Immunoadjuvant effects of polyadenylic:polyuridylic acids through TLR3 and TLR7
International Immunology, Vol. 20, No. 1, pp. 1–9
doi:10.1093/intimm/dxm112
ª The Japanese Society for Immunology. 2007. All rights reserved.
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Immunoadjuvant effects of polyadenylic:polyuridylic
acids through TLR3 and TLR7
Takahiro Sugiyama1,2, Katsuaki Hoshino1, Masuyoshi Saito1, Takahiro Yano1, Izumi Sasaki1,
Chihiro Yamazaki1, Shizuo Akira2 and Tsuneyasu Kaisho1
1
Laboratory for Host Defense, RIKEN Research Center for Allergy and Immunology, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama,
Kanagawa 230-0045, Japan
2
Department of Host Defense, Research Institute for Microbial Diseases and Akira Innate Immunity Project, Exploratory Research
for Advanced Technology, Japan Science and Technology Corporation, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan
Keywords: dendritic cell, double-stranded RNA, immune adjuvant, Toll-like receptor
Abstract
Double-stranded RNA (dsRNA) is produced upon viral infection and can activate innate immunity.
Polyinosinic:polycytidylic acids [poly(I:C)] is a synthetic mimetic of dsRNA and functions through an
endosomal receptor, Toll-like receptor (TLR) 3 or cytosolic receptors. Another type of dsRNA,
polyadenylic:polyuridylic acids [poly(A:U)], can also act as an immune adjuvant, but it remains
unclear how it exhibits its adjuvant effects. Here, we have characterized the adjuvant effects of
poly(A:U). Poly(A:U) could induce both IFN-a and IL-12p40 from murine bone marrow dendritic cells
(DCs). Poly(A:U)-induced IFN-a production depended on a DC subset, plasmacytoid dendritic cell
(pDC), and required TLR7. IL-12p40 was also produced by poly(A:U)-stimulated pDC in a TLR7dependent manner. In addition to pDC, conventional dendritic cell (cDC) also produced IL-12p40 in
response to poly(A:U). This IL-12p40 induction resulted from two cDC subsets, CD24high cDC and
CD11bhigh cDC in a TLR3- and TLR7-dependent manner, respectively. In vivo injection of poly(A:U)
with antigen led to clonal expansion of and IFN-g production from antigen-specific CD81 T cells.
Consistent with the in vitro findings, TLR3 and TLR7 were required for the clonal T-cell expansion.
Notably, TLR3, rather than TLR7, was critical for generating IFN-g-producing CD81 T cells. CD81
T-cell responses induced by poly(A:U) were independent of type I IFN signaling. Our results
demonstrate that poly(A:U) functions as an in vivo immunoadjuvant mainly through TLR3 and TLR7.
Introduction
Nucleic acids can activate innate immunity and function as
potent immune adjuvants through pattern recognition receptors including Toll-like receptors (TLRs) or RIG-I (retinoic
acid-inducible gene-I)-like receptors (1, 2). Single-stranded
RNA (ssRNA) is recognized by TLR7 in mice and TLR7 and
TLR8 in humans (3, 4). DNA with unmethylated CpG motifs
is sensed by TLR9 (5). Nucleic acids can be manipulated
with little contamination of other ingredients and are now
considered to be promisingly applicable to the treatment for
allergy or cancer (6–8).
Double-stranded RNA (dsRNA) is produced in virally
infected cells and sensed by pattern recognition receptors.
Polyinosinic:polycytidylic acids [poly(I:C)] has been widely
used as a synthetic dsRNA which works as a potent immune
adjuvant. Poly(I:C) is recognized not only by an endosomal
receptor TLR3 (9) but also by cytosolic receptors including
RNA helicases such as RIG-I or melanoma differentiationCorrespondence to: T. Kaisho; E-mail:
Transmitting editor: K. Inaba
associated gene 5 (MDA5) (10–12). Gene-targeting experiments have revealed the roles of those receptors in
poly(I:C)-induced in vivo responses (11). TLR3 is essential
for IL-12p40 production, whereas MDA5 is critical for IFN-a
induction. Although poly(I:C) can bind to RIG-I in vitro (10),
RIG-I is dispensable for poly(I:C)-induced type I IFN production in vivo (11). Another type of dsRNA, polyadenylic:polyuridylic acids [poly(A:U)], is also utilized as an immune
modulator (13, 14). When injected with protein or viral antigens into mice, poly(A:U) can promote Th1 generation and
antibody production (13). In vivo target delivery of a tumorassociated epitope to antigen-presenting cells in combination with poly(A:U) leads to regression of tumor growth and
anti-tumor immune effects against antigen-bearing tumor
cells, indicating poly(A:U) as one of promising immunotherapeutic approaches (14). Poly(A:U) has also been used with
moderate success for treating breast cancers with minimum
Received 13 July 2007, accepted 9 October 2007
Advance Access publication 1 November 2007
2 Immunoadjuvant effects of poly(A:U)
side effects (15–17). However, it remains unclear how poly(A:U) activates innate immune cells. Here, we have analyzed the molecular mechanisms on the adjuvant effects of
poly(A:U). Analysis on in vitro bone marrow (BM) dendritic
cells (DCs) revealed that poly(A:U) functioned as TLR3 and
TLR7 agonists, depending on DC subsets. Furthermore, poly(A:U) could augment in vivo antigen-specific CD8+ T-cell
responses, in which both TLR3 and TLR7 are involved.
Methods
Mice
C57BL/6J mice were purchased from CLEA Japan. IFN-a/
bR-deficient mice were purchased from B&K universal (Hull,
UK). TLR3, TLR7 and TLR3/TLR7 double-deficient mice with
C57BL/6 background were established and maintained as
described previously (18, 19). Mice were maintained under
the specific pathogen-free conditions in the animal facility of
the RIKEN Research Center for Allergy and Immunology.
Reagents
Poly(I:C) was purchased from Amersham Bioscience
(Piscataway, NJ, USA). Polyadenylic acid (polyA) and polyuridylic acid (polyU) were purchased from Sigma (St Louis,
MO, USA). Antibodies against CD11c (HL3), CD8a (53-6.7),
CD62L (MEL-14) and IFN-c (XMG1.2) were purchased from
BD Pharmingen (San Jose, CA, USA). Antibodies against
B220 (RA3-6B2), CD24 (M1/69) and CD11b (M1/70) were
purchased from eBioscience (San Diego, CA, USA). AntimPDCA-1 antibody was purchased from Miltenyi Biotec
(Bergisch Gladbach, Germany).
Poly(A:U) was generated by annealing polyA and polyU.
Briefly, the same amounts of each RNA were mixed in the
presence of annealing buffer (20 mM Tris–HCl, 10 mM
MgCl2 and 50 mM NaCl) and incubated at 100C for 5 min.
Then, RNAs were subsequently placed at room temperature
for >15 min and used as poly(A:U). For RNase treatment,
dsRNAs were incubated for 5 h with 1 mg ml 1 of RNaseA
(Roche, Mannheim, Germany).
Generation of BM DCs
BM cells were cultured in the presence of 100 ng ml 1 of human recombinant Fms-like tyrosine kinase 3 ligand (Flt3L)
(PeproTeck, London, UK) for 7–8 days and used as Flt3Linduced BM DCs as described previously (20). To prepare
granulocyte macrophage colony-stimulating factor (GMCSF)-induced BM DCs, BM cells were cultured in the presence of 10 ng ml 1 of mouse recombinant GM-CSF (R&D,
Minneapolis, MN, USA) for 6 days (21).
DC subset sorting
Flt3L-induced BM DCs were stained with FITC–anti-CD11c,
PE–anti-mPDCA-1 and APC–anti-B220. PDCA-1+B220+CD11c+
cells were sorted as plasmacytoid de (...truncated)
