Plasmacytoid Dendritic Cells Delineate Immunogenicity of Influenza
Vaccine Subtypes
Shohei Koyama, et al.
Sci Transl Med 2, 25ra24 (2010);
DOI: 10.1126/scitranslmed.3000759

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RESEARCH ARTICLE
INFECTIOUS DISEASE

Plasmacytoid Dendritic Cells Delineate Immunogenicityof Influenza Vaccine Subtypes
Shohei Koyama,1,2 Taiki Aoshi,3 Takeshi Tanimoto,4 Yutaro Kumagai,1 Kouji Kobiyama,3Takahiro Tougan,3 Kazuo Sakurai,5 Cevayir Coban,1 Toshihiro Horii,3
Shizuo Akira,1* Ken J. Ishii1,3*

A variety of different vaccine types are available for H1N1 influenza A virus infections; however, their immunological mechanisms of action remain unclear. Here, we show that plasmacytoid dendritic cells (pDCs) andtype I interferon (IFN)–mediated signaling delineate the immunogenicity of live attenuated virus, inactivatedwhole-virus (WV), and split virus vaccines. Although Toll-like receptor 7 acted as the adjuvant receptor for the
immunogenicity of both live virus and WV vaccines, the requirement for type I IFN production by pDCs for the
immunogenicity of the vaccines was restricted to WV. A split vaccine commonly used in humans failed to immunize naïve mice, but a pDC-activating adjuvant could restore immunogenicity. In blood from human adults,
however, split vaccine alone could recall memory T cell responses, underscoring the importance of this adjuvant pathway for primary, but not secondary, vaccination.

INTRODUCTION
Vaccination is considered to be the best prophylaxis for influenza
virus infection (1). There are three different types of influenza virus
vaccines: live attenuated influenza virus (LAIV), formalin-inactivatedwhole-virus (WV) vaccine, and ether-treated hemagglutinin (HA)
antigen–enriched virion-free “split” virus (SV) or “subunit” virus (SU)vaccine (2). Among them, SV and SU are the most commonly used inclinics because there are fewer reactogenicities, although LAIV and
WV have been shown to have superior immunogenicity, especiallyin children (3–5). The immunogenicity and efficacy of these vaccines
can be affected by host factors, such as age and immunological status[such as immunodeficiency (6–8)], and viral factors, including the antigenic mismatch between the vaccines and the circulating virus
strains (9). However, the exact mechanisms used by the three typesof vaccine compositions to achieve immunogenicity and how thesemechanisms differ are not fully understood.
It is known that most, if not all, successful vaccines that induce
strong protection against pathogens contain adjuvant components that
activate the innate immune system via specific receptors, including
Toll-like receptors (TLRs), retinoic acid–inducible gene-I (RIG-I)–likereceptors (RLRs), and Nod-like receptors (NLRs) (10–12). These receptors are expressed by antigen-presenting cells, such as dendritic cells(DCs), which mediate the subsequent adaptive immune response by
releasing specific cytokines, such as interferons (IFNs) and interleukins (ILs), and activating antigen-specific T and B cells. The natureof the adjuvant and the specific receptors that are activated on DCs
can determine the type of immune response that is generated (10–12).
Recent studies suggest that TLR7 and a certain NLR that activates the
1

Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka
University, Suita, Osaka 565–0871, Japan. 2Department of Respiratory Medicine, Tohoku
University Graduate School of Medicine, Sendai, Miyagi 980–8575, Japan. 3Department of
Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita,
Osaka 565–0871, Japan. 4The Research Foundation for Microbial Diseases of Osaka
University, Kan-on-ji, Kagawa 768–0061, Japan. 5Department of Chemical Processes andEnvironments, University of Kitakyushu, Kitakyushu, Fukuoka 808–0135, Japan.
*To whom correspondence should be addressed. E-mail: kenishii@biken.osaka-u.ac.jp(K.J.I.); sakira@biken.osaka-u.ac.jp (S.A.)

inflammasome—a collection of components that spur the innate immune system—are involved in controlling adaptive immune responses
to influenza A virus infection (13, 14). Our previous work has characterized a key role for TLR signaling in the immune response to inactivated
WV vaccination (13); however, no comprehensive study directly compares the role of TLRs, NLRs, and RLRs in either live virus or inactivated
WV vaccinations (15). Here, we decipher the mechanism(s) by which thevarious virus preparations drive development of immunogenicity.

RESULTS
Inactivated WV requires TLR7-mediated, but not RLR- or
NLR-mediated, immune activation for its immunogenicity
We first examined whether an inactivated WV preparation of the A/
New Caledonia/20/1999 (NC) (H1N1) strain can immunize mice lacking
TLR7, IPS-1 (IFN-b promoter stimulator–1; an adaptor protein for
RIG-I–mediated type I IFN production), and ASC (apoptotic speckprotein; containing a caspase recruitment domain, an adaptor molecule required for NLRP3 inflammasome activation). All three of thesegenes have been shown to be involved in innate immune recognitionof live influenza virus infection (14, 16–19).
We immunized TLR7-deficient or IPS-1–deficient mice intranasally (i.n.) with WV of NC H1N1 twice at a 2-week interval. One
week after the second injection, the vaccination-induced, virus-specific
B cell and CD4+ T cell adaptive immune responses were analyzed.
TLR7-deficient mice showed virtually no virus-specific immunoglobulin A (IgA) in bronchoalveolar lavage fluid (BALF), no virusspecific IgG in serum (Fig. 1A), and no IFN-g production from CD4+ T
cells specific to a viral nucleoprotein (NP) antigen epitope (NP260–283
specific to I-Ab) (Fig. 1B), whereas IPS-1–deficient mice mounted comparable levels of these responses to wild-type mice (Fig. 1, A and B).
Consistently, the immunized TLR7-deficient mice were not protectedagainst a lethal challenge with the A/Puerto Rico/8/34 (PR) (H1N1)
strain, whereas the immunized IPS-1–deficient mice were protected
to a level comparable to vaccinated wild-type mice (Fig. 1C). We

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RESEARCH ARTICLE
and C) relative to the heterozygous IFNAR2 knockout mice. As aresult, mortality was increased and a significantly larger body weightloss was observed after lethal PR H1N1 challenge in the IFNAR2deficient mice (Fig. 2C). Naïve wild-type and IFNAR2-deficient mice
showed similar susceptibilities to PR H1N1 challenge, consistent witha previous study (23). Similar results were also observed after i.m. immunization (fig. S3, D and E). When naïve IFNAR2-deficient mice
were immunized with live virus, there was no alteration of thevirus-specific serum IgG and IFN-g secretion by virus NP antigenspecific CD4+ T cells (NP260–283 specific to I-Ab) and CD8+ T cells(NP366–374 specific to H-2Db) (fig. S3, F to I), consistent with a previous study (23). Together, these results suggest that TLR7, but not RLRs
or NLRs, is required for immunogenicity of inactivated as well as live
influenza virus vaccination. In addition, the type I IFN receptor–mediatedsignaling pathway was critical for the immunogenic response to WV
but not to the live virus.
Live virus and inactivated WV vaccines induce type I IFNs
through distinct DC types
DCs are a critical component of the innate immune system that recognize
vaccines and mediate the adaptive immune response. There are different
subtypes of DCs that can be divided loosely into conventional myeloid
DCs (mDCs) and plasmacytoid DCs (pDCs), which express different
receptors and secrete different cytokines on activation. To characterize
in more detail the immune response triggered by the WV vaccine andlive virus, we stimulated two types of bone marrow–derived DCs—Flt3

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10
1.0
0.1
10
1.0
0.1

IFN-γ (ng/ml)

Survival rate (%)

BALF
IgA titer (×102)

Serum
Serum
Serum
IgG2a titer (×104) IgG1 titer (×104) tIgG titer (×105)

10
1.0
0.1
10
1.0
0.1
10
1.0
0.1

IFN-γ (ng /ml)

Survival rate (%)

Serum
Serum
Serum
IgG2a titer (×104) IgG1 titer (×104) tIgG titer (×105)

BALF
IgA titer (×102)

The type I IFN receptor is important for the immunogenic
response to inactivated WV vaccine but not to the live virus
Because both the inactivated WV and the live virus require TLR7 fortheir immunogenicity, we sought to identify the downstream effector
molecule(s) involved. Type I A
D
E
B
5
8
IFNs, such as IFN-a and IFN-b,
2
1.0
4
6
are known to have potent adju3
4vant activity (20, 21), and a recent
2
2
1
*
1
0.5
study showed that WV immuni00zation substantially up-regulated
6***
the expression of IFN-inducible
10008genes, such as Cxcl10 (22). There4
NP260 283
NP260 283
6
fore, as a systemic indicator of type(µg/ml)(µg/ml)4
2
WT Tlr7 -/- Ips-1-/WT Asc -/2
I IFN responses, we examined the*00amounts of the cytokine CXCL10
5
in the sera of mice 24 hours afF
C
10
48
ter vaccination. The induction of
100
100
3
6
CXCL10 was significantly reduced
80
80
2
4
1
in TLR7-deficient mice relative to
*
Naive
2
60
60
0
WT
0
wild-type, IPS-1–deficient, and40
40-/Tlr7Na ve(vsWT; P < 0.01)
ASC-deficient mice (Fig. 2A). Si5
10
WT
20
20
-/Ips
1
Asc -/4
8
milar results were obtained for
0
0
3
6
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
messenger RNA (mRNA) analyses
24Days after infection
Days after infectionof IFN-b and CXCL10 in the lung
1
2
*
0
0
(fig. S3A), suggesting that type I
Tlr7 +/+ -/- +/+IFNs might be dominant effector
Asc +/+ -/Ips-1 +/+ +/+ -/molecules in this TLR7-dependent
system. To test this hypothesis di- Fig. 1. TLR7-dependent, but not IPS-1–dependent or ASC-dependent, signaling is required for the induction of protective
rectly, we i.n. immunized mice immune responses by inactivated WV. (A to C) Wild-type (WT), Tlr7-deficient, and Ips-1–deficient mice (n = 9 per group) were
deficient in the IFN-a and IFN- i.n. vaccinated with WV of NC (1.5 mg per mouse) twice, with a 2-week interval. One week after the second vaccination, we
measured titers of antigen-specific mucosal (BALF) IgA and serum total IgG (tIgG), IgG1, and IgG2a (A) and IFN-g production
b receptor 2 (IFNAR2) with WV
by CD4+ T cells (B) by ELISA as described in Materials and Methods. *P < 0.05 versus WT mice. (C) Vaccinated mice (n = 9 per
as in Fig. 1. IFNAR2-deficient mice group) were challenged with 10 × LD (median lethal dose) (2 × 104 PFU per mouse) of lethal influenza virus PR, and their50
failed to induce virus-specific anti- survival was monitored. *P < 0.05 versus vaccinated WT mice. (D to F) WT and Asc-deficient mice (n = 5, each group) werebodies (including BALF IgA and similarly vaccinated, and their antigen-specific antibody responses (D), IFN-g production by CD4+ T cells (E), and survival (WT,
serum IgG) and CD4+ T cell re- n = 7; Asc-deficient, n = 10; naïve, n = 10) (F) were determined by ELISA as described in Materials and Methods. Each bar
sponses (Fig. 2B and fig. S3, B represents the mean (A and D) or mean ± SD (B and E). These results are representative of two independent experiments.
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obtained similar results with intramuscular (i.m.) immunization of thesemice, with respect to total IgG titer and IgG2a titer (fig. S1). In contrast toi.n. immunization, i.m. immunization failed to elicit BALF IgA in either
wild-type or TLR7-deficient mice. There was an enhanced serum IgG1
response in TLR7-deficient mice relative to wild-type mice, but this response failed to protect the TLR7-deficient mice from lethal challenge with
PR H1N1 (fig. S1, A and C). Thus, the inactivated WV vaccine requires
TLR7-mediated, but not RIG-I–mediated, IPS-1 for its immunogenicity,which is consistent with findings for the live virus vaccination (13).
We also immunized ASC-deficient mice with WV (NC H1N1) because a role for the ASC inflammasome in the adaptive immune response
to influenza virus infection is controversial (14, 19). ASC-deficient mice
did not show any defects in their adaptive immune responses to WV immunization relative to wild-type mice (Fig. 1, D and E). Notably, ASC was
not involved in the immunogenicity of live virus immunization, except
for serum IgG1 production, whereas CD8+ T cell responses were notobserved with WV immunization (fig. S2). Immunized wild-type andASC-deficient mice were similarly protected against lethal PR H1N1
virus challenge relative to naïve wild-type mice (Fig. 1F). Therefore,
ASC inflammasome activation was not essential for the induction ofvirus-specific B and CD4 T cell responses to WV.

ligand–generated DCs (FL-DCs; which contain pDCs) and granulocytemacrophage colony-stimulating factor (GM-CSF)–generated DCs(GM-DCs; which contain conventional DCs but no pDCs)—with
the live virus or WV and then measured their IFN-b production by
enzyme-linked immunosorbent assay (ELISA). The live virus strongly
stimulated FL-DCs to produce IFN-b in a TLR7-dependent manner,
whereas production by GM-DCs was in a TLR7-independent manner(Fig. 2D), consistent with our previous study (13). In contrast, WVactivated FL-DCs, but not GM-DCs, produced IFN-b, a process entirely dependent on TLR7 signaling (Fig. 2D), indicating that there isa clear distinction between the live virus and WV in terms of theirabilities to activate DCs to secrete type I IFNs.
To confirm these in vitro observations in vivo, we next depleted
pDCs in vivo to examine the role of these cells in the induction ofadaptive immune responses to live virus and WV, as pDCs are known
to play a key role in bridging the innate and adaptive immune responses (24). Wild-type mice were treated intravenously with an antibody to mPDCA-1 (25) and then immunized 24 hours later with thelive virus or WV. Depletion of pDCs was confirmed in the spleen (fig.
S4). After live virus vaccination, the concentrations of mRNAs
derived from genes involved in induction of the adaptive immune
response, specifically Ifnb, Cxcl10, Il6, and Ccl2, were clearly elevatedin the lung irrespective of treatment with antibody to mPDCA-1(Fig. 2E). In contrast, these transcriptional responses were severely
impaired after WV vaccination in pDC-depleted mice relative toisotype control antibody–treated mice (Fig. 2E). Serum CXCL10
was also reduced in pDC-depleted mice treated with WV but not thelive virus (Fig. 2F). Thus, both live virus and WV induced type I IFNs
predominantly through pDCs in vivo; however, WV was dominantly
recognized by pDCs, whereas live virus could also stimulate other cell
types to activate innate immune responses.
Although it was previously reported that pDC activation is not essential for the induction of adaptive immune responses in live influenza virus
infection (26, 27), pDC depletion specifically rendered the inactivated WV
nonimmunogenic, as measured by virus-specific IgG concentrations inserum (Fig. 3A). In these experiments, mice were treated with an antibody to mPDCA-1 twice at both the primary and the secondary vaccinations. To further examine the more detailed role of pDCs in the
primary and/or secondary vaccinations, we treated mice with an antibody to mPDCA-1 at either the primary or the secondary (boost)vaccination. Virus-specific mucosal IgA, serum IgG (Fig. 3B), andCD4+ T cell IFN-g (Fig. 3C) were significantly impaired when pDCs
were depleted in the primary, but not in the secondary, vaccination
with the inactivated WV. Thus, pDC activation is essential for
inducing B cell and CD4+ T cell responses to the inactivated WV
during primary, but not secondary, vaccination. By sharp contrast,
pDC activation at priming was not required for inducing B celland CD4+ T cell responses with the live virus.

Body weight change (% )BALF IgA titer (×102)

Survival rate (% )Serum CXCL10 (pg/ml)(ng ml)IFN- (ng/ml)

Serum CXCL10 (pg/ml)

WV-loaded pDCs were sufficient to transfer immunogenicity
to naïve mice, which requires intrinsic as well as extrinsic
type I IFN signaling
To further examine the role of pDCs, we performed cell transfer experiments. FL-DCs from wild-type mice were separated into two populations, namely, a B220
A
B
C
(CD45R)–enriched popula200
200
200
tion containing pDCs and a
100
110
*
B220-depleted population
100
80
10
containing virtually no pDCs
8
90
60
100
100
* 100
+/6
*
(as indicated in Fig. 3D).
Ifnar280
40
4Ifnar2 -/The cell populations were70
20
2
(vsIfnar2 +/- ; P = 0.059)0pulsed with WV and injected
0
0
0
0
60
Tlr7 +/+ -/- Ips-1 +/+ -/- Asc +/+ -/Ifnar2 +/- -/0 2 4 6 8 10 12 14
0 1 2 3 4 5 6 7
intravenously into wild-type
Days after infection
Days after infection
mice. The virus-specific IgG
DEF
concentrations elicited by
Live
WV
Live
WV
Live
WV
6.0
2.0
the B220-enriched FL-DCs
Isotype mPDCA-1
Isotype mPDCA-1
WT
200
1000
IsotypeTlr7 -/were significantly higher than
Ifnb
mPDCA-1
800
4.0
those elicited by the B220Cxcl10
1.0
600
depleted population (Fig.
ll6
100
2.0
400
3D). In addition, when we
Ccl2
*
* **
*
200
transferred B220-enriched
Actb
0
0
0
0
FL-DC GM-DC
FL-DC GM-DC
FL-DCs derived from the
IFNAR2-deficient mice (lackFig. 2. Critical roles of type I IFN receptor–mediated signaling and pDC activation in inducing adaptive immune re- ing type I IFN) into wild-type
sponses to the inactivated WV vaccine but not to the live virus. (A) Tlr7-deficient, Ips-1–deficient, Asc-deficient, and control mice or vice versa, virusmice (n = 3 per group) were i.n. vaccinated with WV (3.0 mg per mouse), and CXCL10 production in sera was measured by specific IgG induction was
ELISA after 24 hours. *P < 0.05 versus control mice. (B and C) Control (Ifnar2+/−) and type I IFN receptor–deficient (Ifnar2−/−) significantly impaired in bothmice (n = 8, each group) were i.n. vaccinated with WV (1.5 mg per mouse) as in Fig. 1, and BALF IgA (B) was measured by cases (Fig. 3E). We also conELISA. *P < 0.05 versus control mice. The mice were then infected with lethal influenza PR at 10 × LD50, and their survival firmed thatIFNAR2-deficientand body weight (C) were monitored. *P < 0.05 versus control mice. (D) Type I IFN production by FL-DCs and GM-DCs from
FL-DCs secreted significantly
WT and Tlr7-deficient mice in response to the live virus [multiplicity of infection (MOI) = 10] and WV (5 mg/ml). IFN-b
production was measured by ELISA 24 hours after the stimulation. *P < 0.05 versus WT FL-DC. (E and F) Innate immune less type I IFN relative to that
responses to WV in pDC-depleted mice. Mice were treated with an antibody to mPDCA-1 24 hours before inoculation and of FL-DCs from the heterothen i.n. challenged with the live virus (1 × 105 PFU per mouse) or WV vaccine (5 mg per mouse). The expression of IFN-b, zygous IFNAR2 knockout
CXCL10, IL-6, and CCL2 mRNA in the lungs (E) and CXCL10 in sera (F) 24 hours after vaccination was measured by RT-PCR mice (fig. S5A). When we
tested TLR7 deficiency withand ELISA. *P < 0.05 versus control mice. These results are representative of at least two independent experiments.
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RESEARCH ARTICLE

the same approach, we observed a substantial TLR7 signaling dependency in these FL-DC transfer experiments (fig. S5B). These results
suggest that type I IFN–mediated signaling in pDCs, as well as in
the recipient as yet unidentified cell type(s), is indispensable for eliciting the adaptive immune response to WV.

mice (Fig. 4, C and D). Immunization of TLR9-deficient mice provided
further evidence that the responses induced by the SV+SPG-CpG weredependent on TLR9 but not on TLR7 (fig. S7A).
The protective efficacies of these three types of vaccines were also
examined in mice. WV conferred protection against lethal PR H1N1 virus
challenge in a TLR7-dependent and TLR9-independent manner, whereas
the SV+SPG-CpG provided protection in a TLR9-dependent and TLR7independent manner (Fig. 4E and fig. S7B). Notably, the original SV failed
to provide protection against lethal PR H1N1 virus challenge in any of thegroups of mice examined (Fig. 4E and fig. S7B). We also confirmed thatthe restored protective effect of the SPG-CpG adjuvant was mediated bytype I IFN responses because IFNAR2-deficient mice failed to mountvirus-specific B and T cell responses (fig. S7, C and D) and demonstrated
no improved protection against infection (fig. S7E).
Although the usefulness of SV vaccination in the healthy adult human population has been recognized in many studies, our results are
somewhat contradictory. Therefore, we tested the relevance of theseobservations in a human system. Human peripheral blood mononuclear cells (PBMCs) from healthy volunteers were stimulated with
H1N1 live virus, inactivated H1N1 WV, and H1N1 SV for 24 hours,
and then IFN-a and IFN-g secretion was measured by ELISA. Consistent
with the mice data, IFN-a was secreted with live virus and WV but notwith SV stimulation (Fig. 4F). Depletion of pDCs with BDCA4 microbeads revealed that this IFN-a secretion was totally dependent on
pDC in WV and partially in live virus stimulation (Fig. 4F). On theSerum IgG titer (×10×105)

Serum IgG titer (×10
×105)

B220

Serum IgG titer (×10
×106)

10
1.0
0.1
10
1.0
0.1

10
1.0
0.1
10
1.0
0.1

IFN- (ng/ml)

BALF IgA titer (×102)

Serum IgG titer (×10×105)

Split vaccine does not protect naïve mice, but immunogenicity
can be improved with a pDC-activating adjuvant while itrecalls memory T cells in human adult blood
Currently, the most widely used influenza vaccines in many countries
comprise SV or SU, which mainly consist of purified protein antigens
such as HA and neuraminidase. As mentioned earlier, vaccination ofmice with SV led to significantly lower production of type I IFNs andrelated chemokines, such as CXCL10, at both the mRNA and theprotein levels in the lung and serum, respectively (Fig. 4, A and B).
SV also failed to activate DCs to produce type I IFNs in vitro (fig.
S6A). These data suggest that the intrinsic TLR7 ligand (that is, viralgenomic RNA) was lost during the SV production process. In supportof this idea, removal of the RNA content from WV by ribonucleasetreatment significantly decreased the TLR7-mediated type I IFN production by pDCs (fig. S6B). The reduced immunostimulatory activityof SV was associated with its diminished immunogenicity. Whennaïve mice were immunized with SV at the same dose, adjusted to
the HA content (fig. S6C), as WV, the HA-specific IgG and CD4 T
cell responses were significantly lower than those elicited by WV(Fig. 4, C and D). Together, thesedata strongly suggest that SV losesLive
WV
its built-in TLR7 adjuvant (viral ge- A
B
Isotype
Isotypenome RNA) during purification of
10
5
10
5
mPDCA-1
mPDCA-1
WV, consistent with a recent study
8448
for the H5N1 virus (28).
6
3
3
6
Our results thus far raise the pos422
4
sibility of improving SV immunoge2
1
*
1
2
*
*
*
nicity by adding a pDC-activating
0000
TLR ligand. Because pDCs express
d14
d21
d14
d21
Primary Boost
Primary Boost
both TLR7 and TLR9, we examinedE
EnrichedD
whether addition of a TLR9 ligand C
pDC
WT WT
B220 enriched
Isotype14.1 %
2.0
5
0.8
to the “adjuvant-lost” split vaccine
WT WT
B220 enriched
mPDCA-1
WT Ifnar2 -/B220 depleted
4
would replace the natural TLR71.5
0.6
Ifnar2 -/- WT
3
mediated pDC activation. We used
Depleted
1.0
0.4
***
pDC
2
a second-generation TLR9 ligand of
0.13 %
0.5
0.2
**
**
1
CpG DNA complexed with b-(1→3)**
0
0
D-glucan, namely, schizophyllan (SPG)
0tIgG IgG1 IgG2a
tIgG
IgG1
IgG2a
260 283
mPDCA-1(29). This new TLR9 ligand is more (NPµg/ml)
PrimaryBoostpotent and durable than naked CpG
DNA, and it still retains the TLR9 Fig. 3. Immunogenicity of the inactivated WV, but not of the live virus, depends on pDC activation at theligand activity. Mice were i.n. immu- primary vaccination. (A) The adaptive immune responses to WV (i.n.) were monitored in pDC-depleted mice
nizedwithWV,SV,orSVplustheSPG- (n = 3 per group). Mice were injected with an antibody to mPDCA-1 or isotype control antibody 24 hours before
CpG DNA conjugate (SV+SPG-CpG) each vaccination, and serum IgG concentrations were measured by ELISA at the indicated times. *P < 0.05
and then evaluated for their adaptive versus control mice. (B and C) To determine whether pDCs are more important for priming or boosting after
immune responses. The SV+SPG- vaccination with inactivated WV, we treated one group of mice (n = 3) only with an antibody to mPDCA-1 before
CpG induced robust type I IFN re- the primary vaccination (priming) and another group (n = 3) was treated only before the secondary vaccination+(boosting). BALF IgA and serum IgG 1 week after the boost (B) and IFN-g production from influenza-specific CD4sponses independently of TLR7 (Fig.
T cells (C) were measured by ELISA. *P < 0.05 versus control mice. (D and E) FL-DCs were separated into B220+
4, A and B). Correspondingly, the and B220− DCs by MACS. Each DC was pulsed with WV in vitro, and B220+ or B220− cells (5 × 105) were intraSV+SPG-CpG successfully enhanced venously injected (n = 3 per group). Serum IgG was measured 2 weeks after the injection in WT mice. *P < 0.05
HA-specificBcellandCD4+ Tcellre- versus B220-enriched cells. (D) B220+ cells (1 × 105) from WT and Ifnar2-deficient mice were intravenously
sponses to levels comparable to those transferred into untreated WT and Ifnar2-deficient mice (n = 3 per group), and serum IgG was measured by ELISA.
from WV immunization of wild-type *P < 0.05 versus WT to WT. (E) These results are representative of at least two independent experiments.
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Survival rate (%)

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ND

ND

ND

ND

IFN- (ng/ml)

ND

10
1.0
0.1

10
1.0
0.1
10
1.0
0.1
ND
ND

ND
ND

ND
ND

10
1.0
0.1
10
1.0
0.1
10
1.0
0.1

IFN- (ng/ml)

IFN- (ng/ml)

Anti-HABALF IgA titerCXCL10 (pg/ml)

PG

+S

SV

SV

V

W

+S
P
ve GLi

SV

V
SV

Li

W

ve

C

pG

pG

the immunogenicity and efficacy of influenza inactivated WV vaccinations (Fig. 1 and fig. S1). We identified pDCs as an innate immune celland type I IFNs as humoral factors that are essential for the immunogenicity of the inactivated WV vaccine (Figs. 2 and 3). Although our
results demonstrate an essential role for pDCs in inactivated WV vaccination, other studies have identified a redundant role for pDCs in
antiviral responses to live virus vaccination such as influenza virus(26, 27). In addition, although TLR7 is expressed in a variety of cell
types, including B cells and macrophages, our results strongly suggest
an essential role for pDCs in mediating TLR7-induced innate andadaptive immune responses to inactivated influenza WV vaccination
but not to live virus vaccination.
DISCUSSION
The critical role of pDCs in vaccine priming, but not in boosting, is
Although TLR7 and certain NLRs have been shown previously to be apparent from results of the pDC depletion study, in which pDCs wereinvolved in the induction of adaptive immune responses to influenza A removed from mice before vaccination with inactivated WV (Fig. 3, A tovirus infection (13, 14), the current work represents a comprehensive C). These findings parallel our in vitro data, in which the pDC-containing
study that directly compares the functions of TLRs, NLRs, and RLRs in FL-DC preparation, but not the mDC-dominant GM-DC preparation,
responded to inactivated WV
to produce type I IFNs (Fig.
A
BC
2D); however, these data do
not necessarily exclude the inWV
WV
0.3
100
SV
SV
volvement of other antigenSV+SPG-CpG
SV+SPG-CpG
80
0.2
presenting cells for either
Ifnb
60
priming or boosting. For exCxcl10
400.1
ample, pDCs pulsed with
*
* *
*
* *
Actb
20
inactivated WV in vitro can
Tlr7 -/WT
00
WT
WT
Tlr7 -/Tlr7 -/prime naïve mice (Fig. 3D),
Tlr7-/but the type I IFNs produced
WT
D
E
100
100
3
WV
by these pDCs were required
SV
80
80
Na ve
not only for stimulating the
SV+SPG-CpG
2
WV
60
60
pDCs themselves but also
SV
WV(vs WV; P<0.01) 40
40(vs SV+SPG-CpG; P<0.01)
1
for priming as yet unidentiSV+SPG-CpG
SV
2020*** ******
SV+SPG-CpG
fied cell types of the recipient000mouse (Fig. 3E). This sug0 2 4 6 8 10 12 14
0 2 4 6 8 10 12 14
NP260 283
gests that cross talk exists beDays after infection
Days after infection(µg/ml)Tlr7 -/WT
tween pDCs and the otherFG
as yet unidentified cells via
Experiment 1
Experiment 2
Experiment 1
Experiment 2
type I IFN–mediated signal2.0
5.0
2.0
2.0
PBMC
PBMC
ing. In addition, it will be of
PBMC-BDCA4
PBMC-CD4
4.0
1.5
1.5
1.5
PBMC-CD8interest to examine whether
3.0
other DCs, such as mDCs,
1.0
1.0
1.0
2.0
are involved in this inter0.5
0.5
0.5
1.0
cellular cross talk in such a00
00
way that pDCs can transfer
None Live WV SV
Live
WV
SV
SV
None Live WV SV
Live
WV
SV
SV
S-OIV
S-OIV
the flu antigens to mDCs or
Fig. 4. Immunogenicity differences between WV and SV depend on type I IFN production via pDC activation in naïve hosts that pDCs can present antibut not in primed hosts. (A and B) WT and Tlr7-deficient mice were i.n. vaccinated with three different vaccines as described gens directly to T cells and/in Materials and Methods. After 24 hours, IFN-b and CXCL10 expression in the lungs (A) and CXCL10 production in the sera or B cells. We note the dis(B) was measured by RT-PCR and ELISA. *P < 0.05 versus WT mice immunized with WV. (C and D) To compare the immu- tinct regulation of type I IFN
nogenicities of the three vaccines, we i.n. vaccinated WT (n = 9) and Tlr7-deficient (n = 6) mice as described in Fig. 1 and induction by inactivated WV
determined antigen (HA)–specific BALF IgA, serum total IgG (C), and IFN-g production by CD4+ T cells (D) by ELISA. *P < 0.05 vaccination, which differedversus WT mice immunized with WV. (E) Vaccinated mice were challenged with the PR strain at 10 × LD50, and their survival from IFN induction by live
rates were determined as described in Materials and Methods. *P < 0.05 versus WT mice immunized with WV or control
virus vaccination. Indeed, almice. These results are representative of at least two independent experiments. (F and G) IFN-a and IFN-g production in
response to inactivated influenza vaccines in human PBMCs. PBMCs from healthy volunteers were stimulated with live NC though type I IFNs were invirus, WV, and SV. Total PBMCs and pDC-depleted PBMCs (PBMC-BDCA4) were stimulated with live NC virus (0.01 MOI), WV duced by both inactivated(1.0 mg/ml), and SV (0.5 mg/ml), and IFN-a production was measured by ELISA (F). Total PBMCs and CD4- or CD8-depleted and live virus vaccinations,
PBMCs were stimulated with WV and SV of NC (10 and 5 mg/ml, respectively) and SV of swine-origin influenza virus (SV S-OIV) inactivated WV vaccines ac(5 mg/ml). IFN-g production was measured by ELISA (G). These results are from 2 representatives of 10 volunteers.
tivated only TLR7 on pDCs,
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other hand, both WV and SV induced IFN-g secretion comparably in
the PBMC preparations even when the SV made from the swine-origin
H1N1 A/California/04/2009 strain was used. Results obtained after
CD4+ and CD8+ T cell depletion of the PBMC preparations revealed
that virus-specific IFN-g secretion was produced mainly by CD4+ T
cells in live virus and WV stimulation (Fig. 4G). These results suggestthat SV could efficiently stimulate memory T cell responses without
type I IFNs in a naturally (or seasonally) influenza virus–exposed
human population.

RESEARCH ARTICLE
MATERIALS AND METHODS
Animals, cells, viruses, and reagentsThe generation of Tlr7-, Ips-1–, Ifnar2-, and Tlr9-deficient mice, either
on a 129/Ola × C57/BL6 or on a C57/BL6 background, has been described previously (13, 46). ASC-deficient mice were a gift from V. M.
Dixit (47).
All animal experiments were performed in accordance with theinstitutional guidelines for the Osaka University animal facility.
Purified influenza viruses, H1N1 (PR and NC), a recombinant HAprotein of PR, and both inactivated WV and split vaccines of NC
were prepared as previously described (48). Both types of vaccines
were derived from the NC strain. Briefly, the viruses were purified
from allantoic fluid by filtration (0.45 mm) followed by sedimentation through a linear sucrose gradient. For formalin-inactivated WV
vaccines, purified viruses were treated with 0.1 to 0.2% formalin at
4°C for a week. For the ether-split vaccines (SV), the viruses weremixed with an equal volume of ether and then incubated for 30 min
at room temperature with stirring. The mixture was centrifuged (3000rpm, 15 min), and the aqueous phase was collected and evaporated.
CpG DNA forming a triple helix with SPG, a natural polysaccharide
composed of b-(1→3)-D-glucan, was used as the second-generation
TLR9 ligand as previously described (29, 49, 50). DCs were prepared
as described previously. Briefly, bone marrow cells were cultured in
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
calf serum, 100 mM 2-mercaptoethanol, and human Flt3 ligand(100 ng/ml) (PeproTech) or murine GM-CSF (10 ng/ml) (PeproTech)for 7 to 9 days to use as FL-DCs and GM-DCs.
Influenza virus infection and vaccination
For influenza virus infection or vaccination, mice were anesthetized and administered i.n. with 30 ml of phosphate-buffered saline(PBS) (15 ml for each nares) containing serial amount of influenza
NC viruses and vaccines. Mice were infected with 1 × 105 to 2 × 105
plaque-forming units (PFU) of virus per mouse or vaccinated withWV (1.5 to 3.0 mg per mouse) or SV (0.75 mg per mouse) with orwithout SPG-CpG (30 mg per mouse) twice at a 2-week interval.
For the analysis of protection, mice were infected with the indicated doses of lethal PR strain.
Measurement of innate immune responses
Reverse transcription polymerase chain reaction (RT-PCR) was
performed to measure mRNA expression levels of type I IFNs, cytokines, and chemokines using the RNA of the stimulated cells as
previously described (13). Protein concentrations of IFN-a, IFN-b,
and CXCL10 in the culture supernatants of the stimulated cells
were measured using ELISA kits (IFN-a and IFN-b, PBL Biomedical Laboratories; CXCL10, R&D Systems).
Plasmacytoid DC depletion and cell transfer
Plasmacytoid DCs were depleted by intravenous injection of antibody to mPDCA-1 (500 mg) (Miltenyi Biotec) 24 hours before live
virus infection or inactivated WV vaccination.
FL-DCs were separated into two populations, B220-enriched
and B220-depleted population, by B220 antibody MACS microbeads(Miltenyi Biotec) according to the manufacturer’s protocol to obtain
B220-enriched FL-DC. Each cell population was incubated with WV(5 to 10 mg/ml) for 3 hours, and 1 × 105 to 5 × 105 cells per mousewww.ScienceTranslationalMedicine.org

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whereas live virus activated both TLR7 on pDCs and other TLR7independent pathways in the other cells, possibly mDCs (Fig. 2, D
and E). Therefore, although TLR7, pDCs, and type I IFNs all were essential for inactivated WV vaccination, pDCs and type I IFNs were notessential for live virus vaccination.
The adaptor ASC is a critical component of NLRP3 inflammasome (30). In contrast to type I IFN responses, ASC-dependent inflammasome activation has been shown to play a critical role in thesurvival of the mice challenged with live influenza virus (14, 18, 19).
However, the requirement for inflammasome activation to induce
influenza-specific adaptive immune responses has been controversial(14, 19). Our data indicate that ASC-dependent inflammasome activation is dispensable for inducing adaptive immune responses to
WV and live virus, except for IgG1 production in live virus vaccination (Fig. 1 and fig. S2). Concurrent analysis comparing three innate immune signaling pathways, TLR, NLR, and RLR, enabled us
to elucidate that the TLR-dependent pathway dominantly controlled
the T helper 1–type protective immunity elicited by WV and live
virus vaccination.
Although SV, which is now used as the first choice for influenza
vaccination in many countries, was not protective in naïve mice, its
decreased immunogenicity was fully restored by adding a new
TLR9 ligand that stimulates pDCs to secrete type I IFNs (Fig. 4,
A to E, and fig. S7). These data above further support the notionthat pDC activation and their type I IFN production play a criticalrole in the induction of inactivated influenza vaccine immunogenicity in naïve hosts. These results might explain in part the wellknown fact that the efficacy of adjuvant-less SV is lower in young
children than in adults (7), in which SV is simply boosting thememory T and/or B cell responses. This is further supported byour results obtained using human PBMCs (Fig. 4, F and G), whichsuggest that most human adults have virus-specific CD4+ T cells that
produce IFN-g in response not only to seasonal flu viruses but also to
the novel swine H1N1 virus. Our results also indicate that memory T
cells react to both internal proteins, such as those in SV, and a wide
spectrum of influenza virus surface antigens, such as those on swineorigin H1N1 (31, 32) and H5N1 (33). The age distribution of theaffected population in swine-origin H1N1 and H5N1 infections,which was limited to the young, might reflect the importance ofmemory T cells established by recurrent exposure to seasonal influenza live viruses and vaccines (34, 35)
LAIVs activate both influenza-specific IgA-secreting B cells andcytotoxic CD8+ T cells (36), which provides certain advantages overinactivated vaccines including WV and SV. Although WV is now
unavailable for seasonal influenza, it is cost-effective and can induce heterosubtypic protection not only against a challenge by
H1N1 (Fig. 1C and fig. S1C) but also against H5N1 (37, 38), as with
LAIV (39). In addition, recent progress in manufacturing techniques could reduce the adverse event rate in i.m. WV immunization(37, 38) to yield results that are quite different from those of pastclinical trials (3, 40). An i.n. WV immunization may produce a sufficient combination of efficacy, safety, and utility for both seasonal
and pre-pandemic vaccines (41–45).
Together, analysis of the molecular and cellular mechanisms ofdifferent influenza vaccines provides useful information for improving vaccine immunogenicity and efficacy, as well as for choosing an appropriate form of influenza vaccine with a rational safety
approach.

RESEARCH ARTICLE

Confirmation of pDC depletion in spleen by flow
cytometric analysis
After Fc blocking with an antibody to CD16/32, isolated spleen cells were
stained with fluorescein isothiocyanate (FITC)–conjugated antibody to
CD11c, phycoerythrin (PE)–conjugated antibody to CD45R/B220, andallophycocyanin-conjugated antibody to mPDCA-1 (Miltenyi Biotec)
for 30 min at room temperature and washed with PBS containing 1%
bovine serum albumin. Just before fluorescence-activated cell sorting(FACS) analysis using FACSCalibur and CellQuest software (BD Biosciences), 7-aminoactinomycin D (BD Biosciences) was added.
Measurement of antigen-specific T and B cell responses
After two i.n. vaccinations, B cell–mediated humoral responses
were measured as immunoglobulin production by ELISA using
goat antibody to mouse total IgG, IgG1, IgG2a, and IgA conjugatedto horseradish peroxidase (Southern Biotech) as previously described (1). T cell–mediated cellular responses were monitored bymeasuring NP260–283/I-Ab–specific or NP366–374/H-2Db–specificIFN-g secretion of splenocytes and the frequency and cytotoxicityof H-2Db–specific CD8 T cells as described previously (13).
Preparation of human PBMCs for cytokine analysis
PBMCs were obtained from 10 healthy adult volunteers (30 to 50
years old, 6 males and 4 females). All of the experiments using human PBMCs were approved by the Institutional Review Board of
the Research Institute for Microbial Diseases, Osaka University.
Cells were purified from heparinized blood by density centrifugation using Ficoll-Paque Plus (Amersham). Human pDCs, CD4, or
CD8 T cells were depleted with BDCA4 and CD4 or CD8 antibody
MACS microbeads (Miltenyi Biotec), respectively, according to the
manufacturer’s protocol. Plasmacytoid DC depletion was confirmed by FACS analysis staining with FITC-conjugated antibody
to BDCA2 and PE-conjugated antibody to CD123 (Miltenyi Biotec).
PBMCs or pDC-depleted PBMCs (1 × 106 to 2 × 106 cells) were stimulated with each influenza vaccine at the indicated concentration.
Twenty-four hours later, IFN-a and IFN-g (R&D Systems) were measured in supernatants by ELISA according to their manufacturers’
protocol.
Statistical analysis
Statistical significance (P < 0.05) between groups was determinedusing the Student’s t test. A survival curve was generated using KaplanMeier methodology, and the susceptibility of mice after infection wascompared using the log-rank test.

SUPPLEMENTARY MATERIAL
www.sciencetranslationalmedicine.org/cgi/content/full/2/25/25ra24/DC1
Materials and Methods
Fig. S1. TLR7-dependent, but not IPS-1–dependent, signaling is required for the induction ofprotective immune responses with inactivated WV vaccine by i.m. immunization.
Fig. S2. ASC-dependent inflammasome activation was dispensable for adaptive immune response to influenza virus infection, except for systemic IgG1 production.
Fig. S3. Type I IFN receptor–mediated signaling was indispensable for adaptive immune response to WV but not to the live virus.

Fig. S4. Plasmacytoid DC depletion by mPDCA-1 antibody was confirmed in spleen.
Fig. S5. Type I IFN interaction between pDCs and other immune cells was required for WV
vaccine immunogenicity.
Fig. S6. Different manner of type I IFN response to WV vaccine and split vaccine is dependent
on the presence of the viral genome RNA.
Fig. S7. Indispensable role of type I IFN–mediated signaling in vaccination with split vaccine
plus SPG-CpG.
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Y. Kawaoka, and T. Nakaya for providing critical materials and helpful suggestions. Funding:Japan Society for the Promotion of Science Research Fellowships for Young Scientists (S.K.);
Knowledge Cluster Initiative (K.J.I. and T.H.); Grant-in-Aid for Scientific Research (KAKENHI)(K.J.I. and C.C.); Target protein project (K.J.I. and S.A.); Ministry of Education, Culture, Sports,
Science and Technology; and Core Research for Evolutional Science and Technology, Japan
Science and Technology Agency (K.S. and K.J.I.). Competing interests: Some of the authors
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K.S., S. Shinkai, N. Shimada, K.J.I., and S.A. Applicant ERATO, SORST/JST JP2006-282148. The
authors declare no other competing interests.
Submitted 22 December 2009
Accepted 11 March 2010
Published 31 March 201010.1126/scitranslmed.3000759
Citation: S. Koyama, T. Aoshi, T. Tanimoto, Y. Kumagai, K. Kobiyama, T. Tougan, K. Sakurai,
C. Coban, T. Horii, S. Akira, K. J. Ishii, Plasmacytoid dendritic cells delineate immunogenicity
of influenza vaccine subtypes. Sci. Transl. Med. 2, 25ra24 (2010).

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