Megan E. Sheehy, Adrian B. McDermott, Scott N. Furlan, Paul Klenerman and Douglas F. Nixon

Cytotoxic T cell (CTL) activity has traditionally been assessed by a standard ⁵¹Chromium release assay, which usually requires an autologous Epstein-Barr virus transformed B lymphoblastoid cell line. However, when assessing cross clade CTL responsiveness from vaccinees or HIV clade disparate infected subjects it is often impractical to obtain more than one sample of blood. In order to maximize the information on HIV specific CTL responses in these subjects, we have developed a novel CTL killing assay based upon flow cytometry. This fluorometric analysis of T-lymphocyte antigen-specific lysis (FATAL) assay uses 2 dyes to label the target cell population, and cells which are killed lose their dye. This assay is sensitive, specific and can be used in an autologous system to test for HIV specific CTL responses in a single PBMC population.