A second method, which has the advantage that the cells being analyzed are not killed in the process, is called cytokine capture. This technique uses hybrid antibodies, in which the two separate heavyand light-chain pairs from different antibodies are combined to give a mixed antibody molecule in which
Cytokine-speciÿc antibodies are bound to the surface of a plastic well
Activated T cells are added to the well. These T cells are a mixture of cells with different effector functions
Cytokine secreted by some activated T cells is captured by the bound antibody
The captured cytokine is revealed by a second cytokine-speciÿc antibody, which is coupled to an enzyme, giving rise to a spot of insoluble colored precipitate
Fig. A.26 Cytokine-secreting cells can be identified by intracellular cytokine staining. Fluorochrome-labeled antibodies can be used to detect the cytokines secreted by activated T cells after the cytokine molecules have been allowed to accumulate inside the cell. The accumulation of cytokine molecules to a high enough concentration for efficient detection is achieved by treating the activated T cells with inhibitors of protein export. In such treated cells, proteins destined to be secreted are instead retained within the endoplasmic reticulum (left panel). These treated cells are then fixed, to cross-link the proteins inside the cell and in the cell membranes, so that they are not lost when the cell is permeabilized by dissolving the cell membrane in a mild detergent (center panel). Fluorochromelabeled antibodies can now enter the permeabilized cell and bind to the cytokines inside the cell (right panel). Cells labeled in this way can also be labeled with antibodies that bind to cell-surface proteins to determine which subsets of T cells are secreting particular cytokines.
Cytokine-speciÿc antibodies penetrate the cell to bind the intracellular cytokine molecules The cell is ÿxed and permeabilized with mild detergents Activated T cells are treated with an inhibitor that blocks protein export, allowing cytokines to accumulate in the ER the two antigen-binding sites recognize different ligands (Fig. A.27). In the bispecific antibodies used to detect cytokine production, one of the antigen-binding sites is specific for a T-cell surface marker, while the other is specific for the cytokine in question. The bispecific antibody binds to the T cells through the binding site for the cell-surface marker, leaving the cytokine-binding site free. If that T cell is secreting the particular cytokine, it is captured by the bound antibody before it diffuses away from the surface of the cell. It can then be detected by adding to the cells a fluorochrome-labeled second antibody specific for the cytokine.
A third method for identifying which T cells in a population produce a particular cytokine utilizes cytokine gene reporter mice. In these lines of mice, a cDNA clone encoding a readily detectable protein (the ‘reporter’ protein) is inserted into the 3ʹ untranslated region of the targeted cytokine gene downstream of a sequence known as an internal ribosome entry site (IRES). The IRES sequence allows translation of the reporter protein from the same mRNA as that encoding the cytokine; thus, the reporter protein is produced only when the cytokine mRNA is expressed (Fig. A.28). Common reporter proteins for this application are fluorescent proteins, such as green fluorescent protein (GFP). In fact, the GFP commonly used for this purpose contains