Application protocol

Expansion of antigen-specific cytokine-secreting T cells

This application protocol describes how to expand antigen-specific T cells which have been isolated using the MACS® Cytokine Secretion Assay after restimulation with antigen. These T cells are therefore of a memory phenotype. The memory phenotype, particularly for CD8+ cells, is heterogeneous. It contains cells, which divide rapidly in response to antigen-“central memory cells” and cells, which exhibit recall effector function, such as cytotoxicity-“effector memory cells”. These subsets are not mutually exclusive, e.g., cells can divide rapidly but still lyse antigen-loaded targets. The mixture of memory cells isolated with the MACS Cytokine Secretion Assay is entirely dependent on the antigen the T cells responded to, e.g., immune responses against chronic virus infections are often characterized by a pre-dominance of effector memory T cells. As a general rule, central memory cells have a higher proliferation rate in vitro, compared to effector memory cells. Therefore, the rate of proliferation of T cells expanded with this method will depend on the memory phenotype of the cells that have been isolated. This protocol has been optimized to expand both central and effector memory T cells very efficiently. However, the individual results obtained may vary depending on the antigen and the Cytokine Secretion Assay used to isolate the cells. This protocol has been established for the expansion of CMV-specific, IFN-γ–secreting T cells. It may require optimization when working with other antigens or expanding cells which were isolated according to the secretion of cytokines other than IFN-γ.

Protocol

This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step. 

This expansion system is designed for in vitro expansion of cytokine-secreting, antigen-specific T cells. The antigen-specific T cells in peripheral blood mononuclear cells (PBMCs) or leukapheresis are restimulated with antigen. The responding cells are magnetically isolated, according to their secretion of cytokines, using the MACS Cytokine Secretion Assay. The non-cytokine secreting cell fraction, i.e., the unlabeled flow through, is used as a source of antigen-presenting cells (referred to as “feeder cells” throughout the protocol) after irradiation or treatment with mitomycin C. The cytokine-secreting cells and the feeder cells are co-cultured in vitro in the presence of interleukin 2 (IL-2).

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Principle of in vitro expansion of T cells isolated with the MACS Cytokine Secretion Assay

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. 
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