Identification of a potential pharmacological sanctuary for HIV type 1 in a fraction of CD4(+) primary cells

Abstract

We have identified a subset of HIV-susceptible CD4(+)CCR5(+) cells in human PBMCs that can efficiently exclude protease inhibitors (PI) due to high P-glycoprotein (P-gp) efflux activity. Phenotypically these cells are heterogeneous, include both T and non-T cells, and some display markers of memory cells. Cells with high P-gp represent 16-56% (median = 37.3) of all CD4(+)CCR5(+) cells in healthy donors, and are selectively depleted in HIV-1-infected individuals (4.1-33%, median = 10.1). A fraction of primary cells productively infected by HIV-1, in vitro, have high P-gp pump activity, demonstrating that infection does not inhibit P-gp function. In agreement with these data, HIV-susceptible cells expressing high levels of P-gp require higher levels of PI for complete inhibition of virus spread. We conclude that the PI concentrations achieved in plasma could be suboptimal for full inhibition of virus spread in high P-gp cells, indicating that they may represent a pharmacological sanctuary for HIV-1.

FIG. 1.

Analysis of P-gp efflux pump in human primary PBMCs. (A) PBMCs were loaded with Rh123 dye, washed, and resuspended in Rh123-free medium. The amount of intracellular Rh123 dye was monitored by flow cytometry in the FL1 detector at time 0 (immediately after loading with Rh123) and after 3 h of incubation in Rh123-free medium. Prior to flow cytometric analysis, the cells were stained with anti-CD3, CD4, CD8, and CD56 monoclonal antibodies. Cell populations in the histograms were gated according to their staining with the following antibodies: T lymphocytes, CD3⁺: NK, CD3⁻CD56⁺; CD4⁺, CD3⁺CD4⁺; CD8⁺, CD3⁺CD8⁺. The arrow in the histogram with the CD4⁺ cells highlights the presence of a CD4⁺ subset with high levels of P-gp activity (Rh123 free). (B) Four color flow cytometric analysis of cells loaded with Rh123 and stained with CD3APC, CD4PerCP, and CD56PE antihuman monoclonal antibodies. Dot plot to the right displays the phenotype of gated cells expressing CD4 and high P-gp activity. (C) Four color flow cytometric analysis of cells loaded with Rh123 and stained with CD4PerCP, CD45ROAPC, and CCR5PE antihuman monoclonal antibodies. CD4⁺ cells were gated based on their CCR5 expression. The phenotype and P-gp activity of the gated cells (R3, CD4⁺CCR5⁺; R4, CD4⁺CCR5⁻) are shown in the corresponding dot plots to the right. Numbers in the quadrants represent the percentage of cells within the gated populations.

FIG. 2.

P-gp activity in primary CD4⁺CCR5⁺ cells and PBMCs of healthy donors and HIV-infected individuals. PBMCs were loaded with Rh123 and the amount of intracellular dye was measured by flow cytometry after 3 h of incubation in Rh123-free medium. Cells were stained with anti-CD4, anti-CCR5, and anti-CD45RO antibodies prior to flow cytometric analysis. (A) Identification of a progressively depleted population of memory CD4⁺CCR5⁺ lymphocytes with high P-gp activity in HIV⁺ individuals. Examples of two patients compared to a healthy donor (left panel). Density plots show P-gp activity on gated CD4⁺CCR5⁺ primary lymphocytes. CD45RO was used as a memory marker. (B) Comparison of the percentage of P-gp high cells within the CD4⁺CCR5⁺ population in healthy donors (n = 9) and HIV-infected individuals (n = 27). The decrease of the P-gp high, CD4⁺CCR5⁺ population in HIV-infected individuals is significant (p = 0.0004, unpaired t test not assuming equal variances). (C) Rh123 remaining in total PBMCs of uninfected donors and HIV-infected individuals. Bars represent the amount of intracellular Rh123 remaining in PBMC samples, 3 h after washing out the dye, as percentage of the initial intracellular Rh123 on loading. The average measurements and standard deviations for 11 healthy uninfected donors and 33 HIV-1 patients are presented.

FIG. 3.

Identification of a subset of HIV-1-infected PBMCs with high P-gp pump activity. (A) PBMCs from healthy donors were infected with HIV-1 molecular clones tagged with GFP. At the peak of viral infection the cells were loaded with the rhodamine derivative TMRE, and P-gp activity was monitored by measuring the decrease in intracellular dye 3 h after loading. Infected cells were identified by GFP expression. Two populations of infected cells with high and low P-gp activity (boxed), respectively, were identified based on their TMRE efflux. (B) Prior to flow cytometric analysis, the cells were stained with anti-CD56 and anti-CD3 mAbs. The histograms show P-gp activity in infected cell subsets identified by their phenotype on staining with the monoclonal antibodies as in Fig. 1B. Numbers in the histograms represent the percentage of the gated cells included within the indicated markers (high and low P-gp activity).

FIG. 4.

Suboptimal antiviral effects of ritonavir in primary lymphocytes with high P-gp activity. Primary CD4⁺ T cells from a healthy donor were sorted according to high or low P-gp activity, as judged by their Rh123 intracellular content after 3 h of incubation in medium without the dye. (A) Dot plots of the cells showing CD4 expression and rhodamine content before (left) and after (right) sorting. (B) Effect of ritonavir (5 or 50 nM) on HIV-1 production in the two sorted cell populations is shown in (A).

FIG. 5.

Inhibition of P-gp activity by protease inhibitors. PBMCs were stained with anti-CD3 and CD56 antibodies after 3 h of incubation in Rh123 free medium in the presence of the indicated PI concentrations. Gates were set for CD3⁺ (T lymphocytes) and CD3⁻CD56⁺ (NK) cells. Histograms show the number of cells with the corresponding amounts of intracellular Rh123 in the presence of increasing concentrations of either indinavir (A) or ritonavir (B). Numbers within each histogram represent the percentage of cells able to pump out the dye in the presence of the indicated amounts of PIs. Dose-dependent inhibition of P-gp activity by the PIs is shown in (C).