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. 2017 Jul 7;2(13):eaai8071.
doi: 10.1126/sciimmunol.aai8071.

Constitutive Resistance to Viral Infection in Human CD141 + Dendritic Cells

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Free PMC article

Constitutive Resistance to Viral Infection in Human CD141 + Dendritic Cells

Aymeric Silvin et al. Sci Immunol. .
Free PMC article

Abstract

Dendritic cells (DCs) are critical for the launching of protective T cell immunity in response to viral infection. Viruses can directly infect DCs, thereby compromising their viability and suppressing their ability to activate immune responses. How DC function is maintained in light of this paradox is not understood. By analyzing the susceptibility of primary human DC subsets to viral infections, we report that CD141+ DCs have an innate resistance to infection by a broad range of enveloped viruses, including HIV and influenza virus. In contrast, CD1c+ DCs are susceptible to infection, which enables viral antigen production but impairs their immune functions and survival. The ability of CD141+ DCs to resist infection is conferred by RAB15, a vesicle-trafficking protein constitutively expressed in this DC subset. We show that CD141+ DCs rely on viral antigens produced in bystander cells to launch cross-presentation-driven T cell responses. By dissociating viral infection from antigen presentation, this mechanism protects the functional capacity of DCs to launch adaptive immunity against viral infection.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
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 FACS (B) Susceptibility of blood DCs to infection by HIV-2. GFP expression in blood DC subsets that were sorted and infected for 48hr with GFP-coding HIV-2(JK) at MOIGHOST X4R5=0.4. (C) Quantification as in (B) (n=14 independent donors combined from 7 independent experiments). (D) Susceptibility of blood DCs to infection by HIV-1 and impact of co-delivered Vpx protein that degrades SAMHD1. GFP expression in blood DC subsets that were sorted and infected for 48hr 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 (D) (n=4 independent donors combined from 2 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 1hr. Analysis was performed 4hr after infection. (G) Quantification as in (F) in DCs at 24hr post-infection (n=8; 7 donors combined from 7 independent experiments, including 1 donor repeated 2 times; ANOVA).
Fig. 2
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 CCF4 product indicates viral fusion with target cells as a result of cleavage of the cell-loaded CCF4 substrate by the viral-contained beta-lactamase (BlaM). (B) Quantification as in (A) (n=8 donors combined from 4 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 Bar = 10µm. (D) Quantification of the GFP density in GFPlow regions as in (C), shown for one representative donor (top) and average for 5 donors (bottom; combined from 2 independent experiments). (E) Levels of influenza virus receptors on blood DCs. SNA and MAA binding on CD1c+ and CD141+ DCs (representative of 2 independent experiments). (F) GFP expression in blood DC subsets that were sorted and infected for 48hr with GFP-coding lenti(H1N1) Vpx at MOIGHOST X4R5=1. (n=4 independent donors combined from 2 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 2 independent experiments). Viruses were not spinoculated. (H) GFP expression in blood DC subsets that were sorted and infected for 24hr 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 24hr with HSV-1-GFP at MOI=25 as in (H). (n=10 combined from 4 experiments). (J) GFP expression in blood DC subsets that were sorted and infected for 24hr with 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 24hr with VSVeGFP at MOI=16 as in (J). (n=5 combined from 2 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 5 experiments).
Fig. 3
Fig. 3. RAB15 mediates resistance to HIV and influenza virus entry
(A) RAB15 expression in blood DCs in the public dataset E-TABM-34 (left panel), measured by RNA-seq (average number of read counts for 3 independent donors; middle panel), and measured by RT-qPCR (n=6 independent donors; right panel). (B) Viral infection of THP-1 cells over-expressing RAB15 or RAB5A. Viral expression and live cell frequency in THP-1 cells expressing BFP-RAB15, BFP-RAB5A or BFP following infection with GFP-encoding HIV-2(JK) (MOI=1.2) 48hr post infection, GFP-tagged FluA(PR8) (2 µg/ml) 24hr post infection, GFP-encoding HSV-1-GFP (MOI=0.016) 24hr post infection and GFP-encoding VSVeGFP (MOI=25) 24hr post infection. (C) Localization of BFP, BFP-RAB15 or BFP-RAB5A (green) and GM130 (red) in THP-1 cells. (scale bar=5µm). (D) Viral particle localization with GM130 in CD141+ DCs. Staining of GFP and GM130 in CD141+ DCs 12hr after infection with lenti(H1N1) iGFP (top) and HIV-1(V3R5) iGFP (bottom) viral particles that contain GFP proteins (scale bar=5µM). (E) Quantification of HIV-1(V3R5) iGFP viral particles localization with GM130 as in (D). One representative donor (left panel) and average for 5 donors combined from 2 independent experiments (right panel). (F) Quantification of lenti(H1N1) iGFP viral particles localization with GM130 as in (D). One representative donor (left panel) and average for 5 donors combined from 2 independent experiments (right panel). (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 3 independent donors). (H) GFP expression in CD34-derived CD141+ DCs 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 3 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 3 independent experiments).
Fig. 4
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 48hr alone or in the presence of a viral inhibitors AZT and NVP (n=7 donors combined from 4 independent experiments; MOI=0.4). (B) Expression of TLR7, TLR8 and actin in lysates from blood DC subsets. Blood DC subsets from 3 independent donors were combined at the same ratio and the equivalent of 500,000 cells were loaded. One experiment is shown. Circles indicate protein sizes that match cleaved C-terminal domain of TLR8 or TLR7; 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 cells frequency in CD1c+ DCs and CD141+ DCs infected with GFP-encoding HIV-1(BaL) Vpx for 48hr 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 3 independent donors were combined at the same ratio and the equivalent of 500,000 cells were 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 (1.3µg/ml) for 48hr. (F) Quantification of CD86 expression as in (E) (n=3 donors from 1 experiment representative of 2 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 1hr and incubated for 24hr alone or in the presence of Amantadine (n=5 donors for GFP, n=4 donors for IP-10, combined from 2 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 24hr alone or in the presence of the furin inhibitor DC1 (n=5 donors from 2 independent experiments).
Fig. 5
Fig. 5. Non-redundant 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 MIP-1β, TNF-α, IFN-γ and IL-2 by a SL9-specific CD8+ T cell line exposed to blood DCs infected for 48hr by HIV-1SL9(BaL) Vpx (MOI=0.8) alone or in the presence of viral inhibitors AZT and NVP (n=3 donors combined from 3 independent experiments). (B) IFN-γ concentration in culture supernatants of M1- and NS1-specific CD8+ T cell lines exposed for 18hr to DCs that were treated with or without chloroquine for 30min and then pulsed with FluA(PR8) (MOI=2) for 1hr (n=3 donors combined from 3 experiments). (C) Down-regulation of HLA-ABC in HIV-infected DCs. HLA-ABC expression in Gag+ and Gag- CD1c+ DCs 48h 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, 24hr after infection with GFP-encoding influenza virus. (E) GFP expression and viability as in (D) (n=4; 3 or 4 different donors combined from 3 independent experiments). (F) Down-regulation of HLA-ABC and CD86 in influenza virus-infected DCs. GFP, HLA-ABC and CD86 expression in DAPI-negative CD1c+ DCs analyzed prior and 12hr after infection with GFP-encoding influenza virus. (G) HLA-ABC expression as in (F) (n=4; 4 different donors combined from 3 independent experiments). (H) CD86 expression as in (F) (n=4; 4 different donors combined from 3 independent experiments). (I) T cell stimulation by infected vs. bystander CD1c+ DCs. GFP expression in bulk CD1c+, sorted GFP+CD1c+ and GFP-CD1c+ DCs 24hr after infection with GFP-encoding influenza virus (top panel). CFSE levels and FluM1-tetramer binding on CD8+ T cells after 7-day cocultures at 1:30 and 1:100 DC:T ratio (bottom panel). (J) Summary of the percentage of FluM1-tetramer binding CFSElow CD8+ T cells at 7 days after coculture as in (I) (n=2; 2 different donors combined from 2 independent experiments).
Fig. 6
Fig. 6. DC subset cooperation for activation of antiviral T cells
(A) Stimulation of a 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+ DCs and CD141+ DCs and frequency of CD8+ T cells positive for TNF and IFN-γ expression, 48hr after infection with GFP-encoding HIVSL9(G) Vpx (MOI=1) (n=3 combined from 2 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 4hr 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 18hr to DCs 12hr after infection with GFP-tagged FluA(PR8) (MOI=2; n=3, combined from 2 experiments).
Fig. 7
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 1hr. Analysis was performed at 4hr after infection. (B) Quantification as in (A) at 4, 24hr after infection (n=2 donors from 2 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 3 independent experiments). (E) Detection of GFP in CD1c+ DCs and CD141+ DCs in the draining lymph nodes of infected humanized mice as in C, one representative sample and combined data (n=3 from 3 independent experiments). (F) FACS plots illustrating the GFP and surface HA staining on lung DCs from 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 panel (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 (grey shaded) were stained with anti-HA as the control. (Each sample was a pool of 3 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 3–6 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 3–6 mice; representative of two independent experiments). (J) HA expression in draining LN DCs. Total GFP+ DCs as gated in panel (I) were analyzed for the expression of surface HA (red line). Isotype staining (grey 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.

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