Fig 1.

(A to D) GM-CSF and TNF-α reactivate latent virus in a primary cell model for HIV-1 infection of CD34⁺ HPCs. (A) Time course for latency experiments. (B) Schematic diagrams of the HIV-1 reporter viruses used for these studies. Viruses were pseudotyped with the VSV-G envelope (center) or the CXCR4-tropic HIV-1 HXB envelope (right). The shading of vpr and vpu in HXB-ePLAP indicates that these genes are defective in full-length HXB. Filled rectangles represent genes that have been added to or deleted from the wild-type viral clone. (C) Flow cytometric analysis of cells sorted for reactivation protocols. (Top) Cord blood-derived HPCs infected with HXB-ePLAP/VSV-G and PLAP⁻ cells isolated by magnetic sorting; (bottom) cells infected with NL4-3-ΔGPE-GFP/VSV-G and GFP⁻ cells isolated by flow sorting. The percentages of live cells that are PLAP⁺ or GFP⁺ are given in each panel. Live cells were gated using forward scatter (FSC) and side scatter (SSC) parameters. (D) Flow cytometric analysis of cells for which results are shown in panel C after overnight incubation with STIF medium or GM-CSF (100 ng/ml) and TNF-α (2.5 ng/ml). CD34⁺ cells are gated on at the left; then PLAP or GFP expression in uninfected, STIF-treated, or GM-CSF- and TNF-α-treated cells is shown on the right. Numbers indicate the percentages of cells within the labeled gate. Live cells were gated based on FSC, SSC, and 7-AAD. (E) CD3⁺ T cells are not present in the CD34⁺ HPC population. Cord blood-derived HPCs were sorted and infected with NL4-3-ΔGPE-GFP/VSV-G as outlined in panel A. CD3 and CD34 staining was assessed on day 6 following flow sorting and overnight incubation in STIF medium. Live cells were gated using FSC, SSC, and 7-AAD. Data are representative of two independent experiments, one each using cord blood- or bone marrow-derived HPCs. (F to H) Latent infection that can be reactivated with GM-CSF and TNF-α occurs in immature CD133⁺ HPCs. (F) Flow cytometric analysis of CD133⁺ cells treated with HXB-ePLAP/VSV-G and sorted to remove actively infected (PLAP⁺) cells. The percentages of live cells are given in each quadrant. Live cells were gated using forward scatter and side scatter. (G) Flow cytometric analysis of cells sorted as in panel F and then stimulated overnight with STIF medium or GM-CSF and TNF-α. Live cells were defined by FSC, SSC, and 7-AAD. Numbers indicate the percentages of cells falling within the indicated gate. (H) Quantitation of reactivation in CD133⁺ cells (cells sorted for CD133 prior to stimulation) relative to that in cells incubated in STIF medium. Means and standard errors for 4 independent experiments are shown. The asterisk indicates a significant difference (P = 0.01) from the expected fold increase of 1 by a 1-sample t test.