Blockade of granzyme B reduces viral cross-class serpin inhibition of T cell induced T cell apoptosis.

<p>Jurkat T cells were treated with PMA and ionophore (PI) and the level of granzyme B expressed was measured by IEPDase assay and caspase 3 and 7 activity by DEVDase assay (<b>A</b>). Increased granzyme B (GzmB, p<0.001) secreted by these cells and was inhibited by treating the cells with an intracellular inhibitor of granzyme B, ZAAD-CMK (p<0.001) or anti-granzyme B antibody (p<0.001) (<b>A</b>). The medium containing granzyme B from PI treated T cells (CTLm) was applied to naive HUVECs to induce apoptosis (<b>B</b>). Treatment with Serp-2, but not CrmA, reduced caspase activity in CTLm treated HUVECs (<b>B</b>). The CTLm was also applied to naive T cells in culture to induce apoptosis and increased levels of caspase 3 and granzyme B were observed as IEPDase activity (<b>C</b>, p<0.0001) and DEVDase activity (<b>D</b>, p<0.0001) respectively. Treatment with Serp-2 reduced both granzyme B (<b>C</b>, p<0.01) and caspase 3 (<b>D</b>, p<0.0009) activities significantly. Antibody to granzyme B (GzmB) blocked Serp-2 mediated reductions in CTLm induced granzyme B (<b>C)</b> when compared to Serp-2 treatment alone (p<0.0004), but with a still significant decrease (p<0.005) when compared to CTLm treatment alone. Antibody to granzyme B also blocked the Serp-2 mediated decrease in caspase 3 (<b>D</b>, p<0.149) when compared to CTLm activation. This Serp-2 mediated inhibition of CTLm induced granzyme B activity was also blocked by incubation of cells with antibody to perforin (<b>C</b>, p = 0.412) but not the caspase 3 activity (<b>D</b>, p<0.0193). The results shown here represent mean ± SE from 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance (ANOVA) with secondary Fishers least significant difference and Mann Whitney analysis.