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Introduction
Certain molecular structures that are present in
pathogens (pathogen-associated molecular patterns [PAMP]) are
recognized by innate immune cells via pattern recognition
receptors. The cells are activated upon recognition of PAMP
and trigger the generation of optimal adaptive immune
responses. The bacterial genome, compared to vertebrate
DNA, contains a higher frequency of unmethylated
deoxy-cytidyl-deoxyguanosine (CpG) dinucleotides. Small
oligodeoxynucleotides (ODN) with unmethylated CpG
dinucleotides (CpG ODN) are able to perfectly mimic the
immunostimulatory activity of bDNA[1,2]. CpG ODN are
known to stimulate innate and adaptive immunity because
of their interesting immunostimulatory properties in a
number of vertebrate, such as stimulating B-cell
proliferation[3_5], enhancing the expression and synthesis of
cytokines[6], and promoting natural killer (NK) cell
cytotoxicity[7]. Multiple studies have shown that CpG ODN-induced activation is
presumably a 2-step process. First, CpG ODN, independent
of their sequence, are recognized by different
receptors[1,2,8_10]; Mac-1 surface immunoglobulins and scavenger receptors
are possibly involved in this process. The cellular uptake
of ODN is sequence independent, but can be influenced
dramatically by backbone modifications. Phosphorothioated
modifications can enhance cellular uptake and increase
immunostimulatory activity and half-time of CpG
ODN[9,11]. In addition, runs of at least 4 guanosines have been reported
to enhance the cellular uptake of ODN due to the fact that
base-quartet-stabilized, 4-stranded helices called tetraplexes
are formed[10,12]. Second, CpG ODN trigger the
motif-dependent recognition of Toll-like receptor 9 (TLR-9) within the
endosome and initiate immunocellular activation.
Immunostimulatory activities of CpG ODN depend on
their structural and chemical characteristics. A number of
researchers have studied the relationship between the CpG
ODN structure and their immunostimulatory properties. CpG
ODN were structurally and functionally divided into 3 types
in previous studies for humans: A type (also known as D
type), B type (also known as K type), and C type. A-type
CpG ODN is capable of activating human plasmacytoid
dendritic cells (pDC) to produce large amounts of type I IFN
(α/β) and strongly activates NK cells. It has mixed
phospho-diester/phosphorothioate backbones and contains a single
hexameric purine/pyrimidine/CG/purine/pyrimidine motif
flanked by self-complementary bases that form a stem-loop
structure capped at the 3'-end by a poly G
tail[13]. B-type CpG ODN primarily activates B cells, resulting in their
proliferation and antibody secretion. It has phosphorothioate
backbones and encodes multiple TCGTT and/or TCGTA
motifs[13,14]. C-type CpG ODN optimally consist of a
stimulatory hexameric CpG motif (5'-TCGTCGTT-3') linked by a T
spacer to GC-rich palindromic sequences
(5'-CGGCGCGCG-CCG-3')[15]. The C-type CpG ODN very strongly stimulates B
cells as well as type I IFN secretion in
vitro in human peripheral blood mononuclear cells.
In vivo, it is a strong, Th1-inducing adjuvant. In addition, another C-type CpG ODN is
reported to display a B-type CpG ODN structure feature at
the 5'-end and A-type CpG ODN structure feature at the
3'-end and shares the activities of both A- and B-type CpG
ODN[16]. Furthermore, the sequence of the optimal human
hexamer motif was reported to be
5'-GTCGTT-3'[17] and the optimal mouse motif was 5'-GACGTT-3'
[1].
Despite the previously mentioned reports, more detailed
and systematic structure-activity relationship (SAR)
studies of CpG ODN are still lacking. In this study, we tried to
reveal some changes of the immunostimulatory properties
of CpG ODN induced by the modifications of the ODN
structure, for instance, additions and deletions of the
functional CpG motifs, modification of the distance and the sites
of CpG motifs, and the addition of self-complementary
sequences. We recruited CpG 1826, an effective
immuno-stimulatory B-type CpG ODN for murine, as the positive
control and the template of the SAR study. This ODN was a
strong B-cell stimulator, but a weaker inducer of
IFN-α[1,18,19]. Various modifications on the first-class structural level
were performed on the basis of ODN1826, and the
immuno-stimulatory activities of the structurally-modified CpG ODN
were comprehensively investigated, including the ability to
stimulate mouse B cell proliferation, the ability to induce
cytokines (interleukin [IL]-6, IL-12, and IFN-α) secretion and
the ability to induce NK cell killing activity, and the changes
of the expression of various lymphocyte surface molecules.
Finally, the relationship between the CpG ODN structures
and their immunostimulatory properties were primarily
concluded.
Materials and methods
CpG ODN Purified, single-stranded, phosphorothioated
ODN containing CpG motifs were synthesized by Sangon
Biotech Company (Shanghai, China). The sequences used
in this study are listed in Table 1, where ODN1 was the intact
1826, positive control, and the template; ODN 2_12 were the
sequences modified based on the structure of 1826; and
ODN13_15 were negative controls.
Cells and cell culture Spleen cells from 4- to 8-week-old
C57BL/6 mice were cultured in RPMI-1640 medium
supplemented with 10% (v/v) fetal bovine serum (GIBCO, Carlsbad,
CA, USA) and antibiotics (100 IU/mL penicillin and 100
µg/mL streptomycin). Mouse B cells were obtained by
centrifugation over Lympholyte M (CEDARLANE Laboratories,
Hornby, Ontario, Canada) as described
previously[1]. Yeast artificial chromosome-1 (YAC-1) cells were kindly provided
by Prof Wen-xia ZHOU (Beijing
Institute of Pharmacology and Toxicology, Beijing, China).
Cytokine ELISA The mouse spleen cells were plated
onto 24-well dishes (3×106 per well). The CpG ODN,
dissolved in TE buffer, were added to the cultured cells at a final
concentration of 0.05, 0.25, and 1.0 µmol/L, respectively. The
cells were cultured at 37 °C in a humidified incubator with 5%
CO2, and the culture supernatants (SN) were collected at the
appointed time-points. If not used immediately, the SN were
stored at -20 °C until the assay. The amounts of murine IL-6,
IL-12, and IFN-α in the SN were measured using
commercially available ELISA kits (R&D Systems, Minneapolis, MN,
USA). The experiments were performed 2 or 3 times for each
CpG ODN in triplicate for each concentration.
Proliferation assays The proliferation of mouse B cells
was determined by a [3H]thymidine incorporation assay as
described previously[1]. Briefly, the spleen cells
(5×105/well) were plated onto Costar 96-well plates
(Corning Incorporated Corning, New York, NY, USA) and stimulated with CpG
ODN at various concentrations (0.05, 0.25, and 1.0 µmol/L)
or controls for 56 h, and then 0.5 µCi of
[3H] thymidine was added to each well. The cells were harvested after
incubation for another 16 h and the radioactivity was measured
using a MicroBeta liquid scintillation counter (PerkinElmer,
Boston, MA, USA). All assays were performed 4 times.
Measurement of NK-mediated cytotoxicity The NK
cytotoxicity of mouse spleen cells stimulated by CpG ODN
was assessed by standard 4 h 51Cr-release assays as
previously described[7]. Briefly, the mouse spleen cells were
incubated with CpG ODN (0.25 μmol/L) for 36 h and then
harvested as effector cells. One million YAC-1 cells, used as
target cells, were incubated with 50 µCi of
51Cr for 1 h at
37 °C, then washed several times and incubated for 4 h with
the effector cells (E:T ratio, 50:1). Thereafter, the SN were
harvested and the radioactivity was measured using a
MicroBeta liquid scintillation counter (PE, Boston, MA,
USA). The results were expressed as the percentage of
specific lysis in terms of mean±SD of the results read in triplet
wells. The following formula was used: percent specific
lysis (%)=(experimental counts-target cell spontaneous
release counts)/(maximal release counts-target cell
spontaneous release counts)×100. Spontaneous lysis was
measured from wells containing only target cells, whereas
maximum lysis was measured from the wells containing target
cells incubated with 10% SDS.
The cytotoxicity of mouse spleen cells stimulated by CpG
ODN to the B16 cells was measured using B16 cells as target
cells as described earlier.
Abs and flow cytometry The mouse spleen cells were
incubated with or without CpG ODN (0.25 µmol/L) for 48 h,
and then the B cells, pDC, and NK cells were analyzed by
two-color staining on a FACS Calibur flow cytometer (BD
Pharmingen, Franklin lakes, New Jersey, USA). For B
cells, FITC-labeled anti-CD19 and PE-labeled anti-CD80 mAbs were
used. For pDC, FITC-labeled anti-CD11c and PE-labeled
anti-Iab mAbs were used. For the NK cells, FITC-labeled
anti-CD94 fmAbs were used (BD Pharmingen, Franklin
lakes, NJ, USA).
Animals Female BALB/c mice (6_8 weeks old) were used
for all the experiments and were purchased from
Experimental Animal Center of Academy of Military Medical Sciences
(Beijing, China; certificate No SYKX2002-001).
Statistical analysis Data are shown as mean±SD.
Statistically significant differences were determined by Student's
t-test. Differences were considered statistically significant
when P<0.05.
Results
Production of cytokines induced by CpG ODN In the
present study, we investigated the production of several
cytokines, including murine IL-6, IL-12 (P40), and
IFN-α in immunocytes stimulated by CpG ODN. The positive control
ODN1826 (ODN1) is a typical B-class CpG ODN. It consists
of 2 stimulatory hexameric CpG motifs
(5'-TCGACGTT-3') linked by a CCT spacer and a TpC dinucleotide at the 5'-end.
ODN1826 induced strong IL-6 secretion in a
dose-dependent manner. When its concentration
reached 0.25 µmol/L, the induced IL-6 reached 4651±301 pg/mL (Figure 1A). ODN2,
ODN4, ODN5, and ODN7_12 also stimulated IL-6
production to a different extent, but the IL-6-inducing activity of all
those sequences were weaker than that of ODN1826. ODN10,
which had the most obvious effect on the upregulation of
IL-6, can modulate the IL-6 level to 4579±334 pg/mL at
a higher concentration of 1.0 µmol/L. ODN3, which had a
5'-end, self-complementary palindrome and lost almost all
IL-6-inducing activity, indicating that a free 5'-end was necessary
for a CpG ODN to possess immunostimulatory activity.
There was no obvious IL-6-inducing activity in the negative
control sequences, ODN12_15.
With regards to IL-12, the tendency of
sequence-dependent stimulatory activity was similar to that of IL-6. ODN4,
ODN5, ODN7, ODN10, ODN11, as well as ODN1826, induced
relatively high levels of IL-12 P40 (Figure 1B). However,
ODN1826 was no longer the strongest stimulator for IL-12
production. At the highest studied concentration of 1.0
μmol/L, ODN1826 treatment upregulated IL-12 P40 secretion to
145±12 pg/mL. At the same dose, ODN4, ODN7, ODN10,
and ODN11 stimulated IL-12 P40 production to 172±14,
188±14, 229±17, and 184±16 pg/mL, respectively
(P<0.05).
ODN1826 was a relatively weaker IFN-α stimulator (90±7
pg/mL at the dose of 1.0 µmol/L) compared to its
IL-6-indu-ing activity in this study. Our findings were consistent with
those reported in other studies (Figure
1C)[15]. Among all the ODN studied, the strongest
IFN-α stimulators (the ODN were at a dose of 1.0 µmol/L) were partly similar to the induction of
IL-6 and IL-12 production. The IFN-α stimulators
were ODN4 (14 ±15 pg/mL), ODN5 (97±10 pg/mL), ODN7 (180±14
pg/mL), ODN8 (115±13 pg/mL), ODN10 (135±6 pg/mL) and
ODN11 (150±14 pg/mL). Structural information
(Table 1) demonstrated that, ODN4, ODN7, ODN10, and ODN11 all
had a 3'-end, self-complementary palindrome motif,
indicating that a 3'-end palindrome structure is important for CpG
ODN-induced IL-12 and IFN-α production.
Effect of CpG ODN on B cell proliferation
According to Krug et al, A-class ODN were poor stimulators of human B
cell proliferation. In contrast, B- and C-class ODN efficiently
stimulated B cell proliferation[20]. In our study, B-class
ODN1826 induced the upregulation of B cells to a moderate
extent. ODN5, ODN6, ODN8, and ODN9 stimulated B cell
proliferation with comparable efficiency and in a dosedependent manner. However, ODN4, ODN7, ODN10, and
ODN11 had a more obvious effect on stimulating the
proliferation of B cells than ODN1826 (Figure 2).
NK cell activation Another important activity of CpG
ODN is the activation of NK cells[7]. ODN with a 3'-end,
self-complementary palindrome, such as ODN4, ODN7, ODN10,
and ODN11 induced more efficient NK cytotoxicity (Figure 3).
Flow cytometry analysis CD19 is the characteristic
surface marker of B cells, and the activation of B cell
proliferation is characterized by the upregulation of cell surface
molecules, such as costimulatory molecules CD80. CpG ODN
can also activate pDC and promote its maturation, which can
determined by the upregulation of MHC II molecules
(Iab) on the membrane of pDC. CD11c can be detected as the
characteristic surface marker of pDC. In the present study, we
determined the effect of CpG ODN on different immune cells
by flow cytometry analysis on the basis of those marker
molecules (Figure 4). The results showed that all CpG ODN,
with the exception of ODN3, upregulated the expression level
of CD80 on CD19-positive cells, in which ODN1826, ODN4,
ODN7, ODN10, and ODN11 were especially efficient. As to
the expression of Iab on CD11c-positive cells and CD94
expression on NK cells, the status was similar; the most
potent CpG ODN were still ODN4, ODN7, ODN10, and ODN11.
SAR analysis The results showed that the optimal
stimulatory hexameric CpG motif for murine was
5'-TCGACGTT-3', which was consistent with previously published
studies[15]. Changing the hexameric motif from GACGTT to GTCGTT
weakened the immunostimulatory activity of ODN. The CpG
island without the flank of dinucleotide seemed to have no
immunostimulatory efficiency, as was the case with ODN15.
The number of CpG motifs was not a vital factor for
immuno-stimulatory activity. Adding a hexameric CpG motif (ODN9)
contributed no significance to immunostimulatory activity.
In ODN11, the number of hexameric CpG motifs decreased to
1, but the activity of this ODN improved. The improvement
of the activity might be derived from the 3'-end palindrome
tail.
Owning to lack of a TpC
dinucleotide[21] at the 5'-end, the immune-stimulatory effects of ODN6 and ODN8 were
weakened. The optimal distance between 2 hexameric CpG
motifs seemed to be 3 nucleotides. Modification of the
distance to a shorter (ODN5, the 2 motifs were connective) or a
longer (ODN6, the 2 motifs spaced by an octanucleotide)
one both weakened the comprehensive immunostimulatory
activity.
The palindrome was essential to the immunostimulatory
potency of a CpG ODN. The results showed that all the
ODN with the best general immunostimulatory activity were
that with self-complementary palindrome sequences, such
as ODN4, ODN7, ODN10, and ODN11. However, the
palindrome structures had to be at the 3'-end of the sequence.
Once the palindrome moved to the 5'-end, like ODN3, ODN12,
and ODN15, immunostimulatory activity was almost totally
lost.
Discussion
In the present study, we tried to explore a new role of the
SAR of CpG ODN and find the optimal structure for mouse
spleen cells. The positive control and the template ODN1826
was the optimal B-class CpG ODN reported in previously
published studies. It consists of 2 stimulatory hexameric
CpG motifs (5'-TCGACGTT-3') linked by a CCT spacer and a
TpC dinucleotide at the 5'-end. ODN1826 was a strong
stimulator for IL-6 and IL-12 production and a relatively weak
inducer for IFN-α secretion. ODN1826 efficiently promoted
B cell proliferation and partially promoted the maturation of
pDC, but it lacked NK cell activation.
Various structural modifications were preformed on the
basis of the template ODN1826. Some modifications
introduced remarkable activity promotion to the CpG ODN, for
instance, adding the palindrome to the sequence. However,
some structural changes failed to improve the
immunostimu-latory potency, such as modifications of the CpG number
and the distance between functional CpG motifs or simply
changing the motif sites. Previous
studies[17,19,21], in which conclusions was drawn that a TpC dinucleotide at the 5'-end
was important for the potency of a CpG ODN, was partly
supported in our study. ODN6 lacked the 5'-TpC
dinucleo-tide, which had weaker stimulatory potency than ODN1826,
but the lack of potency could be compensated by an
addition of a 3'-end CpG-rich palindrome tail, this is why ODN8
possess comparable immunostimulatory activity
vs 1826.
All ODN with a palindrome sequence could be classified
as C-class CpG ODN[15]. Our results indicated that C-class
ODN possessed combined immune effects of A- and B-class
CpG ODN[7,13,20]. C-class ODN strongly stimulated B cell
proliferation, NK cell activation, and IFN-α production.
However, not all C-class CpG ODN had immunostimu-latory
potency. It was very important to arrange the site of the
palindromes; self-complementary palindrome at the 3'-end
normally accompanied with satisfactory immunostimu-latory
potency. In contrast, a 5'-end, self-complementary
palindrome is usually useless, like ODN3 and ODN12. A previous
study[22] showed that the free 5'-end of the CpG ODN
sequence was important for immunostimulatory potency. The
5'-end palindrome destroyed the free end; this may have
been responsible for the lost of the immunostimulatory
activity of ODN3 and ODN12.
However, ODN10, one of the most potent ODN involved
in our study, possessed a holistic, self-complementary
palindrome structure
(5'-TCCATGACGTTTTAAAACGTCA-TGGA-3'). In theory, the sequence would form a large
hairpin structure, so it was hard to keep a single chain 5'-end.
Despite this, the comprehensive immunostimulatory
activity of ODN10 was distinguished. Furthermore, in another
study, a cyclic CpG ODN without any free ends had potent
immunostimulatory efficiency (data not shown). This result
challenged the hypothesis that the free 5'-end was essential
for retaining the immunostimulatory activity for a CpG ODN.
Undoubtedly, more investigations still needed to be carried
out in order to elucidate this.
The analysis of cytokine secretion is a traditional method
for the SAR of CpG ODN. Generally, a certain cytokine can
be secreted by several immune cells, for example
IFN-α
secretion by pDC or cells of the mononuclear phagocyte
system, so we could not identify which cells were influenced
by CpG ODN. Thus, the more efficient and accurate method
of flow cytometry was used as a powerful tool in the study
for the determination of the cell-specific activation of ODN.
As we known, CD19 was the characteristic surface marker of
B cells, and the activation of B cell was characterized by the
upregulation of cell-surface molecules, such as costimulatory
molecules CD80. CD11c was the characteristic surface marker
of pDC, and one of the mature markers of pDC was the
upregulation of Iab expression on pDC. The results showed
that both B-class CpG ODN and C-class CpG ODN could
upregulate the level of CD80 expression on CD19-positive
cells. C-class CpG ODN induced a much higher level of
Iab expression on CD11c-positive cells than B-class CpG ODN.
The CD94 expression on NK cells was similar to the
Iab expression on pDC. The results obtained via flow cytometry
and cytokine analysis were consistent.
All of the cellular immune effects of CpG ODN in
murine were believed to result directly and indirectly from
activating TLR-9-expressing pDC and B
cells[23_28]. Never-theless, B cell and pDC activation seemed to need different
structure characteristics[28,29]. An explanation for this is that
pDC activation was associated with ODN that can form
secondary structures, such as the dimeric C-class and the
multimeric A-class. These higher-ordered structures might
induce TLR-9 cross-linking, promote the recruitment of one
or more additional cofactors or adaptor proteins into the
TLR-9-signalling complex, and/or alter the intracellular
compartmentalization of the ODN[30,31]. Thus, the relationship
between the primary and higher-level structure of CpG ODN
and its accessibility, recognition, and combination with
TLR-9 still need further study.
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