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,
*
The Holden Cancer Center and Departments of
Internal Medicine and
Urology, University of Iowa, Iowa City, IA 522421; and
Veterans Affairs Medical Center, Iowa City, IA 52242
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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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, IFN-
, and TNF-)
(1, 2, 3, 4, 5, 6, 7). CpG ODN can also function as potent immune
adjuvants (8). In a number of murine systems, CpG ODN
enhances the Ag-specific Ab response, shifting it toward Abs of the
IgG2a isotype (indicating a Th1 response) and induces development of
enhanced CTL activity (8, 9, 10, 11, 12, 13). Dendritic cells (DCs) are potent APCs and are creating considerable excitement in their ability to induce a cellular immune response (reviewed in Refs. 14, 15, 16, 17). Studies in animal models demonstrate that pulsing DCs with Ag can result in Ag-specific immune responses (18, 19, 20). Recent clinical trials suggest DC therapy may be effective in humans as well (15, 21, 22).
Extensive evidence indicates that the ability of DCs to induce a CTL response is dependent on T cell help (23, 24, 25, 26). In fact, there is extensive cross-talk between CD4+ T cells and DCs. Presentation of class II-restricted epitopes by DCs to CD4+ T cells results in activation of the CD4+ cells (24). CD4+ T cells also express CD40 ligand, and further activate DCs by signaling via CD40. This, in turn, enhances the ability of the DC to activate class I-restricted T cells. This effect is particularly important for naive T cells. Indeed, activation of DCs by CD40 ligand enhances the ability of the DCs to prime CTLs in the absence of CD4+ cells (27, 28, 29).
CpG ODN have been shown to be able to activate DCs in both the human and murine systems as indicated by induction of cytokine production, including IL-12 as well as other soluble factors, and up-regulation of class I MHC, class II MHC, CD80, and CD86 (30, 31, 32). In addition, there are reports of possible direct costimulatory effects of CpG ODN on T cells (2, 9, 33). However, little is known about which of these changes contributes most significantly to the development of an enhanced cellular response. We therefore evaluated how treatment of DCs with CpG ODN impacts on development of an enhanced class I-restricted T cell response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 and C3H female mice were purchased from Harlan Sprague Dawley (Indianapolis, IN). All mice were housed at the animal care unit of the University of Iowa and used at 8–10 wk of age. Naive C57BL/6 OT-1 mice that are transgenic for the TCR which recognizes MHC class I H-2Kb-restricted OVA 257–264 (SIINFEKL) in H-2Kb (34) were obtained from Tim Ratliff (University of Iowa). C57BL/6-IL-12 knockout mice (35, 36, 37, 38) were obtained from The Jackson Laboratory (Bar Harbor, ME). In CTL assays, EL4 cells (murine T cell lymphoma that is H-2Kb restricted and thus C57BL/6 mouse syngeneic) and/or EG7 cells (EL4 cells which contain a plasmid which expresses OVA) were used as targets (39). In select experiments, C57BL/6 mice were infected with an adenoviral vector that expressed OVA. Mice were injected i.p. with 1 x 108 PFU/mouse with the adenovirus designated Ad5TrF-OVA (5 x 1010 PFU/ml or 1.2 x 1012 particles/ml) generated by the University of Iowa Virus Core Lab.
In vitro generation of DCs
DCs were generated using a modification of a previously described approach (40). Bone marrow cells were obtained by flushing the pelvis, femurs, and tibias of C57BL/6 wild-type or IL-12 knockout mice. RBCs were lysed using 0.83% ammonium chloride solution. T cells were removed by complement-mediated lysis using a mixture of anti-CD3 (145.2C11), anti-CD4 (GK1.5), and anti-CD8 (53.6.7) Abs. B cells were removed by panning using a flask coated with anti-B220 (6B2) Ab. Remaining cells were allowed to adhere overnight. Nonadherent cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 µM 2-ME, 10 U/ml penicillin, 10 µg/ml glutamine supplemented with 1000 U/ml murine GM-CSF (R&D Systems, Minneapolis, MN), and 1000 U/ml murine IL-4 (PeproTech, Rocky Hill, NJ). Media was changed at 4 or 5 days, and DCs were harvested and used on day 7. DC phenotype and morphology was confirmed by flow cytometry and scanning electron microscopy.
CpG ODN treatment and Ag pulsing of DCs
A panel of CpG ODN were purchased commercially (Operon Technologies, Alemeda, CA). All ODN had a nuclease-resistant phosphorothioate backbone and were confirmed to have undetectable endotoxin content before use. The immunostimulatory CpG ODN designated 1826 (5'-TCCATGACGTTCCTGACGTT-3'), which has proven to be an effective adjuvant in our previous studies (13), was used as the immunostimulatory agent. CpG ODN 1982, a non-CpG-containing ODN (5'-TCCAGGAGTTCTCTCAGGTT-3'), and CpG ODN 1845, which is identical to 1826 except that the cytosine residues are methylated, were used as controls. Freshly prepared DCs were treated with 5–6 µg/ml CpG ODN for 12–48 h, washed, and then pulsed with 100 µg/ml to 5 mg/ml OVA (Sigma, St. Louis, MO), 2–200 µg/ml SIINFEKL peptide (Research Genetics, Huntsville, AL), or unsupplemented media for 12–24 h and washed twice before use.
Generation and measurement of CTL activity
DCs (5 x 104–1 x
106) generated and treated as above were injected
i.p. into C57BL/6 mice. After 
9 days, spleens from immunized mice
were harvested, homogenized, and RBCs lysed with 0.83% ammonium
chloride solution. Splenocytes from naive C57BL/6 mice were
homogenized, RBCs lysed, incubated with 100 µg/ml SIINFEKL for 3
h at 37°C, and irradiated with 3000 rad. These peptide-pulsed,
irradiated splenocytes (2 x 106/ml) were
then washed twice and mixed with splenocytes (2 x
106/ml) from immunized mice in a 1:1 ratio in 10
ml 10% culture medium (CM) in a 25-cm2 flask and
incubated at 37°C for 5 days. After the 5-day restimulation,
lymphocytes (effectors) were obtained by centrifugation over Histopaque
1083 (Sigma). Effector cells were then incubated at various E:T ratios
with 51Cr-labeled target cells. Target cells
included EL4 cells (Ag negative), EL4 cells incubated with 100 µg/ml
SIINFEKL for 1 h at 37°C and washed twice, or EG7 cells.
Supernatant was harvested after 6 h and release counts were
determined using a Beckman gamma 5500 counter (Beckman Coulter,
Fullerton, CA). All samples were run in triplicate. Percent specific
lysis was determined according to the following equation: (experimental
cpm - minimum cpm)/(maximum cpm - minimum cpm) x
100.
Phenotypic analysis
DCs were stained with FITC-conjugated anti-MHC class I (anti-H2Kb), anti-B7-1 (1G10), anti-B7-2 (GL-1), and anti-SIINFEKL-H2Kb (25-D1.16) (41) using standard techniques. T cells were stained with anti-CD3 (145.2C11), anti-CD4 biotin (GK1.5), anti-CD8 biotin (53.6.7), and anti-B220 biotin (6B2). Propidium iodide staining was used to exclude dead cells. Stained cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mansfield, MA). Appropriate isotype controls were used.
Cytokine analysis
Supernatant from T cell cultures with DCs were analyzed for the
presence of IFN- by ELISA using commercially available kits
(PharMingen, San Diego, CA) (2, 42). All samples were
tested in triplicate.
CpG ODN treatment of purified T cells
Spleens were harvested from C57BL/6 or C3H mice and homogenized, and the lymphocytes were separated by centrifugation over Histopaque 1083 (Sigma). These lymphocytes were panned for 1 h at 4°C, first with a mixture of Abs against B cells, MHC class II, and heat-stable Ag and then with a mixture against Fc receptors, erythroid cells, and B cells (courtesy of Tom Waldschmidt, University of Iowa). The resulting T cells were labeled with anti-CD5 (positive staining) and 6B2 (negative staining) for sorting on an Epics IV flow cytometer (Coulter, Miami, FL). T cells were >99.5% pure as demonstrated by FACS. One hundred thousand T cells were incubated with 5 µg/ml CpG ODN or control ODN. In select samples, 10 µg/ml anti-CD3 or 100,000 unfractionated irradiated splenocytes were added. After 48 h, supernatant was harvested and assayed for the presence of IL-2 using an ELISA per the manufacturer’s protocol (Endogen, Cambridge, MA). Proliferation was determined by scintillation counting after labeling for 6 h with 1 µCi [3H]thymidine (Amersham, Arlington Heights, IL). All samples were tested in triplicate.
In vitro Ag presentation by DCs and T cell activation
Splenocytes from C57BL/6 wild-type, TCR-transgenic OT-1 mice (34), or mice infected with an adenovirus that expresses OVA were finely minced and RBCs were lysed using 0.83% ammonium chloride solution. Adherent cells were removed by plastic adherence. B cells were depleted by panning with anti-B220 (6B2) Ab-coated flasks.
In select experiments, purified CD8+ and CD4+ T cells were obtained by magnetic bead separation of T cells using either anti-CD8 (53.6.7) or anti-CD4 (GK1.5) magnetic beads according to the manufacturer’s specifications (MiniMACS; Miltenyi Biotec, Auburn, CA). All samples were passed over the column twice and were 99% pure as determined by flow cytometry. In experiments where indicated, anti-CD40 Ab (kindly provided by Gail Bishop, University of Iowa) at 10 µg/ml was used in lieu of CD4+ T cells.
DCs generated and treated as above were added to T cells for 48 h at a T cell:DC ratio of 100:1. For proliferation assays, 1 µCi/well [3H]thymidine (Amersham) was added 6 h before harvest. To confirm that thymidine uptake was not due to DCs, control wells containing DCs alone were included. DCs alone demonstrated no significant thymidine uptake. Parallel samples were run without the addition of [3H]thymidine, and supernatant was harvested for cytokine analysis. All tests were performed in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Before exploring the cellular mechanisms involved, it was
important to confirm that treatment of DCs with CpG ODN enhances the
immune response in the system under investigation. Therefore, initial
studies evaluated whether CpG ODN enhances development of a CTL
response following immunization with DCs in vivo. C57BL/6 mice were
immunized with DCs incubated with 10% CM, CpG ODN, or CpG ODN and
SIINFEKL. After 9 days, spleens were harvested and incubated with
irradiated naive splenocytes that had been incubated with SIINFEKL.
After 5 days, live cells were harvested and the standard CTL assay was
performed with targets as indicated. As illustrated in Fig. 1
, CpG ODN significantly enhanced the
ability of Ag-pulsed DCs to induce a CTL response against either EL4
cells incubated with peptide (Fig. 1A) or against EG7 cells
that also express target peptide (Fig. 1B). Preincubation of
DCs with CpG ODN before injection was needed to induce the enhanced CTL
response. When DCs were pulsed with peptide, then coinjected in vivo
with CpG ODN, no enhanced CTL were observed (data not shown).
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As outlined above, previous reports suggest that CpG ODN may
contribute to T cell activation by supplying a costimulatory signal
directly to the T cells in the absence of other cell types. To assess
this further, CpG ODN was added to sort-purified naive T cells in the
presence or absence of CD3 cross-linking. Proliferation of T cells was
then determined. As illustrated in Fig. 2, CpG ODN had no specific effect on T
cell proliferation with or without CD3 cross-linking. Both CpG and
non-CpG ODN had modest, nonspecific effects on the proliferation of T
cells after CD3 cross-linking. Proliferation of T cells was seen in the
positive control when anti-CD3 and APCs in the form of radiated
splenocytes were added. Similar results were found using IL-2
production to detect T cell activation or CD3-based bispecific Abs to
activate T cells (data not shown) (43). These data
indicate that CpG ODN do not have direct effects on T cell activation
in the presence or absence of CD3 cross-linking.
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We next evaluated whether treatment of DCs with CpG ODN enhances
their ability to activate class I-restricted T cells. DCs were treated
with CpG ODN and pulsed with SIINFEKL peptide. Unfractionated T cells
from naive C57BL/6 OT-1 mice, which are transgenic for a TCR that
recognizes OVA254–263 peptide (SIINFEKL) in
H-2Kb, were incubated with DCs obtained from
wild-type C57BL/6 mice. T cell proliferation was determined using a
standard [3H]thymidine assay and IFN-
production as determined by ELISA. As illustrated in Fig. 3, A and B, peptide
pulsed-DC induced both T cell proliferation and IFN- secretion. This
effect was enhanced significantly by treatment of DCs with CpG ODN. No
effect was seen when control, nonstimulatory, ODN were used (data not
shown). CpG ODN had no effect on DC-induced proliferation of T cells in
the absence of peptide, demonstrating that the effect of CpG ODN was Ag
specific. This response was dose dependent, with increased T cell
activation occurring with increased doses of peptide (Fig. 3C). These studies confirm that CpG ODN enhances the ability
of DCs expressing the target peptide to activate class I-restricted T
cells.
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We next sought to determine whether CpG ODN impacts on the ability
of DCs pulsed with soluble protein Ag to induce activation of class
I-restricted T cells. DCs were treated with CpG ODN as outlined above
and pulsed with soluble OVA. Naive OT-1 T cells were then added, and T
cell proliferation and activation were measured as outlined above. As
illustrated in Fig. 4, CpG ODN enhanced
the ability of OVA-pulsed DCs to induce proliferation (Fig. 4A) as well as production of IFN- secretion (Fig. 4B) by class I-restricted T cells. No effect was seen with
control ODN (data not shown).
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The studies illustrated in Figs. 3
and 4 involved unfractionated T
cells from OT-1 mice. These included CD4+ T
cells, which could have supplied T cell help. We therefore used
magnetically purified CD4+ and
CD8+ T cells to assess the role of T cell help in
the activation of MHC class I-restricted T cells by CpG ODN-treated
DCs. CpG ODN-treated DCs pulsed with either protein (Fig. 5A) or peptide (Fig. 5, B and C) induced a low level of activation of
purified class I-restricted CD8+ T cells in an
Ag-restricted manner in the absence of T cell help. The addition of T
cell help significantly enhanced activation. Fig. 5B
(measuring IFN- production) and Fig. 5C (measuring
proliferation) show similar findings using the MHC class I
immunodominant epitope of OVA (SIINFEKL) as the Ag. Non-CpG ODN
controls, not shown in Fig. 5, B and C, revealed
results similar to those in Fig. 5A. T cell help could be
supplied by either Ag-nonspecific T cells (wild-type CD4 T cells), CD40
ligation via anti-CD40 Ab, or by Ag-specific T cells (T cells
obtained from mice infected with an adenovirus that expressed OVA
referred to as Ad-OVA CD4 T cells). Specific T cell help was most
effective at enhancing CD8+ activation. Overall,
these results demonstrate that CpG ODN treatment of DCs can enhance the
ability of DCs to activate CD8+ T cells in the
absence of T cell help. However, T cell help further enhances this
activation. Although help can be Ag nonspecific in this system, the
CD8+ T cell response is enhanced most extensively
when specific T cell help is supplied.
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Previous studies demonstrated that CpG ODN can enhance MHC
expression, increase expression of costimulatory molecules, and enhance
production of soluble factors by DCs and other APCs (6, 32). We explored the relative importance of these changes in the
ability of CpG ODN-treated DCs to induce activation of class
I-restricted T cells. In initial studies, we compared the ability of
fixed and viable peptide-pulsed DCs to activate T cells. CpG ODN
treatment of DCs had no significant effect on the amount of peptide
presented in class I as demonstrated by flow cytometry using an
Ab specific for the SIINFEKL-MHC I complex, and fixation did not
alter expression of this complex (data not shown). However, fixation of
DCs after exposure to CpG ODN eliminated their ability to enhance
IFN- secretion by class I-restricted T cells (Fig. 6). Thus, fixation of peptide-pulsed and
CpG ODN-treated DC does not alter the ability of DCs to present class
I-restricted Ag, but fixation does alter the ability of CpG
ODN-stimulated DCs to activate class I-restricted T cells. This
suggests that CpG ODN enhances the ability of DCs to activate class
I-restricted T cells by enhancing DC production of cytokines or other
soluble factors.
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Given the central role played by IL-12 in the induction of a CTL
response, DCs from IL-12 knockout mice were used to assess whether CpG
ODN induction of IL-12 secretion by DCs is responsible for the ability
of CpG ODN to enhance DC activation of class I-restricted T cells. DCs
were generated from wild-type C57BL/6 and from
C57BL/6-IL-12-/- mice treated with CpG ODN and
pulsed with OVA as outlined above. DCs from wild-type mice and
IL-12-/- mice were similar in their ability to
activate T cells in the absence of CpG ODN. In contrast, the ability of
CpG ODN to enhance class I-restricted T cell activation by DCs was
significantly blunted when DCs from IL-12-/-
mice were used (Fig. 7). This effect was
not complete. CpG ODN did have some stimulatory effects with the
IL-12-/- DC. Thus, the ability of CpG ODN to
enhance class I-restricted T cell activation is due partially, but not
completely, to enhanced production of IL-12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bacterial DNA contains sequences, termed CpG motifs, that have immunostimulatory properties (1, 2). Previous studies by our group and others have demonstrated that synthetic ODN containing such motifs function well as immune adjuvants as indicated by their ability to enhance development of a cellular immune response following immunization with either protein Ag or peptide (8, 46). The current studies were designed to help us understand the cross-priming effects of CpG ODN by exploring the direct and indirect cellular effects of CpG ODN on class I-restricted T cells, with a particular focus on how CpG ODN impacts on the Ag-presenting capabilities of DCs.
In contrast to some previous reports (9, 33, 46), our studies failed to demonstrate a direct effect of CpG ODN on T cells in vitro. There are a number of possible explanations for the differences between our results and those reported by Lipford et al. (9). Different ODN sequences were used in the two sets of studies. Growing evidence suggests that non-CpG sequences can have significant immunostimulatory effects. Thus, the direct effect of ODN on T cells reported by Lipford et al. (9) could have been due to non-CpG effects. Alternatively, even trace contamination of the T cell population with APCs could impact on T cell activation induced by CpG ODN.
Because of the lack of evidence for a direct effect of CpG on T cells in our studies, we focused on how CpG ODN impacts on the interaction between T cells and APCs. As a model, we used bone marrow-derived DCs and T cells from transgenic mice (OT-1) (34) that express a TCR that recognizes an immunodominant OVA peptide (SIINFEKL) (47) complexed with class I MHC (H-2Kb). This model allowed us to evaluate the response to either purified peptide or protein Ag and therefore enabled us to assess the importance of cross-priming (after pulsing with protein Ag) and T cell help (after pulsing with peptide) in the activation of class I-restricted T cells.
The interaction between class I-restricted T cells, APCs such as DCs, and CD4+ T cells is quite complex. Our studies indicate that CpG ODN-treated DCs can activate MHC class I-restricted T cells to a modest degree in a CD4+ T cell-independent manner. The addition of T cell help significantly augmented DC activation of CD8+ T cells. As expected, Ag-activated CD4+ T cells obtained from mice primed with an adenoviral vector that expressed OVA were most effective in providing help. CD4+ T cells obtained from naive mice or CD40 ligation by anti-CD40 Abs were also effective, albeit to a more modest degree, in providing help. It is important to point out that the media used in these studies contained calf serum. CD4+ cells obtained from naive mice could have recognized serum proteins presented in DC class II and could have contributed the needed T cell help even in the absence of class II-restricted OVA peptides.
Our results corroborate and expand work from other laboratories which have suggested that CpG ODN can enhance development of CTL by a completely Th-independent mechanism. Vabulas et al. (48) have recently found that CpG ODN can enhance the development of CTL after immunization with SIINFEKL in both CD40-/- and MHC class II-/-. These results are not inconsistent with our results outlined above since nonspecific T cells could have contributed to the development of CTL in the mice immunized with CpG ODN plus SIINFEKL. Similarly, Cho et al. (49) found CTL could be generated after immunization of MHC class II-/- and CD4-/- mice with Ova-CpG ODN conjugates. Although our studies confirm that CpG ODN-treated DCs can activate CTL in the absence of T cell help, the addition of help greatly augmented CTL activation.
There are a number of possible explanations for the apparent discrepancy between our results and those outlined above. First, our studies and those by Vabulas et al. (48) and Cho et al. (49) evaluated different responses. We evaluated in vitro activation of class I-restricted cells by measuring proliferation and cytokine production of transgenic CD8+ T cells soon after exposure of the cells to Ag-pulsed DCs. In contrast, the other investigators evaluated CTL by harvesting lymphocytes after in vivo immunization and restimulating the cells in long-term culture with Ag and IL-2. It is possible that CD8+ T cells exposed in vivo to Ag in the absence of T cell help can be restimulated with IL-2 in longer term culture to become Ag-specific CTL (as found by Vabulas et al. (48) and Cho et al. (49)). This possibility is currently under investigation in our laboratory. Second, the mechanisms responsible for supplying T cell help are much more complex in vivo compared with our in vitro studies using purified cell populations. As has been described by other investigators, we found that CD40 cross-linking could substitute for T cell help and allow DCs to more potently activate class I-restricted cells. It is likely other signals supplied to the DCs in vivo can play a similar role, and sensitivity to these signals could have been altered by CpG ODN. These signals could have contributed to detection of CTL after immunization with CpG ODN and Ag in CD40-/-, CD4-/-, and MHC class II-/- mice. Ongoing studies are exploring the nature of the T cell help required by CpG ODN-treated APCs.
Our studies also demonstrate that CpG ODN enhances the ability of bone marrow-derived DCs to activate class I-restricted T cells after DCs are pulsed with soluble protein Ag. A variety of mechanisms could be responsible for the enhanced ability of CpG ODN-treated, Ag-pulsed DCs to cross-prime class I-restricted T cells. CpG ODN could have enhanced Ag processing, increased expression of class I MHC (and peptide), enhanced expression of costimulatory molecules, or enhanced production of immunostimulatory cytokines by the DCs.
Enhanced T cell activation was lost when DCs pulsed with Ag and treated
with CpG ODN were fixed before being used to stimulate T cells. In
addition, much, but not all, of the enhanced T cell activation seen
with CpG ODN-treated DCs was lost when DCs from
IL-12-/- mice were used. These results indicate
the primary mechanism responsible for the effect of CpG ODN on DCs was
to enhance the production of immunostimulatory cytokines, with IL-12
playing a particularly important role. Our previous studies
(13) and studies by others have demonstrated that CpG ODN
markedly enhances production of IL-12 by bone marrow-derived DCs.
Production of IL-12 by DCs is known to play a role in the proliferation
and clonal expansion of T cells and their production of IFN-
(50). Kranzer et al. (51) recently reported
that production of type I IFN and IL-12 by APCs plays a key role
in the activation of T cells when the TCR is artificially
cross-linked. The finding that induction of IL-12 plays a key role in
the response of CD8 cells to Ag presented by CpG ODN-treated DCs fits
well with these observations. Other cytokines were likely involved as
well. Recent reports suggest that IL-18 functions synergistically with
IL-12 (52, 53) and may provide a compensatory mechanism in
the absence of IL-12.
As described above, the ability of CpG ODN to enhance development of a class I-restricted response is based largely on induction of cytokine production by the DCs. However, APCs found in situ are likely to be very different functionally from the bone marrow-derived DCs used in the current study. APCs in tissues have not been exposed to supraphysiologic concentrations of cytokines and are unlikely to express such high levels of costimulatory molecules. Thus, despite our results, the adjuvant effect of CpG ODN in vivo could be related, at least in part, to enhancement of Ag uptake or induction of phenotypic changes by APCs as well as by induction of cytokine production by the APCs.
In conclusion, previous studies have demonstrated that CpG ODN are potent immune adjuvants that can enhance development of a Th1 immune response. In the studies outlined above, we demonstrate that CpG ODN can enhance cross-priming, as indicated by enhanced ability of CpG ODN-treated DCs pulsed with soluble protein Ag to induce a class I-restricted T cell response. Although CpG ODN-treated DCs appear to be able to activate class I-restricted T cells in a T cell-independent manner, some degree of T cell help significantly augments CTL activation. Although much of this enhanced response is due to increased production of IL-12, other immunostimulatory cytokines likely also play a role. Further work is needed to determine the nature of the T cell help required by CpG ODN-treated DCs.
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Footnotes |
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2 T.L.W. and S.K.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. George J. Weiner, Holden Cancer Center, University of Iowa, 5970 ZJPP, 200 Hawkins Drive, Iowa City, IA 52242.
4 Abbreviations used in this paper: CpG ODN, cytosine-phosphorothioate-guanine oligodeoxynucleotide; DC, dendritic cell; CM, culture medium.
Received for publication June 15, 2000. Accepted for publication September 5, 2000.
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 S.-J. Yeo, J.-G. Yoon, and A.-K. Yi Myeloid Differentiation Factor 88-dependent Post-transcriptional Regulation of Cyclooxygenase-2 Expression by CpG DNA: TUMOR NECROSIS FACTOR-{alpha} RECEPTOR-ASSOCIATED FACTOR 6, A DIVERGING POINT IN THE Toll-LIKE RECEPTOR 9-SIGNALING J. Biol. Chem., October 17, 2003; 278(42): 40590 - 40600. [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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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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S.-J. Yeo, D. Gravis, J.-G. Yoon, and A.-K. Yi Myeloid Differentiation Factor 88-dependent Transcriptional Regulation of Cyclooxygenase-2 Expression by CpG DNA: ROLE OF NF-{kappa}B AND p38 J. Biol. Chem., June 13, 2003; 278(25): 22563 - 22573. [Abstract] [Full Text] [PDF]
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 U. Kumaraguru, C. D. Pack, and B. T. Rouse Toll-like receptor ligand links innate and adaptive immune responses by the production of heat-shock proteins J. Leukoc. Biol., May 1, 2003; 73(5): 574 - 583. [Abstract] [Full Text] [PDF]
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 T.-Y. Kim, H.-J. Myoung, J.-H. Kim, I.-S. Moon, T.-G. Kim, W.-S. Ahn, and J.-I. Sin Both E7 and CpG-Oligodeoxynucleotide Are Required for Protective Immunity against Challenge with Human Papillomavirus 16 (E6/E7) Immortalized Tumor Cells: Involvement of CD4+ and CD8+ T Cells in Protection Cancer Res., December 15, 2002; 62(24): 7234 - 7240. [Abstract] [Full Text] [PDF]
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L. Sfondrini, D. Besusso, M. T. Zoia, M. Rodolfo, A. M. Invernizzi, M. Taniguchi, T. Nakayama, M. P. Colombo, S. Menard, and A. Balsari Absence of the CD1 Molecule Up-Regulates Antitumor Activity Induced by CpG Oligodeoxynucleotides in Mice J. Immunol., July 1, 2002; 169(1): 151 - 158. [Abstract] [Full Text] [PDF]
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P. Martin, G. M. del Hoyo, F. Anjuere, C. F. Arias, H. H. Vargas, A. Fernandez-L, V. Parrillas, and C. Ardavin Characterization of a new subpopulation of mouse CD8alpha + B220+ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential Blood, June 28, 2002; 100(2): 383 - 390. [Abstract] [Full Text] [PDF]
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 G. Parmiani, C. Castelli, P. Dalerba, R. Mortarini, L. Rivoltini, F. M. Marincola, and A. Anichini Cancer Immunotherapy With Peptide-Based Vaccines: What Have We Achieved? Where Are We Going? J Natl Cancer Inst, June 5, 2002; 94(11): 805 - 818. [Abstract] [Full Text] [PDF]
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P. Riedl, D. Stober, C. Oehninger, K. Melber, J. Reimann, and R. Schirmbeck Priming Th1 Immunity to Viral Core Particles Is Facilitated by Trace Amounts of RNA Bound to Its Arginine-Rich Domain J. Immunol., May 15, 2002; 168(10): 4951 - 4959. [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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T. Storni, F. Lechner, I. Erdmann, T. Bachi, A. Jegerlehner, T. Dumrese, T. M. Kundig, C. Ruedl, and M. F. Bachmann Critical Role for Activation of Antigen-Presenting Cells in Priming of Cytotoxic T Cell Responses After Vaccination with Virus-Like Particles J. Immunol., March 15, 2002; 168(6): 2880 - 2886. [Abstract] [Full Text] [PDF]
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I. Miconnet, S. Koenig, D. Speiser, A. Krieg, P. Guillaume, J.-C. Cerottini, and P. Romero CpG Are Efficient Adjuvants for Specific CTL Induction Against Tumor Antigen-Derived Peptide J. Immunol., February 1, 2002; 168(3): 1212 - 1218. [Abstract] [Full Text] [PDF]
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Y. Kawarada, R. Ganss, N. Garbi, T. Sacher, B. Arnold, and G. J. Hammerling NK- and CD8+ T Cell-Mediated Eradication of Established Tumors by Peritumoral Injection of CpG-Containing Oligodeoxynucleotides J. Immunol., November 1, 2001; 167(9): 5247 - 5253. [Abstract] [Full Text] [PDF]
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B. R. Blazar, A. M. Krieg, and P. A. Taylor Synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides are potent stimulators of antileukemia responses in naive and bone marrow transplant recipients Blood, August 15, 2001; 98(4): 1217 - 1225. [Abstract] [Full Text] [PDF]
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