Costimulatory pathways mediate monocyte-dependent lymphocyte apoptosis in HIV.

Examination of annexin V binding, an indicator of early apoptosis, on lymphocytes from HIV+ people immediately after isolation showed that both CD4(+) and CD8(+) T cells were apoptotic, whereas B cell apoptosis was induced mainly after incubation. CD8(+) T cell apoptosis correlated with fewer CD4(+) T cells, but not the level of viremia. To determine potential mechanisms for apoptosis, we examined FasL expression, which was dramatically elevated on CD14(+) monocytes; however, antibody to FasL did not reproducibly inhibit apoptosis. Rather, CD8(+) T cell apoptosis was caused by antigen-presenting cells because removal of monocytes or addition of antibodies to CD80 and CD86 reduced apoptosis. B cell apoptosis also involved costimulatory signals delivered by T cells but not monocytes. A unique CD8(bright)CD28(dim) T cell population died after costimulation by monocytes. Because this population was increased in patients with undetectable viremia, abnormal antigen-presenting cells may contribute to continued CD8(+) T cell exhaustion by inducing apoptosis.

Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells.

Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.