Active recombinant GzmC delivered by perforin causes target cell death.

(A) Target cell viability. YAC1 cells were treated with a fixed dose of purified perforin (Pfn; 1 μg) or recombinant granzymes alone as negative controls. Cells were treated with 50 nM rGzmB, rGzmBSer183Ala, rGzmC, or rGzmCSer184Ala plus perforin and were incubated for 1 hour at 37°C. Next, samples were plated using limiting dilution in duplicate 96-well microplates and were incubated for 14 days at 37°C. The percentage of total plated cells that formed clones is plotted as the mean ± SEM. This experiment was repeated 4 times with similar results. (B) (top row) YAC1 cells (10⁵) were treated with nothing, perforin, rGzmB, or rGzmC alone for 1 hour at 37°C. Forward scatter properties and 7-AAD staining were quantified by flow cytometry. (bottom rows) YAC1 cells, treated with perforin plus increasing doses of rGzmB or rGzmC for 1 hour at 37°C, were stained with 7-AAD and analyzed by flow cytometry. (C) Dose-response. (Left panel) Percentages of 7-AAD^lo target cells, obtained from panel C, are shown for increasing doses of granzymes. (Right panel) At the end of the 1-hour assay, target cells were plated in duplicate using limiting dilution. The percentage of total plated cells that yielded clones was quantified at 14 days. This experiment was repeated 4 times with similar results.