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HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits

Abstract

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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Figure 1: Infected primary macrophages transfer Nef to B cells and inhibit T cell–dependent class switching.
Figure 2: Nef is sufficient for macrophage-like cells to acquire class switch-inhibiting functions.
Figure 3: Nef uses multiple motifs to stimulate conduit formation in macrophage-like cells.
Figure 4: Infected primary macrophages transfer Nef to B cells through long-range actin-propelled conduits.
Figure 5: THP-1 cells inhibit class switching by trafficking membrane and vesicular Nef through Vav and small GTPases.
Figure 6: Nef inhibits class switching in GCs but not extrafollicular areas.
Figure 7: The ΔNef HIV-1 elicits more virus-specific IgG2, IgA1 and IgA2 than does wild-type HIV-1 regardless of viral load.

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Acknowledgements

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.).

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Authors and Affiliations

Authors

Contributions

W.X. and P.A.S. designed and did research; B.H and K.C. did research and discussed data; J.S.S., W.B.D., A.Cha., D.M.K. and A.Chi. provided samples and discussed data; M.S., T.J.K. and R.W.S. provided reagents and did research; S.C.B. provided clinical data; L.C.-G. did electron microscopy; and A.Ce. designed research, discussed data and wrote the paper.

Corresponding author

Correspondence to Andrea Cerutti.

Supplementary information

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

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