Research ArticleDENDRITIC CELLS

Constitutive resistance to viral infection in human CD141+ dendritic cells

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Science Immunology  07 Jul 2017:
Vol. 2, Issue 13, eaai8071
DOI: 10.1126/sciimmunol.aai8071
  • Fig. 1 Preferential infection of CD1c+ DCs by HIV-1, HIV-2, and influenza virus.

    (A) Absolute cell number of sorted DC subsets (n = 25 donors). Total DCs were enriched by negative selection with magnetic beads (input) and sorted by fluorescence-activated cell sorting (FACS). (B) Susceptibility of blood DCs to infection by HIV-2. GFP expression in blood DC subsets that were sorted and infected for 48 hours with GFP-coding HIV-2(JK) at a multiplicity of infection (MOI)GHOST X4R5 = 0.4. (C) Quantification as in (B) (n = 14 independent donors combined from seven independent experiments). (D) Susceptibility of blood DCs to infection by HIV-1 and impact of codelivered Vpx protein that degrades SAMHD1. GFP expression in blood DC subsets that were sorted and infected for 48 hours with GFP-coding HIV-1(BaL) (MOI = 0.8), HIV-1(BaL) Vpx (MOI = 0.4), HIV-1(NL4-3) (MOI = 0.6), or HIV-1(NL4-3) (MOI = 0.3). Viruses were not spinoculated in this experiment. (E) Quantification as in (D) (n = 4 independent donors combined from two independent experiments). Viruses were not spinoculated in this experiment. (F) Susceptibility of blood DCs to infection by influenza virus. GFP expression in sorted CD1c+ and CD141+ DCs pulsed with NS1-GFP H1N1 influenza virus [GFP-tagged FluA(PR8); MOI = 2] for 1 hour. Analysis was performed 4 hours after infection. (G) Quantification as in (F) in DCs at 24 hours after infection (n = 8; seven donors combined from seven independent experiments, including one donor repeated two times; analysis of variance). FSC, forward scatter. ***P < 0.001, ****P < 0.0001.

  • Fig. 2 Resistance of CD141+ DCs to HIV and influenza virus infection at the level of viral fusion.

    (A) HIV-1 fusion assay in blood DCs. Viral fusion revealed by CCF4 fluorescence in blood DCs after infection with HIV-1(BaL) (MOI = 0.8) or HIV-1(NL4-3) (MOI = 0.6) containing a BlaM-Vpr fusion protein. Fluorescence of the CCF4 product indicates viral fusion with target cells as a result of cleavage of the cell-loaded CCF4 substrate by the virus-contained BlaM. (B) Quantification as in (A) (n = 8 donors combined from four independent experiments). (C) Staining of GFP proteins contained in viral particles and CD44 in CD1c+ and CD141+ DCs after infection with HIV-1(V3R5) iGFP containing GAG-iGFP and GFP-Vpr fusion proteins, alone or in the presence of viral entry inhibitors MVC and TAK-779. Scale bars, 10 μm. (D) Quantification of the GFP density in GFPlow regions as in (C), shown for one representative donor (top) and average for five donors (bottom; combined from two independent experiments). (E) Levels of influenza virus receptors on blood DCs. SNA and MAA binding on CD1c+ and CD141+ DCs (representative of two independent experiments). (F) GFP expression in blood DC subsets that were sorted and infected for 48 hours with GFP-coding lenti(H1N1) Vpx at MOIGHOST X4R5 = 1 (n = 4 independent donors combined from two independent experiments). Viruses were not spinoculated. (G) Viral fusion revealed as in (A) by CCF4 fluorescence in blood DCs after infection with lenti(H1N1) (MOI = 1) containing a BlaM-Vpr fusion protein (n = 4 donors combined from two independent experiments). Viruses were not spinoculated. (H) GFP expression in blood DC subsets that were sorted and infected for 24 hours with HSV-1–GFP at MOI = 25 (one representative donor). (I) Quantification of GFP expression and frequency of live cells in blood DC subsets that were sorted and infected for 24 hours with HSV-1–GFP at MOI = 25 as in (H) (n = 10 combined from four experiments). (J) GFP expression in blood DC subsets that were sorted and infected for 24 hours with enhanced GFP–expressing VSV (VSVeGFP) at MOI = 16 (one representative donor). (K) Quantification of GFP expression and frequency of live cells in blood DC subsets that were sorted and infected for 24 hours with VSVeGFP at MOI = 16 as in (J) (n = 5 combined from two experiments). (L) Viral fusion revealed as in (A) by CCF4 fluorescence in blood DCs after infection with lenti(G) (MOI = 10) containing a BlaM-Vpr fusion protein (n = 13 combined from five experiments). ns, not significant; SSC, side scatter. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 RAB15 mediates resistance to HIV and influenza virus entry.

    (A) RAB15 expression in blood DCs in the public data set E-TABM-34 (left), measured by RNA-seq (average number of read counts for three independent donors; center), and measured by RT-qPCR (n = 6 independent donors; right). (B) Viral infection of THP-1 cells overexpressing RAB15 or RAB5A. Viral expression and live cell frequency in THP-1 cells expressing BFP-RAB15, BFP-RAB5A, or BFP after infection with GFP-encoding HIV-2(JK) (MOI = 1.2) 48 hours after infection, GFP-tagged FluA(PR8) (2 μg/ml) 24 hours after infection, GFP-encoding HSV-1–GFP (MOI = 0.016) 24 hours after infection, and GFP-encoding VSVeGFP (MOI = 25) 24 hours after infection. (C) Localization of BFP, BFP-RAB15, or BFP-RAB5A (green) and GM130 (red) in THP-1 cells. Scale bars, 5 μm. (D) Viral particle localization with GM130 in CD141+ DCs. Staining of GFP and GM130 in CD141+ DCs 12 hours after infection with lenti(H1N1) iGFP (top) and HIV-1(V3R5) iGFP (bottom) viral particles that contain GFP proteins (scale bar, 5 μm). (E) Quantification of HIV-1(V3R5) iGFP viral particle localization with GM130 as in (D). One representative donor (left) and average for five donors combined from two independent experiments (right). (F) Quantification of lenti(H1N1) iGFP viral particle localization with GM130 as in (D). One representative donor (left) and average for five donors combined from two independent experiments (right). (G) Frequencies of CD141+ and CD11c+ cells in CD34+ cells that were expanded, transduced with LacZ or RAB15 shRNA #4 or #5 lentivectors, and differentiated (representative of three independent donors). (H) GFP expression in CD34-derived CD141+ and CD11c+ DCs after infection of total CD34-derived cells with GFP-encoding HIV-1(BaL) Vpx or HIV-1(H1N1) Vpx (n = 3 combined from three independent experiments). (I) Role of RAB15 in the resistance of CD141+ DCs to viral infection. GFP expression in CD34-derived CD141+ DCs that were first transduced with the indicated shRNA lentivectors after infection of total CD34-derived cells with GFP-encoding HIV-1(BaL) Vpx or HIV-1(H1N1) Vpx (n = 4 combined from three independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 Distinct innate response to HIV and influenza virus determined by viral infection in blood DC subsets.

    (A) Response of blood DCs to HIV and role of viral replication. GFP and CD86 expression and IP-10 production by sorted blood DCs after infection with GFP-encoding HIV-1(BaL) Vpx for 48 hours alone or in the presence of viral inhibitors azidothymidine (AZT) and nevirapine (NVP) (n = 7 donors combined from four independent experiments; MOI = 0.4). (B) Expression of TLR7, TLR8, and actin in lysates from blood DC subsets. Blood DC subsets from three independent donors were combined at the same ratio, and the equivalent of 500,000 cells was loaded. One experiment is shown. Circles indicate protein sizes that match the cleaved C-terminal domain of TLR8 or TLR7 and arrowheads indicate protein sizes that match full-length TLR8 or TLR7 (53). (C) Response of blood DCs to HIV-1(Vpx) with or without the furin inhibitor DC1 that prevents maturation of TLR7 and TLR8 proteins. GFP, CD86, and IP-10 expression, and live cell frequency in CD1c+ DCs and CD141+ DCs infected with GFP-encoding HIV-1(BaL) Vpx for 48 hours alone or in the presence of AZT and NVP, with or without the furin inhibitor DC1 (MOI = 0.8). (D) Expression of cGAS and actin in lysates from blood DCs subsets. Blood DC subsets from three independent donors were combined at the same ratio, and the equivalent of 500,000 cells was loaded (representative of two independent experiments). (E) Role of cGAS in the response of CD1c+ DCs to HIV infection. Inhibition of cGAS expression in CD1c+ DCs. GFP and CD86 expression in CD1c+ DCs that were transduced with shRNA lentivectors against LacZ or cGAS and subsequently infected with GFP-encoding HIV-1(BaL) Vpx (MOI = 1) or transfected with cGAMP (1.3 μg/ml) for 48 hours. (F) Quantification of CD86 expression as in (E) (n = 3 donors from one experiment representative of two independent experiments). (G) Response of blood DCs to influenza virus. GFP expression and IP-10 production by sorted blood DCs pulsed with GFP-tagged FluA(PR8) or UV-inactivated GFP-tagged FluA(PR8) for 1 hour and incubated for 24 hours alone or in the presence of amantadine (n = 5 donors for GFP and n = 4 donors for IP-10, combined from two independent experiments; MOI = 2). (H) Response of blood DCs to GFP-tagged FluA(PR8) with or without the furin inhibitor DC1. CD86 expression and IP-10 production by CD1c+ and CD141+ DCs infected with GFP-tagged FluA(PR8) for 24 hours alone or in the presence of the furin inhibitor DC1 (n = 5 donors from two independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 Nonredundant antigen presentation by CD1c+ DCs infected with HIV and influenza virus.

    (A) Stimulation of a CD8+ T cell line by infected DCs. Combined intracellular expression of macrophage inflammatory protein–1β, tumor necrosis factor–α (TNF-α), IFN-γ, and interleukin-2 by an SL9-specific CD8+ T cell line exposed to blood DCs infected for 48 hours by HIV-1SL9(BaL) Vpx (MOI = 0.8) alone or in the presence of viral inhibitors AZT and NVP (n = 3 donors combined from three independent experiments). (B) IFN-γ concentration in culture supernatants of M1- and NS1-specific CD8+ T cell lines exposed for 18 hours to DCs that were treated with or without chloroquine for 30 min and then pulsed with FluA(PR8) (MOI = 2) for 1 hour (n = 3 donors combined from three experiments). (C) Down-regulation of HLA-ABC in HIV-infected DCs. HLA-ABC expression in Gag+ and Gag CD1c+ DCs 48 hours after infection with HIV-2(JK) nef+ (MOI = 1) or HIV-1(AD8) nef+ Vpx (MOI = 0.8) (n = 3). (D) GFP expression and viability in CD1c+ DCs at 4, 12, and 24 hours after infection with GFP-encoding influenza virus. (E) GFP expression and viability as in (D) (n = 4; three or four different donors combined from three independent experiments). (F) Down-regulation of HLA-ABC and CD86 in influenza virus-infected DCs. GFP, HLA-ABC, and CD86 expression in DAPI (4′,6-diamidino-2-phenylindole)–negative CD1c+ DCs analyzed before and 12 hours after infection with GFP-encoding influenza virus. (G) HLA-ABC expression as in (F) (n = 4; four different donors combined from three independent experiments). (H) CD86 expression as in (F) (n = 4; four different donors combined from three independent experiments). (I) T cell stimulation by infected versus bystander CD1c+ DCs. GFP expression in bulk CD1c+, sorted GFP+CD1c+, and GFP-CD1c+ DCs 24 hours after infection with GFP-encoding influenza virus (top). Carboxyfluorescein diacetate succinimidyl ester (CFSE) levels and FluM1-tetramer binding on CD8+ T cells after 7-day cocultures at 1:30 and 1:100 DC/T ratio (bottom). (J) Summary of the percentage of FluM1-tetramer binding CFSElow CD8+ T cells at 7 days after coculture as in (I) (n = 2; two different donors combined from two independent experiments). MFI, mean fluorescence intensity. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 6 DC subset cooperation for activation of antiviral T cells.

    (A) Stimulation of an SL9-specific CD8+ T cell line by HLA-A2 CD1c+ DCs mixed with HLA-A2+ CD141+ DCs after infection with HIV-1, outline of the experiment. (B) GFP expression in CD1c+ and CD141+ DCs and frequency of CD8+ T cells positive for TNF and IFN-γ expression 48 hours after infection with GFP-encoding HIVSL9(G) Vpx (MOI = 1) (n = 3 combined from two experiments). (C) Stimulation of M1- and NS1-specific CD8+ T cell lines by HLA-A2 DCs mixed with HLA-A2+ CD141+ DCs after infection with influenza virus, outline of the experiment. (D) GFP expression in mixed CD1c+ and CD141+ DCs 4 hours after infection with GFP-tagged FluA(PR8). (E) IFN-γ concentration in culture supernatants of M1- and NS1-specific CD8+ T cell lines exposed for 18 hours to DCs 12 hours after infection with GFP-tagged FluA(PR8) (MOI = 2; n = 3 combined from two experiments). **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 7 Resistance of CD141+ DCs to influenza virus in vivo in humanized mice.

    (A) Infection of human lung DC subsets. GFP expression in CD1c+ and CD141+ DCs sorted from human lungs and pulsed with GFP-tagged FluA(PR8) (MOI = 2) for 1 hour. Analysis was performed at 4 hours after infection. (B) Quantification as in (A) at 4 and 24 hours after infection (n = 2 donors from two independent experiments). (C) Intranasal infection of humanized mice by GFP-tagged FluA(PR8), outline of the experiment. (D) Detection of GFP in CD1c+ DCs and CD141+ DCs in the lungs of infected humanized mice as in (C), one representative sample and combined data (n = 5 from three independent experiments). (E) Detection of GFP in CD1c+ DCs and CD141+ DCs in the draining LNs of infected humanized mice as in (C), one representative sample and combined data (n = 3 from three independent experiments). (F) FACS plots showing the GFP and surface HA staining on lung DCs from phosphate-buffered saline (PBS) treatment or GFP-tagged FluA(PR8) infection of humanized mice as in (C). GFP+ HA and GFP+HA+ cells were examined for the expression of CD1c and CD141 (representative of two independent experiments). (G) Total GFP+ DCs as gated in (F) were further defined as CD1c+ and CD141+ DCs and analyzed for the expression of surface HA (red line). DC subsets from PBS-treated lungs (gray shaded) were stained with anti-HA as the control (each sample was a pool of three mice; representative of two independent experiments). (H) The same gating as in (F) on draining LN DCs from PBS treatment or GFP-tagged FluA (PR8) infection (each sample was a pool of three to six mice; representative of two independent experiments). (I) The same gating as in (F) on draining LN DCs from PBS treatment or GFP-tagged FluA (PR8) infection (each sample was a pool of three to six mice; representative of two independent experiments). (J) HA expression in draining LN DCs. Total GFP+ DCs as gated in (I) were analyzed for the expression of surface HA (red line). Isotype staining (gray shaded) and HA staining from PBS-treated LN DCs (blue line) as the control (representative of two independent experiments). (K) Model for T cell activation by CD141+ DCs that depends on productive infection of bystander CD1c+ DCs for the endocytic enveloped viruses tested. *P < 0.05, **P < 0.01.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/13/eaai8071/DC1

    Materials and Methods

    Fig. S1. Preferential infection of CD1c+ DCs by HIV-1, HIV-2, and influenza virus, related to Fig. 1.

    Fig. S2. Resistance of CD141+ DCs to HIV and influenza virus infection at the level of viral fusion, related to Fig. 2.

    Fig. S3. RAB15 mediates resistance to HIV and influenza virus entry, related to Fig. 3.

    Fig. S4. Infection of CD1c+ DCs selectively is required for cytosolic sensing of HIV and influenza virus, related to Fig. 4.

    Fig. S5. Infection of CD1c+ selectively results in nonredundant antigen presentation of HIV and nonstructural influenza virus antigen, related to Fig. 5.

    Fig. S6. DC subset cooperation for activation of antiviral T cells, related to Fig. 6.

    Fig. S7. Resistance of CD141+ DCs to influenza virus in humanized mice, related to Fig. 7.

    Table S1. List of antibodies used in the study.

    Table S2. List of plasmids used in the study.

    Table S3. List of primers to generate HIV-2(JK).

    Table S4. List of primers used for gene expression analysis.

    Table S5. Raw data used to generate all graphs that have n < 25.

  • Supplementary Materials

    Supplementary Material for:

    Constitutive resistance to viral infection in human CD141+ dendritic cells

    Aymeric Silvin, Chun I. Yu, Xavier Lahaye, Francesco Imperatore, Jean-Baptiste Brault, Sylvain Cardinaud, Christian Becker, Wing-Hong Kwan, Cécile Conrad, Mathieu Maurin, Christel Goudot, Santy Marques-Ladeira, Yuanyuan Wang, Virginia Pascual, Esperanza Anguiano, Randy A. Albrecht, Matteo Iannacone, Adolfo García-Sastre, Bruno Goud, Marc Dalod, Arnaud Moris, Miriam Merad, A. Karolina Palucka,* Nicolas Manel*

    *Corresponding authors. Email: karolina.palucka{at}jax.org (A.K.P.); nicolas.manel{at}curie.fr (N.M.)

    Published 7 July 2017, Sci. Immunol. 2, eaai8071 (2017)
    DOI: 10.1126/sciimmunol.aai8071

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Preferential infection of CD1c+ DCs by HIV-1, HIV-2, and influenza virus, related to Fig. 1.
    • Fig. S2. Resistance of CD141+ DCs to HIV and influenza virus infection at the level of viral fusion, related to Fig. 2.
    • Fig. S3. RAB15 mediates resistance to HIV and influenza virus entry, related to Fig. 3.
    • Fig. S4. Infection of CD1c+ DCs selectively is required for cytosolic sensing of HIV and influenza virus, related to Fig. 4.
    • Fig. S5. Infection of CD1c+ selectively results in nonredundant antigen presentation of HIV and nonstructural influenza virus antigen, related to Fig. 5.
    • Fig. S6. DC subset cooperation for activation of antiviral T cells, related to Fig. 6.
    • Fig. S7. Resistance of CD141+ DCs to influenza virus in humanized mice, related to Fig. 7.
    • Table S1. List of antibodies used in the study.
    • Table S2. List of plasmids used in the study.
    • Table S3. List of primers to generate HIV-2(JK).
    • Table S4. List of primers used for gene expression analysis.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S5 (Microsoft Excel format). Raw data used to generate all graphs that have n < 25.

    Files in this Data Supplement:

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