 (2014)
ISSN: 1932-6203
Popis: 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.
Databáze: OpenAIRE
Externí odkaz: