3, 4 Furthermore, this assay, by itself, does not provide a measurement of T cell effector function or activity. 1, 2 Tetramer-based enumeration is expensive however, and requires a unique tetramer per antigen to be tested, which is problematic particularly in the field of personalized cancer neo-antigen discovery. To measure the fraction of antigen-specific CD8 + T cells in a T cell population, which is important in measuring immune responses and immunodominance in vivo as well as in the expansion of antigen-specific T cells in culture, peptide/MHC (major histocompatibility complex) tetramers are often used. Multiple types of experiments designed to quantify the number of antigen-specific CD8 + T cells and measure the cytotoxic activity of a T cell population are routinely used to characterize CD8 + responses however, many standard immune monitoring assays have limitations with respect to sample size (or cell number) requirements, sample processing, assay time or cost that make detailed characterization of immune responses problematic. While this study investigated single T cell cytotoxicity rates against simple targets with subsequent cell sorting, future studies will involve measuring T cell mediated cytotoxicity in more complex cellular environments, enlarging the arrays to identify very rare antigen specific T cells, and measuring single cell CD4 + and CD8 + T cell proliferation.Īntigen-specific, cytotoxic CD8 + T cell responses are an essential component in the adaptive immune system’s ability to control both viral infections and cancer. All of these expressed high-avidity T cell receptors for M1p/HLA*02:01 tetramers, and 2 of the 3 receptors were sequenced. Microrafts with highly active CD8 + T cells were individually transferred to wells of a 96-well plate, using a needle-release device coupled to the microscope. The rates of target cell death among the individual CD8 + T cells varied greatly however, individual T cells maintained their rates of cytotoxicity throughout the time course of the experiment enabling rapid identification of highly cytotoxic CD8 + T cells.
Target cell killing, measured by fluorescence microscopy, was quantified in each microraft. Individual microrafts on a 70 × 70 array were loaded with on average 1 CD8 + cell from the culture and a population of M1p presenting target cells. We have developed a microraft array methodology that automatically measures the ability of individual T cells to kill a population of target cells and viably sorts specific cells into a 96-well plate for expansion.Ī human T cell culture was generated against the influenza M1p antigen. The simultaneous measurement of T cell function with recovery of individual T cells would greatly facilitate characterizing antigen-specific responses both in vivo and in model systems.
0 kommentar(er)
