CXCR4 utilization is sufficient to trigger CD4+ T cell depletion in HIV-1-infected human lymphoid tissue

Abstract

The human chemokine receptors CCR5 and CXCR4 have emerged as the predominant cofactors, along with CD4, for cellular entry of HIV-1 in vivo whereas the contribution of other chemokine receptors to HIV disease has not been yet determined. CCR5-specific (R5) viruses predominate during primary HIV-1 infection whereas viruses with specificity for CXCR4 (R5/X4 or X4 viruses) often emerge in late stages of HIV disease. The evolution of X4 viruses is associated with a rapid decline in CD4+ T cells, although a causative relationship between viral tropism and CD4+ T cell depletion has not yet been proven. To rigorously test this relationship, we assessed CD4+ T cell depletion in suspensions of human peripheral blood mononuclear cells and in explants of human lymphoid tissue on exposure to paired viruses that are genetically identical (isogenic) except for select envelope determinants specifying reciprocal tropism for CXCR4 or CCR5. In both systems, X4 HIV-1 massively depleted CD4+ lymphocytes whereas matched R5 viruses depleted such cells only mildly despite comparable viral replication kinetics. These findings demonstrate that the coreceptor specificities of HIV-1 are a causal factor in CD4+ T cell depletion ex vivo and strongly support the hypothesis that the evolution of viral envelope leading to usage of CXCR4 in vivo accelerates loss of CD4+ T cells, causing immunodeficiency.

Figure 1

Reciprocal utilization of CXCR4 and CCR5 by paired, isogenic HIV-1 strains differing in envelope determinants. COS-7 cells were transfected with plasmids encoding CD4 alone or CD4 in combination with CCR5 or CXCR4. Cells subsequently were infected with pseudotype NL4–3 or 49–5. After 48 h, luciferase expression was assessed by enzymatic activity (relative light units). (a) Transfection with CD4 (black bars) alone or with CD4/CXCR4 (red bars). (b) Transfection with CD4 (black bars) alone or with CD4/CCR5 (blue bars).

Figure 2

CD4+ T lymphocyte depletion in PBMC cultures by paired HIV-1 strains. Shown are CD4+/CD8+ ratios in PBMC infected with NL4–3 (red bars) or 49–5 (red/blue bars) after a 10-day infection. The ratios are expressed as a percentage of that in a control (uninfected) PBMC culture. (Inset) The concentration of p24 in culture medium of PBMC infected with NL4–3 (red circles) or 49–5 (red/blue squares). These experiments are representative of three independent experiments using PBMC isolated from different donors. Color code: An individual color was assigned to each viral isolate as shown under the bar graph. The bar colors for isogenic chimeras correspond to the colors of parental viruses whereas the color of the edges correspond to the sources of Env sequences.

Figure 3

CD4+ T cell depletion and viral replication in human tonsillar tissue infected ex vivo by matched HIV-1 strains. (a) CD4+ T cell depletion, as assessed by CD4+/CD8+ ratio on day 12 after infection. For each measurement, cells were isolated from 6–10 tissue blocks and were analyzed by flow cytometry. Mean values (± SEM) are shown from experiments with tissues from three to six different donors infected in parallel with a panel of tested viruses. (b) Viral replication, as assessed by p24 values in the histoculture supernatants. Tissues from three to five different donors were infected as indicated, and viral replication was monitored. To compare replication kinetics in tissues from different donors, the absolute value for viral replication for a given donor tissue was normalized by the maximum value of viral replication for the entire tested panel. Mean values ± SEM are shown for experiments with tissues from three to six different donors. (Inset) Absolute value of viral replication of the same histocultures, as assessed by measurements of p24 concentration in the culture medium measured on day 12 after infection. For each measurement, medium bathing six blocks of tissue was pooled. Presented are mean values ± SEM for experiments with tissues from three to six different donors. The same tissue blocks were used for measurements presented in a and b. The color code is the same as in Fig. 2.

Figure 4

CD4+ T lymphocyte depletion in human spleen histocultures by paired HIV-1 strains. Mean CD4+/CD8+ ratios from duplicate samples are shown from an experiment performed with human spleen histocultures infected with NL4–3 or 49–5. The p24 concentrations of these viruses in the culture medium over time are indicated as in Fig. 3 (Inset). The color code is the same as in Fig. 2.

Figure 5

Viability of purified CD4+ T lymphocyte cultures after infection by paired HIV-1 strains. CD4+ T lymphocyte cultures were infected with NL4–3 or 49–5. Viability of the cultures also was assessed 10 days after infection by counting cells that excluded trypan blue, and results are expressed as a percentage of the viable cell counts in the uninfected control cultures; this experiment was performed in duplicate, and mean values are shown. (Inset) The p24 concentrations of these viruses in the culture medium over time. The color code is the same as in Fig. 2.