Interleukin-7 induces expression of latent human immunodeficiency virus type 1 with minimal effects on T-cell phenotype

Abstract

Latent human immunodeficiency virus type 1 (HIV-1) persists even in patients treated with antiretroviral therapy. New treatment strategies are therefore needed to eradicate this latent viral reservoir without reducing immune cell function. We characterize the interleukin-7 (IL-7)-induced stimulation of primary human T cells and thymocytes and demonstrate, using the SCID-hu model, that IL-7 induces substantial expression of latent HIV while having minimal effects on the cell phenotype. Thus, IL-7 is a viable candidate to activate expression of latent HIV and may facilitate immune clearance of latently infected cells.

FIG. 1.

Effect of IL-7 on human thymocytes. (A) Anti-CD3, anti-CD28 costimulated normal peripheral blood lymphocytes, either alone (right panel) or treated with N-butyrate (left panel), are shown with the cell cycle status indicated in each quadrant, as an example of the assay used in panel B, and throughout this work. DNA is depicted on the vertical axis, and RNA is depicted on the horizontal axis. The position of the vertical axis marks the division between the G_1A and G_1B stages of the cell cycle and is determined by the N-butyrate-treated cells, which are blocked at the G_1A-to-G_1B transition. Positions indicating stages in the cell cycle are illustrated in the right panel. (B) Four-color cell cycle analysis was performed on fetal thymocytes in whole-lobe thymic organ culture either alone or in the presence of IL-7 for 4 days. Three independent experiments were performed, of which one representative is shown. Top row, CD4SP and CD8SP cell populations were backgated (arrows) and analyzed for the presence of IL-7-induced changes in cell cycle progression (bottom row).

FIG. 2.

Effect of IL-7 on HIV-1 latency. (A) CD4SP thymocytes or pooled peripheral blood lymphocytes (PBL) and splenocytes were isolated from infected (HIV_NL-r-HSAs or HIV_JR-CSF) SCID-hu Thy/Liv implants and were cultured for three days in the presence of Protease Inhibitor, and under the following stimulation conditions: unstimulated (white), costimulated (grey), IL-7 (black), and IL-2 (striped). HIV-1 p24^gag protein concentrations in the supernatants were measured to assess virus production. Each grouping of four bars represents one experiment. Unstimulated peripheral cells fell below the limit of detection (6 pg/ml). (B) Expression of virus-encoded reporter (mu CD24) indicating activation of latent virus. Mature CD4 SP thymocytes from Nl-r-HSAs infected implants were treated for 3 days without stimulation (US) or with IL-2, IL-7, or anti-CD3/CD28 costimulation as indicated. Quadrant gates were set on isotype controls.

FIG. 3.

Effect of IL-7 on surface phenotype of quiescent human lymphocytes. Total, CD45RA⁻, or CD45RO⁻ quiescent lymphocytes were cultured in the presence or absence of IL-7. Developmental and stimulation markers were examined using flow cytometric methods. (A) Total, CD45RA⁻, or CD45RO⁻ quiescent lymphocytes were cultured for 4 days as described previously and then analyzed for expression of CD45RA (x axis) and CD45RO (y axis) in the presence (bottom row) and absence (middle row) of IL-7, as well as on day 0 (top row). (B) CD45RA⁻/4⁻, CD45RA⁻/8⁻, CD45R0⁻/4⁻, and CD45R0⁻/8⁻ quiescent lymphocytes were isolated and cultured under the following stimulation conditions: unstimulated, IL-7, IL-2, or anti-CD3 and anti-CD28 costimulation for 3 days. Lymphocyte subsets were then analyzed for changes in expression of CCR7 (x axis) and CD62-L (y axis).

FIG. 4.

Effect of IL-7 on cell cycle status of quiescent human lymphocytes. Quiescent CD45RA⁻/CD4⁻, CD45RA⁻/CD8⁻, CD45R0⁻/CD4⁻, and CD45R0⁻/CD8⁻ lymphocytes were isolated, and cultured under the following stimulation conditions: unstimulated, IL-7, IL-2, or anti-CD3 and anti-CD28 costimulation. After 3 days, cells were stained to detect DNA (y axis) and RNA (x axis) and analyzed to determine cell cycle status. Control stimulations performed in the presence of cell cycle inhibitor (N-acetyl butyric acid) and were used to set quadrant positions (bottom row). Seven similar experiments were performed.