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Mechanism and Function of a Newly
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J Immunol 2000; 164:944-953; ;
doi: 10.4049/jimmunol.164.2.944
http://www.jimmunol.org/content/164/2/944
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References
Gunther Hartmann and Arthur M. Krieg
Mechanism and Function of a Newly Identified CpG DNA
Motif in Human Primary B Cells1
Gunther Hartmann*‡ and Arthur M. Krieg2*†§
T
he CpG dinucleotides are underrepresented and selectively methylated in vertebrate DNA. In contrast, CpG
dinucleotides are present at the expected frequency and
are unmethylated in bacterial DNA (1). The recognition of unmethylated CpG dinucleotides within specific flanking bases is believed to be an ancestral nonself pattern recognition mechanism
used by the innate immune system to detect DNA of microbes or
viruses (2). In mice, optimal immune activation requires a CpG
motif in which an unmethylated CpG dinucleotide is flanked by
two 5⬘ purines and two 3⬘ pyrimidines (3, 4). DNA containing this
CpG motif (CpG DNA) activates murine macrophages (5– 8), murine dendritic cells (9, 10), murine NK cells (7, 11, 12), and murine
B cells (13–15).
CpG DNA is known to be an excellent immune adjuvant in
various murine disease models and to drive Th1 immune responses
(16 –21). Thus, CpG DNA might be useful for immunotherapy of
allergy, infectious disease, and cancer (20, 22–24). The potent adjuvant activity of CpG DNA in mice is most likely based on its
stimulatory effects on dendritic cells and B cells. Although CpG
effects in mice are well characterized, information regarding the
human system is limited. Many CpG phosphorothioate oligode-
*Department of Internal Medicine, University of Iowa, Iowa City, IA 52242; †Veteran
Affairs Medical Center, Iowa City, IA 52246; ‡CpG ImmunoPharmaceuticals GmbH,
Hilden, Germany; and §CpG ImmunoPharmaceuticals, Inc., Wellesly, MA 02481
Received for publication July 27, 1999. Accepted for publication October 28, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by Grant Ha 2780/1-1 of the Deutsche Forschungsgemeinschaft (G.H.). A.M.K. was supported by grants from the Department of Veterans
Affairs and National Institutes of Health (NIH) Grant P01-CA66570. Services were
provided by the University of Iowa Flow Cytometry Facility, and by the Diabetes and
Endocrinology Center (NIH DK25295). Support was received from CpG ImmunoPharmaceuticals GmbH (Hilden, Germany) and CpG ImmunoPharmaceuticals, Inc.
(Wellesly, MA).
2
Address correspondence and reprint requests to Dr. Arthur Krieg, Department of
Internal Medicine, University of Iowa, 540 EMRB, Iowa City, IA 52242. E-mail
address: [email protected]
Copyright © 2000 by The American Association of Immunologists
oxynucleotides (ODNs)3 with strong stimulatory activity in the
mouse system show only low activity on human immune cells
(A. M. Krieg, unpublished observations). In human PBMCs, synthetic phosphodiester ODNs with hexamer palindromic sequences
containing a central CpG dinucleotide have been described as inducing IFN-␣ synthesis (25). To date, the most active phosphorothioate oligonucleotide reported to stimulate human B cells is a
27-mer ODN (DSP30) originally designed as an antisense ODN to
the rev gene of HIV (26 –28). A 21 mer (27) and a 15 mer (29),
both 3⬘-truncated forms of the original anti-rev 27 mer, were
equally active as the complete 27 mer. DSP30 contains the sequence 5⬘-TCGTCG-3⬘ at its 5⬘ end, suggesting the hypothesis
that repeating TCG motifs may activate human B cells. However, phosphorothioate ODNs containing (TCG)n showed a
lower activity than DSP30 did (29). Furthermore, testing of
phosphorothioate ODNs which were not related to DSP30 but
contained multiple copies of various mouse 6-mer CpG motifs
(GACGTT; TGACGTT; GACGTC; and TGACGTC) were also
less active than DSP30 (29). A non-CpG-specific effect mediated
by the phosphorothioate backbone was suggested to be mainly
responsible for the proliferative response in these studies. NonCpG-specific activation of human PBMCs by the phosphorothioate
backbone has been described earlier (30). Thus, although human
cells can be stimulated by a variety of ODNs, the requirement for
CpG dinucleotides and the optimal flanking bases, if any, remain
unclear.
The identification of a potent human CpG motif is critical for the
transfer of therapeutic strategies derived from animal models to
clinical settings. We recently demonstrated that CpG DNA induces
activation of human monocytes (31) and dendritic cells (32), effects which are distinct from LPS-mediated effects. The goal of the
present study is to systematically search for an optimal human
CpG motif, to determine its functional effects on human B cells,
and to elucidate its molecular mechanism of action.
3
Abbreviations used in this paper: ODN, oligodeoxynucleotides; ERK, extracellular
receptor kinase; JNK, c-Jun N-terminal kinase; ATF-2, activating transcription factor-2; MFI, mean fluorescence intensity; MHC-II, MHC class II; FSC, forward light
scatter; CFSE, (5-(and-6-) carboxyfluorescein diacetate succinimidyl ester; SAPK,
stress-activated protein kinase.
0022-1767/00/$02.00
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The vertebrate immune system recognizes bacterial DNA based on the presence of unmethylated CpG-dinucleotides in particular
base contexts (“CpG motifs”). In contrast to mice, knowledge about CpG-mediated effects on human B cells is poor. In the present
study we identify and determine an optimal human CpG motif. A phosphodiester oligonucleotide containing this motif strongly
stimulated CD86, CD40, CD54, and MHC class II expression, IL-6 synthesis, and proliferation of primary human B cells. These
effects required internalization of the oligonucleotide and endosomal maturation. The molecular mechanism of action of this CpG
motif was associated with the sustained induction of the NF-␬B p50/p65 heterodimer and of the transcription-factor complex AP-1.
Transcription-factor activation by CpG DNA was preceded by increased phosphorylation of the stress kinases c-Jun N-terminal
kinase and p38, and of activating transcription factor-2. In contrast to CpG, signaling through the B cell receptor led to activation
of extracellular receptor kinase and to phosphorylation of a different isoform of c-Jun N-terminal kinase. These studies define the
structure of a highly active human CpG motif and characterize its molecular mechanism of action in primary human B cells. The
Journal of Immunology, 2000, 164: 944 –952.
The Journal of Immunology
945
Table I. ODN panel used a
Name
Sequence 5⬘ to 3⬘
PE 2079
TCG ACG TTC CCC CCC CCC CC
Middle base
PE 2100
PE 2082
PE 2080
TCG GCG TTC CCC CCC CCC CC
TCG CCG TTC CCC CCC CCC CC
TCG TCG TTC CCC CCC CCC CC
5⬘ Flanking base
PE 2105
PE 2107
PE 2104
GCG TCG TTC CCC CCC CCC CC
ACG TCG TTC CCC CCC CCC CC
CCG TCG TTC CCC CCC CCC CC
3⬘ Flanking base
PE 2098
PE 2099
PE 2083
TCG TCG CTC CCC CCC CCC CC
TCG TCG GTC CCC CCC CCC CC
TCG TCG ATC CCC CCC CCC CC
First CpG deleted
Second CpG deleted
PE 2108
PE 2106
CTG TCG TTC CCC CCC CCC CC
TCG TCA TTC CCC CCC CCC CC
Methylation
PE 2095
PE 2094
TZG TZG TTC CCC CCC CCC CC
TCG TCG TTC CCC CCC ZCC CC
Non-CpG control of 2080
PE 2078
PE 2101
TGC TGC TTC CCC CCC CCC CC
GGC CTT TTC CCC CCC CCC CC
PS form of 2080
Additional CpG motifs
Best PS
Methylated 2006
PS 2116
PE 2059
PS 2006
PS 2117
TCG
TCG
TCG
TZG
Human CpG motif
TCG
TCG
TCG
TZG
TTC
TTT
TTT
TTT
CCC
TGT
TGT
TGT
CCC
CGT
CGT
ZGT
CCC CC
TTT GTC GT
TTT GTC GT
TTT GTZ GTT
a
PE, phosphodiester; PS, phosphorothioate; bold, base exchange; bold Z, methylated cytidine; underlined, CpG
dinucleotides.
Materials and Methods
ODNs and DNA
Unmodified (phosphodiester) and modified nuclease-resistant (phosphorothioate) ODNs were purchased from Operon Technologies (Alameda, CA)
and Hybridon Specialty Products (Milford, MA). The sequences used are
provided in Table I. Escherichia coli DNA and calf thymus DNA were
purchased from Sigma (St. Louis, MO). Genomic DNA samples were purified by extraction with phenol-chloroform-isoamyl alcohol (25/24/1) and
ethanol precipitation. DNA was purified from endotoxin by repeated extraction with Triton X-114 (Sigma) and tested for endotoxin using the LAL
assay (BioWhittaker, Walkersville, MD; lower detection limit 0.1 EU/ml)
and the high-sensitivity assay for endotoxin described earlier (lower detection limit 0.0014 EU/ml) (31). Endotoxin content of DNA samples was
below 0.0014 U/ml. E. coli and calf thymus DNA were made single
stranded before use by boiling for 10 min and then cooling on ice for 5 min.
DNA samples were diluted in TE buffer using pyrogen-free reagents.
Cell preparation and cell culture
Human PBMCs were isolated from peripheral blood of healthy volunteers
by Ficoll-Paque density gradient centrifugation (Histopaque-1077, Sigma)
as described (33). Cells were suspended in RPMI 1640 culture medium
supplemented with 10% (v/v) heat-inactivated (56°C, 1 h) FCS (HyClone,
Logan, UT), 1.5 mM L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml
streptomycin (all from Life Technologies, Grand Island, NY) (complete
medium). All compounds were purchased endotoxin tested. Viability was
determined before and after incubation with ODN by trypan blue exclusion
(conventional microscopy) or by propidium iodide exclusion (flow cytometric analysis). In all experiments, 96 –99% of PBMCs were viable. Cells
(final concentration, 1 ⫻ 106 cells/ml) were cultured in complete medium
in a 5% CO2-humidified incubator at 37°C. Different ODNs (see Table I;
concentration as indicated in the figure legends), LPS (from Salmonella
typhimurium; Sigma), or anti-IgM were used as stimuli. Chloroquine (5
␮g/ml; Sigma) was used to block endosomal maturation/acidification. At
the indicated time points, cells were harvested for flow cytometry as described below.
For signal transduction studies, human primary B cells were isolated by
immunomagnetic cell sorting using the VARIOMACS technique (Miltenyi
Biotec, Auburn, CA) as described by the manufacturer. In brief, PBMCs
obtained from buffy coats of healthy blood donors (Elmer L. DeGowin
Blood Center, University of Iowa) were incubated with a microbeads-con-
jugated Ab to CD19 and passed over a positive selection column. Purity of
B cells was higher than 95%. After stimulation, whole cellular extracts
(Western blot) and nuclear extracts (EMSA) for signal-transduction studies
were prepared at the indicated time points.
Flow cytometry
Staining of surface Ags was performed as previously described (34). mAbs
to HLA-DR were purchased from Immunotech (Marseille, France). All
other Abs (mAbs to CD19 (B43), CD40 (5C3), CD54 (HA58), and CD86
(2331 (FUN-1)) were purchased from PharMingen (San Diego, CA).
IgG1,␬ (MOPC-21) and IgG2b,␬ (27–32, 35–37) were used to control for
specific staining. Intracellular cytokine staining for IL-6 was performed as
described (31). In brief, PBMCs (final concentration, 1 ⫻ 106 cells/ml)
were incubated in the presence of brefeldin A (final concentration, 1 ␮g/ml;
Sigma). After incubation, cells were harvested and stained using a FITClabeled mAb to CD19 (B43), a PE-labeled rat anti-human IL-6 mAb
(MQ2-6A3, PharMingen), and the Fix and Perm Kit (Caltag Laboratories,
Burlingame, CA). Flow cytometric data of 5000 cells/sample were acquired on a FACScan (Becton Dickinson Immunocytometry Systems, San
Jose, CA). Nonviable cells were excluded from analysis by propidium
iodide staining (2 ␮g/ml). Data were analyzed using the computer program
FlowJo (version 2.5.1, Tree Star, Stanford, CA).
Proliferation assay
CFSE (5-(and-6-) carboxyfluorescein diacetate succinimidyl ester; Molecular Probes, Eugene, OR) is a fluorescein-derived intracellular fluorescent
label which is divided equally among daughter cells upon cell division.
Staining of cells with CFSE allows both quantification and immunophenotyping (PE-labeled Abs) of proliferating cells in a mixed-cell suspension.
Briefly, PBMCs were washed twice in PBS, resuspended in PBS containing CFSE at a final concentration of 5 ␮M, and incubated at 37°C for 10
min. Cells were washed three times with PBS and incubated for 5 days as
indicated in the figure legends. Proliferating CD19-positive B cells were
identified by decreased CFSE content using flow cytometry.
Preparation of whole-cell, nuclear, and cytosolic protein
extracts
For Western blot analysis, whole-cell extracts were prepared. Primary B
cells were treated with medium, the phosphodiester ODNs 2080 (CpG) or
2078 (non-CpG) at 30 ␮g/ml, or anti-IgM (10 ␮g/ml) as indicated. Cells
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Starting sequence
946
were harvested, washed twice with ice-cold PBS containing 1 mM
Na3VO4, resuspended in lysis buffer (150 mM NaCl, 10 mM Tris (pH 7.4),
1% Nonidet P-40 (NP-40), 1 mM Na3VO4, 50 mM NaF, 30 mg/ml leupeptin, 50 mg/ml aprotinin, 5 mg/ml antipain, 5 mg/ml pepstatin, and 50
␮g/ml PMSF), incubated for 15 min on ice, and spun at 14,000 rpm for 10
min. The supernatant was frozen at ⫺80°C. For the preparation of nuclear
extracts, primary B cells were resuspended in hypotonic buffer (10 mM
HEPES/KOH (pH 7.9), 10 mM KCl, 0.05% NP-40, 1.5 mM MgCl2, 0.5
mM DTT, 0.5 mM PMSF, 30 mg/ml leupeptin, 50 mg/ml aprotinin, 5
mg/ml antipain, and 5 mg/ml pepstatin). After 15 min incubation on ice, the
suspension was centrifuged at 1000 ⫻ g for 5 min. The pelleted nuclei were
resuspended in extraction buffer (20 mM HEPES (pH 7.9), 450 mM NaCl,
50 mM NaF, 20% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM
PMSF, 30 mg/ml leupeptin, 50 mg/ml aprotinin, 5 mg/ml antipain, and 5
mg/ml pepstatin) and incubated on ice for 1 h. The nuclear suspension was
centrifuged for 10 min at 16,000 ⫻ g at 4°C. Supernatant was collected and
stored at ⫺80°C. Cytosolic extracts for the CpG-binding protein studies
were prepared from unstimulated Ramos cells, which were lysed with hypotonic buffer as described for the preparation of the nuclear extract. After
centrifugation, the supernatant was removed as cytoplasmic fraction and
stored at ⫺80°C. Protein concentrations were measured using a Bradford
protein assay (Bio-Rad, Hercules, CA) according to the manufacturer’s
instructions.
Equal concentrations of whole-cell protein extracts (25 ␮g/lane) were
boiled in SDS sample buffer (50 mM Tris-Cl (pH 6.8), 1% 2-ME, 2% SDS,
0.1% bromphenol blue, and 10% glycerol) for 4 min before being subjected
to electrophoresis on a 10% polyacrylamide gel containing 0.1% SDS
(SDS-PAGE). After electrophoresis, proteins were transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). Blots were blocked
with 5% nonfat dry milk. Specific Abs against the phosphorylated form of
extracellular receptor kinase (ERK), c-Jun N-terminal kinase (JNK), p38,
and activating transcription factor-2 (ATF-2) were used (New England
Biolabs, Beverly, MA). Blots were developed in enhanced chemiluminescence reagent (Amersham International, Aylesbury, U.K.) according to the
manufacturer’s recommended procedure.
EMSA
To detect the DNA-binding activity of the transcription factor AP-1 and
NF-␬B, nuclear extracts (1 ␮g/lane) were analyzed by EMSA using the
dsODNs 5⬘-GAT CTA GTG ATG AGT CAG CCG GAT C-3⬘ containing
the AP-1 binding sequence and the NF-␬B upstream regulatory element
(URE) from the c-myc promotor region 5⬘-TGC AGG AAG TCC GGG
TTT TCC CCA ACC CCC C-3⬘ as probes. ODNs were end labeled with
T4-polynucleotide kinase (New England Biolabs) and [␥-32P]ATP (Amersham, Arlington Heights, IL). Binding reactions were performed with 1 ␮g
nuclear protein extract in DNA-binding buffer (10 mM Tris-HCl (pH 7.5),
40 mM MgCl2, 20 mM EDTA, 1 mM DTT, 8% glycerol, and 100 – 400 ng
of poly(dI-dC) with 20,000 – 40,000-cpm-labeled ODN in 10 ␮l total volume). Specificity of the NF-␬B bands was confirmed by competition studies with cold ODNs from unrelated transcription-factor binding sites (10 –
100 ng). For the supershift assay, 2 ␮g of specific Abs for c-rel, p50, and
p65 (Santa Cruz Biotechnology, Santa Cruz, CA) were added into the
reaction mixture for 30 min before the radiolabeled probe was added. After
incubation for 30 min at room temperature, loading buffer was added and
the probes were electrophoresed on a 6% polyacrylamide gel in Tris-borate-EDTA running buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA
(pH 8.0)). Gels were dried and then autoradiographed.
Statistical analysis
Data were expressed as means ⫾ SEM. Statistical significance of differences was determined by the unpaired two-tailed Student t test. Differences
were considered statistically significant for p ⬍ 0.05. Statistical analyses
were performed by using StatView 4.51 software (Abacus Concepts,
Calabasas, CA).
Results
Identification of a potent human CpG motif for activating
primary human B cells
Phosphorothioate ODNs containing the murine CpG motif
GACGTT (for example ODN 1826) and used at concentrations
which are highly active on murine B cells (3) show little or no
immunostimulatory activity on human immune cells (data not
shown). Added at high concentrations, phosphorothioate ODNs
FIGURE 1. CpG DNA induces expression of CD86 on human primary
B cells. Freshly isolated PBMCs (1 ⫻ 106 cells/ml) were incubated in the
presence of the CpG ODN 2080 and the non-CpG control ODN 2078
(added at 0, 4, and 18 h; 30 ␮g/ml at each time point). After 2 days CD86
expresssion was measured on CD19-positive B cells by flow cytometry.
One of more than 10 representative experiments is shown.
stimulate human B cells in a CpG-independent manner (29). This
likely is due to the phosphorothioate modification of the ODNs.
The use of bacterial DNA or unmodified phosphodiester ODNs
avoids the sequence-independent background activity of the phosphorothioate backbone. We found that B cells express increased
levels of the costimulatory molecule CD86 (B7-2) in response to
CpG DNA but not in response to non-CpG control DNA (Fig. 1).
Assuming that bacterial DNA contains human CpG motifs, we
tested the effect of highly purified E. coli DNA (31) on human
primary B cells. Repeated addition (at 0, 4, and 8 h) of 30 ␮g/ml
E. coli DNA resulted in maximal expression of CD86 on human B
cells within 2 days. Higher concentrations showed no further increase. Calf thymus DNA (non-CpG control DNA) did not activate
human B cells. High concentrations of LPS (1 ␮g/ml, 1000-fold
more than needed for maximal activation of human monocytes)
only slightly activated B cells above background (mean fluorescence intensity (MFI) CD86: E. coli DNA, 3.4 ⫾ 0.9; calf thymus
DNA, 1.5 ⫾ 0.4; LPS 1.6 ⫾ 0.4; medium only, 0.9 ⫾ 0.1; n ⫽ 4).
In earlier studies on B cell activation in mice, we found that a
CpG-dinucleotide flanked by two 5⬘ purines and two 3⬘ pyrimidines is optimal for a phosphodiester ODN to be active, and that
the 6-mer motif 5⬘-GACGTT-3⬘ is best (2, 3). Furthermore, testing
all 16 possible dimers at the 5⬘ end of a CpG ODN, we found that
the dimer 5⬘-TpC-3⬘ added to the activity of the ODN. The presence of 3⬘-polypyrimidine sequences after the 6-mer CpG motif
further enhanced the effect of the ODN (3).
Using this information we designed a 20-mer phosphodiester
ODN with a TpC dinucleotide at the 5⬘ end preceding the optimal
murine CpG motif 5⬘-GACGTT-3⬘ and followed by a poly C tail
(2079, 5⬘-TCG ACG TTC CCC CCC CCC CC-3⬘). This ODN, if
added to human primary B cells under the same conditions found
to be optimal for E. coli DNA (repeated addition at 0, 4, and 18 h;
30 ␮g/ml for each time point), stimulated high levels of CD86
expression on human primary B cells after 2 days (Fig. 2). To
determine the structure-function relationship of the CpG motifs,
we replaced the bases adjacent to the CpG dinucleotides while
maintaining the two CpG dinucleotides within the sequence. Exchange of the adenine located between both CpG dinucleotides by
thymidine (2080) resulted in slightly higher activity (Fig. 2). Replacement by guanosine (2100) or cytidine (2082) at this position
showed no major changes compared with 2079. In contrast, replacement of the thymidine 3⬘ to the second CpG dinucleotide by
the purines guanosine (2099) or adenine (2083) resulted in a major
drop in activity of the ODN, whereas the pyrimidine cytidine
caused only a minor decrease (Fig. 2). The thymidine immediately
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Western blot analysis
IDENTIFICATION OF A POTENT HUMAN CpG MOTIF
The Journal of Immunology
947
5⬘ to the first CpG dinucleotide was also critical. Replacement of
the thymidine by any other base (e.g., 2105, guanosine; 2107, adenine; or 2104, cytidine) led to a marked decrease in activity of the
ODN. Elimination of the first (2108) or the second (2106) CpG
dinucleotide also partially reduced the activity (Fig. 2).
Consistent with the B cell activation by bacterial but not by
vertebrate DNA, an ODN in which the cytidines of the CpG
dinucleotides were methylated (2095) was not stimulatory (Fig. 2).
Methylation of an unrelated cytidine (2094) did not change the
potency to activate B cells. An ODN with an inversion of both
CpG dinucleotides to GpC (2078) and another non-CpG control
ODN derived from 2080 (2101; Table I and data not shown) did
not stimulate CD86 expression above background (Fig. 2).
The addition of more 5⬘-GTCGTT-3⬘ CpG motifs to the phosphodiester ODN containing the 8-mer duplex CpG motif (5⬘TCGTCGTT-3⬘, 2080) did not further enhance CD86 expression
on B cells (2059). An ODN with the same sequence as 2080 but
FIGURE 3. Up-regulation of CD40,
CD54, and MHC-II expression on human primary B cells. PBMCs were incubated for 2 days with the CpG ODN
2080 and its control 2078 (added at 0, 4,
and 24 h; 30 ␮g/ml at each time point).
Expression of CD40, CD54, and
MHC-II on CD19-positive B cells was
examined by flow cytometry. Data are
shown as means of three independent
experiments with PBMCs from different
donors. The values of 2080 (MFI) are set
to 100% for each experiment. Error bars
indicate the SEM. ⴱ, p ⬍ 0.001 (comparison of the no addition sample to
2080; absolute values).
with a phosphorothioate backbone showed no activity above background (2116). This was surprising because the phosphorothioate
backbone has been reported to greatly stabilize ODNs and to enhance CpG-induced stimulation (2). Therefore, we performed further structure-function analyses of phosphorothioate ODNs containing the 5⬘-GTCGTT-3⬘ and 5⬘-TCGTCGTT-3⬘ motifs, which
showed that an active phosphorothioate ODN required the presence of additional CpG motifs (2006) (Fig. 2, and Hartmann et al.,
manuscript in preparation). Although the maximal activities of
phosphodiester and phosphorothioate ODNs were similar, much
lower concentrations of the phosphorothioate ODN (0.6 ␮g/ml)
were sufficient to produce maximal activity, most likely due to the
higher nuclease stability of the phosphorothioate backbone. The
effect of 2006 at this concentration was CpG-specific because a
control ODN with the same sequence but with methylated cytidines (2117) did not induce CD86 expression on primary B cells
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FIGURE 2. Identification of a potent human CpG motif. PBMCs were incubated with a panel of phosphodiester ODNs (see Table I) or E. coli DNA
(added to PBMCs at 0, 4, and 18 h; 30 ␮g/ml at each time point), with phosphorothioate ODNs (added to PBMCs at 0 h; 0.6 ␮g/ml) and LPS (1 ␮g/ml).
After 2 days, induction of CD86 expression on human primary B cells (CD19-positive cells) was examined by flow cytometry. The phosphodiester ODNs
without a CpG dinucleotide (2078) are inactive. The ODN with the 8-mer motif 5⬘-TCG TCG TT-3⬘ (2080) containing two CpG dinucleotides shows the
highest activity. Replacement of the bases flanking the two CpG dinucleotides (5⬘ position, middle position, 3⬘ position) reduces the activity of this
sequence. Both CpG dinucleotides within the 8-mer CpG motif are required for optimal activity (2108 and 2106). Cytidine methylation of the CpG
dinucleotides (2095) abolishes the activity of 2080, whereas methylation of an unrelated cytidine (2094) does not. The addition of two CpG motifs into
the sequence of 2080 resulting in 2059 does not further increase the activity of the phosphodiester ODN. The sequence of 2080 with a phosphorothioate
backbone (2116) is inactive. Additional CpG motifs are required for a potent phosphorothioate ODN (2006). Data are shown as means of four independent
experiments using different donors. The values of 2080 (MFI) are set to 100% for each experiment. Error bars indicate the SEM. ⴱ, p ⬍ 0.001 (comparison
of the no addition sample to 2080; absolute values).
948
IDENTIFICATION OF A POTENT HUMAN CpG MOTIF
(MFI CD86: 2006, 8.4 ⫾ 1.2; 2117, 2.4 ⫾ 0.3; medium only,
2.3 ⫾ 0.2; n ⫽ 3).
Purified B cells isolated from peripheral blood by immunomagnetic cell sorting were activated by CpG DNA to the same extent
as unpurified B cells within PBMCs (data not shown). Thus, activation of B cells is a primary response and not a secondary effect
caused by cytokines secreted by other cells.
Induction of CD40, CD54, and MHC class II (MHC-II)
expression and IL-6 synthesis by CpG DNA
In addition to the costimulatory molecule CD86, the functional
state of B cells is characterized by other surface markers. For example, activated T helper cells stimulate B cells by CD40 ligation,
ICAM-1 (CD54) mediates binding to other immune cells, and
MHC-II is responsible for Ag presentation. We found that B cell
expression of CD40, CD54, and MHC-II was up-regulated by the
CpG ODN 2080 (Fig. 3). The non-CpG control ODN 2078 (CpG
inverted to GpC) showed no activity compared with medium
alone.
IL-6 synthesis by human primary B cells in response to CpG
DNA was examined by intracellular staining and flow cytometry.
Within the first 6 h, 18.3% of B cells incubated in the presence of
the CpG phosphorothioate ODN 2006 (4 ␮g/ml) accumulated IL-6
compared with 3.4% of cells incubated with medium only (data not
shown).
Sequence-dependent induction of B cell proliferation by CpG
DNA
When PBMCs were incubated for 5 days in the presence of 2080
(added at 0, 4, and 18 h and every subsequent morning), it was
intriguing that a subpopulation of lymphocytes increased in cell
size (forward light scatter (FSC)) and became more granular (side
light scatter) (Fig. 4, upper panel). To examine whether this subpopulation represented proliferating B cells, we stained freshly isolated PBMCs with CFSE at day 0 and incubated them for 5 days
with 2080 as above. CFSE is a fluorescent molecule that binds
irreversibly to cell proteins. Each cell division decreases CFSE
stain by 50% (35). Cells staining low with CFSE (proliferating
cells) were found to be mainly CD19-positive B cells (Fig. 4, middle panel). The ODN 2080 induced 60 –70% of CD19-positive B
cells to proliferate within 5 days. The control ODN 2078 induced
less than 5% of B cells to proliferate. Proliferating B cells (CFSElow) showed a larger cell size (FSC) and higher granularity (Fig.
4, lower panel).
Proliferating B cells expressed higher levels of CD86 than nonproliferating cells (data not shown). In agreement with this finding,
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FIGURE 4. CpG DNA induces proliferation of
human primary B cells. PBMCs were freshly isolated, stained with CFSE (see Materials and Methods), and subsequently incubated in the presence of
the CpG ODN 2080 and its control 2078 (added at 0,
4, and 24 h; 30 ␮g/ml at each time point). After 5
days, cells were stained with CD19 (B cell marker)
and examined by flow cytometry. In the presence of
2080, a subfraction of the PBMCs show enhanced
cell size (FSC) and granularity (side light scatter)
compared with the non-CpG control (upper panel).
Proliferating cells are identified by a decreased
brightness with the CFSE stain (middle panel). Cells
with an increased cell size (upper panel) are identical with the proliferating cells (CFSE-low, lower
panel). The upper and middle panels show whole
PBMCs; the lower panel only shows CD19-positive
B cells. The results are representative of more than
six independent experiments.
The Journal of Immunology
the ODN panel tested above for induction of CD86 expression
resulted in an almost identical pattern of B cell proliferation (Fig.
5). Replacement of the 3⬘ thymidine reduced activity more than
changing the thymidine in the middle position. Methylation of the
CpG dinucleotides (2095) blocked activity, whereas methylation
of an unrelated cytidine (2094) did not influence the capacity to
induce B cell proliferation. Activity of E. coli DNA was lower than
activity of 2080. LPS did not induce B cell proliferation.
FIGURE 6. CpG DNA stimulates rapid and sustained NF-␬B binding
activity. Primary B cells were isolated from peripheral blood by immunomagnetic beads (see Materials and Methods) and were incubated in the
presence of the CpG ODN 2080 and its control 2078 (30 ␮g/ml). Cells
incubated for a total of 18 h received ODNs at 0 and 4 h (30 ␮g/ml each).
At the indicated time points, cells were harvested and nuclear extracts were
prepared. Equal concentrations of protein (1 ␮g/lane) were analyzed on a
6% polyacrylamide minigel for NF-␬B-binding activity. Specificity of the
NF-␬B band (arrow on left) is examined by adding cold ODNs (NF-␬B,
c-myc). The components of the NF-␬B heterodimer are identified with
specific Abs c-rel, p50, and p65 (arrows on right). The experiment was
repeated three times with similar results.
B cell activation requires endosomal maturation/acidification of
CpG DNA
It has been shown earlier that chloroquine, an inhibitor of endosomal acidification, blocks CpG-mediated stimulation of murine
APCs and B cells, but does not influence LPS-mediated effects (8,
15, 36). We found that the addition of 5 ␮g/ml chloroquine completely blocked CpG DNA-mediated induction of CD86 expression on primary B cells (MFI CD86: 2006, 4.7 vs 1.4; E. coli DNA,
3.4 vs 1.4; medium only, 0.9; n ⫽ 4). Furthermore, chloroquine
completely inhibited the induction of B cell proliferation by the
phosphorothioate ODN 2006 measured with the CFSE proliferation assay as well as with the standard. These results suggest that,
as with murine cells, activation of human B cells by CpG DNA
requires the uptake of DNA in endosomes and subsequent endosomal acidification.
CpG DNA stimulates rapid and sustained NF-␬B binding
activity in human B cells
Because the CpG motif requirement for maximal B cell activation
is substantially different between mice (GACGTT) and humans
(TCGTCGTT), we were interested in whether the basic intracellular signaling events are comparable. This information will help
to extrapolate from mouse studies to responses of the human immune system. Rapid induction of NF-␬B binding activity has been
found earlier in murine B cells and macrophages (6, 15). To investigate the NF-␬B response to CpG DNA in humans, human
primary B cells were isolated from peripheral blood by immuno-
magnetic cell sorting and incubated with the CpG ODN 2080, the
non-CpG control ODN 2078, or medium. At the indicated time
points, cells were harvested and nuclear extracts were prepared. In
the presence of CpG ODN, NF-␬B binding activity was increased
within 1 h and maintained up to 18 h (latest time point examined)
(Fig. 6). The non-CpG control ODN 2078 did not show enhanced
NF-␬B activity compared with cells incubated with medium only.
The NF-␬B band was identified by cold competition and shown to
consist of p50 and p65 subunits by supershift assay (Fig. 6).
Rapid and sustained stimulation of DNA-binding activity of
transcription factor AP-1
The AP-1 transcription factor is involved in the regulation of immediate early genes and cytokine expression (37). In murine B
cells, AP-1-binding activity is induced in response to CpG DNA
(13). To determine whether this transcription factor would also be
induced by CpG DNA in humans, we examined AP-1 DNA-binding activity in human primary B cells. Cells were incubated with
the CpG ODN 2080 or the control ODN 2078. At the indicated
time points, nuclear extracts were prepared and the AP-1-binding
activity was analyzed by EMSA. AP-1-binding activity was enhanced within 1 h (Fig. 7A) and increased up to 18 h (latest time
point examined), showing a sustained response.
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FIGURE 5. CpG-specific induction of human B cell proliferation.
PBMCs were stained with CFSE and incubated in the presence of different
ODNs. Phosphodiester ODNs and E. coli DNA were added at 0 h, 4 h, and
each morning of the subsequent 3 days (30 ␮g/ml at each time point). The
phosphorothioate ODN 2006 was added at 0 h (0.6 ␮g/ml). After 5 days,
cells were stained with CD19 (B cell marker) and examined by flow cytometry. The percentage of CFSE-low CD19-positive cells of all CD19positive cells (proliferating B cells) was determined. Results represent the
means of two independent experiments from two different donors. Error
bars indicate SEM.
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950
CpG DNA induces rapid phosphorylation of JNK, ATF-2, and
p38, but not of ERK
Because AP-1 activity is induced by many stimuli (38), we were
interested in signal-transduction pathways upstream of AP-1. The
AP-1 transcription-factor complex integrates different mitogen-activated protein kinase pathways (37). Western blots were performed using whole-cell extracts from primary B cells incubated
with the CpG ODN 2080, the control 2078, or medium only. Specific Abs to the phosphorylated form of JNK, p38, ATF-2, and
ERK were used. Strong induction of JNK phosphorylation was
found 30 and 60 min after exposure to CpG DNA, whereas the
non-CpG ODN showed no activity above background (Fig. 7B).
The protein kinase p38, another stress-activated protein kinase
(SAPK), was also phosphorylated in response to CpG DNA within
60 min. ATF-2, a substrate of both p38 and JNK (39) and a component of the AP-1 complex, showed weak phosphorylation after
30 min which increased after 60 min. CpG DNA failed to induce
substantial phosphorylation of ERK. In contrast, anti-IgM, stimulating the B cell receptor, did trigger phosphorylation of ERK.
Anti-IgM activated different isoforms of JNK than CpG DNA did
(Fig. 7B).
Discussion
CpG DNA has developed into an exciting tool to activate and to
steer immune responses toward Th1 in mice. However, most
ODNs which activate mouse cells show only weak activation of
human immune cells, and to date, an optimal human CpG motif
has not been demonstrated. We now report the discovery of a
potent human CpG motif and the characterization of its effects and
mechanisms of action on human primary B cells. DNA containing
this CpG motif strongly stimulated primary human B cells to proliferate, to produce IL-6, and to express increased levels of CD86,
CD40, CD54, and MHC-II. It increased DNA-binding activity of
the transcription factors NF-␬B and AP-1, as well as phosphorylation of the stress-activated protein kinases JNK and p38 and the
transcription factor ATF-2. B cell signaling pathways activated by
CpG DNA were different from those activated by the B cell receptor, which activated ERK and a different isoform of JNK, but
did not activate p38 and ATF-2. Blockade of endosomal maturation with chloroquine abolished these effects.
The use of phosphodiester ODNs facilitates the identification of
an active CpG motif. Base exchanges within the most potent 8-mer
CpG motif (5⬘-TCGTCGTT-3⬘) diminished the activity of the
ODN. The thymidines at the 5⬘ and the 3⬘ positions of this motif
were more critical than the thymidine at the middle position. An
adenine or guanosine at the middle position only slightly decreased
the activity but resulted in a CpG dinucleotide flanked by two 5⬘
purines and two 3⬘ pyrimidines, a 6-mer sequence which was previously identified to be the optimal murine CpG motif (2). The
significant change compared with the mouse motif is the additional
requirement of a 5⬘ TC adding a second CpG. Although the human
CpG motif is still active in mice (data not shown), the murine
6-mer CpG motif alone is not sufficient to produce high activity in
humans. This argues for a refinement of the CpG recognition
mechanism in primates.
CpG effects on B cells were strictly CpG specific. Replacement
of CpG dinucleotides with GpC dinucleotides as well as cytidine
methylation of the CpG dinucleotides abolished B cell activation.
The methylation of an unrelated cytidine did not change the activity of the CpG ODN. Due to degradation by nucleases, detection
of immune stimulation by phosphodiester ODNs required that they
be added several times at relatively high concentration (30 ␮g/ml).
Even at these high concentrations, phosphodiester ODNs without
CpG dinucleotides showed no background activity.
The development of CpG DNA as a practical drug requires nuclease resistance such as is conferred by the phosphorothioate
backbone which protects DNA from rapid degradation in vivo (2).
Of note, our studies demonstrate that one human CpG motif within
a phosphodiester ODN (2080) is sufficient to produce the maximal
effect and that additional CpG motifs (2059) did not further enhance the activity. Surprisingly, the phosphorothioate modification
completely abolished the immunostimulatory activity of an ODN
with only one human CpG motif (2116), and additional CpG motifs were required to regain activity (2006). Furthermore, phosphorothioate ODNs with the murine CpG motif highly stimulatory to
murine immune cells showed only low activity in human B cells
(our unpublished results). Thus, the use of the phosphorothioate
backbone requires the optimal CpG motif, whereas phosphodiester
ODNs still show some activity if the motif is slightly changed.
This suggests that the specificity or affinity of the interaction between the CpG DNA and a putative CpG binding protein is reduced by the phosphorothioate backbone. This is supported by
EMSA studies in which we have been unable to detect binding of
phosphorothioate CpG ODNs to a putative CpG-binding protein
(data not shown) under conditions where phosphodiester CpG
DNA binds well.
The nuclease-resistant phosphorothioate backbone dramatically
reduced the concentration of an ODN required for maximal activity (0.6 ␮g/ml). At this concentration, B cell stimulation was CpGspecific and little background stimulation by the phosphorothioate
backbone was found. Others have reported that, in contrast to
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FIGURE 7. CpG DNA induces increased DNA-binding activity of transcription factor AP-1 and phosphorylation of p38, ATF-2, and JNK but not
ERK. Primary B cells isolated from PBMCs by immunomagnetic beads
were incubated in the presence of the CpG ODN 2080, its non-CpG control
2078, or anti-IgM (signaling through B cell receptor) as indicated. A, Equal
amounts of nuclear extracts (1 ␮g protein/lane) were analyzed by EMSA
(6% polyacrylamide minigel) for AP-1-binding activity. The experiment
was repeated three times with similar results. B, Cells were harvested after
60 min and whole-cell extracts were loaded on 10% SDS-PAGE minigel
(20 ␮g/lane) and analyzed by Western blot. Specific Abs to phosphoroylated (p) p38, ATF-2, JNK, and ERK were used. CpG DNA and anti-IgM
show phosphorylation of different isoforms of JNK (arrows).
IDENTIFICATION OF A POTENT HUMAN CpG MOTIF
The Journal of Immunology
Acknowledgments
We thank Ae-Kyung Yi and Micho Gravis for helpful discussions.
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when added repeatedly and in concentrations up to 100 ␮g/ml (29).
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putative CpG-binding protein and on how the recognition of CpG
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