Label target cells with Na2 51CrO4 Killed cells release radioactive chromium Add cytotoxic T cells to labeled target cells
Fig. A.38 Cytotoxic T-cell activity is often assessed by chromium release from labeled target cells. Target cells are labeled with radioactive chromium as Na251CrO4, washed to remove excess radioactivity, and exposed to cytotoxic T cells. Cell destruction is measured by the release of radioactive chromium into the medium, detectable within 4 hours of mixing target cells with T cells.
the DNA of the target cells is rapidly fragmented and retained in the filtrate, while large, unfragmented DNA is collected on a filter, and one can measure either the release of these fragments or the retention of 3H-thymidine in chromosomal DNA. These assays provide a rapid, sensitive, and specific measure of the activity of cytotoxic T cells.
An alternative to these in vitro cytotoxicity assays is to measure target-cell killing by cytotoxic T cells in intact experimental animals. This assay is generally performed with mice that have been infected with a pathogen known to induce a strong cytotoxic T-cell response, such as a virus. Target cells are incubated with the antigenic peptide, which will bind to MHC class I on the target-cell surface. These cells are then incubated with a low concentration of the fluorescent dye CFSE (see Section A-26). A control population of cells that is not given the antigenic peptide is incubated with a high concentration of CFSE, allowing these cells to be distinguished from the antigen-bearing target cells. The two cell populations are mixed 1:1 and injected into the experimental animals. Four hours later, spleen cells are recovered from the animals and analyzed by FACS, allowing the specific target-cell lysis to be calculated from the ratio of the two CFSE-labeled cell populations (Fig. A.39).
Target cells and control cells are labeled with different concentrations of dye, mixed 1:1, and injected into mice
Fig. A.39 Assay for cytotoxic T-cell activity using CFSE-labeled target cells. To detect cytotoxic T-cell activity in intact experimental animals, mice that have been infected with a virus are injected with a mixture of target cells labeled with the fluorescent dye CFSE. One group of target cells is pre-incubated with a viral peptide that binds to MHC class I on the target cells; this group of cells is labeled with a low concentration of CFSE. A second group of cells is incubated with a control (nonviral) peptide and is labeled with a high concentration of CFSE. The two groups of cells are mixed together in a 1:1 ratio, and injected into the infected mice. After 4 hours, the mice are sacrificed and the target cells are recovered and analyzed by flow cytometry. Examination of the ratio of the two target-cell populations provides a measure of specific lysis of viral peptide-coated target cells.
A-29 Assays for CD4 T cells.
CD4 T-cell functions usually involve the activation rather than the killing of cells bearing specific antigen. The activating effects of CD4 T cells on B cells or macrophages are mediated in large part by cytokines, which are released by the T cell when it recognizes antigen. Thus, CD4 T-cell function is usually studied by measuring the type and amount of cytokine released. Because different effector T cells release different amounts and types of cytokines, one can learn about the effector potential of a T cell by measuring the proteins it produces.
Cytokines can be detected by their activity in biological assays of cell growth, where the cytokines serve either as growth factors or as growth inhibitors. A more specific assay is a modification of ELISA known as a capture or sandwich ELISA (see Section A-4). In this assay, the cytokine is characterized by its ability to act as a bridge between two monoclonal antibodies reacting with different epitopes on the cytokine molecule. Cytokine-secreting cells can also be detected by ELISPOT (see Section A-22).