FIG. 5.

Bystander CD4 T cells undergo apoptosis when cocultured with X4 HIV-infected cells or upon exposure to cell-free supernatants from X4 HIV-infected cultures. (A) Schematic representation of the experimental setup. CD4 T cells were isolated from uninfected ex vivo human lymphoid cultures and labeled with CFSE. A total of 150,000 CFSE-labeled CD4-enriched cells (more than 90% CD4 T cells) were added to human lymphoid cultures ex vivo from the same donor, which had been infected for 6 days with the indicated HIV-1 strains. Cells were cultured in the presence or absence of the viral reverse transcriptase inhibitor AZT (5 μM) for 6 days longer. The medium was changed every 3 days, and AZT was readded to the respective cultures. (B) After 6 days of coculture, apoptosis in CFSE-positive CD4 T cells was determined by using annexin V staining. In parallel, infection of the CFSE-positive CD4 T cells was analyzed by intracellular p24 staining (inset). (C) Viral replication was monitored in the same cultures by assessing the p24 concentration in the culture supernatant at days 3, 6, 9, and 12 after infection by using an anti-p24 ELISA. AZT was added at days 6 and 9 to the indicated samples. Shown are the mean values (n = 3) with standard errors of the means from a representative experiment from among at least three experiments with different donor tissue. (D) Culture supernatants of human lymphoid cultures ex vivo infected with the R5 strain 49-5 or the X4 strain NL4-3 were collected at days 6 and 9, sterile filtered, and diluted 1:1 with fresh medium. The concentration of p24 in the diluted supernatants was determined by using an anti-p24 ELISA and adjusted to 45 ng/ml, and the supernatants were added to uninfected cultures of cells from the same donor. The cells were cultured for an additional 6 days in the presence or absence of 5 μM AZT, and apoptosis was determined by using annexin V staining. Shown are the mean values (n = 3) with standard errors of the means from a representative experiment from among two experiments with different donor tissue.