Expression of active caspase-3 is not a reliable apoptosis marker in stimulated CD4+ T-cells

One of the common methods for flow cytometry apoptosis detection is the intracellular active caspase-3 analysis. Still, in stimulated CD4+ T-cells this molecule expression may not be a sign of the guaranteed cell death. The aim of the present work was to analyze the active caspase-3 expression in stimulated CD4+ T-lymphocytes and to compare it with other widely used apoptosis and activity markers of CD4+ T-cells. We stimulated human peripheral blood mononuclear cells with anti-CD3/anti-CD28 antibodies for 48 hours and studied the expression of active caspase-3, activation (CD25), proliferation (Ki-67), and apoptosis (PO-PRO/PI and AnnexinV/PI) markers with flow cytometry. We found that under stimulation, the proportion of caspase-3-positive lymphocytes among CD4+CD45RA- T-cells increased by 5.7 times (P0.05). The relative number of cells expressing active caspase-3 was associated with activation (R=0.878; P0.05). Based on the data obtained, we conclude that the use of active caspase3 as the only marker to study apoptosis of stimulated CD4+ T-lymphocytes leads to false-positive results that mainly reflect cell activation. Reliable data requires the introduction of additional parameters that take into account both the induction of programmed cell death and lymphocyte activity.