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,
,
*
Department of Veterans Affairs Medical Center, Iowa City, IA 52246;
Department of Internal Medicine and the University of Iowa Holden Cancer Center, University of Iowa College of Medicine, Iowa City, IA 52242;
Coley Pharmaceutical Group, Wellesley, MA 02481; and
Loeb Health Research Institute, Ottawa Hospital and University of Ottawa, Ottawa, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has recently become apparent that bacterial DNA has potent
immunostimulatory properties. Vertebrate DNA has low levels of CpG
dinucleotides and most of the cytokine residues are methylated. In
contrast, the genomes of bacteria, viruses, and retroviruses are not
methylated and may or may not show CpG suppression (3, 4).
It is now well established that humans and other vertebrates may detect
unmethylated CpG dinucleotides in particular base contexts ("CpG
motifs") as a sign of danger or infection. Such CpG DNA, or synthetic
oligodeoxynucleotides
(ODN)3 containing
these CpG motifs, activate dendritic cells, monocytes, and macrophages
to secrete Th1-like cytokines and to induce Th1 T cell responses,
including the generation of cytotoxic T lymphocytes
(5, 6, 7, 8, 9, 10, 11). In conjunction with adherent cell-derived
cytokines, CpG DNA strongly stimulates NK cells to secrete IFN-
and
to have increased lytic activity (12, 13, 14). CpG DNA also
directly activates B cells to proliferate, secrete IL-6, and Ig and to
have increased cell surface expression of costimulatory molecules
(3, 9, 15). In contrast to LPS and other immune
stimulatory agents that have been evaluated, CpG DNA shows an extremely
strong pattern of Th1-like cytokine induction and can even be used for
immunotherapy of Th2-mediated allergic disease in experimental models
(16, 17, 18). Teleologically, it seems likely that immune
recognition of CpG DNA has evolved as a defense against intracellular
pathogens, thus providing a rationale for the observed skewing of its
effects toward Th1 activation (19).
Since Th1-like immune activation and NK cells are thought to be desirable for cancer immunotherapy (20, 21), we tested a variety of CpG ODN to identify which type of CpG motif is optimal for activating these responses. There was little correlation between the ability of a CpG motif to induce cytokine secretion and its ability to induce NK activity. Moreover, although several CpG ODN were active as sole immunotherapeutic agents in two tumor models, different motifs were optimal in each model. CpG ODN 1585 was optimal against B16 melanoma and its effects were dependent on NK cells. CpG ODN 1826 was optimal in a lymphoma model and its effects appeared to require NK (early) and T cells (late). These results illustrate the potent therapeutic potential of various CpG motifs and suggest that distinct CpG motifs can be custom-tailored for each desired immune effect.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Virus-free 4- to 6-wk-old C57BL/6 (B6), SCID, and beige mice, as
well as IL-2, IL-4, IFN-, IL-12, TAP, perforin, and
2-microglobulin knockouts (all on a C57BL/6
background) were purchased from The Jackson Laboratory (Bar Harbor,
ME); type I IFNR (IFN-IR) knockout mice (on a 129J background) were
generously supplied by Dr. T. Waldschmidt (Department of Pathology,
University of Iowa, Iowa City, IA). All mice were housed in the
specific pathogen-free facility at the University of Iowa Animal
Care Unit.
Oligodeoxynucleotides
Phosphorothioate-modified ODN and chimeric ODN were provided by
Coley Pharmaceutical Group (Wellesley, MA). Chimeric ODN have
phosphorothioate linkages between the first two bases on the 5' end and
the last five bases on the 3' end with the remaining, central, bases
connected by phosphodiester linkages; we have previously shown this
arrangement to be optimal (3, 22). The sequences used are
provided in Table I
. ODN were found to have undetectable endotoxin
levels using the limulus amebocyte lysis assay (BioWhittaker,
Walkersville, MD; lower detection limit, 0.1 endotoxin
units/ml). For in vitro assays, ODN were diluted in TE buffer
(10 mM Tris (pH 7.0) and 1 mM EDTA) and stored at -20°C. For in vivo
use, ODN were further diluted in PBS (0.1 M PBS, pH 7.3) and stored at
4°C. All dilutions were conducted using pyrogen-free reagents.
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Spleens were removed from 6- to 12-wk-old female C57BL/6 mice
and cultured at 5 x 106/ml with the
indicated ODN for 4 h (TNF-
) or 24 h (IL-6, IFN-,
IL-12), the supernatants were harvested, and cytokines were detected by
ELISA as previously described (14, 23). To
evaluate CpG-induced B cell proliferation, spleen cells were depleted
of T cells with anti-Thy-1.2 and complement treatment; viable cells
were obtained by centrifugation over Lympholyte M (CEDARLANE
Laboratories, Hornby, Ontario, Canada) and cultured with the indicated
ODN. At 44 h, the cultures were pulsed for 4 h with 1 µCi
of [3H]thymidine as described previously
(3).
Tumor models
The B16.F1 melanoma and EL4 T cell lymphoma cell lines, which are syngeneic to B6 mice, were maintained in vitro (24, 25). Tumor cells were harvested and injected, i.p. (B16.F1) or s.c. (EL4), into B6 mice at the doses indicated in the figure legends. Various ODN were administered at the indicated time points i.p. at doses of 100 µg/mouse, unless otherwise indicated. Mice were checked daily for tumor growth and for survival. There were 5–10 mice in each group, unless otherwise indicated. NK cell depletion was done as previously described (26).
Statistical analysis
Survival curves were estimated using the Kaplan-Meier method (27). Hypotheses were tested using the log rank test (28). All statistical tests were performed using the SAS system (SAS System for Windows, version 8.0; SAS Institute, Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that ODN containing CpG motifs
trigger the activation of B cells, NK cells, and dendritic cells, favor
a Th1-like cytokine production, and function as very effective vaccine
adjuvants (3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 29, 30, 31). To date, these effects have
been thought to be a general property of CpG ODN, and there has been no
evidence for the existence of CpG motifs with fundamentally different
types of immune effects (32, 33, 34). To determine whether
there may be different subsets of CpG motifs with distinct profiles of
immune activity, we tested >300 ODN, with different types of CpG
motifs, for their ability to induce cytokine secretion (IL-6, IL-12,
IFN-, and TNF-), for their ability to stimulate B cell
proliferation, and for their ability to induce NK cell killing
activity. We found that many CpG ODN with a completely
phosphorothioate-modified backbone (S-ODN) were potent inducers of
cytokine secretion and B cell proliferation with significant effects at
nanomolar concentrations (representative ODN are depicted in Table I
). Chimeric CpG ODN (in which the flanks
were phosphorothioate-modified but the center was native
phosphodiester; SOS-ODN) were also effective at inducing cytokine
secretion and B cell proliferation albeit at higher concentrations. For
these ODN, there was little apparent correlation between the ability of
an ODN to induce cytokine secretion or B cell proliferation and its
ability to augment NK cell activity (Table I). Of the ODN initially
tested, the optimal CpG ODN for activating NK cells had a chimeric
backbone with a single copy of the palindromic motif TCAACGTTGA in the
middle phosphodiester portion of the ODN and poly(G) sequences on both
the 5' and 3' phosphorothioate ends (Table I).
CpG ODN increase survival of B6 mice with B16 melanoma
We have previously shown that CpG ODN, while not effective on
their own, synergized with mAb in inducing the regression of tumor in a
B cell lymphoma model (35). We wondered whether a CpG ODN
could be effective on its own in a murine melanoma tumor model. The B16
melanoma was chosen because it is known to be susceptible to
MHC-nonrestricted cytotoxicity (36, 37, 38, 39, 40, 41). The B16 cell line
grown in our laboratory is NK resistant but is susceptible to lysis by
lymphokine-activated killer cells (data not shown). Because of the B16
susceptibility to activated NK cells, we first tested the effect of the
NK-optimized CpG ODN 1585 in C57BL/6 (B6) mice, which are syngeneic to
the B16 melanoma. In our hands, 100% of the mice die from progressive
tumor when challenged with as few as 1000 tumor cells. B6 mice received
various amounts of the B16 tumor i.p. on day 0. The CpG or control ODN
was administered on days 0, 3, and 7 and weekly thereafter. As shown in
Fig. 1, we found that the CpG ODN 1585
significantly prolonged (with a p < 0.001 for all of
Fig. 1 panels comparing 1585 vs PBS or non-CpG ODN) survival of mice
that received the very high challenge dose of 1.5 x
106 tumor cells (Fig. 1A). When
smaller numbers of tumor cells were used, the mice survival improved
progressively with a resultant 80% long-term survival of the mice that
received 1 x 104 tumor cells and the CpG
ODN 1585 (Fig. 1D). It is worth noting that the duration of
survival of mice receiving PBS only was almost identical regardless of
the tumor dose tested in Fig. 1, demonstrating the high tumorigenic
potential of the B16 tumor. Using a 5 x 103
tumor challenge dose and pooling identical experiments, we found that,
using a pool of 25 mice with PBS challenge, the mean survival time was
25.16 ± 0.84 (mean ± SEM) days, the mean survival of the
control CpG group was 35.53 ± 2.12, and all 25 mice in the 1585
group were long-term survivors (i.e., 0% mortality). We next attempted
to determine the optimal dose and frequency of CpG ODN. B6 mice were
given 5 x 103 tumor cells on day 0 and then
were given only one injection, on day 0 as well, of various doses of
CpG ODN 1585 or control ODN (2118). As shown in Fig. 2, 100 µg of 1585 was optimal and
resulted, in this experiment, in a 100% survival. Interestingly,
higher doses (300 µg) were less effective and resulted in a survival
rate similar to that of lower doses (30 µg).
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CpG ODN have been shown to stimulate a variety of immune cell
types. For example, some CpG ODN are very potent mitogens for B cells
and can induce the activation of APC (3, 7, 8, 42, 43). It
has also been suggested that CpG ODN can activate T lymphocytes
(10, 11). The ability of CpG ODN to activate APC could
potentially promote the induction of CTL against the tumor. To
determine whether T or B cells were required for this protection,
C57BL/6 SCID mice were treated with CpG ODN 1585 and challenged with
the B16 melanoma using the same regimen as in Fig. 1. Similar survival
rates were seen in CpG-treated SCID mice compared with wild-type mice
at each tumor dose (Fig. 3). These data
demonstrate that neither T nor B cells are necessary for the observed
survival induced by CpG ODN in this model. Interestingly, not all CpG
ODN were equally effective. When the CpG ODN 1826 (optimal B and
dendritic cell-inducing motif; Table I and data not shown) and the CpG
ODN 2006 (also optimal for B and dendritic cell activation) were
tested, using SCID (Fig. 4) or B6 (data
not shown) mice, we found CpG 1585 to be optimal whereas CpG 1826 was
intermediate and CpG 2006 was similar to the non-CpG control ODN. Also
shown in Fig. 4 is that, when 5 x 103 tumor
cells were used, CpG ODN 1585 induced a 100% survival of the SCID
mice, similar to the immunocompetent B6 mice. In data not shown,
TAP-1 and 2-microglobulin knockout mice
were protected by CpG 1585 to a similar degree as normal B6 mice, again
suggesting that CD8 and NKT lymphocytes are not needed for the
protective effect.
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The data shown above demonstrated that B and T cells are not
necessary for the observed therapeutic effect. It remained possible,
however, that T cells might play a role in tumor protection in
immunocompetent mice. We reasoned that if this were the case, we should
be able to demonstrate immunological memory; i.e., mice that survived
the tumor challenge should be able to reject a second tumor challenge
in an accelerated fashion. Therefore, mice that had been treated with
CpG ODN 1585 and failed to establish tumor (tumor free for >100 days
after the initial tumor challenge) were rechallenged later with 1
x 104 tumor cells each. The tumor challenge dose
was given 2–3 mo after the last dose of CpG ODN to allow for the
protective CpG ODN effect to "wear off." Age-matched control B6
mice were also given the same dose. As shown in Fig. 5, mice previously "cured" by CpG ODN
had a slightly prolonged survival (with a mean survival time of 38.9
days compared with 26.0 days for the control mice) but there were no
long-term survivors and no "cures." Thus, CpG ODN-mediated
resistance to the B16 melanoma does not lead to the generation of
significant Ag-specific memory responses. Indeed, in data not shown,
adoptive transfer of splenocytes from cured mice failed to protect
naive mice against B16 tumor challenge.
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The data presented so far strongly suggested that T cells and B
cells are not necessary for the observed therapeutic effect, the
implication being that NK cells are the cells of import. However, SCID
mice, in addition to having normal NK cells, have normal APC including
dendritic cells. We, therefore, examined whether NK cells were
necessary for the CpG ODN effect. SCID mice were given 100 µg of
anti-NK1.1 Ab, or control Ig, on days -4 and -1. On day 0, the
mice were give 2 x 104 B16 cells i.p.; some
mice also received CpG ODN 1585 as above. Anti-NK1.1 Ab was given i.p.
twice weekly until the end of the experiment (we have previously shown
that this regimen results in near total depletion of NK cells
(26)). As seen in Fig. 6, the control mice whose NK cells were depleted died more rapidly than
the control mice that received the control Ig. Similarly, the mice that
received CpG ODN 1585 and anti-NK1.1 had 100% mortality in a time
frame identical to the control mice. The mice that received CpG ODN
1585 and control Ig had a 100% survival. These data demonstrate that
it is indeed the NK cells that are responsible for the observed
therapeutic effect in this model. To confirm these results further,
normal B6 mice were depleted of NK cells and the effect of CpG ODN was
examined. In data not shown, we found that NK cell depletion in the
wild-type mice also resulted in abolishing the CpG protective
effect.
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It is clear from the above data that the effects of CpG in the B16
melanoma model are dependent on NK cells. It remained to be seen,
however, as to whether CpG acted by inducing cytokine secretion by NK
cells and/or dendritic cells or whether the protective effect was
actually mediated by the lytic capacity of NK cells. We first examined
the ability of CpG to induce NK cell killing activity in vivo in
various knockout mice. CpG was injected in the footpad and the draining
lymph nodes were obtained 2 days later and used as effectors against
YAC-1 tumor target cells (we have previously shown (13)
that this approach was optimal for the in vivo induction of NK cell
killing activity by CpG). As shown in Fig. 7, CpG was able to induce NK cell
activity in normal B6 mice as well as in IL-2, IL-4, IFN-, IL-12,
and 2-microglobulin knockout mice.
Interestingly, mice whose receptor for type I IFN has been inactivated
(IFNI-R) did not augment their NK cell activity. These data suggested
that the induction of type I IFNs may be the pivotal step in the
protection conferred by CpG. It has been known for some time that type
I IFN can augment the lytic potential of NK cells without necessarily
inducing their proliferation (44). We sought to determine
whether NK lytic activity is necessary for rejection of the B16
melanoma. We tested beige mice whose fresh NK killing activity is
barely detectable but this activity can be readily augmented with the
proper stimulation (45, 46). As shown in Fig. 8A, CpG induced significant
protection in beige mice with 80% long-term survival
(p < 0.0001). Perforin knockout mice (whose NK
cells cannot kill except through the relatively less efficient Fas
pathway; 47), on the other hand, had only 20% long-term
survivors. These findings strongly suggest that it is the ability of
CpG ODN to augment the killing activity of NK cells, perhaps by the
induction of type I IFN, that is responsible for the rejection of B16
melanoma in this model.
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The toughest challenge for any type of tumor therapy is to cure
established tumors, rather than simply preventing the generation of a
new tumor. To test whether CpG ODN treatment could accomplish this,
mice were challenged with 1 x 104 B16
melanoma cells on day 0; 100 µg of CpG ODN 1585 or control ODN 2118
was given starting 3 days afterward. The ODN were also given on days 7,
10, and 14 and weekly thereafter for 2 mo. As shown in Fig. 9, treatment with the CpG ODN 1585 led to
a 60% survival rate, while the mice given a control ODN or PBS all
died between days 20 and 41 (1585 vs PBS had a p <
0.01, 2118 vs PBS had a p of 0.8 and 2118 vs 1585 had a
p of 0.006). The surviving mice lived with no evidence of
recurrent tumor for >100 days, when the experiment was terminated.
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To assess whether the above results can be generalized to other
tumors, we examined the effect of CpG ODN in a lymphoma model using the
EL4 tumor cells, which are also syngeneic to B6 mice. Similar to B16,
EL4 cells are resistant to killing by fresh NK cells but are
susceptible to killing by activated NK cells. B6 mice were inoculated
s.c. with 107 EL4 cells on day 0 and treatment
with 100 µg of several different CpG ODN given i.p. weekly beginning
on day 2 (i.e., days 2, 9, 16, etc.). All mice developed tumor by day
12. However, tumor growth was slower in those mice treated with CpG ODN
1826 (data not shown) and overall survival was significantly prolonged
(Fig. 10A). In repeat
studies, between 30 and 50% of mice treated with CpG ODN in this
manner rejected the tumor and remained tumor free thereafter (Fig. 10B). A number of interesting differences were found when
comparing results from the B16 model to those from the EL4 model. The
CpG ODN 1585, which was more effective than the CpG ODN 1826 in the B16
model, was not effective in the EL4 model. Unlike the B16 tumor, it
appeared that the EL4-challenge surviving mice were able to develop
memory since they failed to develop tumor upon rechallenge (arrow in
Fig. 10B), suggesting a memory response.
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To assess the relative contribution of NK and T cells for the
rejection of EL4 induced by the CpG ODN 1826, SCID mice were examined.
In contrast to the B16 model, no antitumor activity against EL4 was
seen (Fig. 11), suggesting that T cells
are necessary and that NK cells, if needed, are not sufficient for the
therapeutic effect. Surprisingly, however, NK cell depletion, using
anti-NK1.1 as above, in normal B6 mice abrogated the protective
effect of CpG (Fig. 11). Thus, in contrast to the B16 model, both NK
and T cells are required for the antitumor effect in the EL4 model.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies performed using phosphodiester or phosphorothioate
backbone ODN suggested that a single type of optimal CpG motif mediates
these protean effects (3, 32, 33, 34). Our present results
show that the effects of CpG DNA are actually more complicated. There
are at least two classes of CpG ODN, with distinct profiles of immune
activity depending on the ODN backbone and on the presence of another
kind of DNA sequence motif, the G quartet or poly(G) sequence. The
nuclease-resistant phosphorothioate backbone greatly improves the
stability of an optimal CpG ODN and its ability to activate B cells and
dendritic cells, and its ability to induce cytokine production
(3, 8, 22, 62) (Table I). On the other hand, the
phosphorothioate backbone reduces the ability to activate NK cells and
thus may be less useful for tumor immunotherapy applications that
depend on these effector cells (Table I) (13). Since the
beneficial stabilizing effects of the phosphorothioate backbone can be
obtained by modification of just the 5' and 3' ends of the ODN
(22), we have tested this type of chimeric ODN backbone.
Poly(G) sequences consist of four or more consecutive guanines in a
row, or two or more regions of three consecutive guanines, and are
known to bind to the macrophage scavenger receptor and to improve ODN
uptake and IFN- production (63), but to interfere with
cell proliferation (64, 65).
The experiments reported in this manuscript indicate that the
combination of a CpG motif with a chimeric backbone along with poly(G)
sequences on the 5' and 3' flanks yields an ODN which has relatively
little B cell-, TNF--, or IL-12-stimulating effects, but which is
extremely active at inducing NK cell lytic activity (Table I). NK cells
have long been thought to be important in tumor surveillance. By virtue
of their ability to secrete IFN-, among other cytokines, NK cells
are also thought to influence adaptive immunity (66, 67).
Melanoma is a tumor model that has lent itself to the examination of
various models of immunotherapy. For human melanoma, several
tumor-specific Ags have been described and are known to induce T cell
responses (68, 69). On the other hand, the B16 murine
melanoma is thought to be amenable to treatment both by activated T or
NK cells (37, 39).
Since we have shown that CpG ODN activate NK cells both in vivo and in
vitro (13), we examined their effect on the survival of
mice with melanoma. The data presented in this report clearly
demonstrate that, by virtue of their ability to activate NK cells, some
CpG ODN are quite effective, as the sole therapeutic agent, at
preventing the development of B16 melanoma and, more interestingly, at
rejecting an established B16 tumor. As little as one dose of CpG ODN
1585 is as protective against tumor challenge as repeated doses (Fig. 2). Moreover, there appeared to be nonlinear dose response since a
300-µg dose of CpG was less effective than doses of 100 or 30 µg.
This is similar to our previous findings with CpG in a
Listeria model and probably relates to the observation that
high doses of CpG prime mice for the sepsis syndrome
(70, 71, 72).
Interestingly, it appears that the protection afforded by CpG ODN against the B16 melanoma is mediated by innate rather than adaptive immunity since SCID mice rejected the tumor just as efficiently as normal mice. Moreover, no "memory" appears to have developed in the cured immunocompetent mice. In data not shown, we were not able to transfer protection by adoptive immune transfer using spleen cells from the cured immunocompetent mice. All of these results point to innate immunity as being the system of import in this model.
The two major cellular components of innate immunity are NK cells and
dendritic cells. In theory, dendritic cells could be the cells of
import in this model. However, our data with the in vivo depletion of
NK cells, clearly demonstrate that NK cells are the essential cells.
These results do not exclude the possibility that DC may help to
activate NK cells as shown in other antitumor models where NK cells can
slow tumor growth, but do not cause tumor regression (73).
Interestingly, it has been suggested that it is the NKT, rather than
the classical NK cells, that are the cells of import in rejecting the
B16 melanoma in an IL-12-induced tumor rejection model (37, 41). Our data suggest that NKT (equivalent data obtained in
TAP-1 and 2-microglobulin knockout mice; data
not shown) cells are not necessary for the therapeutic effect of CpG
ODN in this model.
It appears that IL-12 is also not necessary for the therapeutic effect
in this model. Using SCID mice, we found IL-12 (which is dependent on
NKT cells for its therapeutic effect (37, 41)) to be less
efficient than CpG ODN at rejecting the B16 tumor and we had no
long-term survivors (data not shown). Moreover, the ability of a CpG
ODN to induce IL-12 did not correlate with its ability to stimulate NK
cell activity or to improve long-term survival. These data suggest that
the antitumor effect of CpG ODN in this model is not mediated through
the IL-12 pathway and that the IL-12 pathway is not essential for the
CpG ODN beneficial effect. Indeed, IL-12 knockout mice gave a response
similar to the normal B6 mice (data not shown), suggesting that IL-12
is not necessary for the protective effect. Surprisingly, the induction
of IFN- secretion also did not correlate with CpG-induced
protection, suggesting that the therapeutic effects in this model may
be mediated through distinct NK activation pathways. Our studies with
the various knockout mice indicate that type I IFN may be the cytokine
of import since IFNI-R knockout mice were the only cytokine/cytokine
receptor knockout mice that failed to augment their NK cell activity in
response to CpG either in vivo (Fig. 7) or in vitro (data not shown).
Moreover, it appears that it is the lytic potential of NK cells (rather
than their ability to secrete various cytokines) which is protective in
this model, since perforin knockout mice yielded significantly less
long-term survivors (20 vs 80%). NK cells can kill through the
perforin pathway or through the Fas/Fas ligand pathway with the
perforin pathway being dominant in cytokine-activated NK cells
(47). It is possible that the 20% survival seen in the
perforin knockout mice is due to killing through the Fas pathway,
although we were unable to demonstrate significant staining of B16 with
anti-CD95 Ab nor were we able to demonstrate augmentation of CD95
on B16 melanoma cultured with CpG in vitro (data not shown). Moreover,
we examined the effect of CpG on the expression of other surface
markers on B16 cells after culture with CpG ODN for 1–3 days; we found
no change in the expression of class I or class II MHC, CD40, CD40
ligand, CD28, CD80, or CD86 (data not shown).
The results in the B16 model are in marked contrast to results in the
EL4 T cell lymphoma model. Whereas CpG ODN 1585 was optimal in the B16
melanoma model, CpG ODN 1826 was more effective in the EL4 model.
Although CpG 1826 is optimal for B cell induction, it does induce some
NK cell activity (Table I) and, perhaps by virtue of cytokine
induction, it appears to induce some T cell activity (data not shown).
Therefore, it was not too surprising to find that 1826 requires NK
cells for its anti-EL4 activity although NK cells were not, by
themselves, sufficient for the therapeutic effect. Although we have not
directly demonstrated that T cells are needed for this effect, one can
infer that T cells play a major role in the EL4 model since no
therapeutic effect was seen in SCID mice (which have NK and dendritic
cells but lack T or B cells). Furthermore, we found that cured mice
reject a subsequent tumor challenge, suggesting a memory response. The
effectors of this memory response remain to be established. One’s
initial suspicion would be a T cell response. However, it is possible
that Abs might play a role in this response as well. Since the CpG
motifs that work best in EL4 are also those that are optimal for B cell
stimulation, it is possible that these ODN induce a significant
anti-EL4 Ab. Depending on the Ab induced, one can envision at least
two mechanisms. Complement-fixing Abs could kill the tumor via that
pathway; noncomplement-fixing Abs could kill EL4 by Ab-dependent
cellular cytotoxicity. Ongoing studies are exploring these
possibilities more extensively. It is likely that the mechanism through
which NK cells contribute to the antitumor response in the EL4 model is
different from that in the melanoma model, where it is clear that the
lytic activity of NK cells is pivotal. It is likely that the role of NK
cells in the EL4 model does not rely on their lytic potential but
relies on their ability to secrete certain cytokines (such as IFN-,
for example) which may facilitate the activation of T cells that lead
to rejection of EL4. These possibilities are currently under
investigation.
Interestingly, the results in these two models also differ from our previous findings in a murine B cell lymphoma model (35). In the B cell lymphoma model, CpG ODN alone were not effective at inhibiting tumor growth, although some CpG ODN were synergistic with antitumor mAb and gave an impressive survival rate of 80%. The 38C13 lymphoma used in that model is not susceptible to NK or activated NK cell lysis. Interestingly, Carpentier et al. (74) reported that a phosphorothioate CpG ODN was effective in slowing the growth of a murine neuroblastoma model and that this effect seemed to be dependent on NK cells.
For more than a century, it has been clear that bacterial extracts such as Coley’s toxins contain potent compounds with the potential to induce regression of established tumors in humans (1, 2). Although it remains to be established whether the CpG motifs in bacterial DNA are the "missing link" to Coley’s toxins, the data presented in this report clearly demonstrate that CpG ODN can be effective as antitumor agents. Importantly, our findings suggest that different immunotherapeutic strategies will be required for different tumors and that one may need to design different CpG ODN for inducing different types of desired responses. A more complete understanding of the antitumor immune responses needed for cancer therapy, and further dissection of the immunologic response to CpG ODN, should allow for the development of more rational and effective therapeutic strategies based on these promising new classes of potent immunologic agents.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Zuhair K. Ballas, Department of Internal Medicine, C42-E13, GH, University of Iowa, Iowa City, IA 52242. E-mail address: ballasz{at}uiowa.edu
3 Abbreviation used in this paper: ODN, oligodeoxynucelotide.
Received for publication June 4, 2001. Accepted for publication August 23, 2001.
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References |
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14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623.-galactosylceramide. J. Immunol. 163:2387.-dependent mechanisms. Proc. Natl. Acad. Sci. USA 96:6970. production by NK cells and increases sensitivity to endotoxin. J. Immunol. 156:4570.
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 U. Bhan, G. Trujillo, K. Lyn-Kew, M. W. Newstead, X. Zeng, C. M. Hogaboam, A. M. Krieg, and T. J. Standiford Toll-Like Receptor 9 Regulates the Lung Macrophage Phenotype and Host Immunity in Murine Pneumonia Caused by Legionella pneumophila Infect. Immun., July 1, 2008; 76(7): 2895 - 2904. [Abstract] [Full Text] [PDF]
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 R. B. Anderson, G. J. Cianciolo, M. N. Kennedy, and S. V. Pizzo {alpha}2-Macroglobulin binds CpG oligodeoxynucleotides and enhances their immunostimulatory properties by a receptor-dependent mechanism J. Leukoc. Biol., February 1, 2008; 83(2): 381 - 392. [Abstract] [Full Text] [PDF]
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 J. N. Kochenderfer and R. E. Gress A Comparison and Critical Analysis of Preclinical Anticancer Vaccination Strategies Experimental Biology and Medicine, October 1, 2007; 232(9): 1130 - 1141. [Abstract] [Full Text] [PDF]
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J. Li, W. Song, D. K. Czerwinski, B. Varghese, S. Uematsu, S. Akira, A. M. Krieg, and R. Levy Lymphoma Immunotherapy with CpG Oligodeoxynucleotides Requires TLR9 Either in the Host or in the Tumor Itself J. Immunol., August 15, 2007; 179(4): 2493 - 2500. [Abstract] [Full Text] [PDF]
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 J. N. Kochenderfer, J. L. Simpson, C. D. Chien, and R. E. Gress Vaccination regimens incorporating CpG-containing oligodeoxynucleotides and IL-2 generate antigen-specific antitumor immunity from T-cell populations undergoing homeostatic peripheral expansion after BMT Blood, July 1, 2007; 110(1): 450 - 460. [Abstract] [Full Text] [PDF]
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N. Durakovic, V. Radojcic, M. Skarica, K. B. Bezak, J. D. Powell, E. J. Fuchs, and L. Luznik Factors governing the activation of adoptively transferred donor T cells infused after allogeneic bone marrow transplantation in the mouse Blood, May 15, 2007; 109(10): 4564 - 4574. [Abstract] [Full Text] [PDF]
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C. Bourquin, L. Schmidt, V. Hornung, C. Wurzenberger, D. Anz, N. Sandholzer, S. Schreiber, A. Voelkl, G. Hartmann, and S. Endres Immunostimulatory RNA oligonucleotides trigger an antigen-specific cytotoxic T-cell and IgG2a response Blood, April 1, 2007; 109(7): 2953 - 2960. [Abstract] [Full Text] [PDF]
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R. R. Flores, K. A. Diggs, L. M. Tait, and P. A. Morel IFN-{gamma} Negatively Regulates CpG-Induced IL-10 in Bone Marrow-Derived Dendritic Cells J. Immunol., January 1, 2007; 178(1): 211 - 218. [Abstract] [Full Text] [PDF]
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J. N. Kochenderfer, C. D. Chien, J. L. Simpson, and R. E. Gress Synergism between CpG-Containing Oligodeoxynucleotides and IL-2 Causes Dramatic Enhancement of Vaccine-Elicited CD8+ T Cell Responses J. Immunol., December 15, 2006; 177(12): 8860 - 8873. [Abstract] [Full Text] [PDF]
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 W. Kozak, S. Wrotek, and A. Kozak Pyrogenicity of CpG-DNA in mice: role of interleukin-6, cyclooxygenases, and nuclear factor-{kappa}B Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2006; 290(4): R871 - R880. [Abstract] [Full Text] [PDF]
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L. Chaperot, A. Blum, O. Manches, G. Lui, J. Angel, J.-P. Molens, and J. Plumas Virus or TLR Agonists Induce TRAIL-Mediated Cytotoxic Activity of Plasmacytoid Dendritic Cells J. Immunol., January 1, 2006; 176(1): 248 - 255. [Abstract] [Full Text] [PDF]
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I. N. Buhtoiarov, H. D. Lum, G. Berke, P. M. Sondel, and A. L. Rakhmilevich Synergistic Activation of Macrophages via CD40 and TLR9 Results in T Cell Independent Antitumor Effects J. Immunol., January 1, 2006; 176(1): 309 - 318. [Abstract] [Full Text] [PDF]
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S. Gregoire, A. S. Bergot, C. Feraudet, C. Carnaud, P. Aucouturier, and M. B. Rosset The Murine B Cell Repertoire Is Severely Selected against Endogenous Cellular Prion Protein J. Immunol., November 15, 2005; 175(10): 6443 - 6449. [Abstract] [Full Text] [PDF]
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G. S. D. Reid, K. She, L. Terrett, M. R. Food, J. D. Trudeau, and K. R. Schultz CpG stimulation of precursor B-lineage acute lymphoblastic leukemia induces a distinct change in costimulatory molecule expression and shifts allogeneic T cells toward a Th1 response Blood, May 1, 2005; 105(9): 3641 - 3647. [Abstract] [Full Text] [PDF]
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 B. Poon, J. T. Safrit, H. McClure, C. Kitchen, J. F. Hsu, V. Gudeman, C. Petropoulos, T. Wrin, I. S. Y. Chen, and K. Grovit-Ferbas Induction of Humoral Immune Responses following Vaccination with Envelope-Containing, Formaldehyde-Treated, Thermally Inactivated Human Immunodeficiency Virus Type 1 J. Virol., April 15, 2005; 79(8): 4927 - 4935. [Abstract] [Full Text] [PDF]
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R. Brummel and P. Lenert Activation of Marginal Zone B Cells from Lupus Mice with Type A(D) CpG-Oligodeoxynucleotides J. Immunol., February 15, 2005; 174(4): 2429 - 2434. [Abstract] [Full Text] [PDF]
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 B. Jahrsdorfer, L. Muhlenhoff, S. E. Blackwell, M. Wagner, H. Poeck, E. Hartmann, R. Jox, T. Giese, B. Emmerich, S. Endres, et al. B-Cell Lymphomas Differ in their Responsiveness to CpG Oligodeoxynucleotides Clin. Cancer Res., February 15, 2005; 11(4): 1490 - 1499. [Abstract] [Full Text] [PDF]
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K. A. Mason, H. Ariga, R. Neal, D. Valdecanas, N. Hunter, A. M. Krieg, J. K. Whisnant, and L. Milas Targeting Toll-like Receptor 9 with CpG Oligodeoxynucleotides Enhances Tumor Response to Fractionated Radiotherapy Clin. Cancer Res., January 1, 2005; 11(1): 361 - 369. [Abstract] [Full Text] [PDF]
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 J. Christiansen and A. K. Rajasekaran Biological impediments to monoclonal antibody-based cancer immunotherapy Mol. Cancer Ther., November 1, 2004; 3(11): 1493 - 1501. [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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J. Vollmer, R. D. Weeratna, M. Jurk, H. L. Davis, C. Schetter, M. Wullner, T. Wader, M. Liu, A. Kritzler, and A. M. Krieg Impact of modifications of heterocyclic bases in CpG dinucleotides on their immune-modulatory activity J. Leukoc. Biol., September 1, 2004; 76(3): 585 - 593. [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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T. Switaj, A. Jalili, A. B. Jakubowska, N. Drela, M. Stoksik, D. Nowis, G. Basak, J. Golab, P. J. Wysocki, A. Mackiewicz, et al. CpG Immunostimulatory Oligodeoxynucleotide 1826 Enhances Antitumor Effect of Interleukin 12 Gene-Modified Tumor Vaccine in a Melanoma Model in Mice Clin. Cancer Res., June 15, 2004; 10(12): 4165 - 4175. [Abstract] [Full Text] [PDF]
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L. Benimetskaya, T. Wittenberger, C. A. Stein, H.-P. Hofmann, C. Weller, J. C. Lai, P. Miller, and V. Gekeler Changes in Gene Expression Induced by Phosphorothioate Oligodeoxynucleotides (Including G3139) in PC3 Prostate Carcinoma Cells Are Recapitulated at Least in Part by Treatment with Interferon-{beta} and -{gamma} Clin. Cancer Res., June 1, 2004; 10(11): 3678 - 3688. [Abstract] [Full Text] [PDF]
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M. B. Rosset, C. Ballerini, S. Gregoire, P. Metharom, C. Carnaud, and P. Aucouturier Breaking Immune Tolerance to the Prion Protein Using Prion Protein Peptides Plus Oligodeoxynucleotide-CpG in Mice J. Immunol., May 1, 2004; 172(9): 5168 - 5174. [Abstract] [Full Text] [PDF]
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 P. M. Campbell, V. Bovenzi, and M. Szyf Methylated DNA-binding protein 2 antisense inhibitors suppress tumourigenesis of human cancer cell lines in vitro and in vivo Carcinogenesis, April 1, 2004; 25(4): 499 - 507. [Abstract] [Full Text] [PDF]
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Y.-F. Chen, C.-W. Lin, Y.-P. Tsao, and S.-L. Chen Cytotoxic-T-Lymphocyte Human Papillomavirus Type 16 E5 Peptide with CpG-Oligodeoxynucleotide Can Eliminate Tumor Growth in C57BL/6 Mice J. Virol., February 1, 2004; 78(3): 1333 - 1343. [Abstract] [Full Text] [PDF]
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O. Duramad, K. L. Fearon, J. H. Chan, H. Kanzler, J. D. Marshall, R. L. Coffman, and F. J. Barrat IL-10 regulates plasmacytoid dendritic cell response to CpG-containing immunostimulatory sequences Blood, December 15, 2003; 102(13): 4487 - 4492. [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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P. Lundberg, P. Welander, X. Han, and E. Cantin Herpes Simplex Virus Type 1 DNA Is Immunostimulatory In Vitro and In Vivo J. Virol., October 15, 2003; 77(20): 11158 - 11169. [Abstract] [Full Text] [PDF]
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K. Lore, M. R. Betts, J. M. Brenchley, J. Kuruppu, S. Khojasteh, S. Perfetto, M. Roederer, R. A. Seder, and R. A. Koup Toll-Like Receptor Ligands Modulate Dendritic Cells to Augment Cytomegalovirus- and HIV-1-Specific T Cell Responses J. Immunol., October 15, 2003; 171(8): 4320 - 4328. [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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L. Beloeil, M. Tomkowiak, G. Angelov, T. Walzer, P. Dubois, and J. Marvel In Vivo Impact of CpG1826 Oligodeoxynucleotide on CD8 T Cell Primary Responses and Survival J. Immunol., September 15, 2003; 171(6): 2995 - 3002. [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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B. J. Weigel, D. A. Rodeberg, A. M. Krieg, and B. R. Blazar CpG Oligodeoxynucleotides Potentiate the Antitumor Effects of Chemotherapy or Tumor Resection in an Orthotopic Murine Model of Rhabdomyosarcoma Clin. Cancer Res., August 1, 2003; 9(8): 3105 - 3114. [Abstract] [Full Text] [PDF]
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J. Baines and E. Celis Immune-mediated Tumor Regression Induced by CpG-containing Oligodeoxynucleotides Clin. Cancer Res., July 1, 2003; 9(7): 2693 - 2700. [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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 C. A. Farman and D. J. Kornbrust Oligodeoxynucleotide Studies in Primates: Antisense and Immune Stimulatory Indications Toxicol Pathol, January 1, 2003; 31(1_suppl): 119 - 122. [Abstract] [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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 A. P. Vicari, C. Chiodoni, C. Vaure, S. Ait-Yahia, C. Dercamp, F. Matsos, O. Reynard, C. Taverne, P. Merle, M. P. Colombo, et al. Reversal of Tumor-induced Dendritic Cell Paralysis by CpG Immunostimulatory Oligonucleotide and Anti-Interleukin 10 Receptor Antibody J. Exp. Med., August 19, 2002; 196(4): 541 - 549. [Abstract] [Full Text] [PDF]
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E. G. Rhee, S. Mendez, J. A. Shah, C.-y. Wu, J. R. Kirman, T. N. Turon, D. F. Davey, H. Davis, D. M. Klinman, R. N. Coler, et al. Vaccination with Heat-killed Leishmania Antigen or Recombinant Leishmanial Protein and CpG Oligodeoxynucleotides Induces Long-Term Memory CD4+and CD8+T Cell Responses and Protection Against Leishmania major Infection J. Exp. Med., June 17, 2002; 195(12): 1565 - 1573. [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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 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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