Contact-dependent communication between immune cells generates protection but also facilitates viral spread. Here we found that macrophages formed long-range actin-propelled conduits in response to negative factor (Nef), a human immunodeficiency virus type 1 (HIV-1) protein with immunosuppressive functions. Conduits attenuated immunoglobulin G2 (IgG2) and IgA class switching in systemic and intestinal lymphoid follicles by shuttling Nef from infected macrophages to B cells through a guanine-exchange factor–dependent pathway involving the amino-terminal anchor, central core and carboxy-terminal flexible loop of Nef. By showing stronger virus-specific IgG2 and IgA responses in patients with Nef-deficient virions, our data suggest that HIV-1 exploits intercellular 'highways' as a 'Trojan horse' to deliver Nef to B cells and evade humoral immunity systemically and at mucosal sites of entry.
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We thank M. Stevenson (University of Massachusetts Medical School) for the ΔNef-ADA plasmid; B. Berkhout (Academic Medical Center) for the ΔNef-LAI plasmid; S.J. Burakoff (New York University) for the Nef-dsRED vector; M.G. Caron, (Duke University) for dominant negative dynamin-K44A and β-arrestin-2–V54D; J.G. Donaldson (National Institutes of Health) for dominant negative ARF6-T27N; Y. Zheng (Cincinnati Children's Hospital Medical Center) for dominant negative RhoA-N19, Cdc42-N17 and Rac1-N17; P. Marignani (Dalhousie University) for dominant negative Vav2-R/S; J.P. Moore (Weill Medical College of Cornell University) for the ΔNef-HIV-1–expressing NL4-3/9-7-dsRed plasmid; A. Pernis (Columbia University) for reagents; and all reagent donors for discussions. Supported by the US National Institutes of Health (AI07621 to W.X.; and R01 AI057653, R01 AI057653-S1 and R01 AI074378 to A.Ce.), The Irma T. Hirschl Charitable Trust (to A.Ce.), the Cornell Comprehensive Cancer Center (Chronic Lymphocytic Leukemia Research Center Award; to A.Ce.), the Ministerio de Ciencia e Innovación (Plan Nacional de Investigación Cientifica, Desarollo e Innovación Tecnológica SAF 2008-02725 to A.Ce.) and the Cancer Research Institute (to P.A.S.).
Supplementary Text and Figures
Supplementary Figures 1–15, Supplementary Tables 1–2 and Supplementary Methods (PDF 3542 kb)
Supplementary Movie 1
Membrane ruffling and protrusions in Nef-containing macrophage-like cells. Three-dimensional animation of a THP-1 macrophage-like cell expressing Nef-eGFP. The movie was generated by acquiring up to 15 XY planes with 0.4 ∼ 0.5 μm Z spacing by confocal microscopy. Three-dimensional views were constructed with maximum projection and exported as 30-40 tiff images. QuickTime Pro software was used to edit images into movies. One of several experiments yielding similar results. (MOV 1681 kb)
Supplementary Movie 2
Nef-containing macrophage-like cells form short-range intercellular bridges. Three-dimensional animation of two THP-1 macrophage-like cells expressing Nef-eGFP. One of several experiments yielding similar results. (MOV 2355 kb)
Supplementary Movie 3
Macrophage-like cells can transfer cytoplasmic material to B cells through both short- and long-range intercellular mechanisms upon activation. Time-lapse animation of macrophage-like THP-1 cells pre-loaded with LysoTracker (green) and co-cultured with IgD+ B cells in the presence of LPS, a microbial product with macrophage- but not B cell-stimulating activity. Live-cell DIC and epifluores cence images were acquired every 20 sec to generate this time-lapse movie. One of 5 experiments yielding similar results. (MOV 4555 kb)
Supplementary Movie 4
HIV-1-infected primary macrophages form long-range Nef-trafficking intercellular conduits. Three-dimensional animation of two primary macrophages infected with HIV-1 ADA and stained for Nef (red) in the presence of the membrane-specific lectin WGA (green). One of several experiments yielding similar results. (MOV 2257 kb)
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Xu, W., Santini, P., Sullivan, J. et al. HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits. Nat Immunol 10, 1008–1017 (2009). https://doi.org/10.1038/ni.1753