Laser light suicide of proliferating virus-specific CD8+ T cells in an in vivo response

Virus-specific CD8+ CTL precursor (CTLp) frequencies are a function of rates of clonal expansion and loss, both of which are difficult to assess in vivo. Proliferating T cells incorporate the nucleoside analogue 5-bromo-2'-deoxyuridine (BrdU), making them sensitive to light-induced apoptotic cell death (suicide). Mice were infected with an influenza A virus, then given 50 mg of BrdU 24 h before sampling. Exposure of freshly isolated, CD8-labeled T cells to the laser beam of the flow cytometer resulted in the elimination of > 90% of the responding CTLp. The effect was obvious for the regional lymph node from day 6 to day 12 after priming, indicating continued cycling over a 7-day interval. Thus, as CTLp frequencies remain fairly constant from 7 to 30 days after infection, the persistent increase (> 30x) in numbers must be accompanied by a very substantial loss of virus-specific CD8+ T cells.