Fig. A.25 The frequency of cytokine-secreting T cells can be determined by the ELISPOT assay. The ELISPOT assay is a variant of the ELISA assay in which antibodies bound to a plastic surface are used to capture cytokines secreted by individual T cells. Usually, cytokine-specific antibodies are bound to the surface of a plastic tissue-culture well and the unbound antibodies are removed (top panel). Activated T cells are then added to the well and settle onto the antibody-coated surface (second panel). If a T cell is secreting the appropriate cytokine, this will then be captured by the antibody molecules on the plate surrounding the T cell (third panel). After a period of time the T cells are removed, and the presence of the specific cytokine is detected using an enzyme-labeled second antibody specific for the same cytokine. Where this antibody binds, a colored reaction product can be formed (fourth panel). Each T cell that originally secreted the cytokine gives rise to a single spot of color, hence the name of the assay. The results of such an ELISPOT assay for T cells secreting IFN-γ in response to different stimuli are shown in the last panel. In this example, T cells from a stem-cell transplant recipient were treated with a control peptide (top two panels) or a peptide from cytomegalovirus (bottom two panels). As can be seen, there are a greater number of spots in the bottom two panels, a clear indication that the patient’s T cells are able to respond to the viral peptide and produce IFN-γ. Photographs courtesy of S. Nowack.
A-22 ELISPOT assay.
A modification of the ELISA antigen-capture assay (see Section A-4), called the ELISPOT assay, is a powerful tool for measuring the frequency of T-cell responses and also provides information about the cytokines produced. Populations of T cells are stimulated with the antigen of interest, and are then allowed to settle onto a plastic plate coated with antibodies against the cytokine that is to be assayed (Fig. A.25). If an activated T cell is secreting that cytokine, the cytokine is captured by the antibody on the plastic plate. After a period of time the cells are removed, and a second antibody against the cytokine is added to the plate to reveal a circle (‘spot’) of bound cytokine surrounding the position of each activated T cell; it is these circles that give the ELISPOT assay its name. By counting each spot and knowing the number of T cells originally added to the plate, one can easily calculate the frequency of T cells secreting that particular cytokine. ELISPOT can also be used to detect specific antibody secretion by B cells, in this case by using antigen-coated surfaces to trap specific antibody and labeled anti-immunoglobulin to detect the bound antibody.
A-23 Identification of functional subsets of T cells based on cytokine production or transcription factor expression.
One problem with the detection of cytokine production on a single-cell level is that the cytokines are secreted by the T cells into the surrounding medium, and any association with the originating cell is lost. Three methods have been devised that allow the cytokine profile produced by individual cells to be determined. The first, that of intracellular cytokine staining (Fig. A.26), relies on the use of metabolic poisons that inhibit protein export from the cell. The cytokine thus accumulates within the endoplasmic reticulum and vesicular network of the cell. If the cells are subsequently fixed and rendered permeable by the use of mild detergents, antibodies can gain access to these intracellular compartments and detect the cytokine. The T cells can be stained for other markers simultaneously, and thus the frequency of IL-10-producing CD25+ CD4 T cells, for example, can be easily obtained.