ExVivo Detection of Antigen Specific T Cells

Originally, the only means of detecting antigen-specific T cells was to separate the T lymphocytes from whole blood and to expose predetermined numbers of cells deposited into wells of microtiter plates to synthetic antigen plus growth stimulatory cytokines, such as interleukin-2 (IL-2) and/or autologous feeder cells [50]. In order to convert this method into an assay for determining the number of antigen-specific cells, sequential dilution of T lymphocytes in 96-well plates, expansion of antigen-specific T cells and subsequent functional assays (e.g., proliferation or cytotoxicity assays) are required to ascertain the number of wells containing antigen-specific clones or lines. Unfortunately, this was found to be a cumbersome procedure which took several days to complete and often produced highly variable results. Furthermore, more recent studies have shown that this so-called "limiting dilution assay" (LDA) often significantly underestimated the "true" in-vivo frequencies of antigen-specific T cells [51-53]. This underestimation most likely occurs partly due to apoptosis of immature antigen-specific T cells in vitro.

Recently, considerable improvement of the determination of antigen-specific T-cell frequencies has been achieved by the development of assay methods requiring only relatively short in-vitro re-stimulation, such as the enzyme-linked immunospot assay (ELISPOT) [54, 55] and intracellular cytokine staining [51]. Details of these and other relevant assays (e.g., CTL cytotoxicity assays) are outlined below.

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