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. 2003 May;77(10):5846-54.
doi: 10.1128/jvi.77.10.5846-5854.2003.

In vivo evolution of human immunodeficiency virus type 1 toward increased pathogenicity through CXCR4-mediated killing of uninfected CD4 T cells

Andreas Jekle  1 , Oliver T Keppler, Erik De Clercq, Dominique Schols, Mark Weinstein, Mark A Goldsmith
Affiliations

In vivo evolution of human immunodeficiency virus type 1 toward increased pathogenicity through CXCR4-mediated killing of uninfected CD4 T cells

Andreas Jekle et al. J Virol. 2003 May.

Abstract

The destruction of the immune system by progressive loss of CD4 T cells is the hallmark of AIDS. CCR5-dependent (R5) human immunodeficiency virus type 1 (HIV-1) isolates predominate in the early, asymptomatic stages of HIV-1 infection, while CXCR4-dependent (X4) isolates typically emerge at later stages, frequently coinciding with a rapid decline in CD4 T cells. Lymphocyte killing in vivo primarily occurs through apoptosis, but the importance of apoptosis of HIV-1-infected cells relative to apoptosis of uninfected bystander cells is controversial. Here we show that in human lymphoid tissues ex vivo, apoptosis of uninfected bystander CD4 T cells is a major mechanism of lymphocyte depletion caused by X4 HIV-1 strains but is only a minor mechanism of depletion by R5 strains. Further, X4 HIV-1-induced bystander apoptosis requires the interaction of the viral envelope glycoprotein gp120 with the CXCR4 coreceptor on CD4 T cells. These results emphasize the contribution of bystander apoptosis to HIV-1 cytotoxicity and suggest that in association with a coreceptor switch in HIV disease, T-cell killing evolves from an infection-restricted stage to generalized toxicity that involves a high degree of bystander apoptosis.

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Figures

FIG. 1.
FIG. 1.
X4 but not R5 HIV-1 induces massive apoptosis among CD4 T cells. Human lymphoid cultures ex vivo were infected for 12 days with the R5 viral strain 49-5 (B and E) or the X4 viral strain NL4-3 (C and F) or kept uninfected (A and D). Apoptosis of CD4 T cells was determined independently by annexin V staining (A to F), caspase-3 activity (A to C), and depolarization of the mitochondrial membrane (D to F). Presented are flow cytometry dot plots from a typical experiment from among three independent experiments using different donor tissues.
FIG. 2.
FIG. 2.
X4 but not R5 HIV-1 induces pronounced apoptosis and depletion among CD4 T cells. Human lymphoid cultures ex vivo were infected with the R5 strain 1/85 or 49-5, the R5X4 strain 7/86, or the X4 strain NL4-3 or kept uninfected. (A) At day 12, apoptosis among CD4 and CD8 T cells was determined by using annexin V staining. (B) CD4 T-cell depletion displayed as CD4/CD8 ratio. (C) Viral replication was monitored in the same infections by assessing the p24 concentration in the culture medium at days 1, 4, 7, 10, and 13 after infection, using an anti-p24 ELISA. Shown are the mean values (n = 3) with standard errors of the means from a representative experiment from among six experiments with different donor tissue.
FIG. 3.
FIG. 3.
Apoptosis exceeds by far the infection level in NL4-3-infected lymphoid cultures ex vivo. CD3+ T cells were isolated from human lymphoid cultures ex vivo infected for 7 days with 49-5 (B and E) or NL4-3 (C and F) or from uninfected controls (A and D). (A to C) Viral infection was determined by HIV-1 RNA in situ hybridization. Representative infected cells are indicated by arrows. (D to F) Apoptosis was determined in parallel samples by using the TUNEL assay. Apoptotic cells are characterized by brown staining, and representative apoptotic cells are indicated by arrows.
FIG. 4.
FIG. 4.
X4 but not R5 strains induce extensive apoptosis in bystander CD4 T cells. Human lymphoid cultures ex vivo were infected for 7 days with the R5 strain 1/85 or 49-5, the R5X4 strain 7/86, the X4 strain NL4-3, or kept uninfected. Apoptosis in productively infected and uninfected CD4 T cells was determined by either annexin V staining (A and C) or caspase-3 activity (B). Intracellular p24 (A and B) or viral RNA (C) was used to determine viral infection. Error bars indicate standard errors of the means.
FIG. 5.
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.
FIG. 6.
FIG. 6.
HIV-1-induced bystander apoptosis critically depends on the gp120-CXCR4 interaction. (A) Human lymphoid cultures ex vivo were infected for 7 days with the R5 strain 1/85 or 49-5, the R5X4 strain 7/86, or the X4 strain NL4-3 or were kept uninfected. Apoptosis among CCR5+ CD4 cells was determined by annexin V staining, and infection was monitored simultaneously by intracellular p24 staining. (B and C) Uninfected, CFSE-labeled, CD4-enriched cells were cocultured with human lymphoid cultures ex vivo infected for 6 days with the X4 HIV-1 strain NL4-3 (B) or LAI (C) in the presence of the CXCR4-specific inhibitor AMD3100, an anti-CXCR4 antibody, or an anti-CCR5 antibody (B) or an anti-gp120 antibody (C). After 6 days of coculture, apoptosis in CFSE-positive CD4 T cells 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 three experiments with different donor tissues.
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