Identification and isolation of antigen-specific cytotoxic T lymphocytes with an automated microraft sorting system.

Antigen-specific cytotoxic CD8+ T cell responses are an essential component in the ability of the adaptive immune system to control viral infections and cancer. There are several immune monitoring assays that have been developed to measure the cytotoxic activity of a T cell population; however, there are limitations in terms of sample size, sample processing, overall assay time, and cost. Furthermore, these methods do not directly measure individual T cell mediated killing, nor do they provide data regarding the temporal dependence of CD8+ T cells. Armistead and colleagues developed a methodology to identify, isolate, and clonally expand antigen-specific CD8+ T cells based upon the time course measurement of the killing of antigen expressing target cells. The CellRaft® Technology was utilized wherein each CellRaft® contained a population of fluorescently-labeled antigen-presenting target cells and one CD8+ T cell. Over the 6-hour time course of the experiment, a cytotoxicity dye was used in the media of the CellRaft™ Array to monitor target cell death over time. The CD8+ T cells that demonstrated a high rate of target cell death were isolated from the CellRaft™ Array to a 96-well plate for clonal expansion and further characterization, including measurement of TCR affinity to a peptide/MHC tetramer and sequencing of the TCRα and TCRβ CDR3 regions.