Accuri C6 Flow Cytometer System
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Published online before print July 6, 2005
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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0505237

Received for publication May 2, 2005.
Revised May 16, 2005.
Accepted for publication June 3, 2005.

Article

HLA-A2 down-regulation on primary human macrophages infected with an M-tropic EGFP-tagged HIV-1 reporter virus

Amanda Brown *{dagger}@, Suzanne Gartner *, Thomas Kawano {dagger}, Nicole Benoit *, and Cecilia Cheng-Mayer {dagger}

*Johns Hopkins University School of Medicine, Department of Neurology, Baltimore, Maryland; and {dagger}Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York

@ To whom correspondence should be addressed. E-mail: abrown76{at}jhmi.edu.


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Abstract

Multiple mechanisms are used by the human immunodeficiency virus type 1 (HIV-1) to interfere with host-cell immune effector functions. The 27-kD Nef protein has been shown to down-modulate specific genes of the major histocompatibility complex class I (MHC-I) on the surface of infected primary T cells, facilitating their escape from lysis by cytolytic T lymphocytes. Macrophages, as the other major immune cell type targeted by the virus, also contribute to the transmission, persistence, and pathogenesis of HIV-1. Yet, whether Nef modulates MHC-I expression on HIV-infected primary macrophages remains unclear. Currently available infectious HIV-1 molecular clones, which express a reporter gene, only infect T cells and/or do not express Nef. To overcome these limitations, we generated macrophage-tropic green fluorescent protein (GFP)-tagged HIV-1 viruses, which express the complete viral genome, and used these to assess the expression of human leukocyte antigen (HLA)-A2 on the surface of productively infected macrophages. The reporter viral genomes were replication-competent and stable, as Nef, p24 antigen, and GFP expression could be detected by immunostaining of infected, monocyte-derived macrophages (MDM) after more than 2 months postinfection. Fluorescein-activated cell sorter analyses of infected macrophages and T cells revealed that although wild-type reporter virus infection induced a statistically significant decrease in the density of surface HLA-A2, down-regulation of HLA-A2 was not seen in cells infected with reporter viruses encoding a frameshift or a single point mutation in Nef at prolines 74P and P80. The impact of Nef on HLA-A2 surface expression in MDM was also confirmed by confocal microscopy. These results suggest that the mechanisms of HLA-A2 down-modulation are similar in primary T cells and macrophages.

Key Words: Nef • MHC • confocal microscopy • immunofluorescence




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A. Brown, H. Zhang, P. Lopez, C. A. Pardo, and S. Gartner
In vitro modeling of the HIV-macrophage reservoir
J. Leukoc. Biol., November 1, 2006; 80(5): 1127 - 1135.
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