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Section of Retroviral Research, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by NK cells, whereas the
other ("K") stimulates cell proliferation and the production of
IL-6 and IgM by monocytes and B cells. The distinct immunostimulatory
properties of K and D ODN can improve the design of CpG-based products
to achieve specific therapeutic goals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify motifs that activate human cells, several hundred
novel ODN were synthesized and studied. The results demonstrate that
human PBMC recognize and respond to two structurally distinct clusters
of CpG motifs. One cluster preferentially induced cell proliferation,
IgM production by B cells, and IL-6 secretion by monocytes/dendritic
cells, whereas the other stimulated IFN- release by NK cells. By
selectively employing these two types of ODN, the immune system can be
manipulated to support specific therapeutic ends.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal PBMC were obtained from the National Institutes of Health Department of Transfusion Medicine (Bethesda, MD). The human myeloma cell line RPMI 8226 (CCL-155; American Type Culture Collection, Manassas, VA) and the NK-92 human NK cell line (a kind gift of Dr. J. Ortaldo, National Cancer Institute, Frederick, MD) were grown in RPMI 1640 supplemented with 10% FCS, penicillin/streptomycin, L-glutamine, HEPES, sodium pyruvate, and 2-ME in a 5% CO2 in-air incubator. Medium for NK-92 cells was supplemented with IL-2 (200 IU/ml; R&D Systems, Minneapolis, MN) and IL-15 (15 ng/ml; Endogen, Boston, MA).
Oligodeoxynucleotides
ODN were synthesized at the Center for Biologics Evaluation and Research core facility. All had <0.1 endotoxin U/ml endotoxin at ODN concentrations of 1 mg/ml.
Antibodies
Abs against human IFN- (Endogen), IL-6 (R&D Systems), and IgM
(Serotec, Oxford, U.K.) were used for ELISA and enzyme-linked
immunospot (ELISPOT) assays. FITC- and/or CyChrome-labeled Abs against
human CD3, CD4, CD14, CD11c, CD16, CD56, CD83, HLA-DR, IL-6, and
IFN- were obtained from BD PharMingen (San Diego, CA) or BD
Biosciences (San Jose, CA) and used as recommended by the manufacturer.
Neutralizing Abs to IL-12 were obtained from R&D Systems, and Abs to
IL-18 were kindly provided by Dr. Howard Young (National Cancer
Institute).
Mononuclear cell preparation
Mononuclear cells were separated by density gradient centrifugation over Ficoll-Hypaque as described (17). Cells were washed three times and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS for 72 h at 5 x 105 cells/well in the presence of 1–3 µM ODN.
ELISA and ELISPOT assays
Ninety-six-well microtiter plates (Millipore, Bedford, MA) were
coated with anti-cytokine Ab or anti-IgM and blocked with PBS-5%
BSA (17, 18). Cytokines and Ig in culture sups or secreted
by individual cells were detected colorimetrically using biotin-labeled
Abs followed by phosphatase-conjugated avidin and then
phosphatase-specific colorimetric substrate. Standard curves were
generated to quantitate ELISA results using known amounts of
recombinant cytokine or purified IgM. The detection limit of the assays
was: 6 pg/ml for IFN-, 20 pg/ml for IL-6, and 10 ng/ml for IgM.
Stimulation index was calculated by the formula: (value for stimulated
cells - background)/(value for unstimulated cells -
background). In cases where cytokine/Ig production was below assay
sensitivity, the lower limit of detection was used to calculate the
stimulation indices. All assays were performed in triplicate.
Proliferation assays
A total of 105 PBMC/well were incubated with 3 µM of ODN for 68 h, pulsed with 1 µCi of [3H]thymidine, and then harvested 4 h later. The proliferation index represents the fold difference between stimulated and unstimulated cells. All assays were performed in triplicate.
Intracellular cytokine staining and flow cytometry
PBMC were cultured for 8 h (K type) or 24 h (D type)
with 3 µM of various ODN. Brefeldin A (20 µg/ml) was added to the
cultures after 2 or 12 h, respectively. Cells were harvested with
warm PBS-0.02% EDTA and washed. PBMC (1 x
106/sample) were fixed and permeabilized using
the Fix & Perm cell permeabilization kit (Caltag, Burlingame, CA) as
recommended by the manufacturer. Cells were then stained with
PE-conjugated anti-IL-6 or anti-IFN- plus specified FITC- or
CyChrome-conjugated Abs against cell surface markers for 30 min in
darkness. After labeling, the cells were washed twice, and 40,000
events per sample were analyzed by FACScan flow cytometry (BD
Biosciences). CellQuest software (BD Biosciences) was used for data
analysis.
Statistical analysis
Statistically significant differences were determined using a two-tail nonparametric Mann-Whitney U test and nonparametric ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Novel ODN were studied for their ability to stimulate human PBMC
to proliferate and/or secrete Ig or cytokines. As seen in Fig. 1
, two structurally distinct ODN classes
were identified that stimulated PBMC from >95% of the donors. Those
of the K type stimulated significantly greater cell proliferation
(p < 0.0001) and induced higher levels of IL-6
(240 vs 85 pg/ml; p < 0.01) and IgM (695 vs 20 ng/ml;
p < 0.0001) than D ODN. In contrast, D ODN were
stronger inducers of IFN- (70 vs 13 pg/ml; p <
0.05).
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Modifications were introduced in various regions of D ODN to
identify the critical sequences and structures that account for the
ability of these ODN to induce IFN-. To standardize results from the
large number of subjects and experiments included in the analysis, the
magnitude of each response is presented as fold increase over cells
from the same subject incubated in medium alone. The general magnitude
of these responses was comparable to that shown in Fig. 1
. D-type ODNs
contain an unmethylated CpG dinucleotide (Fig. 2). Inversion, replacement, or
methylation of the CpG reduces or abrogates reactivity (Fig. 2A, line 1 vs lines 2–6, and
line 7 vs line 8; p <
0.0001). D ODN are stimulatory only if the CpG dinucleotide and its
immediate flanking regions are composed of phosphodiester (shown in
gray) rather than phosphorothioate nucleotides (Fig. 2B,
line 1 vs line 2;
p < 0.001). Because phosphorothioate-modified
nucleotides confer resistance to exonuclease digestion, they are
incorporated at the ends of the ODN to improve activity (Fig. 2B, lines 1 and 5 vs
lines 3 and 4; p =
0.07). Unless otherwise stated, all D ODN studied are
phosphorothioate/phosphodiester chimeras.
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C, lines 1 and 2).
Substituting a Pu for a Py, or vice versa, significantly reduces or
eliminates ODN activity (circled nucleotides in Fig. 2C,
lines 1 and 2 vs lines
6–13; p < 0.0001). By comparison, hexamers
that maintained the PuPyCGPuPy sequence but are
nonself-complementary induce lower levels of IFN- production (Fig. 2C, lines 1 and 2 vs
lines 3–5; p < 0.001).
Sequential deletion experiments show that the minimum length of an
active D ODN is 
18 bp (Fig. 2D; p <
0.01). This finding suggests that sequences outside the central hexamer
might influence D ODN activity. Indeed, stimulation is maximal when the
three bases on each side of the CpG-containing hexamer form a
self-complementary sequence (Fig. 2E, lines
1 and 2 vs lines 3 and 4;
p < 0.0001). Computer modeling of D ODN suggests that
these self-complementary base sequences help form a stem-loop structure
with the CpG dinucleotide at the apex at 37°C (not shown). The ends
of the ODN also contribute to its activity, with the inclusion of more
than four Gs at the 3' end significantly improving function (Fig. 2F, lines 1–3 vs lines
4 and 5; p < 0.001). Thus,
changes in any of the three areas (the central hexamer, the region
flanking the hexamer, or the poly G tail) influence ODN activity.
Type K ODN
K ODN trigger cell proliferation and the secretion of IgM and
IL-6, but little IFN- (Fig. 1). These ODN have a phosphorothioate
backbone and at least one unmethylated CpG dinucleotide (Table IA). As with D ODN,
eliminating the CpG dinucleotide significantly reduces immune
activation (Table IA, line 3 vs
line 4; p < 0.02). Incorporating
multiple CpGs in a single ODN increases immune stimulation (Table IA, lines 1–3). To determine the
minimum length of a stimulatory K ODN, nucleotides were sequentially
deleted from each end. ODN at least 12 bases long consistently induce
strong immune cell activation, whereas shorter ODN are relatively less
active (Table IB, lines 1–5 vs
lines 6–10).
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C, line 2 vs line 3 and
line 4 vs line 5), although
at least one base upstream of the CpG was required (Table IC, line 1 vs line
6). Indeed, a thymidine in the immediate 5' position (Table ID, lines 1 and 2 vs
lines 3–5 and line 6 vs
line 7) and a 3' TpT or a TpA (Table IE, lines 1 and 2 vs
lines 3 and 4 and lines
5 and 6 vs line 7) yielded
the most active K ODN. Modifications >2 bp from the CpG dinucleotide
had relatively less effect on ODN activity (data not shown). Cellular targets of K and D ODN
The phenotype of the cells stimulated to produce cytokine was
determined by combined cell surface and intracytoplasmic staining. As
seen in Table IIA, D ODN
selectively stimulated
CD3-CD16+CD56+CD14-
cells to produce IFN-, consistent with the direct activation of NK
cells. The effect appears to be direct because D ODN do not induce a
significant increase in IL-12 secretion (data not shown). Moreover,
studies using neutralizing anti-IL-12, which reduce the production
of IFN- by PBMCs stimulated with PHA (44% p <
0.05) or with bacillus Calmette-Guérin (77%; p
< 0.05), did not decrease the IFN- production induced by CpG-ODN
(data not shown).
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B). K ODN also stimulated a fraction of
CD19+ B cells to release IL-6.
To confirm these findings, human NK, T, and B cell lines were
tested for their responsiveness to K and D ODN. The NK-92 cell line
responded exclusively to D ODN by secreting IFN-, whereas the human
RPMI 8226 B cell line was stimulated by K ODN to release IL-6 (Fig. 3). Non-CpG ODN did not stimulate either
cell line.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Interest in the immunostimulatory properties of CpG ODN has grown rapidly since their ability to stimulate murine cells to secrete Ig and cytokine was first reported (2, 3, 4, 5). Research in mice established that CpG ODN can function as vaccine adjuvants, as anti-allergens, and as immunoprotective agents (12, 13, 14). Several clinical trials are under way to examine their safety and efficacy for these applications (19).
Although earlier works examined the response of human cells to CpG ODN (7, 16, 20, 21, 22, 23), the current report is the first to establish that different elements of the human immune system respond to two structurally distinct CpG motifs and to establish the "rules" governing recognition of these motifs. Previous studies generally examined the response of a small number of donors to a few DNA sequences that frequently contained multiple CpG motifs (7, 16, 20, 21, 22, 24). This limited their ability to identify the structural basis underlying PBMC stimulation (20, 22). The use of costimulatory factors (such as IL-2) to augment ODN-mediated responses, or cationic lipids to bypass normal cellular uptake, also obscured the unique effects of ODN (20, 25). However, recent work by Hartmann et al. (16, 24) identified multiple TCG repeats as contributing to the stimulation of B and NK cells .
Our work significantly extends those findings by establishing that human PBMC respond to two structurally distinct types of ODN and by describing the key structural elements of each. These results were derived from the study of a large number of normal donors and were confirmed by analysis of monoclonal cell lines. Unmethylated CpG dinucleotides were critical components of both types of ODN because methylation, inversion, or substitution of this dinucleotide reduced or eliminated activity. The use of degradation-resistant phosphorothioate nucleotides improved ODN function, although D motifs were most effective when the CpG and flanking regions were composed of phosphodiester nucleotides. This confirms the findings of Ballas et al. (7) that pure phosphorothioate ODN stimulate human NK cells poorly. Consistent with previous reports, pure phosphorothioate ODN lacking CpG motifs were modestly stimulatory on B cells from some subjects (26).
The CpG motifs at the heart of K and D ODN differ. The optimal K motif contains a thymidine immediately 5' and a TpT or TpA 3'. These findings corroborate and extend the observation by Hartmann et al. (24) and Yamamoto et al. (27) that ODN encoding a 5' TCGTCGTT octamer stimulate the expression of CD86 on B cells. By comparison, optimally active D ODN contain Pu-Py dimers on each side of the CpG. These structural differences may underlie the functional differences between D vs K ODN.
D ODN also differed from K ODN by being longer and requiring that the
CpG-flanking regions be self-complementary. Two-dimensional computer
modeling suggests that the self-complementary sequence facilitates the
formation of a hairpin loop that exposes the CpG at the apex. It is
likely that this stem-loop structure contributes to the recognition of
D ODN because IFN- production declines when the length or binding
strength of the palindrome is reduced (Fig. 2E, and data not
shown). The increased stimulation of NK cells by ODN-containing
palindromes is reminiscent of early findings by Yamamoto et al.
(27) involving stimulatory structures in bacterial DNA.
The inclusion of poly Gs at the 3' end of D ODN may also confer a
structural benefit or may simply improve the efficiency of cellular
uptake (28).
ELISA analysis shows that significant amounts of cytokine are secreted
into the culture supernatant of ODN-activated cells. K and D ODN
activate distinct cell types. Flow cytometric analysis showed that K
ODN activate monocytes and B cells to secrete IL-6, whereas D ODN
stimulate NK cells to secrete IFN-. Studies currently under way
suggest that the differential stimulation is not due to differential
uptake of the ODN, because monocytes and NK cells take up both types of
ODN (M. Gursel, K. Ishii, D. Verthelyi, and D. Klinman, manuscript in
preparation). Furthermore, it appears that the ODN activate their
target cells directly because 1) CpG ODN can stimulate cloned cell
lines to secrete cytokines; 2) cytokine mRNA appears within minutes of
ODN stimulation 4 ; and 3) ELISPOT studies show that the
CpG ODN induced rapid increases (2- to 5-fold) in IL-6- and
IFN--secreting PBMC (5 and 18 h after stimulation, respectively
(data not shown)). Moreover, flow cytometric analysis of cells
stimulated to secrete IFN- by CpG ODN were
CD3- even after 72 h of stimulation,
indicating that the increased IFN- in supernatants is not the result
of a secondary activation of T cells. Although it is possible that the
induction of IFN- secretion by NK cells is mediated by increased
IL-12 or IL-18, our studies using neutralizing anti-IL-12 or
anti-IL-18 confirmed the observations reported by Iho et al.
(25) that induction of IFN- by NK cells is not mediated
by these cytokines . It should be noted that culture conditions can
influence the relative magnitude of the cytokine response induced by K
or D. Thus, care must be taken to eliminate lots of FCS that contain
factors that synergize with ODN activity.
Although the immunostimulatory properties of CpG DNA were only recently described, clinical trials exploring their utility as vaccine adjuvants and anti-allergens have been initiated (19). The distinct immunostimulatory properties of K and D CpG ODN should allow for the design of DNA-based products that support specific therapeutic goals. For example, agents designed to treat allergy or control infection by Th1-sensitive pathogens might benefit from the inclusion of D ODN, whereas vaccines dependent on strong humoral immune responses might benefit from the use of K ODN as adjuvants.
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Acknowledgments |
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Footnotes |
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2 The assertions herein are private ones of the authors and are not to be construed as official or as reflecting the views of the Food and Drug Administration.
3 Address correspondence and reprint requests to Dr. Dennis Klinman, Building 29A, Room 3 D 10, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892.
4 Abbreviations used in this paper: ODN, oligodeoxynucleotides; Pu, purine; Py, pyrimidine; ELISPOT, enzyme-linked immunospot.
Received for publication August 24, 2000. Accepted for publication November 30, 2000.
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 R. Schindler, W. Beck, R. Deppisch, M. Aussieker, A. Wilde, H. Gohl, and U. Frei Short Bacterial DNA Fragments: Detection in Dialysate and Induction of Cytokines J. Am. Soc. Nephrol., December 1, 2004; 15(12): 3207 - 3214. [Abstract] [Full Text] [PDF]
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J. C. Deng, T. A. Moore, M. W. Newstead, X. Zeng, A. M. Krieg, and T. J. Standiford CpG Oligodeoxynucleotides Stimulate Protective Innate Immunity against Pulmonary Klebsiella Infection J. Immunol., October 15, 2004; 173(8): 5148 - 5155. [Abstract] [Full Text] [PDF]
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E. A. Moseman, X. Liang, A. J. Dawson, A. Panoskaltsis-Mortari, A. M. Krieg, Y.-J. Liu, B. R. Blazar, and W. Chen Human Plasmacytoid Dendritic Cells Activated by CpG Oligodeoxynucleotides Induce the Generation of CD4+CD25+ Regulatory T Cells J. Immunol., October 1, 2004; 173(7): 4433 - 4442. [Abstract] [Full Text] [PDF]
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F. Takeshita, K. Suzuki, S. Sasaki, N. Ishii, D. M. Klinman, and K. J. Ishii Transcriptional Regulation of the Human TLR9 Gene J. Immunol., August 15, 2004; 173(4): 2552 - 2561. [Abstract] [Full Text] [PDF]
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 C. C. N. Wu, J. Lee, E. Raz, M. Corr, and D. A. Carson Necessity of Oligonucleotide Aggregation for Toll-like Receptor 9 Activation J. Biol. Chem., August 6, 2004; 279(32): 33071 - 33078. [Abstract] [Full Text] [PDF]
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 S. Rothenfusser, V. Hornung, M. Ayyoub, S. Britsch, A. Towarowski, A. Krug, A. Sarris, N. Lubenow, D. Speiser, S. Endres, et al. CpG-A and CpG-B oligonucleotides differentially enhance human peptide-specific primary and memory CD8+ T-cell responses in vitro Blood, March 15, 2004; 103(6): 2162 - 2169. [Abstract] [Full Text] [PDF]
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B. Spies, H. Hochrein, M. Vabulas, K. Huster, D. H. Busch, F. Schmitz, A. Heit, and H. Wagner Vaccination with Plasmid DNA Activates Dendritic Cells via Toll-Like Receptor 9 (TLR9) but Functions in TLR9-Deficient Mice J. Immunol., December 1, 2003; 171(11): 5908 - 5912. [Abstract] [Full Text] [PDF]
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R. Schirmbeck, P. Riedl, R. Zurbriggen, S. Akira, and J. Reimann Antigenic Epitopes Fused to Cationic Peptide Bound to Oligonucleotides Facilitate Toll-Like Receptor 9-Dependent, but CD4+ T Cell Help-Independent, Priming of CD8+ T Cells J. Immunol., November 15, 2003; 171(10): 5198 - 5207. [Abstract] [Full Text] [PDF]
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 F. C. Hasslung, M. Berg, G. M. Allan, B. M. Meehan, F. McNeilly, and C. Fossum Identification of a sequence from the genome of porcine circovirus type 2 with an inhibitory effect on IFN-{alpha} production by porcine PBMCs J. Gen. Virol., November 1, 2003; 84(11): 2937 - 2945. [Abstract] [Full Text] [PDF]
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 E. Hartmann, B. Wollenberg, S. Rothenfusser, M. Wagner, D. Wellisch, B. Mack, T. Giese, O. Gires, S. Endres, and G. Hartmann Identification and Functional Analysis of Tumor-Infiltrating Plasmacytoid Dendritic Cells in Head and Neck Cancer Cancer Res., October 1, 2003; 63(19): 6478 - 6487. [Abstract] [Full Text] [PDF]
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F. Elias, J. Flo, R. A. Lopez, J. Zorzopulos, A. Montaner, and J. M. Rodriguez Strong Cytosine-Guanosine-Independent Immunostimulation in Humans and Other Primates by Synthetic Oligodeoxynucleotides with PyNTTTTGT Motifs J. Immunol., October 1, 2003; 171(7): 3697 - 3704. [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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H. H. van Ojik, L. Bevaart, C. E. Dahle, A. Bakker, M. J. H. Jansen, M. J. van Vugt, J. G. J. van de Winkel, and G. J. Weiner CpG-A and B Oligodeoxynucleotides Enhance the Efficacy of Antibody Therapy by Activating Different Effector Cell Populations Cancer Res., September 1, 2003; 63(17): 5595 - 5600. [Abstract] [Full Text] [PDF]
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I. Sabroe, R. C. Read, M. K. B. Whyte, D. H. Dockrell, S. N. Vogel, and S. K. Dower Toll-Like Receptors in Health and Disease: Complex Questions Remain J. Immunol., August 15, 2003; 171(4): 1630 - 1635. [Full Text] [PDF]
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J. D. Marshall, K. Fearon, C. Abbate, S. Subramanian, P. Yee, J. Gregorio, R. L. Coffman, and G. Van Nest Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions J. Leukoc. Biol., June 1, 2003; 73(6): 781 - 792. [Abstract] [Full Text] [PDF]
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M. Kerkmann, S. Rothenfusser, V. Hornung, A. Towarowski, M. Wagner, A. Sarris, T. Giese, S. Endres, and G. Hartmann Activation with CpG-A and CpG-B Oligonucleotides Reveals Two Distinct Regulatory Pathways of Type I IFN Synthesis in Human Plasmacytoid Dendritic Cells J. Immunol., May 1, 2003; 170(9): 4465 - 4474. [Abstract] [Full Text] [PDF]
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D. Verthelyi, M. Gursel, R. T. Kenney, J. D. Lifson, S. Liu, J. Mican, and D. M. Klinman CpG Oligodeoxynucleotides Protect Normal and SIV-Infected Macaques from Leishmania Infection J. Immunol., May 1, 2003; 170(9): 4717 - 4723. [Abstract] [Full Text] [PDF]
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S. E. Blackwell and A. M. Krieg CpG-A-Induced Monocyte IFN-{gamma}-Inducible Protein-10 Production Is Regulated by Plasmacytoid Dendritic Cell-Derived IFN-{alpha} J. Immunol., April 15, 2003; 170(8): 4061 - 4068. [Abstract] [Full Text] [PDF]
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A. Krug, S. Rothenfusser, S. Selinger, C. Bock, M. Kerkmann, J. Battiany, A. Sarris, T. Giese, D. Speiser, S. Endres, et al. CpG-A Oligonucleotides Induce a Monocyte-Derived Dendritic Cell-Like Phenotype That Preferentially Activates CD8 T Cells J. Immunol., April 1, 2003; 170(7): 3468 - 3477. [Abstract] [Full Text] [PDF]
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 R. A. Zeuner, D. M. Klinman, G. Illei, C. Yarboro, K. J. Ishii, M. Gursel, and D. Verthelyi Response of peripheral blood mononuclear cells from lupus patients to stimulation by CpG oligodeoxynucleotides Rheumatology, April 1, 2003; 42(4): 563 - 569. [Abstract] [Full Text] [PDF]
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H. Hemmi, T. Kaisho, K. Takeda, and S. Akira The Roles of Toll-Like Receptor 9, MyD88, and DNA-Dependent Protein Kinase Catalytic Subunit in the Effects of Two Distinct CpG DNAs on Dendritic Cell Subsets J. Immunol., March 15, 2003; 170(6): 3059 - 3064. [Abstract] [Full Text] [PDF]
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A. Mazzoni, C. A. Leifer, G. E. D. Mullen, M. N. Kennedy, D. M. Klinman, and D. M. Segal Cutting Edge: Histamine Inhibits IFN-{alpha} Release from Plasmacytoid Dendritic Cells J. Immunol., March 1, 2003; 170(5): 2269 - 2273. [Abstract] [Full Text] [PDF]
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C. Nonnenmacher, A. Dalpke, S. Zimmermann, L. Flores-de-Jacoby, R. Mutters, and K. Heeg DNA from Periodontopathogenic Bacteria Is Immunostimulatory for Mouse and Human Immune Cells Infect. Immun., February 1, 2003; 71(2): 850 - 856. [Abstract] [Full Text] [PDF]
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H. Tsujimura, T. Tamura, and K. Ozato* Cutting Edge: IFN Consensus Sequence Binding Protein/IFN Regulatory Factor 8 Drives the Development of Type I IFN-Producing Plasmacytoid Dendritic Cells J. Immunol., February 1, 2003; 170(3): 1131 - 1135. [Abstract] [Full Text] [PDF]
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H. Yamada, I. Gursel, F. Takeshita, J. Conover, K. J. Ishii, M. Gursel, S. Takeshita, and D. M. Klinman Effect of Suppressive DNA on CpG-Induced Immune Activation J. Immunol., November 15, 2002; 169(10): 5590 - 5594. [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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K. Heckelsmiller, K. Rall, S. Beck, A. Schlamp, J. Seiderer, B. Jahrsdorfer, A. Krug, S. Rothenfusser, S. Endres, and G. Hartmann Peritumoral CpG DNA Elicits a Coordinated Response of CD8 T Cells and Innate Effectors to Cure Established Tumors in a Murine Colon Carcinoma Model J. Immunol., October 1, 2002; 169(7): 3892 - 3899. [Abstract] [Full Text] [PDF]
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M. Zheng, D. M. Klinman, M. Gierynska, and B. T. Rouse DNA containing CpG motifs induces angiogenesis PNAS, June 25, 2002; 99(13): 8944 - 8949. [Abstract] [Full Text] [PDF]
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M. Gursel, D. Verthelyi, I. Gursel, K. J. Ishii, and D. M. Klinman Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide J. Leukoc. Biol., May 1, 2002; 71(5): 813 - 820. [Abstract] [Full Text] [PDF]
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V. Hornung, S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdorfer, T. Giese, S. Endres, and G. Hartmann Quantitative Expression of Toll-Like Receptor 1-10 mRNA in Cellular Subsets of Human Peripheral Blood Mononuclear Cells and Sensitivity to CpG Oligodeoxynucleotides J. Immunol., May 1, 2002; 168(9): 4531 - 4537. [Abstract] [Full Text] [PDF]
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D. Yu, E. R. Kandimalla, Q. Zhao, Y. Cong, and S. Agrawal Immunostimulatory properties of phosphorothioate CpG DNA containing both 3'-5'- and 2'-5'-internucleotide linkages Nucleic Acids Res., April 1, 2002; 30(7): 1613 - 1619. [Abstract] [Full Text] [PDF]
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D. Verthelyi, R. T. Kenney, R. A. Seder, A. A. Gam, B. Friedag, and D. M. Klinman CpG Oligodeoxynucleotides as Vaccine Adjuvants in Primates J. Immunol., February 15, 2002; 168(4): 1659 - 1663. [Abstract] [Full Text] [PDF]
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 I. Sabroe, C.M. Lloyd, M.K.B. Whyte, S.K. Dower, T.J. Williams, and J.E. Pease Chemokines, innate and adaptive immunity, and respiratory disease Eur. Respir. J., February 1, 2002; 19(2): 350 - 355. [Abstract] [Full Text] [PDF]
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F. Takeshita, C. A. Leifer, I. Gursel, K. J. Ishii, S. Takeshita, M. Gursel, and D. M. Klinman Cutting Edge: Role of Toll-Like Receptor 9 in CpG DNA-Induced Activation of Human Cells J. Immunol., October 1, 2001; 167(7): 3555 - 3558. [Abstract] [Full Text] [PDF]
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