Assessing main death pathways in T lymphocytes from HIV infected individuals.

Increased lymphocyte death is a hallmark of human immunodeficiency virus (HIV) infection. Although virological factors have been linked to this phenomenon, increased cell death rates are still observed in treated individuals in which viral replication is halted. To understand the nature of this remaining altered cell death, we have developed a simple and fast assay to assess major cell death pathways in lymphocytes isolated from HIV-infected individuals. The combination of three factors: (i) antibody staining to identify CD3(+) CD4(+) and CD3(+) CD8(+) cells, (ii) assessment of mitochondrial and plasma membrane function using DiOC6(3) or JC-1 probes and vital dyes, and (iii) caspase inhibition, allowed for the quantification of caspase-independent and -dependent cell death in CD4 and CD8 T cells. The latter mechanism was divided in intrinsic and extrinsic apoptotic pathways according to the sensitivity of the dissipation of mitochondrial membrane potential to Z-VAD-fmk or Q-VD-oPH treatment. Our data show similar results for both caspase inhibitors in treated infected individuals, whereas Q-VD-oPH showed a more potent inhibition in viremic individuals, yielding lower levels of intrinsic apoptosis. Comparison of DiOC6(3) and JC-1 probes yielded similar results in CD4 T cells, allowing for a clear definition of death mechanism in these cells. However, in CD8 T-cells, JC-1 showed heterogeneous staining and detected significantly lower levels of cell death with a higher contribution of intrinsic apoptosis. In conclusion, we provide a simple method to assess CD4 T-cell death mechanisms in HIV-infected individuals. The reasons and consequences of mitochondrial heterogeneity in CD8 T-cells require further evaluation.