Measurement of TCell Cytotoxicity

Cytotoxicity, which has been shown to be important in clearing many viral infections [70-72], may be a more reliable indicator of the effector function of antigen-specific CD8+ T cells than cytokine synthesis. It could be important, for example, in controlling HIV-1 pathogenesis, though this has recently been questioned as HIV-1 antigen-specific CD8+ T cells appear to kill infected cells at a slow rate in vivo [43]. As previously alluded to, measuring IFN-y-secreting CD8+ T cells does not provide an estimate of cytotoxic potential, as these two activities can be independent of one another [73]. For instance, virus-specific CTL (CD8+ T cells) populations have been shown to vary in their capacity to secrete IFN-y and/or kill target cells within 5 h ex vivo [74], or after re-stimulation in vitro [75]. However, as with the IFN-y ELISPOT data, results from studies in which CTL cytotoxicity has been quantified during HIV infection are conflicting. Some have reported that ex-vivo CTL cytotoxicity is not detectable [76, 77], whereas others have found it in HIV-1-specific CD8+ T cells from either acute [31] or chronically infected patients [78-83]. This diverse information may possibly be due to the types of cytotoxicity assay employed in these studies. Most have used the bulk lysis assay approach in which cytotoxicity is measured in whole populations of T cells, such as the chromium release assay [85]. Alternatively, caspase activation has been measured in target cells undergoing lysis [86]. Such assays may provide results that are not correlated with other CD8+ T-cell functions, and thus cause misleading interpretations. The introduction of the Lysispot assay [73] to measure the frequencies of CTL in HIV-infected patients should provide more reliable results, as it can be used simultaneously with IFN-y ELISPOT assays. For the Lysispot assay, ELISPOT plates are coated with anti-P-galactosidase and autologous B cells loaded with HIV-1 peptide pools, and transfected with herpes simplex virus-Lac amplicons added as target cells. Each HIV-1-specific CTL migrates in the medium to attack and kill one target cell during an incubation time of 4 h, whereupon P-galactosidase is released and binds to the immobilized anti-P-galactosidase. CTL spots are visualized and enumerated following the sequential addition of a biotinylated anti-P-galactosidase, streptavidin-alkaline phosphatase and substrate mix (Fig. 6.4) [73, 84].

Cytotoxic Assays Ctl

Fig. 6.4 Schematic diagram illustrating the Lysispot assay. Here, ELISPOT plates are coated with anti-^-galactosidase. Autologous B cells are loaded with peptide pools and transfected with herpes simplex virus-Lac amplicons added as target cells. Each pep-tide-specific cytotoxic T lymphocyte (CTL) migrates in the medium to attack and kill one target cell during an incubation time of 4 h. Following B-cell lysis, P-galactosidase is released and binds to the immobilized anti-P-galactosidase. CTL spots are visualized and enumerated following sequential addition of biotinylated anti-^-galactosidase, streptavidin-alkaline phosphatase and substrate mix.

Fig. 6.4 Schematic diagram illustrating the Lysispot assay. Here, ELISPOT plates are coated with anti-^-galactosidase. Autologous B cells are loaded with peptide pools and transfected with herpes simplex virus-Lac amplicons added as target cells. Each pep-tide-specific cytotoxic T lymphocyte (CTL) migrates in the medium to attack and kill one target cell during an incubation time of 4 h. Following B-cell lysis, P-galactosidase is released and binds to the immobilized anti-P-galactosidase. CTL spots are visualized and enumerated following sequential addition of biotinylated anti-^-galactosidase, streptavidin-alkaline phosphatase and substrate mix.

By using the Lysispot and IFN-y ELISPOT assays together, Synder-Cappione and colleagues [84] showed that the relative frequencies of IFN-y-secreting and CTL varied markedly between different HIV peptide pools within the same patient, and some T cells lysed targets without secreting IFN-y. This finding indicates that the measurement of IFN-y production alone is probably insufficient to evaluate the breadth of the HIV-specific T-cell response. The further analysis of the frequency of directly cytotoxic HIV-specific T cells - for which the Lysispot assay provides a sensitive tool - may be of enhanced value, compared with IFN-y production, in assessing the potential efficacy of preventive HIV vaccines.

In addition to the Lysispot assay, novel flow cytometric assays for measuring cellmediated cytotoxicity are being developed. For example, active human CTL can be detected by an increased cell-surface expression of CD107a due to degranulation and the release of granzyme B, an effector of cytotoxicity in target cells [87, 88]. The parallel increase of annexin V binding to target cells due to target cell apoptosis (cell killing) can also be studied within the same assay. This flow cytometric assay has been designated the "LiveCount Assay", and permits measurement of CTL activation and frequency as well as of target cell death in the same sample [88]. Although aimed primarily at the ex-vivo detection of tumor antigen-specific T cells, the LiveCount Assay should also prove useful for detecting/measuring viral antigen-specific CTL.

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