Fig. A.27 Hybrid antibodies containing cell-specific and cytokine-specific binding sites can be used to assay cytokine secretion by living cells and to purify cells secreting particular cytokines. Hybrid antibodies can be made by mixing together heavyand light-chain pairs from antibodies of different specificities, for example, an antibody against an MHC class I molecule and an antibody specific for a cytokine such as IL-4 (first panel). The hybrid antibodies are then added to a population of activated T cells, and bind to each cell via the MHC class I binding arm (second panel). If some of the cells in the population are secreting the appropriate cytokine, IL-4, this is captured by the cytokine-specific arm of the hybrid antibody (third panel). The presence of the cytokine can then be revealed, for example, by using a fluorochrome-labeled second antibody specific for the same cytokine but binding to a different site from the one used by the hybrid antibody (last panel). The labeled cells are analyzed by flow cytometry or are isolated using a fluorescence-activated cell sorter (FACS). Alternatively, the second cytokine-specific antibody can be coupled to magnetic beads, and the cytokine-producing cells isolated magnetically.
An IRES element and the eGFP coding sequences are inserted downstream of the cytokine gene stop codon a point mutation that greatly improves its spectral properties for experimental purposes. This version of GFP is commonly referred to as ‘enhanced GFP,’ or ‘eGFP’ for short. eGFP can be detected by FACS or by fluorescent microscopy using the settings designed to detect the commonly used fluorescent dye FITC. Due to broad utility of these fluorescent proteins, a host of GFP derivatives have been developed by genetic engineering of the original GFP protein. Each derivative has distinct fluorescent properties and therefore can be uniquely identified, allowing these proteins to be used in combination with each other to provide information about multiple cytokines at the same time (Fig. A.29).
Several of these techniques for measuring cytokine expression by T-cell subsets have been adapted to examine the transcription factors expressed by T cells and other lymphocytes, providing an alternative method for identifying functional lymphocyte subsets. In one approach, antibodies specific for lineage-defining transcription factors are used to label permeabilized cells. As was described above for the intracellular cytokine staining assay, the cells can then be examined by flow cytometry or immunofluorescence microscopy. Lines of reporter mice have also been generated, in which the gene locus encoding a transcription factor is modified to express a fluorescent protein, such as eGFP. For both of these approaches, the advantage of using transcription factors to identify lymphocyte subsets is that there is no need to stimulate the cells prior to antibody staining or to assessing reporter protein expression, as lineage-defining transcription factors are constitutively expressed by the cells. Therefore, this approach is more widely used for the identification of T-cell and other lymphocyte subsets in intact tissues by microscopy.