High Efficiency <i>Ex Vivo</i> Cloning of Antigen-Specific Human Effector T Cells

<div><p>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 <i>in vivo</i> 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 <i>in vitro</i>. Here we describe an efficient method for cloning effector T cells <i>ex vivo</i>. 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. <i>Ex vivo</i> effector cell cloning provides a rapid and effective method of deriving antigen-specific T cells clones with traceable <i>in vivo</i> precursor function and phenotype.</p>