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NEOPLASIA
From the University of Minnesota Cancer Center and
Department of Pediatrics, Division of Bone Marrow Transplant,
Minneapolis, MN; Department of Veteran Affairs Medical Center and the
Department of Internal Medicine, University of Iowa College of
Medicine, Iowa City; and the Coley Pharmaceutical Group, Wellesley, MA.
Immunostimulatory cytosine-phophate-guanosine (CpG)-containing
motifs in bacterial DNA are potent immune system activators. Depending
on the bases flanking the CpG motif and on the DNA backbone, CpG
oligodeoxynucleotides (ODNs) can induce relatively more B-cell activation or relatively more natural killer (NK)-cell activation. To
evaluate their antitumor activities, an NK-optimized ODN (1585) and 2 B-cell-optimized ODNs (1826 and 2006) were compared for their ability
to protect naive mice against a lethal acute myelogenous leukemia (AML)
challenge. CpG 2006, but not CpG 1585, administered 2 days before the
AML challenge, allowed mice to survive more than 100 times a lethal
tumor dose. Cell depletion studies showed that protection did not
require T or B cells but depended on NK cells and also on an
NK-independent mechanism. CpG 2006 protected against AML challenge in
both syngeneic and allogeneic bone marrow transplant (BMT) recipients
at both early and late time points after transplantation. Although CpG
1585 had no protective effect on its own, it showed a striking synergy
with CpG 2006 to induce prolonged survival to AML challenge in
allogeneic recipients of T-cell-depleted marrow grafts, exceeding the
survival benefit of donor lymphocyte infusion (DLI). When combined with
DLI, a synergistic effect was observed in recipients of CpG2006 or
2006 + 1585 with 88% of mice surviving long-term. These data are
the first to indicate that the systemic administration of CpG ODNs is a
potent means of inducing therapeutic anti-AML innate immune responses
in naive and BMT recipients.
(Blood. 2001;98:1217-1225) The innate immune system is geared toward providing
a rapid response to foreign pathogens by pattern recognition receptors that distinguish prokaryotic from eukaryotic DNA.1 These
receptors specifically bind to unmethylated
cytosine-phosphate-guanosine (CpG) dinucleotides, enabling bacteria and
other pathogens to stimulate the innate immune system.2-6
Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG
motifs mimic bacterial DNA and can activate immune
responses.2-5 CpG administration results in high T helper
1 (Th1) activity and lower toxicity than complete Freund
adjuvant.7-10 CpG ODNs directly stimulate B lymphocytes to
produce interleukin-6 (IL-6) and IL-103; natural killer
(NK) cells to produce interferon Acute myelogenous leukemia (AML) is the most common form of acute
leukemia in adults. Intensive chemotherapy regimens have led to
remission induction in 70% to 85% of patients. Autologous bone marrow
transplantation (BMT) can result in long-term, disease-free survival,
although such therapy can be associated with morbidity/mortality, and
postremission relapses are frequent.15-17 Although a
graft-versus-leukemia (GVL) effect for AML has been observed in
allogeneic bone marrow transplant (alloBMT) recipients and in
recipients given delayed lymphocyte infusions (DLIs) to treat relapse
after alloBMT,18,19 the graft-versus-host (GVH) effect
that often accompanies the GVL effect is a significant cause of
morbidity and mortality. For patients not undergoing BMT or for those
receiving autologous BMT, alloresponses as a means to achieving
remission are not an option. In these instances, the generation of
tumor-specific immune responses requires the tumor cell or an
antigen-presenting cell (APC) to present tumor antigens to autologous
immune-competent T cells. Tumors usually are poor stimulators of immune
responses. Because endogenous T-cell responses are not sufficiently
potent as to protect against AML recurrence and alloresponses are
associated with graft-versus-host disease (GVHD), alternative
approaches to preventing AML recurrence are needed. CpG ODNs are
attractive candidates for such purposes.
In autologous and especially alloBMT recipients, T cells generated
after BMT may not be fully immunocompetent because of defects in the
thymic microenvironment and other types of injury.20 In
contrast to the prolonged period of time that can be required for
T-cell immune reconstitution, NK-cell recovery after BMT is rapid.21-23 NK cells present early after BMT have been
shown to be potent effectors against myeloid leukemias.23
Because CpG ODNs can be potent stimulators of innate immune system
effector cells, we reasoned that CpG ODNs might provide a means of
achieving an anti-AML response in mice early after BMT prior to
reconstitution of a fully functional adaptive immune system. Our data
demonstrate that CpG ODNs are highly effective in inducing an anti-AML
immune response in naive mice as well as in BMT recipients of either autologous or T-cell-depleted (TCD) allogeneic bone marrow when CpG
ODNs are given as early as 2 weeks after BMT, a time period when T-cell
reconstitution is poor. The combined administration of CpG ODNs along
with mature donor lymphocytes (DLI) completely prevented AML-induced
lethality. These data have important clinical ramifications.
Mice
CpG ODNs
AML challenge C1498, derived as a spontaneous tumor line from a B6 mouse, was grown in RPMI 1640 (Gibco, Grand Island, NY) with 10% heat-inactivated fetal bovine serum (Hyclone, Ogden, UT) as described.24 C1498 is an MHC class I+II AML
line.27 C1498 cells were given intravenously at doses
ranging from 2 × 103 to 2 × 106/mouse.
Necropsies were performed at the time of death. AML tumor involvement
was found in all mice, unless otherwise noted.
Anti-AML responses in naive mice CpG ODNs were given beginning on day 2 or as late as day 6 until day 26 relative to the injection of C1498 cells (range, 2 × 105 to 2 × 106). In some model
systems, administration of CpG ODN must precede challenge with
infectious pathogens by 2 days for optimal effect,28 but,
in other model systems, treatment can be delayed for a week or
longer.29 The duration of the CpG ODN immune effects is 2 weeks or more after the last injection.28 To determine the
effectors responsible for the CpG-induced immune response, mice were
treated with irrelevant rat immunoglobulin G (IgG) or anti-NK 1.1 (clone PK136)- or anti-CD4 (clone GK1.5)- and/or anti-CD8 (clone
2.43)-depleting monoclonal antibodies (mAbs) given
intraperitoneally at a dose of 400 µg each from days 1 and weekly
for 4 weeks that routinely results in more than 95% depletion of the
targeted cell population for more than 1 week beyond discontinuing of
mAb administration.
Anti-AML responses in BMT recipients To generate syngeneic BMT recipients, B6 mice were irradiated with 8.0 Gy total body irradiation (TBI) on day1 and reconstituted with 5 × 106 B6 bone marrow cells on day 0. CpG ODNs
were administered beginning on day 63 and ending on day 90 (CpG 1585)
or day 92 (CpG 2006) after BMT. C1498 was given intravenously on day 80 after BMT. Cohorts received 2 subcutaneous vaccines of irradiated C1498
cells (107) given on days 66 and 73 or no immunizations.
To determine the effects of CpG ODNs given later after alloBMT, B6
recipients were irradiated with 8.0 Gy TBI on day To assess the effects of CpG ODNs on DLI-mediated GVHD and GVL, B6 recipients were given B10.BR TCD bone marrow as described above. On day 17 after BMT, C1498 cells (2 × 105) were given under the cover of CpG ODNs that were administered from days 15 to 42 after BMT. On day 21 after BMT, splenocytes (25 × 106) from B10.BR donors30 were infused. Flow cytometry Cells were incubated with anti-FcR mAb and then mAb directed toward CD4 or CD8, CD19, and Mac1. Cells were analyzed by 2- or 3-color flow cytometry by using fluorescein isothiocyanate-, phycoerythrin-, or biotin-conjugated (along with SA-peridinin chlorophyll protein) mAb (Pharmingen). Irrelevant mAb control values were subtracted from values obtained with relevant values, using a FACScalibur (Becton Dickinson, Mountain View, CA). Forward and side scatter settings were gated to exclude red cells and debris, and 104 cells were analyzed for each determination.Cytokine analysis B6 splenocytes (106/mL) were plated in RPMI 1640 with 10% fetal bovine sera. ODNs were added at 0.3 to 3.0 µg/mL. Supernatants were collected for enzyme-linked immunosorbent assay analysis (IL-6, IL-12, IFN , TNF : all sensitivities
< 50 pg/mL).
Statistical analyses Group comparisons of continuous data were made by Student t test. Survival data were analyzed by life table methods, using the Mantel-Peto-Cox summary of chi-square. Actuarial survival and relapse rates are shown. P values .05 were considered significant.
Delineation of ODN with distinct profiles of immune activation In previous studies we reported that ODNs containing particular arrangements of CpG motifs with a phosphodiester backbone flanked by polyG motifs with a nuclease-resistant phosphorothioate backbone had dramatically enhanced NK-stimulating effects compared with ODNs with all-thioate backbones.31 We have recently reported that the optimal CpG motif for activating murine B cells is GACGTT, whereas that for activating human B cells is GTCGTT.32,33 To investigate the immune-stimulatory effects, in vitro assays were performed using representative ODNs from these 3 classes: NK-optimized ODN 1585, mouse B-cell stimulating-optimized ODN 1826, and human-stimulating ODN 2006. Each ODN stimulated splenic production of proinflammatory cytokines (TNF, IFN, IL-6, and IL-12) (Table
1). Although IL-12 was induced to a
similar degree by all 3 ODNs at high ODN concentrations, IL-12
production at lower ODN concentrations showed the following hierarchy:
CpG 1826 > 2006 > 1585. On the basis of achievable in vivo
concentrations, relevant comparisons would be CpG 1585 at 0.3 µg/mL
versus CpG 2006 at 3.0 µg/mL. CpG 1585 was a more potent inducer of
IFN and TNF production than CpG 1826 or 2006. Consistent with
IFN and TNF expression, CpG 1585 is a more potent inducer of
NK-cell function than the other ODNs (Z. Ballas, personal communication, 2001). In contrast, CpG 1826 and 2006 were more potent inducers of IL-6 production as compared with CpG 1585. B-cell
proliferation roughly correlated with induction of IL-6 expression
(data not shown). These data all were reproduced fully in a second
experiment. We conclude that CpG 2006 is a more potent inducer of the
proinflammatory cytokines IL-12, IL-6, and IFN at biologically
achievable doses as compared with CpG 1585.
Administration of CpG ODNs in vivo is highly effective in stimulating immune responses to AML cells The immune response to a low burden of C1498 cells ( 3 × 104) is dependent on NK cells, whereas
resistance to a higher tumor burden ( 105) is T-cell
dependent.27 To determine whether CpG ODNs might stimulate
an immune response to a higher tumor burden, naive B6 mice were given
CpG ODNs selected as potent stimulators of NK-cell function (CpG 1585)
or as potent stimulators of B-cell and DC function (CpG 1826 or CpG
2006). In vitro, C1498 proliferation was not impaired by exposure to
high concentrations (100 µg/mL) of CpG 1585 or CpG 2006 ODNs (CpG
1826 was not tested) (not shown). B6 recipients challenged with C1498
cells (2 × 105/mouse) succumbed within 5.5 weeks to
tumor (Figure 1). Complete Freund
adjuvant did not significantly affect survival. Recipients given CpG
1585 had a significant (P < .008) extension in survival, although all succumbed to tumor by 7.5 weeks after the challenge. In
contrast, CpG 1826 or CpG 2006 given beginning on day 2 before C1498
challenge protected 70% of mice from a lethal AML dose
(Figure 1).
CpG 2006 rather than CpG 1826 was selected for further study because a
similar effect was observed with these 2 ODNs, and CpG 2006 is immune
stimulatory in humans and mice.32 Although all controls
died within 4 weeks of C1498 challenge (2 × 105 cells),
80% of the recipients treated with CpG 2006 beginning on day
To quantify the magnitude of the CpG 2006 effect, recipients were
challenged with various C1498 doses, ranging from
2 × 104 to 2 × 106 cells (Figure
3). A cohort was given CpG 2006 and
2 × 106 cells. In controls, a dose-response effect of
C1498 cells was evident, as measured by time to mortality. None of the
mice survived a challenge with 2 × 104 cells or
2 × 105 cells. In contrast, 20% of CpG 2006-treated
recipients of 2 × 106 C1498 cells survived long-term,
significantly (P = .002) higher than recipients of
100-fold fewer cells.
To determine which effector cells were required for the antitumor
effects mediated by CpG administration, CpG 2006-treated B6 recipients
of C1498 cells (6 × 105) were given irrelevant mAb or
mAbs to deplete T cells or NK cells (Figure
4A). In contrast to irrelevant
mAb-treated controls that died of tumor by 25 days after infusion, CpG
treatment significantly delayed tumor mortality by 50 days with all
succumbing to tumor by 75 days after infusion (P = .008).
CpG-treated mice receiving 6 × 105 C1498 cells had a
modest but not statistically significant longer mean survival time
(P = .10) as compared with controls receiving 30-fold
fewer C1498 cells, indicating a high level of antitumor activity
conferred by CpG administration. Depletion of NK cells significantly
impaired survival in CpG-treated recipients (P = .0054),
although CpG-treated, NK-depleted mice survived longer than controls
(P = .0006). In contrast, pan-TCD did not adversely affect
survival (P = .14). Additionally, CpG-treated recipients depleted of both NK and T cells had similar survival to CpG-treated recipients depleted of NK cells alone (not shown). These data were
reproduced in other experiments using a lower tumor cell dose
(2 × 105) (not shown). Together, these studies indicate
that the tumor protection conferred by CpG administration is mediated
primarily but not exclusively by NK cells.
Long-term CpG 2006-treated B6 survivors of C1498 challenge (110-148 days) were rechallenged with C1498 cells at the same dose as used for the initial challenge (2 × 105 cells). Despite the potent CpG 2006-induced anti-AML effects, pooled data in 2 experiments revealed that all 12 CpG 2006-treated recipients succumbed to AML lethality within 32 days after rechallenge, a time course similar to that observed with the initial challenge and with concurrent controls (not shown). Subsequent experiments will be designed to determine whether CpG given at AML rechallenge will be as effective as treatment at the time of initial AML challenge. Together, these data indicate a lack of memory-cell responses in CpG-treated recipients and are consistent with NK cells as the major effector cells in CpG 2006-mediated anti- AML responses. Studies were performed in CpG-treated B6-SCID mice to determine whether CpG ODNs could provide an anti-AML effect in T- and B-cell-deficient mice. B6-SCID mice were treated with CpG 2006 or CpG 1585 and challenged with C1498 cells (2 × 105). In B6-SCID mice, known to have heightened NK activity, CpG 2006 but not CpG 1585 significantly delayed the time to AML-induced lethality (Figure 4B). NK-cell depletion of B6-SCID mice reduced the time to AML-induced lethality as compared with non-NK-depleted controls (P = .006), indicating that endogenous NK function in B6-SCID mice provided some anti-AML immune responses (Figure 4B). NK-cell depletion completely abrogated the effect of CpG 1585 in B6-SCID mice. In contrast, although NK-cell depletion largely eliminated the anti-AML effect of CpG 2006, NK-cell depleted, CpG 2006-treated B6-SCID mice survived significantly longer than NK-cell-depleted controls (P = .0008), suggesting that cell types other than T, B, or NK cells stimulated by CpG 2006 contributed to the anti-AML response. The most likely candidate populations are DCs and monocytes, known to be activated by CpG 2006. CpG 2006 but not CpG 1585 induces anti-AML responses in syngeneic BMT recipients Immunotherapy may be most useful in the setting of minimal residual disease as might occur after BMT. However, after BMT, the adaptive immune system is slow to be restored. Initial studies were performed in syngeneic BMT recipients that were challenged with C1498 (105) cells on day 80 after BMT (Figure 5A). Cohorts of mice were treated with CpG 2006 or CpG 1585, beginning on day 64 after BMT or were immunized with irradiated C1498 cells (107 on days 66 and 73 after BMT) prior to AML challenge. Another control group received a lower C1498 dose (104), which is nonlethal to non-BMT recipients. Consistent with post-BMT immune dysfunction, recipients challenged with a typically nonlethal dose of 104 C1498 cells succumbed to leukemia within 2 months after the challenge. BMT recipients challenged with 105 cells died faster, succumbing by 1.5 months after challenge. In contrast, 50% of CpG 2006-treated recipients survived the C1498 cell (105) challenge long-term (> 4.5 months after challenge), indicating a more than 10-fold reduction in AML-induced lethality. Irradiated cellular vaccines were highly effective in inducing AML resistance with 90% of BMT recipients surviving long-term (P = .023 versus CpG 2006).
Surviving recipients were challenged with C1498 cells (105) on day 219 after BMT and were compared with a group of concurrently challenged BMT controls that had not received prior exposure to C1498 cells (Figure 5B). Long-term survivors initially treated with CpG 2006 had no evidence of a memory cell response, as these recipients died at an identical rate as concurrently challenged BMT controls. In contrast, 67% of recipients immunized with irradiated cellular vaccines survived more than 3.5 months after rechallenge. Thus, irradiated cellular vaccines but not CpG 2006 treatment is able to induce a demonstrable anti-AML memory cell response in syngeneic BMT recipients. To determine whether CpG 2006 could induce anti-AML responses at early
times after BMT, syngeneic BMT recipients were challenged on day 14 after BMT with C1498 cells at doses varying from 2 × 103
to 105 (Figure 6). Cohorts
received CpG 2006 beginning on day 12 after BMT. At that time, splenic
analyses were performed on representative mice (n = 5 per group) to
determine the status of immune reconstitution and whether CpG 2006 had
influenced these parameters by the day of C1498 challenge. Comparable
splenic cellularity and very low but similar absolute numbers of T
cells (range, 1.0-1.6 × 106 in individual mice) were
noted in these 2 groups, consistent with peripheral T-cell deficiency.
Recipients challenged with 105 C1498 cells succumbed to
leukemia within 1 month after challenge. Although CpG 2006 delayed the
time to mortality in recipients of 105 cells as compared
with nontreated controls (P = .002), all succumbed to
leukemia by 8 weeks after challenge. Fifty percent of recipients of
104 cells survived long-term and there was no mortality in
recipients of 2 × 103 cells. Eighty percent of CpG
2006-treated recipients of 104 cells survived long-term
(P = .13 versus controls). Because recipients of
104 cells had a superior survival to CpG-treated recipients
of 105 cells (P = .046) and because recipients
of 2 × 103 cells had a superior survival to CpG-treated
recipients of 104 cells (P = .040), these data
are consistent with a less than the 10-fold reduction observed when CpG
2006 was administered later after BMT. Regardless of the degree in
reduction in the tumorigenicity of C1498, it is clear that CpG 2006 can
augment the anti-AML response of syngeneic BMT recipients before the
adaptive immune system can be fully reestablished.
AlloBMT recipients of full MHC-disparate TCD bone marrow require longer
periods of time for functional immune reconstitution as compared with
syngeneic BMT recipients. To determine whether the beneficial effects
of CpG ODN early after syngeneic BMT would translate into an alloBMT
setting, irradiated B6 recipients were reconstituted with B10.BR TCD
bone marrow and then challenged with C1498 (2 × 105
cells) on day 36 after BMT (Figure 7).
Cohorts received no CpG ODN, CpG 1585, or CpG 2006 beginning day 20 after BMT. A cohort of nontreated controls received a lower C1498 dose
(2 × 104 cells). All recipients of
2 × 105 C1498 cells died within 4 weeks, and 90% of
recipients of 10-fold lower cells died within 5.5 weeks after AML
infusion. As compared with controls, CpG 1585-treated recipients of
2 × 105 C1498 cells had a slight increase in survival
(P = .045). However, all recipients succumbed within 6 weeks of challenge, and survival was shorter than controls given
2 × 104 cells (P = .049). As compared with
controls, CpG 2006-treated recipients challenged with
2 × 105 cells survived longer (P = .001)
with all mice succumbing approximately 5 weeks after tumor challenge.
The degree of tumor reduction was estimated to be about 10-fold because
survival of CpG 2006-treated recipients given 2 × 105
cells was similar to controls given 2 × 104 cells
(P = .34).
Studies have shown that the innate system returns earlier than the
adaptive immune system in recipients of allogeneic TCD bone marrow
grafts.20,23 A significant correlation between return of
IL-2-activated NK cells with antimyeloid leukemia cytolytic potential
by 3 weeks after BMT and the reduced risk of relapse has been
reported.23 Because CpG ODNs can activate the innate immune system, we modified the model to analyze the effects of CpG ODNs
on anti-AML immune responses early after alloBMT prior to
reconstitution of the adaptive immune system. Irradiated B6 recipients
were reconstituted with B10.BR TCD bone marrow. Recipients were
challenged with 2 × 105 C1498 cells on day 17 after BMT.
Cohorts were given no CpG ODNs, CpG 1585, CpG 2006, or both CpGs
(Figure 8A). Additional cohorts received
a single DLI dose (25 × 106 cells) on day 21, the
typical day of DLI administration in this model,28 alone
or in combination with CpG ODNs (Figure 8B). Control and CpG
1585-treated recipients of C1498 died at a similar rate with all
succumbing within 31 to 38 days after challenge (Figure 8A). In
contrast, CpG 2006-treated recipients survived significantly longer
than controls (P = .015), requiring 6 months after
challenge for uniform lethality. Three (33%) of 9 recipients that were
autopsied had no gross evidence of tumor. Combined administration of
CpG 1585 + 2006 ODNs resulted in 20% long-term survival (> 8
months after challenge), which was significantly
(P = .001) superior to controls but ultimately
statistically similar (P = .07) to CpG 2006 alone. Two
(22%) of 9 mice that were autopsied had no evidence of tumor.
As compared with nontreated controls, DLI administered 4 days after
C1498 challenge extended survival (P = .0006). However, all died within 6.5 weeks after AML cell injection. DLI was similarly effective as CpG 2006 administration at this period after BMT (P > .50) (Figures 8A versus 8B). Mean weight curves in
DLI-treated groups were modestly but not significantly lower
(P = .08) than the comparable non-DLI-treated groups
(weight curves are not shown). When DLI was combined with CpG 1585, survival was extended greater than with CpG 1585 (P = .001) or DLI (P = .01) alone (Figure
8B), and 4 (44%) of 9 mice died without evidence of tumor in contrast to the uniform detection of tumor in the other 2 groups. Mean weight
curves in the DLI and DLI + CpG 1585 groups were comparable (P = .96). When DLI was combined with CpG 2006, survival
was modestly but not significantly longer than with CpG 2006 alone
(P = .10) and was superior to DLI alone
(P = .004). Moreover, 5 (63%) of 8 mice that were
autopsied had no gross evidence of tumor. DLI-induced GVH was enhanced
by the inclusion of CpG 2006 with DLI as indicated by the clinical
appearance (ruffled fur, hunched posture, cachexia) and lower mean
weight curves in DLI + CpG 2006 with or without CpG 1585 as
compared with DLI (P
Several major findings can be derived from our study. First, CpG 2006 is a potent inducer of anti-AML responses in naive as well as syngeneic BMT recipients. In the setting of alloBMT, CpG 2006 also induces an anti-AML response that is bolstered by the inclusion of CpG 1585 and especially by combining both CpG ODNs with DLI. DLI-induced GVHD responses were increased by CpG 2006 administration. Thus, CpG ODNs are potent antileukemia agents that are effective in naive and BMT recipients with minimal residual disease even in the setting in which mice do not have a fully functional adaptive immune system. We have shown that CpG 2006 but not CpG 1585 ODN pretreatment can reduce tumorigenicity by more than 100-fold in naive mice. CpG ODN was more effective when given 2 days before the AML challenge than concurrently with or following the challenge. To date, the published literature indicates that systemic CpG ODN administration alone may delay but not prevent tumor growth.7,34-40 Because CpG ODNs each have distinct functions on the immune system, extrapolation to studies using CpG ODNs other than the ones used here is not possible. Results with CpG 2006 for the induction of antitumor immune responses have not been reported, although recent unpublished data (Z. Ballas, A.M.K., 2001) indicate that our findings with systemic administration can be observed in other tumor models. Because CpG ODNs in general stimulate the innate immune system, antitumor studies have focused on developing CpG ODNs as adjuvants for tumor-associated antigens or cellular vaccinations. We have shown that systemic CpG ODNs can induce an antilymphoma immune response when combined with fusion proteins consisting of tumor idiotype protein conjugated to either a neoantigen or granulocyte-macrophage colony-stimulating factor or with an antitumor mAb.7,34,35 Others have shown that systemic CpG ODNs can enhance the survival of mice that receive peptide or protein vaccines followed by a melanoma cell challenge.38 Local CpG ODN injection in the vicinity of or at the tumor bed site eradicated tumor and/or led to long-term survival.36,37 In both instances, CpG ODN generated a memory cell response as assessed by rechallenge experiments. In our experiments, NK cells were required for the anti-AML responses provided by CpG 1585 or 2006 ODNs as demonstrated by mAb depletion studies and the use of T- and B-cell-deficient SCID recipients. NK-cell depletion did not completely eliminate the protective effect of CpG 2006 ODN in either wild-type or SCID recipients, and TCD did not adversely affect the antitumor immune responses of CpG 2006-treated wild-type mice. Our studies demonstrated a requirement for NK cells in the optimal AML resistance of mice treated with CpG ODN. T cells were not obligatory for the CpG ODN-facilitated tumor resistance. Consistent with these findings, we could not uncover a memory cell response to AML rechallenge in CpG-treated mice resisting initial AML challenge. Because CpG ODNs are known to stimulate the function of monocytes and DCs, the most likely interpretation of these results is that NK cells are required for optimal antitumor responses, and monocyte/DCs participate by either augmenting indirectly31 or directly41 stimulating NK function or by monocyte/DC-mediated antitumor cytotoxicity.42 CpG ODN 2006 had the greatest antitumor activity in this model, despite
being optimized for activation of human rather than murine cells. This
unexpected observation points to the relative conservation in the
immune recognition of CpG DNA motifs between different species.
Although bacterial and synthetic ODN can induce NK cells to produce
IFN Immature DCs treated with CpG ODN and cocultured with irradiated tumor cells have been shown to provide protection against tumor challenge in vaccinated mice.39 CpG ODNs are known to activate APCs and to enhance the capacity of APCs to stimulate both CD4+ and CD8+ T-cell responses.35,44 We hypothesize that APC stimulation induced by CpG 2006 contributed to the antitumor effects. Although CpG 1585 is a potent stimulator of NK function, it is possible that CpG 2006 stimulation of APCs is responsible for the generation of NK effectors and activated monocytes/DCs that work in concert to resist AML. Consistent with this hypothesis, preliminary data indicate that NK-depleted flt3L knockout mice that have a defect in DC numbers were more susceptible to AML-induced lethality than NK-depleted wild-type mice (unpublished data). An important aspect of our studies was the finding that CpG 2006 ODN administered to syngeneic or allo-TCD bone marrow recipients at early time periods after BMT could augment an anti-AML response. As compared with controls, syngeneic recipients with severe T-cell immune deficiency had a significant survival advantage when given CpG 2006 ODN and challenged with AML cells on day 14 after BMT and when rechallenged later after BMT (day 80). AlloBMT recipients treated with CpG 2006 ODN and challenged on day 36 after BMT also had a significant survival advantage and combined CpG 1585 + 2006 ODNs resulted in 20% long-term survival, even though 2 months or more are required for substantial T-cell immune reconstitution in this setting. In preclinical lymphoma studies, anti-idiotypic mAb given along with a single dose of CpG ODN resulted in 70% versus 40% survival when mAb was combined with 3 doses of IL-2.31 In naive mice, high-dose IL-2 is not particularly effective in inducing AML resistance,24 in contrast to CpG 2006 ODN. If proven to be well tolerated in humans, CpG ODNs would represent an attractive alternative to a continuous infusion of low-dose IL-2. A mainstay of the treatment of leukemia relapse after BMT is the use of DLI. However, DLI is far less effective in acute than chronic leukemia. In our preclinical studies, alloBMT recipients of TCD bone marrow challenged with AML cells on day 17 and then treated on day 21 with DLI had an extended survival, although all died with leukemia. The inclusion of CpG 1585 or CpG 2006 ODNs provided a more potent DLI-facilitated GVL effect and the coadministration of both CpG ODNs with DLI resulted in 88% long-term survival. A potential explanation is the possibility that these CpG ODNs stimulate DLI-derived T-cell and NK-cell effectors, possibly in concert with bone marrow-derived effector cells present at this early time period after BMT. Consistent with the potential effects of CpG ODNs on DLI-derived T cells, recipients given DLI + CpG ODNs had a more severe GVHD reaction than either DLI- or CpG ODN-treated controls. Future studies will be required to determine whether CpG ODNs + DLI differentially affect particular GVHD target organs. Combined administration of DLI + CpG ODNs represents a new approach potentially useful in situations in which DLI alone is incompletely effective in tumor cell reduction. Th1-like responses dominate the rapid response of the innate immune system to foreign antigens. Immunostimulatory bacterial DNA and synthetic CpG ODNs result in Th1 responses.6 Although Th1 responses are desirable for cancer immunotherapy, proinflammatory Th1 responses could be detrimental early after BMT. Studies are in progress to explore the effects of CpG ODNs on GVHD and graft rejection. Regardless, CpG 2006 ODNs given with DLI are highly effective in treating AML. Although mice had clinical GVHD, the GVHD was not of sufficient severity to cause lethality in most recipients. Combined CpG ODN and DLI could be especially useful in patients that have acute leukemia typically refractory to DLI.
Submitted February 7, 2000; accepted April 19, 2001.
Supported in part by grants R01 CA-72669, R01 HL63452, and P01 CA66579 from the National Institutes of Health, by a Career Development Award from the Department of Veterans Affairs, by a grant from the Leukemia Task Force, and by a grant from the Coley Pharmaceutical Group (A.M.K., B.R.B.).
A.M.K. has declared a financial interest in the Coley Pharmaceutical Group, whose product was studied in the present work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Bruce R. Blazar, University of Minnesota Hospital, Box 109 Mayo Bldg, 420 SE Delaware St, Minneapolis, MN 55455; e-mail: blaza001{at}tc.umn.edu.
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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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H. Fujii, J. D. Trudeau, D. T. Teachey, J. D. Fish, S. A. Grupp, K. R. Schultz, and G. S. D. Reid In vivo control of acute lymphoblastic leukemia by immunostimulatory CpG oligonucleotides Blood, March 1, 2007; 109(5): 2008 - 2013. [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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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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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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B. J. Weigel, N. Nath, P. A. Taylor, A. Panoskaltsis-Mortari, W. Chen, A. M. Krieg, K. Brasel, and B. R. Blazar Comparative analysis of murine marrow-derived dendritic cells generated by Flt3L or GM-CSF/IL-4 and matured with immune stimulatory agents on the in vivo induction of antileukemia responses Blood, December 1, 2002; 100(12): 4169 - 4176. [Abstract] [Full Text] [PDF]
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A. R. M. Olbrich, S. Schimmer, K. Heeg, K. Schepers, T. N. M. Schumacher, and U. Dittmer Effective Postexposure Treatment of Retrovirus-Induced Disease with Immunostimulatory DNA Containing CpG Motifs J. Virol., October 11, 2002; 76(22): 11397 - 11404. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
