Department of Immunology, The Scripps Research Institute, La Jolla, California
Correspondence: Tsung-Hsien Chuang, Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail: chuang{at}scripps.edu
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Key Words: innate immunity • NF-
B • IFN-
• MyD88
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Bacterial DNA is a potent stimulus to immune cells. This activity of DNA is assigned to sequence motifs containing unmethylated CpG dideoxynucleotides. This feature provides a major distinction between bacterial and mammalian host DNA [18 19 20 21 ]. Synthetic CpG containing oligodeoxynucleotide (CpG-ODN) mimics the stimulatory effect of bacterial DNA. The CpG-DNA (both bacterial DNA and CpG-ODN) induces B-cell proliferation and activates cells of the myeloid lineage including dendritic cells [22 23 24 25 26 27 28 29 ]. Injection of CpG-DNA in vivo evokes immune responses in mice without the serious pathophysiological changes that follow injection of LPS, suggesting their potential uses in immunotherapy of human diseases [30 , 31 ]. A molecular understanding of cellular recognition of CpG-DNA is only now beginning to emerge [18 , 20 , 32 ]. Herein, we provide data supporting the concept that the species-dependent and sequence-dependent responses to various CpG-ODN reside within TLR9 and that TLR9 functions as a cell-surface protein and uses intracellular signaling cascades that include MyD88, IRAK, and TRAF6.
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and
hIL-1ß were purchased from PeproTech (Rocky Hill, NJ). A palmitylated
synthetic peptide analog of bacterial lipoprotein Pam3Cys was purchased
from Boehringer Mannheim (Indianapolis, IN). Soluble peptidoglycan
(sPGN) was obtained from Dr. R. Dziarski (Indiana University,
Bloomington). Bacterial lipoprotein LipL32 was isolated from
Leptospira interrogans as described
[35
]. Lipoprotein OspA isolated from
Borrelia burgdorferi was obtained from
Dr. T. Sellati (University of Connecticut, Storrs). CpG-ODNs, including
phosphorothioate-modified mCpG, mCpG.1-mCpG.5, hCpG, non-CpG, and the
phosphodiester mCpG.0, were purchased from Research Genetics
(Huntsville, AL). The sequences of mCpG
(TCC,ATG,ACGx,TTC,CTG,ACGx,TT), hCpG
(TCGx,TCGx,TTT,TGT,CGxT,TTT,GTC,GxTT),
and non-CpG (TCC,ATG,AGC,TTC,CTG,AGC,TT) were selected from
previously published work of Krieg and co-workers [18
].
Expression plasmids
Expression vectors hTLR2/pFlag.CMV1 and 
MyD88/pRK7 (aa
152–296) were gifts from Tularik (South San Francisco, CA). Expression
vectors for truncated IRAK1 (aa 1–215), TRAF6 (aa 289–522), and
TRAF2 (aa 87–501) were constructed by subcloning cDNAs encoding the
truncated proteins into a pRK5 mammalian expression vector. These cDNAs
were polymerase chain reaction (PCR)-amplified from cDNA encoding
full-length IRAK1, TRAF6, and TRAF2, respectively. Expression vectors
for hTLR9, mTLR9, mTLR91, and mTLR92 were constructed by PCR
amplification from the cDNA sequence corresponding to the beginning of
the mature peptide to the 3' end, as indicated and subcloned into a
pFlagCMV1 vector (Sigma Chemical Co.). This vector contains sequences
encoding a preprotrypsin signal peptide followed by a Flag epitope tag.
The inserted cDNAs were fused in-frame after the Flag epitope tag. All
cDNA constructs were confirmed by DNA sequencing. Plasmids were
isolated with a Qiagen (Valencia, CA) Endo-free Maxi-prep kit.
Cloning mTLR9
Several expressed sequence tag (EST) cDNAs (accession numbers
AA273731, AA197442, AA162495, and AA451215) encoding partial mTLR9 cDNA
sequence were identified in a search of the DNA databases of NCBI with
a Blast program. These cDNAs cover the 3'-end sequence of the mTLR9. A
rapid amplification of cDNA ends (RACE) method was used to clone cDNA
containing the 5' end of this mTLR9 from a cDNA library, which was
prepared from mouse-spleen polyA+ mRNA using a SmartTM RACE
cDNA amplification kit (Clontech, Palo Alto, CA). The RACE products
were subcloned into a T/A cloning vector (Invitrogen, La Jolla, CA) and
sequenced. Based on the cDNA sequences from the RACE product and the
EST cDNA, primers corresponding to the sequences at both ends of the
mTLR9 were designed. The full-length mTLR9 was PCR-amplified from a
mouse-spleen first-strand cDNA library. This library was synthesized
from polyA+ mRNA using a SuperScriptTM preamplification kit
(Gibco BRL, Gaithersburg, MD). The amplified, full-length cDNA was
subcloned into a T/A cloning vector and sequenced. The cDNA sequence
for mTLR9 is in GenBank under accession number AF314224.
Cell culture, nuclear factor-B (NF-B) reporter assay and flow
cytometry analysis
Human embryonic kidney 293 (HEK293) cells and the murine cell
line, RAW264.7, were cultured in Dulbecco’s modified Eagle’s medium,
supplemented with 10% fetal bovine serum. The HEK293 cells were plated
in six-well plates and transfected on the following day by
Lipofectamine 2000 (Gibco BRL) with indicated amounts of expression
vectors plus 0.1 µg endothelial leukocyte adhesion molecule 1
(ELAM-1) luciferase-reporter plasmid and 0.1 µg ß-galactosidase
plasmid for normalization. The RAW264.7 cells were transfected by
Superfect (Qiagen) with the indicated amount of expression vectors plus
1 µg ELAM-1 luciferase-reporter plasmid and 1 µg ß-galactosidase
plasmid. Twenty-four hours later, the cells were treated with indicated
agonists for an indicated time period. The cells were lysed, and
luciferase activity was determined by using reagents from Promega Corp.
(Madison, WI). Relative luciferase activities were calculated as folds
of induction compared with unstimulated vector control. The data
presented are the mean ± SE (n=3). To
demonstrate that the mTLR9 was expressed on the cell surface, 5 x
105 HEK293 cells expressing TLR9 were dislodged with
phosphate-buffered saline (PBS) plus 2 mM diethylenediaminetetraacetate
(EDTA) and washed with PBS, maintaining all solutions and cells at
4°C. The cells were stained with anti-Flag fluorescein isothiocyanate
(FITC)-conjugated antibody (Sigma Chemical Co.) for 30 min at
4°C and were washed with ice-cold PBS followed by flow cytometric
analysis.
Immunoprecipitation and Western blot analysis
HEK293 cells were transfected with indicated cDNAs. Twenty-four
hours after transfection, cells were washed once with PBS and lysed in
buffer containing of 50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol (DTT), 1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride (PMSF), and 2 µg/ml aprotinin. Cell lysates were
centrifuged, and nuclei were removed. The lysates were incubated with
anti-Flag monoclonal antibody (mAb) M2 (Sigma Chemical Co.) for 4 h at 4°C. Immune complexes were precipitated by the addition of
protein A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). After
extensive washing with lysis buffer, precipitated complexes were
solubilized by boiling in SDS-PAGE sample buffer fractionated by 8%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to nitrocellulose membranes. The membranes were blotted
with anti-Flag mAb. After washes, the blots were incubated with
horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin
G (IgG; Jackson Immuno Research Laboratories, West Grove, PA). The
reactive bands were visualized with the ECL+Plus Western blotting
detection reagents (Amersham Pharmacia Biotech).
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B activationB-dependent E-selectin
(ELAM-1) promoter [37
]. Three different CpG-ODNs used in
these experiments were selected based on findings demonstrated by
others and include those shown to be more selective for murine or human
cells (here termed mCpG and hCpG, respectively) [18
].
The mCpG contains GACGTT hexamer motifs, and hCpG contains
GTCGTT motifs. The sequence of the control ODN (non-CpG) is
identical to the sequence for mCpG except that the CpG
dideoxynucleotides are reversed. In transfected HEK293 cells expressing
mTRL9, NF-B activity was stimulated by mCpG but only marginally
induced by hCpG and not induced by the control ODN. Moreover, a panel
of other microbial products failed to induce activation (Figs. 2A
and 3A). The other stimuli included Re595 LPS, LTA,
sPGN, HKSA, lipoprotein LipL32 isolated from L. interrogans,
OspA isolated from B. burgdorferi, and a palmitylated
synthetic-peptide analog of bacterial lipoprotein Pam3Cys (Fig. 2
A). In contrast, hTLR2-transfected HEK293 cells responded to sPGN, HKSA,
LipL32, OspA, and Pam3Cys but not to the CpG-ODNs (Fig. 2B)
. HEK293
cells expressing hTLR9 responded to the hCpG, weakly to mCpG, and not
to the control ODN (Fig. 2C) .
 View larger version (15K): [in a new window] |
Figure 1. Architecture analysis of mouse and human TLR9. This analysis was
performed by a SMART architecture research computer program
(http://smart.EMBL-heidelberg.de/). SP, Signal peptide; LRRs,
leucine-rich repeats; TM, transmembrane domain; TIR, Toll/IL-1R
cytoplasmic domain. The scale shows amino acid residue numbers. The
mTLR9 cDNA sequence was submitted to GenBank under accession number
AF314224; hTLR9 is under accession number AF245704.
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 View larger version (40K): [in a new window] |
Figure 2. TLR9 mediates CpG-ODN-induced NF-B activation. HEK293 cells were
transfected with 0.2 µg mTLR9 (A, D), 0.2 µg hTLR2 (B), or 0.2 µg
hTLR9 (C) expression vector plus ELAM luciferase-reporter plasmid.
Twenty-four hours later, the transfected cells were treated with 10
ng/ml each hIL-1 and hTNF-, 5 µM each mCpG, hCpG, and non-CpG, 200
ng/ml LPS(Re595), 2 µg/ml each LTA and PGN, 2 x 106
bacteria/ml HKSA, 1 µg/ml each LipL32 lipoprotein from L.
interrogans, OspA lipoprotein from B. burgdorferi, and
Pam3Cys (A–C), or 1 µg/ml chloroquine plus 5 µM mCpG (D) for
6 h. The cells were washed and lysed, and relative luciferase
activities in each sample were determined as described in Materials and
Methods.
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B activation induced by mCpG was blocked by pretreatment of cells
with chloroquine (Fig. 2D)
. This is consistent with the previously
described inhibitory effects of chloroquine on CpG-ODN-induced
activation in macrophages, dendritic cells, and B cells
[38
]. Using explanted cells, others have shown that the
optimal concentration of CpG-ODN varied from submicromolar to
micromolar [18
, 20
]. Herein, the mCpG
concentration required for a maximal NF-B activation in HEK293 cells
expressing mTLR9 was observed at about 3 µM CpG-ODN (Fig. 3
A). This concentration is similar to that required for the optimal
stimulation of RAW264.7 cells using the same luciferase reporter system
(Fig. 3B)
.
 View larger version (23K): [in a new window] |
Figure 3. Concentrations of CpG-ODNs required for the NF-B activation. (A)
HEK293 cells were transfected with 0.2 µg mTLR9 expression vector
plus ELAM luciferase-reporter plasmid. (B) RAW 246.7 cells were
transfected with ELAM luciferase-reporter plasmid. Twenty-four hours
later, the transfected cells were treated with different concentrations
of mCpG, hCpG, and non-CpG as indicated. Six hours later, the cells
were washed and lysed, and relative luciferase activities in each
sample were determined.
|
B
activation (Fig. 4B)
. This effect was selective for TLR9 but not the
common signaling components downstream of TLRs, because neither blocked
IL-1-induced NF-B activation in HEK293 cells (Fig. 4B)
. In other
control experiments not shown, they also failed to inhibit NF-B
activation induced by HKSA or bacterial lipoprotein. The mCpG.3 and
mCpG.4 were activating ligands, although they were less active than
mCpG and mCpG.5 (Fig. 4A)
. These data further support our contention
that the results observed in explanted cells and/or cell lines
demonstrated by others reflect a key role for TLR9 in CpG-ODN-induced
cell activation [39
40
41
].
 View larger version (39K): [in a new window] |
Figure 4. Activation of mTLR9 overexpressed cells by CpG-ODNs containing a
different CpG motif and backbone. HEK293 cells were transfected with
0.2 µg mTLR9 expression vector plus ELAM luciferase-reporter plasmid
for 24 h. (A) The transfected cells were treated with 5 µM mCpG,
mCpG.0, mCpG.1, mCpG.2, mCpG.3, mCpG.4, and mCpG.5. (B) The transfected
cells were treated with 10 ng/ml IL-1 (squares), 5 µM
phosphorothioate-modified mCpG (circles), and phosphodiester mCpG.0
(triangles) plus different concentrations of inhibitory mCpG.1 (closed
symbols) or mCpG.2 (open symbols), as indicated. Six hours later, the
cells were washed and lysed, and relative luciferase activities in each
sample were determined. The nucleotide sequences of these CpG-ODNs are
shown below the figures. The CpG dinucleotide in each CpG-ODN and the
nucleotide base varied from the mCpG are underlined. The mCpG.0
contains a native phosphodiester backbone with nucleotide sequence
identical to mCpG. All the other CpG-ODNs are
phosphorothioate-modified.
|
B activation decreased nearly linearly by increasing
concentrations of mCpG.1 and mCpG.2. In contrast, mCpG.0-induced
activation was blocked more effectively (Fig. 4B) . This result suggests
that the mTLR9 may bind phosphorothioated CpG-ODNs more effectively and
may account for the higher cell uptake rate observed by others
[42
43
44
]. In totality, the data provided here show that
activity of a CpG motif toward TLR9 is determined by the backbone and
specific base sequence.
TLR9 is a cell-surface receptor and uses MyD88, IRAK, and TRAF6 for
CpG-DNA signaling
Although the sequence information for TLR9 suggests that it is a
membrane receptor (Fig. 1)
, direct studies of its cellular localization
and role as a functional signaling molecule are lacking. To provide
information to establish data to support the contention that TLR9 is
indeed a plasma membrane protein and functions in this context as a
signaling receptor, we performed the following experiments. We first
showed that mTLR9 is a cell-surface molecule by using flow cytometry
analysis (Fig. 5 A
). To link this protein to cell activation, we prepared two
mutants of mTLR9 containing deletions in the cytoplasmic domain. The
two mutants termed mTLR91 and mTLR92 represent truncations from
amino acid residues 1001 and 954, respectively (Fig. 5B
, upper panel).
These constructs were transiently expressed in HEK293 cells, and
expression was confirmed by immunoprecipitation and Western blotting
with anti-Flag antibody (Fig. 5B
, bottom panel). Neither mutant
supported CpG-ODN-induced NF-B activation when compared with
wild-type mTLR9 (Fig. 5B
, middle panel). Recent studies have
demonstrated the involvement of signaling molecules downstream of the
TIR domain in CpG-ODN-induced cell activation [45
,
46
]. These signaling molecules include MyD88, IRAK, and
TRAF6. Cells from MyD88 knockout mice are unable to respond to CpG-ODN,
whereas cells from TLR2- and TLR4-deficient mice are unaffected
[46
]. Moreover, CpG-ODN stimulation of JNK activation in
RAW264.7 cells is blocked by overexpression of dominant-negative
mutants of MyD88 and TRAF6 [45
]. To investigate whether
the mTLR9-mediated CpG-ODN activation uses a similar set of downstream
transducers, we cotransfected HEK293 cells with an expression vector
for mTLR9, as well as vector encoding truncations in each of these
three proteins plus a control protein TRAF2: MyD88(152–296),
IRAK(1–215), TRAF6(289–522), and TRAF2(87–501). Here, we
show that mCpG-induced NF-B activation was blocked by overexpression
of MyD88(152–296), IRAK(1–215), and TRAF6(289–522) but not
by TRAF2(87–501) (Fig. 5C)
. These data indicate that mTLR9 uses the
same set of signaling molecules as used by other TIRs for mediating
CpG-DNA signaling.
 View larger version (30K): [in a new window] |
Figure 5. mTLR9 is a cell-surface signaling receptor and uses MyD88, IRAK, and
TRAF6 for CpG-DNA signaling. (A) Flow cytometry analysis of the mTLR9
expression. The mTLR9 on the cell surface was stained with
FITC-conjugated anti-Flag antibody and detected by flow cytometry
analysis. The shaded histogram represents the parental HEK293 cells;
open histogram represents cells expressed with mTLR9. (B, Upper panel)
Schematic illustration of truncated mTLR9 constructs. The cDNAs were
subcloned into a pFlagCMV1 vector. (B, Middle panel) Cytoplasmic
signaling domain of mTLR9 is required for CpG-ODN-induced NF-B.
HEK293 cells were transfected with 0.2 µg different mTLR9 expression
vectors as indicated, plus ELAM luciferase-reporter plasmid.
Twenty-four hours later, the transfected cells were stimulated with 5
µM mCpG. Six hours later, the cells were washed and lysed, and
relative luciferase activities in each sample were determined. (B,
Bottom panel) Expression of the mTLR9 and truncated mTLR9 were
confirmed by immunoprecipitation and Western blotting of the proteins
with anti-Flag antibody. (C) mTLR9 uses a signaling molecule downstream
of the IL-1 receptor to mediate CpG-ODN-induced NF-B activation.
HEK293 cells were transfected with 0.2 µg mTLR9 expression vector and
0.2 µg MyD88 (152–296), IRAK1 (1–215), TRAF6 (289–522),
or TRAF2 (87–501), respectively, plus ELAM luciferase-reporter
plasmid. Twenty-four hours later, the transfected cells were stimulated
with 5 µM mCpG or 20 ng/ml TNF- for 6 h. The cells were
harvested, and relative luciferase activities in each sample were
determined.
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B
activation through intracellular pathways dependent on MyD88, IRAK, and
TRAF6. Thus, expression of TLR9 is sufficient to render HEK293 cells
responsive to various CpG-ODNs in a manner that reflects the same
patterns observed using primary explants of cells and/or continuous
cell lines.
Using the gene-deletion approach, TLR9 has been shown as an essential
element of responses to CpG-DNA in mouse [47
]. However,
the mechanism(s) for species-specific and sequence-dependent activities
of CpG motifs were not defined, and it was unclear whether TLR9 was
sufficient for CpG-DNA signaling. Simultaneously, another study has
revealed that mice lacking the DNA-dependent protein kinase catalytic
subunit (DNA-PKcs) are unable to respond to CpG-DNA
[48
], but the role of DNA-PK in distinction of different
CpG motifs was not addressed. Their results suggested that CpG-DNA
activated cytoplasmic DNA-PK, which directly phosphorylated IB and
activated NF-B in a MyD88-independent way [48
]. At
present, the connection between TLR9 and DNA-PK is not clear. Our data
show an exact parallel between the properties of ectopically expressed
TLR9 and those of the endogenous CpG-DNA receptor. This supports the
contention that TLR9 is the endogenous receptor as well as the first
cellular protein to recognize CpG-DNA. The DNA-PK might be a necessary
but not sufficient molecule downstream of TLR9 for CpG-DNA signaling,
or alternatively, TLR9 and DNA-PK represent two parallel signal
pathways in some cell types.
In addition to TLR9, other TLRs confer species-specific cellular responses. For example, mTLR4 and hTLR4 confer cellular responsiveness to LPS and lipid A. However, only mTLR4 mediates cell activation by a lipid A partial structure, lipid IVa; in fact, lipid IVa is an antagonist in human cells [49 , 50 ]. The structural basis of this specificity is still unclear. In the case of TLR9, a computer analysis has revealed distinct distribution patterns of leucine-rich repeats in the ectodomains of mTLR9 and hTLR9 (Fig. 1) . The CpG-DNA may have direct contact with this region of the receptor. Thus, it will be useful to determine if the sequence differences observed in the ectodomains of m- and hTLR9 account for the differences in ligand specificity in murine versus human TLR9.
TLR9 as well as TLR4 use intracellular signaling components including
MyD88, IRAK, and TRAF6. However, the biological consequences initiated
by these two receptors are different. For example, LPS induces
synthesis of inducible nitric oxide in microphages, but CpG-DNA does
not [25
]. In human peripheral blood monocytes, LPS
rapidly induces TNF- and IL-6, but CpG-DNA stimulation of these
cytokines does not occur for 18 h [51
]. It is
interesting that chloroquine and related compounds block cell
activation by CpG-DNA but do not prevent activation by LPS
[52
]. The chloroquine effects suggest that an endosomal
maturation process might be required for TLR9 signaling. Alternatively,
some specific components in a TLR9 signaling pathway might be inhibited
by chloroquine or related compounds. Clarification of the effects of
chloroquine on LPS and CpG-DNA-induced cell activations might lead to
an understanding of how signals downstream of TLR4 and TLR9 are
differentially regulated.
Received May 21, 2001; revised October 6, 2001; accepted October 9, 2001.
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S. Epelman, D. Stack, C. Bell, E. Wong, G. G. Neely, S. Krutzik, K. Miyake, P. Kubes, L. D. Zbytnuik, L. L. Ma, et al. Different Domains of Pseudomonas aeruginosa Exoenzyme S Activate Distinct TLRs J. Immunol., August 1, 2004; 173(3): 2031 - 2040. [Abstract] [Full Text] [PDF]
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J. Platz, C. Beisswenger, A. Dalpke, R. Koczulla, O. Pinkenburg, C. Vogelmeier, and R. Bals Microbial DNA Induces a Host Defense Reaction of Human Respiratory Epithelial Cells J. Immunol., July 15, 2004; 173(2): 1219 - 1223. [Abstract] [Full Text] [PDF]
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H. Tsujimura, T. Tamura, H. J. Kong, A. Nishiyama, K. J. Ishii, D. M. Klinman, and K. Ozato Toll-Like Receptor 9 Signaling Activates NF-{kappa}B through IFN Regulatory Factor-8/IFN Consensus Sequence Binding Protein in Dendritic Cells J. Immunol., June 1, 2004; 172(11): 6820 - 6827. [Abstract] [Full Text] [PDF]
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S. Cornelie, J. Hoebeke, A.-M. Schacht, B. Bertin, J. Vicogne, M. Capron, and G. Riveau Direct Evidence that Toll-like Receptor 9 (TLR9) Functionally Binds Plasmid DNA by Specific Cytosine-phosphate-guanine Motif Recognition J. Biol. Chem., April 9, 2004; 279(15): 15124 - 15129. [Abstract] [Full Text] [PDF]
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A. A. Cecil and M. J. Klemsz p38 activation through Toll-like receptors modulates IFN-{gamma}-induced expression of the Tap-1 gene only in macrophages J. Leukoc. Biol., March 1, 2004; 75(3): 560 - 568. [Abstract] [Full Text] [PDF]
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S.-J. Yeo, J.-G. Yoon, and A.-K. Yi Myeloid Differentiation Factor 88-dependent Post-transcriptional Regulation of Cyclooxygenase-2 Expression by CpG DNA: TUMOR NECROSIS FACTOR-{alpha} RECEPTOR-ASSOCIATED FACTOR 6, A DIVERGING POINT IN THE Toll-LIKE RECEPTOR 9-SIGNALING J. Biol. Chem., October 17, 2003; 278(42): 40590 - 40600. [Abstract] [Full Text] [PDF]
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J. D. Marshall, E. M. Hessel, J. Gregorio, C. Abbate, P. Yee, M. Chu, G. V. Nest, R. L. Coffman, and K. L. Fearon Novel chimeric immunomodulatory compounds containing short CpG oligodeoxyribonucleotides have differential activities in human cells Nucleic Acids Res., September 1, 2003; 31(17): 5122 - 5133. [Abstract] [Full Text] [PDF]
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A. A. Ashkar, S. Bauer, W. J. Mitchell, J. Vieira, and K. L. Rosenthal Local Delivery of CpG Oligodeoxynucleotides Induces Rapid Changes in the Genital Mucosa and Inhibits Replication, but Not Entry, of Herpes Simplex Virus Type 2 J. Virol., August 15, 2003; 77(16): 8948 - 8956. [Abstract] [Full Text] [PDF]
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S.-J. Yeo, D. Gravis, J.-G. Yoon, and A.-K. Yi Myeloid Differentiation Factor 88-dependent Transcriptional Regulation of Cyclooxygenase-2 Expression by CpG DNA: ROLE OF NF-{kappa}B AND p38 J. Biol. Chem., June 13, 2003; 278(25): 22563 - 22573. [Abstract] [Full Text] [PDF]
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 J. Lee, T.-H. Chuang, V. Redecke, L. She, P. M. Pitha, D. A. Carson, E. Raz, and H. B. Cottam Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: Activation of Toll-like receptor 7 PNAS, May 27, 2003; 100(11): 6646 - 6651. [Abstract] [Full Text] [PDF]
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D. Yu, E. R. Kandimalla, L. Bhagat, J.-Y. Tang, Y. Cong, J. Tang, and S. Agrawal 'Immunomers'--novel 3'-3'-linked CpG oligodeoxyribonucleotides as potent immunomodulatory agents Nucleic Acids Res., October 15, 2002; 30(20): 4460 - 4469. [Abstract] [Full Text] [PDF]
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