SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

DENDRITIC CELLS Copyright 2017

The Authors, some
Constitutive resistance to viral infection in human rights reserved;

CD141+ dendritic cells

exclusive licensee
American Association
for the Advancement
of Science. No claim
Aymeric Silvin,1* Chun I Yu,2,3,4* Xavier Lahaye,1 Francesco Imperatore,5 Jean-Baptiste Brault,6 to original U.S.
Sylvain Cardinaud,7,8 Christian Becker,9 Wing-Hong Kwan,10,11,12 Ccile Conrad,1 Mathieu Government Works
Maurin,1 Christel Goudot,1 Santy Marques-Ladeira,1 Yuanyuan Wang,2 Virginia Pascual,2
Esperanza Anguiano,2 Randy A. Albrecht,10,11 Matteo Iannacone,13 Adolfo Garca-Sastre,10,11,12
Bruno Goud,6 Marc Dalod,5 Arnaud Moris,7 Miriam Merad,14 A. Karolina Palucka,2,3,4 Nicolas Manel1

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 re-
sponses. 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

Downloaded from http://immunology.sciencemag.org/ by guest on September 18, 2017

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.

INTRODUCTION
Dendritic cells (DCs) are a heterogeneous population of antigen-
presenting cells (APCs) essential for the launching of protective T cell
immunity. In humans, DCs include three major subsets with different
phenotypes, tissue localizations, and functions. For example, blood and
lung CD1c+ DCs drive the differentiation of mucosal effector CD8+
T cells in response to influenza virus (1); blood and lung CD141+ DCs
are the most potent in cross-presentation of antigens from dying cells
(26); and blood plasmacytoid DCs (pDCs) rapidly produce abun-
dant type I interferons (IFNs) in response to many viruses (7). This
functional specialization is determined in part by subset-specific patho-
gen recognition receptor expression (8) and by spatiotemporal or-
chestration (911). However, the mechanisms that allow DC subsets
to develop specialized functions remain incompletely understood
1 ease (24). Studies on monocyte-derived DCs (MDDCs) showed that
Immunity and Cancer Department, Institut Curie, PSL Research University, INSERM
U932, 75005 Paris, France. 2Baylor Institute for Immunology Research, Dallas, TX
75204, USA. 3The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032,
USA. 4The Jackson Laboratory, Bar Harbor, ME 04609, USA. 5Centre dImmunologie
de Marseille-Luminy, Aix Marseille University, UM2, INSERM U1104, CNRS UMR7280,
France. 6Institut Curie, PSL Research University, CNRS, UMR144, Molecular Mecha-
nisms of Intracellular Transport, 75005 Paris, France. 7Centre dImmunologie et des
Maladies Infectieuses-Paris, Pierre and Marie Curie University UMRS C7, INSERM
U1135, CNRS ERL 8255, Paris, France. 8INSERM U955, IMRB Equipe-16, Vaccine Re-
search Institute (VRI), F-94010, Creteil, France. 9Division of Pulmonary, Critical Care and
Sleep Medicine, Department of Medicine; and Immunology Institute, Mount Sinai
School of Medicine, New York, NY 10029, USA. 10Department of Microbiology, Icahn
School of Medicine at Mount Sinai, New York, NY 10029, USA. 11Global Health and
Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York,
NY 10029, USA. 12Department of Medicine, Division of Infectious Diseases, Icahn School
of Medicine at Mount Sinai, New York, NY 10029, USA. 13Division of Immunology, Trans-
plantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 20132 Milan,
Italy. 14Precision Immunology Institute, Human Immune Monitoring Center, Tisch Can-
cer institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
*These authors contributed equally to this work.
Present address: INSERM U955, IMRB Equipe-16, VRI, F-94010, Creteil, France.
Present address: Drukier Institute for Childrens Health, Weill Cornell Medical Col-
lege, New York, NY 10021, USA.
Corresponding author. Email: karolina.palucka@jax.org (A.K.P.); nicolas.manel@
curie.fr (N.M.)
(12, 13), especially in the context of viral infection where DCs are sub-
ject to viral infection and are simultaneously key cells that activate
and regulate immune response against viruses.
It is well established that DCs respond to viruses and vaccines
through their innate sensors, allowing them to initiate adaptive an-
tiviral effector T and B cell responses (12, 1417). Extracellular viral
particles engage sensors facing the extracellular and vesicular space,
such as Toll-like receptors (TLRs), and the viral antigens are processed
for presentation on major histocompatibility complex (MHC). Upon
infection, viruses reach the cytoplasm and start replication, which pro-
vides additional antigens for presentation on MHC and activates cy-
tosolic immune sensors (13, 1820). However, viral infection also leads
to irreversible damage of cellular integrity and enables manipulation
of immune responses by the virus (2123). Viral infection of DCs may
also generate inflammatory mediators that can contribute to dis-
HIV-1 infection is restricted by SAMHD1 (25, 26), which prevents
DC activation through the cytosolic DNA sensor cyclic guanosine
monophosphateadenosine monophosphate (cGAMP) synthase (cGAS)
and limits antigen presentation to T cells (19, 27, 28). HIV-2, a virus
with reduced pathogenicity compared with HIV-1 (29), abrogates
SAMHD1 restriction through its Vpx protein (27). Accordingly, Vpx
sensitizes HIV-1 infection in MDDCs and restores the ability of
MDDCs to recognize the virus (19, 30). pDCs use a Vpx-independent
mechanism of resistance to HIV-1 infection (31) and respond to viral
particles via a TLR7-dependent endosomal pathway (32). In mouse
models, lung DCs respond to influenza virus infection (33) and
migrate to the draining lymph nodes (LNs) (34), where they do not
appear to be infected (35). Even in the absence of viral replication,
murine pDCs respond to influenza virus through TLR7 (3639), whereas
bone marrowderived DCs use the cytosolic nucleic acid sensor
retinoic acid-inducible gene I (RIG-I) (21). In humans, infection of
blood DCs by influenza virus impairs their cross-presentation ability
(37, 38), and pDCs resist influenza virus infection through an unknown
mechanism (37).

Silvin et al., Sci. Immunol. 2, eaai8071 (2017) 7 July 2017 1 of 12

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE
The following question thus arises: How do DCs balance the need
to acquire antigen with avoidance of viral targeting and its detrimental
consequences to promote protective antiviral immunity? To address
this question, we used functional in vitro and genetic approaches in
human bloodderived and tissue-resident DCs to evaluate DC subtype
specific functional responses to two human pathogenic RNA viruses:
HIV and influenza virus.
RESULTS
Differential susceptibility of human DC subsets to HIV and
influenza virus infection
Blood CD1c+ DCs, CD141+ DCs, and pDCs were sorted from healthy
donors (Fig.1A and fig. S1A) and exposed to viruses. Upon infecting
the cells with titrated doses of a CCR5-tropic HIV-2 reporter virus
that encodes green fluorescent protein (GFP) instead of Nef [molecu-
lar clone JK7312As, HIV-2(JK) herein] (fig. S1B) (40), CD1c+ DCs be-
came infected at titers similar to those of susceptible reporter
GHOST cells (fig. S1C). CD141+ DCs and pDCs showed substantially
lower frequencies of GFP-expressing cells (4.6- and 2-fold, respective-
ly) (Fig.1, B and C, and fig. S1, C and D). As expected, an intact Vpx
gene was required for infection of all DC subsets by HIV-2, indicat-
ing that SAMHD1 is an active restriction factor in all blood DC sub-
sets (fig. S1, E and F). All blood DCs were refractory to infection by
both CXCR4-tropic HIV-1 [HIV-1(NL4-3)] and CCR5-tropic HIV-
1 [HIV-1(BaL)], which lacks Vpx (Fig.1, D and E, fig. S1G). Including
the Vpx protein in viral particles increased the susceptibility of CD1c+
DCs to HIV-1 infection, but CD141+ DCs and pDCs remained re-
sistant (Fig.1, D and E, fig. S1, G to I).
We next examined infection with influenza A virus. Blood CD1c+
and CD141+ DCs were infected with recombinant live influenza virus
H1N1 carrying a GFP reporter gene in the NS1 segment [FluA(PR8)
herein] (41). CD1c+ DCs became infected, whereas CD141+ DCs were
comparatively resistant (Fig.1, F and G). CD1c+ DCs but not CD141+
DCs expressed the nonstructural viral protein NS1 (fig. S1J). Viral ti-
trations confirmed the resistance of CD141+ DCs to influenza virus
infection (fig. S1K). Thus, human DC subsets show differential suscep-
tibility to infection with influenza virus, HIV-2, and HIV-1 with Vpx.
Constitutive inhibition of HIV and influenza virus entry in
CD141+ DCs and pDCs
Type I IFNs are known to induce a broad antiviral state in cells. Gene
expression analysis confirmed earlier studies on expression of IFN-
stimulated genes (ISGs) in CD1c+ DCs but not in CD141+ DCs (fig.
S2A) (42). To test whether type I IFN production contributed to the
resistance of CD141+ DCs to infection, we used recombinant B18R,
a soluble protein encoded by vaccinia virus that neutralizes type I IFN
(fig. S2, B and C) (43). B18R did not rescue infection of CD141+ DCs
with influenza virus or infection of CD141+ DCs and pDCs with HIV-1
(fig. S2, B and C). Thus, the resistance of CD141+ DCs was indepen-
dent of type I IFNinduced antiviral state.
We next examined steps of the viral infection cycle. We measured
the ability of HIV to fuse with the cell membrane using the beta-
lactamase (BlaM)-Vpr reporter assay (Fig.2A) (44). HIV-1(BaL) and
HIV-1(NL4-3) fused efficiently with CD1c+ DCs but not with CD141+
DCs and pDCs (Fig.2, A and B, and fig. S2D). All DC subsets expressed
the HIV receptors CD4, CCR5, and CXCR4 (fig. S2E) at variable lev-
els that did not correlate with the resistance of the cells, suggesting
that reduced receptor levels were not responsible for the reduced effi-
Silvin et al., Sci. Immunol. 2, eaai8071 (2017) 7 July 2017
ciency of viral fusion in CD141+ DCs and pDCs (fig. S2E). The quan-
tity of cell-associated HIV-1 p24 protein was also similar between DC
subsets at the time of the fusion assay, indicating that lower viral in-
ternalization also does not account for reduced fusion (fig. S2F).
We next examined the localization of a GFP-labeled virus,
HIV-1(V3R5) Gag-iGFP (interdomain GFP), within the cells. In CD1c+
DCs, GFP-enriched puncta and diffuse, low-level staining in the cy-
tosol were observed, suggestive of viral fusion that resulted in GFP
dispersion from viral particles (Fig.2C and fig. S2G). Using CCR5 an-
tagonists that prevent viral fusion but preserve incoming viral parti-
cles, we confirmed that the diffuse cytosolic staining was abrogated,
whereas the GFP-enriched puncta remained, indicating retention of
the virus in endocytic compartments (Fig.2, C and D, and fig. S2H). In
CD141+ DCs, the virus exclusively accumulated in puncta, whereas
diffuse cytosolic staining was not detectable, consistent with an inhibi-
tion of viral fusion in this subset (Fig.2, C and D, and fig. S2H). In
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pDCs, the resolution was too low to unequivocally distinguish cyto-
plasmic staining. We next measured SAMHD1 protein levels after
infection because its degradation induced by Vpx also requires viral
fusion. In CD1c+ DCs, SAMHD1 was degraded in most cells (fig. S2,
I and J). In contrast, most CD141+ DCs and pDCs remained positive
for SAMHD1 (fig. S2, I and J). We also evaluated the progression of
the viral cycle by measuring the amount of reverse-transcribed DNA
species. All viral DNA species were reduced in CD141+ DCs and pDCs
compared with CD1c+ DCs (fig. S2K). These results indicate that HIV
is endocytosed in all DC subsets, but in CD141+ DCs and pDCs, viral
fusion and entry are inhibited.
Next, we examined at which step infection by the influenza virus is
inhibited in CD141+ DCs. CD1c+ and CD141+ DCs showed similar
levels of 2,3- and 2,6-linked sialic acid, which are receptors for in-
fluenza virus entry (Fig.2E). To determine whether viral fusion was
inhibited, we used H1N1-pseudotyped lentivector [lenti(H1N1)].
Infection of CD141+ DCs and pDCs by lenti(H1N1) was reduced
compared with that of CD1c+ DCs, recapitulating our results using
FluA(PR8) (Fig.2F and fig. S2L). Using the BlaM-Vpr assay, we found
that CD1c+ DCs fused efficiently with the virus, whereas viral fusion
was reduced in CD141+ DCs and pDCs (Fig.2G and fig. S2L). Thus,
CD141+ DC and pDC resistance to influenza virus can be partly at-
tributed to reduced fusion of the virus with the cell, similar to that ob-
served with HIV.
Broad resistance of CD141+ DCs to infection by
enveloped viruses
Because resistance occurs after endocytosis but before viral fusion,
we examined how the viral envelope could influence infection of
DC subtypes. Vesicular stomatitis virus (VSV) and herpes simplex
virus (HSV-1) are enveloped endocytic viruses, modified vaccinia
Ankara (MVA) is a membrane-fusing enveloped live-attenuated vac-
cine strain (45, 46), and adenovirus (AdV) is a nonenveloped virus.
CD141+ DCs and pDCs were largely resistant to infection-induced
cell death by GFP reporter VSV (47) and HSV, and the fraction of
GFP+ cells was reduced twofold compared with CD1c+ DCs (Fig.2,
H to L, and fig. S2M). CD141+ DCs and pDCs were also largely re-
sistant to infection by VSV glycoprotein (VSVG)pseudotyped len-
tivector (fig. S1, E, F, and K) and to viral fusion mediated by VSVG
(Fig.2L and fig. S2N). In contrast, all DC subsets were readily in-
fected with MVA and GFP reporter AdV (fig. S2, O and P). Thus,
CD141+ DCs and pDCs preferentially resist infection by endocytic
enveloped viruses.
2 of 12
SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

A B trafficking (48). We found that RAB15 was

C
6 10 7 the only RAB selectively expressed in both
CD1c+ DCs
Control HIV-2(JK) CD141+ DCs and pDCs (Fig.3A and fig.
CD141+ DCs
4 107
Sorted cell numbers

2 107 0.4 32 S3A). RNA sequencing (RNA-seq) and

CD1c+ 80 **** 4.6 ****
5 10 6
DCs
reverse transcription quantitative poly-
60 merase chain reaction (RT-qPCR) analy-
4 106

% GFP+ cells
sis confirmed the selective expression of
3 106 CD141+ 0.7 6.2
40 RAB15 in CD141+ DCs and pDCs and its
2 106 DCs paucity in CD1c+ DCs (Fig.3A). We first

FSC
1 10 6 20 tested the impact of ectopic expression of
3 105 GFP
0 0
RAB15 (Fig.3B). In CD1c+ DCs, transduc-
Input CD1c+ CD141+ HIV-2(JK) + + tion of a GFP-RAB15 lentivector resulted
DCs DCs in low expression of GFP-RAB15 and a
D HIV-1(BaL) HIV-1(NL4-3)
small but significant decrease in infection
Control HIV-1(BaL) Vpx HIV-1(NL4-3) Vpx
with H1N1- and VSVG-pseudotyped vi-
ruses. To obtain cells with higher levels of

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1.8 20 0.8 12
CD1c+ 0.4 RAB15 ectopic expression, we transduced
DCs monocytic THP-1 cells with blue fluores-
cent protein (BFP)RAB15 or either BFP
alone or the endosomal guanosine triphos-
0.6 0.2 0.2 0.3 0.3 phatase (GTPase) BFP-RAB5A as controls
CD141+ and sorted high from low BFP expressers.
DCs RAB15 overexpression in THP-1 cells re-
FSC

duced viral infection and infection-induced

GFP cell death after exposure to HIV-2(JK), in-
E CD1c + DCs
F G fluenza virus, VSV, HSV-1 (Fig.3B), and
CD141+ DCs CD1c+ DCs lentivectors pseudotyped with VSVG or
CD141 DCs+
9 19 Control FluA (PR8) H1N1 (fig. S3B). Expression of RAB15 AN1,
25 16 23 15 *** ***
**** **** 0.1 31 a splicing variant of RAB15 that lacks the
20 CD1c+
11 CXC domain required for membrane bind-
% GFP+ cells

% GFP+ cells

10 DCs 50 **** ****

15 ing (49), completely and specifically ab-
40 rogated infection and fusion by influenza
cells

10 0.04 1.5
5 CD141+ 30 virus H1N1 and H1N1-pseudotyped virus
5
% GFP+

DCs (fig. S3, C to J).

FSC

20
0 0 To understand how RAB15 limits viral
GFP 10
HIV-1(BaL)
HIV-1(BaL) Vpx
HIV-1(BaL)
HIV-1(BaL) Vpx

HIV-1(NL4-3)
HIV-1(NL4-3) Vpx
HIV-1(NL4-3)
HIV-1(NL4-3) Vpx

infection, we first examined its localization.

0 In THP-1 cells, BFP-RAB15 colocalized
FluA(PR8) + + with the Golgi marker GM130 (Fig.3C).
We then investigated the localization of
HIV-1(BaL) and H1N1-pseudotyped len-
tivector [lenti(H1N1)] in CD1c+ DCs and
Fig. 1. Preferential infection of CD1c+ DCs by HIV-1, HIV-2, and influenza virus. (A) Absolute cell number of CD141+ DCs and found that the two vi-
sorted DC subsets (n = 25 donors). Total DCs were enriched by negative selection with magnetic beads (input) and ruses were enriched in GM130+ compart-
sorted by fluorescence-activated cell sorting (FACS). (B) Susceptibility of blood DCs to infection by HIV-2. GFP ex- ments, specifically in CD141+ DCs (Fig.3,
pression in blood DC subsets that were sorted and infected for 48 hours with GFP-coding HIV-2(JK) at a multiplicity D to F, and fig. S3, K and L). Last, we sought
of infection (MOI)GHOST X4R5 = 0.4. (C) Quantification as in (B) (n = 14 independent donors combined from seven in- to test whether endogenous RAB15 con-
dependent experiments). (D) Susceptibility of blood DCs to infection by HIV-1 and impact of codelivered Vpx pro- tributed to the resistance of CD141+ DCs
tein that degrades SAMHD1. GFP expression in blood DC subsets that were sorted and infected for 48 hours with to viral infection. To circumvent the chal-
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). lenge of low numbers of CD141+ DCs that
Viruses were not spinoculated in this experiment. (E) Quantification as in (D) (n = 4 independent donors combined
can be isolated from human blood (Fig.
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
1A), we generated CD141+ DCs ex vivo
influenza virus [GFP-tagged FluA(PR8); MOI = 2] for 1 hour. Analysis was performed 4 hours after infection. (G) Quan- from CD34+ hematopoietic progenitor
tification as in (F) in DCs at 24 hours after infection (n = 8; seven donors combined from seven independent experi- cell cultures (50). This system also generates
ments, including one donor repeated two times; analysis of variance). FSC, forward scatter. ***P < 0.001, ****P < 0.0001. CD11c+ DCs that are similar to MDDCs
(Fig.3G). Compared with CD11c+ DCs,
RAB15 limits viral infection in CD141+ DCs CD141+ DCs showed partial resistance to HIV-1(BaL) and lenti(H1N1)
We next searched for differentially expressed genes that regulate en- infection (Fig.3H). In contrast, short hairpin RNA (shRNA)mediated
docytosis in CD141+ DCs and pDCs compared with CD1c+ DCs. RAB knockdown of endogenous RAB15 significantly increased the fre
proteins are key coordinators of endocytic pathways and vesicular quency of infected cells for HIV-1(BaL) and H1N1-pseudotyped

Silvin et al., Sci. Immunol. 2, eaai8071 (2017) 7 July 2017 3 of 12

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

Fig. 2. Resistance of CD141+ DCs A CD1c+ DCs

HIV-1(BaL) HIV-1(NL4-3) C D
to HIV and influenza virus infection GFP Overlay
Control BlaM BlaM
at the level of viral fusion. (A) HIV-1
0.2 60 29 HIV-1(V3R5) 10000
fusion assay in blood DCs. Viral fusion CD1c+

in GFPlow regions GFPlow regions

Average GFP density GFP density in
iGFP
revealed by CCF4 fluorescence in blood DCs 8000

CCF4 product

per cell
DCs after infection with HIV-1(BaL) 6000
(MOI = 0.8) or HIV-1(NL4-3) (MOI = 4000
CD141+ 1.1 4.1 5.0
HIV-1(V3R5)
0.6) containing a BlaM-Vpr fusion pro- 2000
DCs iGFP
tein. Fluorescence of the CCF4 product + MVC 0
indicates viral fusion with target cells CCF4 substrate + TAK 5000 ****
as a result of cleavage of the cell-loaded 4000

per donor
B CD141+ DCs
CCF4 substrate by the virus-contained 3000 ns
GFP Overlay
BlaM. (B) Quantification as in (A) (n = 11 2000
80 **** 40 8
8 donors combined from four inde-

%CCF4 product + cells

%CCF4 product + cells

**** HIV-1(V3R5) 1000

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pendent experiments). (C) Staining of iGFP 0
60 30
GFP proteins contained in viral par- HIV-1(V3R5) iGFP + + + +
+ + MVC + TAK + +
ticles and CD44 in CD1c and CD141 40 20
DCs after infection with HIV-1(V3R5) CD1c+ CD141+
HIV-1(V3R5) DCs DCs
iGFP containing GAG-iGFP and GFP- 20 10 iGFP
Vpr fusion proteins, alone or in the + MVC
presence of viral entry inhibitors MVC 0 0 + TAK
+ + + +
and TAK-779. Scale bars, 10 m. (D) Quan- HIV-1(BaL) BlaM HIV-1(NL4-3) BlaM GFP CD44
tification of the GFP density in GFPlow GFP
regions as in (C), shown for one rep- E F 60 4.4 G 1.9

%CCF4 product + cells

** 60 ****
resentative donor (top) and average
for five donors (bottom; combined
%GFP+ cells
40 40
from two independent experiments).
(E) Levels of influenza virus recep-
20 20
tors on blood DCs. SNA and MAA
%Max

+ +
binding on CD1c and CD141 DCs
(representative of two independent 0 0
SNA MAA lenti lenti lenti lenti
experiments). (F) GFP expression in (H1N1) (H1N1) (H1N1) (H1N1)
+
CD1c DCs
blood DC subsets that were sorted Vpx Vpx BlaM BlaM
+
CD141 DCs
and infected for 48 hours with GFP- CD1c+ CD141+ CD1c+ CD141+
coding lenti(H1N1) Vpx at MOIGHOST DCs DCs DCs DCs
X4R5 = 1 (n = 4 independent donors H HSV-1 J VSV L lenti(G)
combined from two independent ex- CD1c+ DCs CD141+ DCs CD1c+ DCs CD141+ DCs Control BlaM
periments). Viruses were not spinocu-
+ 2.6 58
lated. (G) Viral fusion revealed as in CD1c
DCs
(A) by CCF4 fluorescence in blood DCs CCF4 product
19 4.2 83 27
after infection with lenti(H1N1) (MOI =
1) containing a BlaM-Vpr fusion pro- GFP GFP CD141+ 0.3 4.0
tein (n = 4 donors combined from two I K DCs
50 ** 100 **
independent experiments). Viruses
%GFP+ cells

40
%GFP+ cells

80 CCF4 substrate
were not spinoculated. (H) GFP expres-
30 60
sion in blood DC subsets that were
20 40
sorted and infected for 24 hours with **** ****
HSV-1GFP at MOI = 25 (one repre- 10 20 100
sentative donor). (I) Quantification of 0 0
%BlaM+ cells

ns 80
GFP expression and frequency of live *
%In FSC SSC gate

***
cells in blood DC subsets that were 100 100 ns 60
***
%Live cells

sorted and infected for 24 hours with 80 80 40

HSV-1GFP at MOI = 25 as in (H) (n = 60 60
20
10 combined from four experiments). 40 40
(J) GFP expression in blood DC sub- 0
20 20 lenti(G) + + + +
sets that were sorted and infected
0 0 NH4Cl + +
for 24 hours with enhanced GFP HSV-1 + + VSV + +
expressing VSV (VSVeGFP) at MOI =
16 (one representative donor). (K) Quan-
tification 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.

Silvin et al., Sci. Immunol. 2, eaai8071 (2017) 7 July 2017 4 of 12

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

A **
lentivector in CD34-derived CD141+ DCs
10 *** 100 10
(Fig.3I and fig. S3M). CD141+ cell differenti-

RAB15/ACTB
*** **
expression
Affymetrix

(2Cp method)
1

RNA-seq
ation was not affected (Fig.3G and fig. S3N).
RAB15

RAB15

counts
10 0.1

CD141+ DCs sense HIV and influenza

0.01
1 1 0.001
in the absence of infection

1+ s
s

s
14 Cs

s
14 Cs

s
s

C
C

C
C

C
C The resistance of CD141+ DCs to HIV and

C c +D
pD

pD
D

D
D

D
pD
+

1+

1+
+
1c

1c

1
14
influenza virus infection prompted us to test

D
D

D
D

D
C
C

C
B
C

C
THP-1 BFP THP-1 BFP-RAB15 THP-1 BFP-RAB5A
ns ns the role of innate sensing in the overall re-
* 20 40 ns
60 ns
100 * **** sponse to viral exposure. In response to
%GFP+ cells

**** *
15 * *
%HA+ cells

%GFP+ cells

%GFP+cells
* ** **
80
40 ** 30 ** HIV-2(JK) or HIV-1(BaL) Vpx, CD1c+ DCs
10 ns 60
20 * up-regulated CD86 and produced IP-10,
20 * 5 40
** 10 20 indicating activation of an innate immune
0 0 0
100 80 ns 100 ** ns **
0
100
response in the DCs, and this induction
ns ns * *
%Cells in FSC

* ns
80 * ** was reduced by inhibitors of viral infection
%Live cells

80

%Live cells
60 80

%Live cells
SSC gate

60 60 60 (Fig.4A and fig. S4, A and B). CD141+ DCs

Downloaded from http://immunology.sciencemag.org/ by guest on September 18, 2017

40
40 40 40
20 20 and pDCs also responded to HIV-2(JK) or
20 20
0 0 0 0
HIV-1(BaL) Vpx, as shown by the induc-
HIV-2(JK) + + + FluA(PR8) + + + HSV-1 + + + VSV + + + tion of IP-10 in both subsets and CD86 in
C D
BFP GM130 y
Overlay
GFP GM130 overlay CD141+ DCs, but the response was not af-
THP-1
BFP lenti(H1N1) fected by viral infection inhibitors (Fig.4A
and fig. S4, A and B). These results suggest
CD141+
THP-1 DCs GFP GM130 overlay
a functional dissociation of innate sensing
BFP-RAB15
from viral infection selectively in CD141+
HIV-1(V3R5) DCs and pDCs.
THP-1
BFP-RAB5A We next addressed the contribution of
E F endosomal TLRs to innate sensing of HIV.
100 **** 100 100 100
Average % GFP in

Average %GFP in

**
pDCs respond to HIV-1 RNA through TLR7
GM130 regions

GM130 regions
GM130 regions

GM130 regions

80 80 * 80 80 ****
per donor
per donor

%GFP in
%GFP in

(32), and TLR8 can signal in response to

per cell
per cell

60 60 60 60
40 40 40 40 HIV-derived RNA (51). We detected expres-
20 20 20 20
0 0 0 0 sion of TLR8 protein in CD1c+ and CD141+
CD1c+ CD141+ CD1c+ CD141+ CD1c+ CD141+ CD1c+ CD141+ DCs but not pDCs, whereas TLR7 protein
DCs DCs DCs DCs DCs DCs DCs DCs
G NI shRNA LacZ H 15 80 I 1.5 * 80 *
was expressed in all DC subsets (Fig.4B and
25 17 * fig. S4C). We used a furin antagonist (DC1)
%GFP+ cells

%GFP+ cells
%GFP+ cells

%GFP+ cells

60 60
10 1.0
22 16 40 40
that inhibits TLR7 and TLR8 activity by pre-
shRNA shRNA
5
20
0.5
20
venting cleavage (52, 53). DC1 prevented re-
RAB15 #4
20
RAB15 #5
17 0 0 0.0 0
sponses of pDCs to CL264, a TLR7 agonist
CD11c+ DCs +
CD141+ DCs +
+
+
+
+
shRNA LacZ + +
shRNA RAB15 #4 + +
+


+
(fig. S4D). Furin inhibition abrogated the
response of CD141+ DCs and pDCs to in-
CD11c

21 22 Control HIV-1(BaL) lenti(H1N1) shRNA RAB15 #5 + + +

Vpx Vpx
CD141
Control HIV-1(BaL)
Vpx
lenti(H1N1)
Vpx
fection with HIV-1(BaL) Vpx but not the re-
sponse of CD1c+ DCs (Fig.4C and fig. S4E).
Fig. 3. RAB15 mediates resistance to HIV and influenza virus entry. (A) RAB15 expression in blood DCs in the This prompted us to test the putative role
public data set E-TABM-34 (left), measured by RNA-seq (average number of read counts for three independent do- of cGAS in CD1c+ DCs. cGAS was detectable
nors; center), and measured by RT-qPCR (n = 6 independent donors; right). (B) Viral infection of THP-1 cells overex-
at different levels in all DC subsets (Fig.4D
pressing 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 g/ml)
and fig. S4F). shRNA-mediated knockdown
24 hours after infection, GFP-encoding HSV-1GFP (MOI = 0.016) 24 hours after infection, and GFP-encoding of cGAS (fig. S4G) (27) prevented CD86 up-
VSVeGFP (MOI = 25) 24 hours after infection. (C) Localization of BFP, BFP-RAB15, or BFP-RAB5A (green) and regulation by CD1c+ DCs after infection by
GM130 (red) in THP-1 cells. Scale bars, 5 m. (D) Viral particle localization with GM130 in CD141+ DCs. Staining of GFP HIV-1(BaL) Vpx (Fig.4, E and F). Responses
and GM130 in CD141+ DCs 12 hours after infection with lenti(H1N1) iGFP (top) and HIV-1(V3R5) iGFP (bottom) viral to cGAMP, a product of cGAS, and suscep-
particles that contain GFP proteins (scale bar, 5 m). (E) Quantification of HIV-1(V3R5) iGFP viral particle localization tibility to infection by the virus remained in-
with GM130 as in (D). One representative donor (left) and average for five donors combined from two independent tact upon cGAS knockdown (Fig.4, E and F,
experiments (right). (F) Quantification of lenti(H1N1) iGFP viral particle localization with GM130 as in (D). One repre- and fig. S4H). Thus, the response of CD1c+
sentative donor (left) and average for five donors combined from two independent experiments (right). (G) Fre- DCs to HIV-1 requires infection for cyto-
quencies of CD141+ and CD11c+ cells in CD34+ cells that were expanded, transduced with LacZ or RAB15 shRNA #4
solic sensing, whereas CD141+ DCs respond
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
to HIV-1 in the absence of infection.
or HIV-1(H1N1) Vpx (n = 3 combined from three independent experiments). (I) Role of RAB15 in the resistance of We next examined DC responses to in-
CD141+ DCs to viral infection. GFP expression in CD34-derived CD141+ DCs that were first transduced with the indicated fluenza virus using ultraviolet (UV) inactiva-
shRNA lentivectors after infection of total CD34-derived cells with GFP-encoding HIV-1(BaL) Vpx or HIV-1(H1N1) Vpx tion or amantadine to inhibit influenza virus
(n = 4 combined from three independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. entry and infection (Fig.4G) (54). CD1c+ DCs

Silvin et al., Sci. Immunol. 2, eaai8071 (2017) 7 July 2017 5 of 12

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

A B C CD1c+ DCs
CD1c+ DCs CD141+ DCs

s
C DC
s
CD141+ DCs

D DC
80

pD 1 +

IP-10 (pg/ml) %CD86+ cells %GFP+ cells

+
80 60

s
14
1c
D
40

C
% GFP+ cells
60 130 kDa 20
40 -TLR7 0
70 kDa
80 ****ns
**** * **
20 55 kDa 60
-actin
0 35 kDa 40
80 **** *** 20
% CD86+ cells

ns 0
60 130 kDa
-TLR8 104 ns
** ***
* 103 * *
40 100 kDa
102
20 55 kDa
-actin 101

Downloaded from http://immunology.sciencemag.org/ by guest on September 18, 2017

35 kDa
0 100
104 D s 100 ****
******** s C
IP-10 (pg/ml)

%Live cells
C D 80
103 * ns
+ D 1
+
60
ns
s
1c C 14 40
102 C
D pD C
D
20
70 kDa 0
-cGAS HIV-1(BaL) Vpx +++ ++
101 55 kDa
HIV-1(BaL) Vpx + + + + 55 kDa AZT + NVP +
-actin Furin inhibitor + + +
AZT + NVP + + 35 kDa

E HIV-1(BaL) G CD1c+ DCs H CD1c+ DCs

Control Vpx cGAMP CD141+ DCs CD141+ DCs
31 2.5 41 41 90 7 ****
****
LacZ 40 **** 100 * *
****
shRNA

%CD86+ cells
%GFP+ cells

30 80 ** **
66 0.1 10 8 2.9 0
60
28 1.2 16 16 88 8 20
cGAS 40
CD86

10
shRNA 20
70 0.4 44 24 3.8 0 0 0
GFP
F LacZ shRNA 800 ***
** 2000 * **
cGAS shRNA ****
IP-10 (pg/ml)

600 ns ns
IP-10 pg/ml

ns 1500
100
%CD86+ cells

** 400 1000
80
60 200 500
40 0 0
20 FluA(PR8) + + + + FluA(PR8) + + + +
0 UV-inactivated FluA(PR8) + + DC1 + +
+ + Amantadine + +
Serum-free media
+ +
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 load-
ed. 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 re-
sponse 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 g/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 expres-
sion 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.

Silvin et al., Sci. Immunol. 2, eaai8071 (2017) 7 July 2017 6 of 12

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

were susceptible to influenza virus infection, whereas CD141+ DCs
were largely resistant. CD1c+ and CD141+ DCs induced IP-10 in re-
sponse to influenza virus, and this was inhibited by UV treatment
or amantadine, indicating that an intact virus is required for infec-
tion (Fig.4G). IFNs were induced in CD1c+ DCs and were below limit
of detection in CD141+ DCs (fig. S4I), but the ability of B18R to re-
duce CD86 induction in response to influenza virus suggests that IFNs
were also induced in CD141+ DCs (fig. S2M). To determine the con-
tribution of TLR sensing to DC activation and response, we moved
to serum-free medium to limit serum-induced CD141+ DC activation.
In these conditions, CD141+ DCs expressed CD86 and induced IP-10
in response to influenza virus, and both were inhibited by DC1 treat
ment (Fig.4H). These results show that CD141+ DCs are capable of
innate, TLR-mediated sensing of influenza virus despite being resistant
to overall infection.
Class I MHC and CD86 expression were down-regulated in GFP+
CD1c+ DCs 12 hours after infection, at a time when most cells were
alive (Fig.5, F to H). Thus, influenza virus inhibits immunostimula-
tory molecule expression in infected CD1c+ DCs in a cell-intrinsic
manner and subsequently induces cell death, whereas noninfected
bystander CD1c+ DCs remain unaltered. This raised the possibility
that in a culture of CD1c+ DCs partially infected after exposure to the
virus, noninfected CD1c+ DCs may also contribute to antigen presen-
tation. To test this idea, we sorted GFP+ and GFP CD1c+ DCs after
infection with influenza virus and measured the expansion of autolo-
gous M1-specific CD8+ T cells. GFP CD1c+ DCs were more potent
at expanding T cells than GFP+ DCs (Fig.5, I and J). Thus, at least
in vitro, viral infection in CD1c+ DCs is required for antigen presen-
tation, but the infection simultaneously impairs T cell stimulation
capacity at the cellular level.

Downloaded from http://immunology.sciencemag.org/ by guest on September 18, 2017

DC infection determines T cell responses to HIV DC subset cooperation for induction of antiviral
and influenza virus T cell responses
Because viral infection was essential for innate sensing in CD1c+ DCs CD141+ DCs are thought to be specialized in cross-presentation of an-
but not CD141+ DCs, we examined how infection could determine tigens from necrotic cells, but whether cross-presentation is an es-
antigen presentation to T cells by the DCs. For HIV, we used a CD8+ sential mode of antigen presentation by these cells is unknown (55).
T cell line specific to a structural antigen of HIV-1 (SL9 in Gag). Our findings suggest that because CD141+ DCs resist viral infection,
CD1c+ DCs activated SL9-specific CD8+ T cells more efficiently than their ability to stimulate virus-specific T cells may require cross-
CD141+ DCs after infection by HIV-1 with Vpx (Fig.5A and fig. presentation of viral antigens produced in bystander cells. To test this
S5A). T cell activation was abrogated when viral infection was blocked idea, we mixed human leukocyte antigen (HLA)A2 CD1c+ DCs
by RT inhibitors. pDCs did not activate CD8+ T cells after infection with HLA-A2+ CD141+ DCs, infected cells with HIV-1 or influenza
(fig. S5B). All DC subsets activated CD8+ T cells in response to a syn- virus, and measured T cell responses in the mixed culture (Fig.6, A
thetic SL9 peptide (fig. S5C). Thus, infection of CD1c+ DCs by HIV is and C). In the mixed culture, viral infection was largely restricted to
essential for viral antigen presentation to CD8+ T cells. HLA-A2 CD1c+ DCs (Fig.6, B and D, and fig. S6), recapitulating the
To examine antigen presentation by blood DCs in response to in- situation with purified DC subsets. HLA-A2+ CD141+ DCs were in-
fluenza virus, we generated CD8+ T cell lines specific to the structural efficient at stimulating T cells after infection with influenza virus and
protein M1 and nonstructural protein NS1 (fig. S5, D and E). M1 is HIV. The presence of HLA-A2 CD1c+ DCs during infection rescued
present in viral particles and is expressed in infected cells. CD1c+ DCs the ability of HLA-A2+ CD141+ DCs to efficiently present HIV-1 or in-
induced activated M1-specific CD8+ T cells more efficiently than fluenza virus antigen (Fig.6, B and E). These in vitro results support
CD141+ DCs (Fig.5B). NS1 is present in productively infected cells but the notion that CD141+ DCs depend on viral antigen produced in
not in the viral particles. Only CD1c+ DCs induced IFN- in NS1- bystander-infected cells, such as CD1c+ DCs, for efficient T cell
specific CD8+ T cells (Fig.5B). To test the requirement for viral infec- stimulation.
tion, we used the endosomal acidification inhibitor chloroquine, which
inhibits influenza virus fusion. Chloroquine inhibited infection of Resistance of CD141+ to influenza virus infection in vivo
CD1c+ DCs by FluA(PR8) and the M1- and NS1-specific T cell activa- To validate the findings in tissue-resident DCs, we infected sorted
tion, but it did not inhibit activation of M1-specific T cells by CD141+
human lung DCs with influenza virus. Similar to blood-derived DCs,
DCs in response to viral particles (Fig.5B). Chloroquine had no effecthuman lung CD1c+ DCs were susceptible to FluA(PR8) infection ex
on peptide presentation by CD1c+ DCs (fig. S5, F and G). Thus, viral vivo, whereas lung CD141+ DCs were comparatively resistant (Fig.7,
infection is required in CD1c+ DCs for efficient virus-specific CD8+ A and B). To determine susceptibility to infection in vivo, we infected
T cell activation to both structural and nonstructural viral antigens. In
humanized mice with GFP reporter influenza virus through the in-
contrast, CD141+ DCs are resistant to viral infection, but this restricts
tranasal route (Fig.7C). In the lung, both CD1c+ and CD141+ DCs
became GFP+ over time, likely reflecting productive infection in CD1c+
their presentation on MHC to the antigens present in the viral particles.
DCs and antigen capture in CD141+ DCs (Fig.7D and fig. S7A), al-
Virus infection and antigen presentation are mutually though we cannot exclude the idea that CD141+ DCs may carry
+
exclusive in CD1c DCs some undetectable level of intracellular and postfusion viral material
We next sought to understand how CD1c+ DCs could simultaneously that failed to replicate. In contrast, only CD141+ DCs carried GFP+
activate CD8+ T cells and sustain a viral infection. In HIV-1infected material in the draining LNs (Fig.7E and fig. S7B). To confirm that
cells, the Nef protein down-regulates class I MHC (22). Using Nef- CD1c+ DCs were infected while CD141+ DCs were resistant to infec-
expressing HIV-1 and HIV-2, we confirmed that class I MHC is down- tion, we analyzed the cell-surface expression of hemagglutinin (HA)
regulated in a cell-intrinsic manner in infected CD1c+ DCs but not on GFP+ DCs as a measure of cellular viral infection. Lung GFP+HA
in uninfected bystander CD1c+ DCs (Fig.5C). We next examined the cells contained both CD1c+ and CD141+ DCs, whereas GFP+HA+
impact of influenza virus infection on viability and immunostimu- cells contained mainly CD1c+ DCs (Fig.7F). Rare CD141+ events
latory molecule expression. In response to influenza virus infection, were detected among lung GFP+HA+ cells, which may be remaining
GFP+ CD1c+ DCs preferentially died after 24 hours (Fig.5, D and E). doublets and are unlikely to represent CD1c+ DCs acquiring CD141

Silvin et al., Sci. Immunol. 2, eaai8071 (2017) 7 July 2017 7 of 12

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

Fig. 5. Nonredundant antigen presentation by B M1-specific NS1-specific

A
CD1c+ DCs infected with HIV and influenza vi- 15 CD8+ T cells CD8+ T cells
rus. (A) Stimulation of a CD8+ T cell line by infected * 103 **** **** **** ****

%Positive T cells
DCs. Combined intracellular expression of mac-
rophage inflammatory protein1, tumor necro- 10 *

IFN- (pg/ml)
sis factor (TNF-), IFN-, and interleukin-2 by 102 *** ns ns ns
an SL9-specific CD8+ T cell line exposed to blood
DCs infected for 48 hours by HIV-1SL9(BaL) Vpx 5 ns
(MOI = 0.8) alone or in the presence of viral in- 101
hibitors AZT and NVP (n = 3 donors combined
from three independent experiments). (B) IFN- 0
HIV-1SL9(BaL) Vpx + + + + 100
concentration in culture supernatants of M1- and AZT + NVP + + + + FluA (PR8) + + + + + + + +
NS1-specific CD8+ T cell lines exposed for 18 hours Peptide + + + +
Chloroquine + + + + + + + +
to DCs that were treated with or without chloro-
quine for 30 min and then pulsed with FluA(PR8) C HIV-2(JK) nef+ HIV-1(AD8) nef+ Vpx D 0 hour 4 hours
(MOI = 2) for 1 hour (n = 3 donors combined from Gag+ Gag Gag+ Gag 0.1 0.04 1 0.1
three experiments). (C) Down-regulation of HLA-

Downloaded from http://immunology.sciencemag.org/ by guest on September 18, 2017

ABC in HIV-infected DCs. HLA-ABC expression in
Gag+ and Gag CD1c+ DCs 48 hours after infec-
HLA-ABC
C HLA-ABC 99 0.02 74 24
tion with HIV-2(JK) nef+ (MOI = 1) or HIV-1(AD8)
nef+ Vpx (MOI = 0.8) (n = 3). (D) GFP expression 300 * 350 12 hours 24 hours
and viability in CD1c+ DCs at 4, 12, and 24 hours **

HLA-ABC /
250 300 9.6 7.0 18 19
HLA-ABC /

isotype
isotype

after infection with GFP-encoding influenza virus. 250

MFI
200
MFI

200

DAPI
(E) GFP expression and viability as in (D) (n = 4; 150 150
three or four different donors combined from three 100 100 60 23 52 8.9
independent experiments). (F) Down-regulation HIV-2(JK) nef+ + + HIV-1(AD8)
Gag
Gag +
+
+ + GFP
of HLA-ABC and CD86 in influenza virus-infected nef Vpx Gag Gag+

DCs. GFP, HLA-ABC, and CD86 expression in DAPI

E F 0 hour 12 hours G H
(4,6-diamidino-2-phenylindole)negative CD1c+ 95 0 70 25
DCs analyzed before and 12 hours after infection 30 10 5
105 *
HLA-ABC
%GFP+

MFI HLA-ABC
with GFP-encoding influenza virus. (G) HLA-ABC 20 *
cells

MFI CD86
expression as in (F) (n = 4; four different donors 10
combined from three independent experiments). 4.9 0.02 1.8 1.6 104 104
0
(H) CD86 expression as in (F) (n = 4; four different 99 0.02 70 27
30
%GFP+
DAPI+

donors combined from three independent ex-

cells

periments). (I) T cell stimulation by infected ver-

20 103 103
CD86

sus bystander CD1c+ DCs. GFP expression in bulk 10 GFP + GFP +

0 0.1 0 1.4 0.7
CD1c+, sorted GFP+CD1c+, and GFP-CD1c+ DCs
24 hours after infection with GFP-encoding in-
Time (hours) 0 4 12 24 GFP
fluenza virus (top). Carboxyfluorescein diacetate I CD1c+ DCs
succinimidyl ester (CFSE) levels and FluM1-tetramer FluA FluA, GFP FluA,GFP+ J
binding on CD8+ T cells after 7-day cocultures at 0.2 44 23 85
1:30 and 1:100 DC/T ratio (bottom). (J) Summary 10
of the percentage of FluM1-tetramer binding
CFSElow CD8+ T cells
SSC

% FluM1-tetramer+

CFSElow CD8+ T cells at 7 days after coculture as GFP

in (I) (n = 2; two different donors combined from 0 0.03 2.2 0.02 2.3 0.03
. 0.4 0.02
two independent experiments). MFI, mean fluo- 5
1:30
rescence intensity. *P < 0.05, **P < 0.01, ***P < 0.001, DC:T ratio
****P < 0.0001. 0.2 99 11 86 18 79 8 91
FluM1-tetramer

0 0.03 1.1 0.03 1.8 0.02 0.9 9

1:100
0
BulkGFP GFP+ BulkGFP GFP+
DC:T ratio
1:30 DC/ T ratio 1:100
0.2 99 15 83 20 78 22 76
CFSE

after infection (Fig.6D). Analysis of HA expression within lung GFP+ DISCUSSION

CD141+ DCs and GFP+CD1c+ DCs confirmed the much-reduced level Infection of DCs with viruses can be considered a dilemma for the
of HA expression on CD141+ DCs (Fig.7, G and H). In the draining immune system: It allows activation of the innate and adaptive im-
LNs, GFP+ CD141+ DCs remained uninfected (Fig.7, I and J). These mune response, but it simultaneously facilitates manipulation by the
results in tissue-resident DCs recapitulate our in vitro observations viruses of the immune system itself. We show that this paradox is re-
on differential susceptibility of DC subsets to viral infection, thereby solved by dissociation of viral infection from viral antigen presentation
supporting the notion that the functional specialization of DC subsets to T cells across DC subsets. Blood and lung DC subsets display dif-
for viral infection applies to both circulating blood DCs and tissue- ferential susceptibility and selective resistance to infection with influ-
resident DCs. enza virus (blood and lung DCs), as well as HIV, VSV, and HSV (blood

Silvin et al., Sci. Immunol. 2, eaai8071 (2017) 7 July 2017 8 of 12

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

A Donor HLA-A2+ Donor HLA-A2 C Donor HLA-A2+ Donor HLA-A2 because of the infection. In contrast,
CD141+ DCs resist viral entry and rely
on virus-infected CD1c+ DCs (and other
CD1c + CD141 +
CD1c + CD1c + CD141 +
CD1c + cell types) as source of antigen for cross-
presentation to promote activation of T cells
(Fig.7K). We propose that this resistance
Mix Mix of CD141+ DCs to viral entry is a mecha-
FluA(PR8)
nism to avoid the deleterious effects of
HIVSL9(G) Vpx
viral infection while preserving the func-
48 hours 12 hours
tional capacity of DCs to launch T cell
+
SL9-restricted CD8 T cells M1- or NS1-restricted CD8+ T cells stimulation and subsequent cell-mediated
6 hours 18 hours immunity.
IFN- secretion Given the critical role of DCs in the
IFN- intracellular staining
induction of antiviral immunity, it is re-
B D markable that CD141+ DCs resist such
100 No DCs Control FluA (PR8) GFP+
a broad range of viruses. Nonetheless, our

Downloaded from http://immunology.sciencemag.org/ by guest on September 18, 2017

0.3 17 4.1 0.4
CD1c+ DCs CD1c+ observations do not preclude the possi-
80 CD141+ DCs &
CD141+ DCs

bility of variable infectivity of individual

CD141
CD141 +
%GFP in

60 CD1c+ & viruses toward CD141+ DCs, given that

FSC

DCs 0.4 95
CD141+ DCs additional virus-specific factors and mech
GFP CD1c
40 anisms could play an additive role. In-
20 fluenza virus and VSV are pH-dependent
3.3 and require viral internalization followed
E
0 **** by acidification in the endosomes (56).
****
100 Entry of HIV-1 is pH-independent (57),
8000 but endocytosis before viral fusion has
IFN- (pg/ml)

80 6000
been extensively documented in primary
CD1c+ DCs

M1-specific CD4+ target cells (5860). HIV receptor

%GFP in

60 CD8+ T cells 4000 levels are reduced on CD141+ DCs com-

40 pared with CD1c+ DCs, but receptor levels
2000
did not correlate with the pattern of re-
No DCs
20 + 0 sistance among subsets and thus are not
CD1c DCs
+
CD141 DCs the dominant factor explaining the broad
0
+
CD1c & 5.6 resistance of CD141+ DCs.
**** ***
8 4X
+
CD141 DCs Our results demonstrate an essential
**
**** 8000 role for RAB15 in the resistance of CD141+
%Positive T cells

6 DC (and pDC). RAB15 appears to define

IFN- (pg/ml)

6000
a distinct antiviral resistance pathway
NS1-specific
4 +
CD8 T cells 4000 based on constitutive and selective expres-
sion in CD141+ DCs and pDCs. HIV-1
2000
2 and influenza-pseudotyped virus accu-
0 mulated in GM130+ compartments in
0 FluA (PR8) + + + + CD141+ DCs. This correlates with the
+ + + + +
CD1c DCs A2 A2 A2 + localization of RAB15 after ectopic ex-
A2 A2 A2+ A2 A2 A2+ +
CD141 DCs A2 + A2+ pression, suggesting that viral particles
A2+ A2+ A2+ A2+ undergo a retrograde transport and/or
+
Fig. 6. DC subset cooperation for activation of antiviral T cells. (A) Stimulation of an SL9-specific CD8 T cell line by
accumulation into the Golgi that impairs
HLA-A2 CD1c+ DCs mixed with HLA-A2+ CD141+ DCs after infection with HIV-1, outline of the experiment. (B) GFP ex- their fusogenic capacity in a RAB15-
+ + +
pression in CD1c and CD141 DCs and frequency of CD8 T cells positive for TNF and IFN- expression 48 hours after dependent manner. The transit and mat-
infection with GFP-encoding HIVSL9(G) Vpx (MOI = 1) (n = 3 combined from two experiments). (C) Stimulation of M1- and uration of viral envelope glycoproteins and
NS1-specific CD8+ T cell lines by HLA-A2 DCs mixed with HLA-A2+ CD141+ DCs after infection with influenza virus, out- cellular receptors during their biogenesis,
line of the experiment. (D) GFP expression in mixed CD1c+ and CD141+ DCs 4 hours after infection with GFP-tagged in the absence of recognized Golgi-to-Golgi
FluA(PR8). (E) IFN- concentration in culture supernatants of M1- and NS1-specific CD8+ T cell lines exposed for 18 hours fusion, support the idea that the Golgi ap-
to DCs 12 hours after infection with GFP-tagged FluA(PR8) (MOI = 2; n = 3 combined from two experiments). **P < paratus may be a privileged compartment
0.01, ***P < 0.001, ****P < 0.0001. with intrinsic resistance to viral envelope
protein-mediated fusion. It is possible that
DCs). CD1c+ DCs are permissive to viral fusion by influenza virus and other endocytic enveloped viruses, such as respiratory syncytial vi-
HIV, support viral infection and cytosolic sensing, and generate viral rus, evade this mechanism of antiviral resistance (61, 62). Neutral-
antigen that is needed to launch T cell responses but also rapidly die ization of type I IFN had a marginal ability to rescue infection in

Silvin et al., Sci. Immunol. 2, eaai8071 (2017) 7 July 2017 9 of 12

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

Fig. 7. Resistance of CD141+ DCs to A B C

Lung CD1c+ DCs FluA (PR8) intranasal
influenza virus in vivo in humanized Control FluA(PR8) Lung CD141+ DCs
mice. (A) Infection of human lung DC 2.52 20.2 4 hours 24 hours
Lung 40 4.8
40 ** 4.6
subsets. GFP expression in CD1c+ and + **
CD1c

%GFP+ cells

%GFP+ cells
CD141+ DCs sorted from human lungs DCs 30 30
3d
and pulsed with GFP-tagged FluA(PR8)
Lung 2.04 3.81 20 20 Lungs and draining LNs harvested
(MOI = 2) for 1 hour. Analysis was per-
formed at 4 hours after infection. (B) Quan CD141+ 10 10
tification as in (A) at 4 and 24 hours after DCs

FSC
Staining for DC subsets and
0 0 FACS analysis of GFP and HA
infection (n = 2 donors from two inde- GFP FluA(PR8) + + + +

pendent experiments). (C) Intranasal D E F 0 0

infection of humanized mice by GFP- Lungs 3 days
Draining LNs 3 days PBS
PBS FluA 0
tagged FluA(PR8), outline of the exper- PBS FluA lung
+ 0.2 6.0 15 ns 10 ** GFP+HA GFP+HA+
iment. (D) Detection of GFP in CD1c DCs CD1c+ ** ** CD1c+
0 0.4 ns ** 2.2 1.0
+ 8 12 3.7
and CD141 DCs in the lungs of infected

%GFP+ cells
%GFP+ cells
DCs DCs FluA
10 4.6
6 lung
humanized mice as in (C), one repre- #1 72 81
0 6.3 8.4 4
sentative sample and combined data CD141+
5
CD141+
0
1.6 1.0
2

Downloaded from http://immunology.sciencemag.org/ by guest on September 18, 2017

17 4.3
(n = 5 from three independent experi- DCs DCs
FluA

CD141
0

GFP
0 lung 3.9
ments). (E) Detection of GFP in CD1c+
GFP

+ +

GFP
+ +
FluA (PR8) FluA (PR8) #2 63 66
+ FSC
DCs and CD141 DCs in the draining LNs FSC
HA CD1c
of infected humanized mice as in (C), one
representative sample and combined
G FluA (PR8), GFP+ H I J
PBS
data (n = 3 from three independent ex- Isotype control 4
0 0 FluA (PR8)
periments). (F) FACS plots showing the CD1c+ CD141+

MFI (HA / Isotype)

*
GFP+ DCs DCs 3 PBS 0 (GFP+ gate)
GFP and surface HA staining on lung DCs dLN
10 +
GFP HA GFP HA + + PBS
from phosphate-buffered saline (PBS) FluA 2
0.3 0 86 0 Isotype
treatment or GFP-tagged FluA(PR8) in- lung #1 73 FluA 0.4 control
1

CD141
+
fection of humanized mice as in (C). GFP dLN

GFP
10 14 0
HA and GFP+HA+ cells were examined FluA 0
CD141

lung #2 Lung Lung dLN HA CD1c

for the expression of CD1c and CD141 70 CD1c+ CD141+ DCs
(representative of two independent ex- CD1c HA
DCs DCs HA
periments). (G) Total GFP+ DCs as gated K Endocytic enveloped virus
in (F) were further defined as CD1c+ and CD1c+ DCs Influenza virus, HIV-2... CD141+ DCs
CD141+ DCs and analyzed for the ex- Permissive to viral fusion Constitutive resistance to viral fusion
pression of surface HA (red line). DC sub-
sets from PBS-treated lungs (gray shaded) Cytosolic sensing Furin-dependent
were stained with anti-HA as the con- endocytic sensing
Cytokines, IFN-I
trol (each sample was a pool of three
mice; representative of two indepen-
dent experiments). (H) The same gating RAB15

as in (F) on draining LN DCs from PBS

treatment or GFP-tagged FluA (PR8) in-
fection (each sample was a pool of three Virus-encoded immune suppressors Cross-presentation
Viral antigen
to six mice; representative of two inde- Virus-induced cell death of viral antigens
synthesis
pendent experiments). (I) The same gat-
ing 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.

pDCs, which contrasts with the prevailing view that the resistance
of pDCs to viral infection would be mediated by autocrine type
I IFN production.
Although we have identified a key role for RAB15 in promoting
resistance of CD141+ DCs to viral infection, how resistance is achieved
at the biochemical level remains to be determined. It is possible that
CD1c+ DCs express specific gene programs that render them suscep-
tible to viral infection in a way that overcomes RAB15 activity. An
interesting scenario is that ISG expression in CD1c+ DCs is not suffi-
cient to impose an antiviral state and that CD1c+ DCs are equipped
with specific programs that enable viral replication. A similar concept
has been proposed for marginal zone macrophages (63). Alternatively,
RAB15 might depend on specific effectors and cofactors that are also
expressed selectively in CD141+ DCs that remain to be identified. How
RAB15 may increase virus localization in GM130+ compartments and
how the Golgi apparatus may limit virus-mediated fusion thus need
further study. Other RAB GTPases previously linked to RAB15 activ-
ity and to HIV and influenza virus infection could also be implicated
(6466). In addition, we find that the vaccine strain MVA poxvirus
infects all DC subsets, presumably through efficient fusion at the
cell surface (46). The ability to bypass RAB15-mediated resistance
may be a desirable property for the immunogenicity of live-attenuated
vaccines, such as MVA, because it would maximize production of vi-
ral antigens and triggering of cytosolic sensors within the APCs.

Silvin et al., Sci. Immunol. 2, eaai8071 (2017) 7 July 2017 10 of 12

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE
How DCs deal with viral infection and perform antigen presenta-
tion is highly organized in space and time (1, 9, 10, 55). Here, we have
demonstrated that a mechanism enabling this dual function is a disso-
ciation of these two events. To ensure that antigen is presented, CD141+
DCs are protected from viral infection and use cross-presentation.
This paradigm may allow a better understanding of the induction of
protective immunity against viruses and live-attenuated vaccines and
sheds light on the existence of constitutive mechanisms of resistance
to viral infection.
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.
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Acknowledgments: We thank A.L. Lucido, S. Amigorena, and P. Benaroch for critical reading
of the manuscript. Some reagents were provided by the NIH AIDS Reagent Program. We thank
our healthy volunteers; J. Fay; P. Metang; the Clinical Core, the Apheresis Core, the Flow
Cytometry Core, the Imaging Core, and the Genomic Core at Baylor Institute for Immunology
Research; the Flow Cytometry facility, and V. Fraisier from the Cell and Tissue Imaging
(PICT-IBiSA) at Institut Curie, a member of the French National Research Infrastructure
France-BioImaging (ANR10-INBS-04). S. Whelan initially provided VSVeGFP to M.I. Funding:
A.S. was successively supported by a Ministre de lEducation Nationale de la Recherche et de
Technologie fellowship and by Sidaction. S.C. was supported by Sidaction. This work was
supported by the ATIP-Avenir program; the Ville de Paris Emergence program; European FP7
Marie Curie Actions; Labex VRI (ANR-10-LABX-77); Labex DCBIOL (ANR-10-IDEX-0001-02 PSL*
and ANR-11-LABX-0043); the ACTERIA Foundation; Fondation Schlumberger pour lEducation
et la Recherche (FSER) and European Research Council grant 309848 HIVINNATE (to N.M.);
ANRS (France REcherche Nord & Sud Sida-hiv Hpatites, to N.M. and A.M.); Baylor Research
Institute (BRI, to A.K.P.); NIH grants U19AI057234 and U19AI089987 (to A.K.P.), R01DA033773
(to A.G.-S.), 1U19 AI 118610-01 (to M. Merad), U19AI117873 (to M. Merad, R.A.A., and A.G.-S.),
and UO1AI095611 (to M. Merad, A.G.-S., and A.K.P.); and the Center for Research on Influenza
Pathogenesis (NIAID CEIRS HHSN272201400008C). Author contributions: A.S. and C.I.Y.
performed most of the experiments. X.L., F.I., and S.C. performed parts of the HIV experiments.
C.B. performed the experiment with lung DCs. J.-B.B. performed parts of the RAB15
experiments. W.-H.K. performed parts of the influenza virus experiments. C.C. provided
technical help. M. Maurin developed image analysis scripts. C.G., S.M.-L., and Y.W. performed
bioinformatics analysis. V.P. and E.A. directed the RNA-seq experiments. R.A.A. contributed to
influenza virus experiments. M.I. provided reagents and expertise in the experiments involving
VSV. A.G.-S. provided influenza virus reagents and expertise. B.G. provided expertise on RAB
proteins. M.D. directed the HIV experiments with F.I. A.M. directed the HIV experiments with
S.C. M.M. designed parts of the study. C.I.Y., A.S., A.G.-S., and M. Merad contributed to
manuscript writing. A.K.P. and N.M. designed the study and wrote the manuscript. Competing
interests: The authors declare that they have no competing interests. Data and materials
availability: The data for this study have been deposited in the National Center for
Biotechnology Information database with accession number GSE76511.
Submitted 15 August 2016
Resubmitted 9 March 2017
Accepted 17 May 2017
Published 7 July 2017
10.1126/sciimmunol.aai8071
Citation: A. Silvin, C. I. Yu, X. Lahaye, F. Imperatore, J.-B. Brault, S. Cardinaud, C. Becker, W.-H. Kwan,
C. Conrad, M. Maurin, C. Goudot, S. Marques-Ladeira, Y. Wang, V. Pascual, E. Anguiano, R. A. Albrecht,
M. Iannacone, A. Garca-Sastre, B. Goud, M. Dalod, A. Moris, M. Merad, A. K. Palucka, N. Manel,
Constitutive resistance to viral infection in human CD141+ dendritic cells. Sci. Immunol. 2,
eaai8071 (2017).
12 of 12
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, Ccile Conrad, Mathieu Maurin, Christel Goudot, Santy Marques-Ladeira, Yuanyuan Wang, Virginia
Pascual, Esperanza Anguiano, Randy A. Albrecht, Matteo Iannacone, Adolfo Garca-Sastre, Bruno Goud, Marc Dalod, Arnaud
Moris, Miriam Merad, A. Karolina Palucka and Nicolas Manel

Sci. Immunol. 2, eaai8071.

DOI: 10.1126/sciimmunol.aai8071

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Divided, they conquer
Dendritic cells (DCs) play a crucial role in priming T celldriven antiviral responses. Silvin et al. have examined the
paradox of how virus-infected DCs retain the ability to drive adaptive immune responses. In response to endocytic
viruses, they found CD1c+ DCs to be susceptible to infection and death, whereas CD141+ DCs were not. They report
that viral resistance of CD141 + DCs was conferred by the expression of an endocytic guanosine triphosphatase, RAB15,
and that transfer of antigen from infected CD1c + DCs by CD141+ DCs allowed these virus-resistant DCs to prime T cell
responses. By documenting a division of labor between DC subsets that separates antigen acquisition from antigen
presentation, Silvin et al. offer a solution to this long-standing puzzle.

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