Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.

Oligodeoxynucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN))4 can activate B cells, monocytes, and NK cells and induce production of a wide variety of cytokines (IL-6, IL-10, IL-12, IL-18, IFN-α, IFN-γ, and TNF-α) (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.

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 × 108 PFU/mouse with the adenovirus designated Ad5TrF-OVA (5 × 1010 PFU/ml or 1.2 × 1012 particles/ml) generated by the University of Iowa Virus Core Lab.

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.

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.

DCs (5 × 104–1 × 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 × 106/ml) were then washed twice and mixed with splenocytes (2 × 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) × 100.

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.

Supernatant from T cell cultures with DCs were analyzed for the presence of IFN-γ by ELISA using commercially available kits (PharMingen, San Diego, CA) (2, 42). All samples were tested in triplicate.

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.

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.

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. 1,A) or against EG7 cells that also express target peptide (Fig. 1 B). 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).

FIGURE 1.

CpG ODN enhances the ability of peptide-pulsed DCs to induce a CTL response. DCs (5 × 104–1 × 106) were injected into mice after incubation with either 10% CM, SIINFEKL (100 μg/ml), or SIINFEKL and CpG ODN (6 μg/ml). Nine days later, immune splenocytes were incubated with naive, irradiated, Ag-pulsed splenocytes for 5 days. Targets (EL4, EL4 incubated with SIINFEKL, or EG7) were labeled with 51Cr and incubated with effector cells in ratios indicated. A, Percent specific lysis when EL4 plus SIINFEKL were used as targets. B, Percent specific lysis when EG7 were used as targets. The key indicates treatment of DCs used in immunization. All samples were run in triplicate.

FIGURE 1.

CpG ODN enhances the ability of peptide-pulsed DCs to induce a CTL response. DCs (5 × 104–1 × 106) were injected into mice after incubation with either 10% CM, SIINFEKL (100 μg/ml), or SIINFEKL and CpG ODN (6 μg/ml). Nine days later, immune splenocytes were incubated with naive, irradiated, Ag-pulsed splenocytes for 5 days. Targets (EL4, EL4 incubated with SIINFEKL, or EG7) were labeled with 51Cr and incubated with effector cells in ratios indicated. A, Percent specific lysis when EL4 plus SIINFEKL were used as targets. B, Percent specific lysis when EG7 were used as targets. The key indicates treatment of DCs used in immunization. All samples were run in triplicate.

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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.

FIGURE 2.

CpG ODN has no detectable direct effect on T cells in the absence of APCs. Highly purified T cells (1 × 105) were cultured with or without CpG ODN. T cells were also further stimulated by cross-linking CD3 and by adding irradiated splenocytes. All samples were run in triplicate. Error bars represent SEM.

FIGURE 2.

CpG ODN has no detectable direct effect on T cells in the absence of APCs. Highly purified T cells (1 × 105) were cultured with or without CpG ODN. T cells were also further stimulated by cross-linking CD3 and by adding irradiated splenocytes. All samples were run in triplicate. Error bars represent SEM.

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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-γ production as determined by ELISA. As illustrated in Fig. 3, A and B, peptide pulsed-DC induced both T cell proliferation and IFN-γ 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. 3 C). These studies confirm that CpG ODN enhances the ability of DCs expressing the target peptide to activate class I-restricted T cells.

FIGURE 3.

CpG ODN enhances the ability of peptide (SIINFEKL)-pulsed DCs to activate MHC class I-restricted T cells. DCs were treated with CpG ODN (6 μg/ml) for 12 h and pulsed with SIINFEKL (2–200 ng/ml) for 12 h. Washed DCs (5 × 103) were then cultured for 48 h with T cells (5 × 105) obtained from spleens of OT-1 mice which have TCRs that recognize SIINFEKL-H2kb. A, Proliferative response was measured by a 6-h pulse with [3H]thymidine. B, IFN-γ production was measured in culture supernatants by a standard ELISA. C, IFN-γ production by T cells cultured with DCs that were pulsed with 2, 20, or 200 ng/ml SIINFEKL. All samples were run in triplicate. Error bars represent SEM.

FIGURE 3.

CpG ODN enhances the ability of peptide (SIINFEKL)-pulsed DCs to activate MHC class I-restricted T cells. DCs were treated with CpG ODN (6 μg/ml) for 12 h and pulsed with SIINFEKL (2–200 ng/ml) for 12 h. Washed DCs (5 × 103) were then cultured for 48 h with T cells (5 × 105) obtained from spleens of OT-1 mice which have TCRs that recognize SIINFEKL-H2kb. A, Proliferative response was measured by a 6-h pulse with [3H]thymidine. B, IFN-γ production was measured in culture supernatants by a standard ELISA. C, IFN-γ production by T cells cultured with DCs that were pulsed with 2, 20, or 200 ng/ml SIINFEKL. All samples were run in triplicate. Error bars represent SEM.

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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. 4,A) as well as production of IFN-γ secretion (Fig. 4 B) by class I-restricted T cells. No effect was seen with control ODN (data not shown).

FIGURE 4.

CpG ODN enhances the ability of DCs pulsed with soluble protein Ag (OVA) to activate MHC class I-restricted T cells. DCs were treated with CpG ODN (5 μg/ml) for 12 h and pulsed with soluble OVA (100 μg/ml to 5 mg/ml) for 12 h. Washed DCs (5 × 103) were then cultured for 48 h with T cells (5 × 105) obtained from spleens of OT-1 mice which have TCRs that recognize SIINFEKL-H2kb. A, Proliferative response was measured by a 6-h pulse with [3H]thymidine. B, IFN-γ production was measured in culture supernatants by a standard ELISA. All samples were run in triplicate. Error bars represent SEM.

FIGURE 4.

CpG ODN enhances the ability of DCs pulsed with soluble protein Ag (OVA) to activate MHC class I-restricted T cells. DCs were treated with CpG ODN (5 μg/ml) for 12 h and pulsed with soluble OVA (100 μg/ml to 5 mg/ml) for 12 h. Washed DCs (5 × 103) were then cultured for 48 h with T cells (5 × 105) obtained from spleens of OT-1 mice which have TCRs that recognize SIINFEKL-H2kb. A, Proliferative response was measured by a 6-h pulse with [3H]thymidine. B, IFN-γ production was measured in culture supernatants by a standard ELISA. All samples were run in triplicate. Error bars represent SEM.

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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. 5,A) 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. 5,B (measuring IFN-γ production) and Fig. 5,C (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. 5 A. 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.

FIGURE 5.

Although CpG ODN treatment of Ag-pulsed DCs can result in activation of class I-restricted T cells in the absence of T cell help, the presence of T cell help will further enhance activation. DCs were incubated for 24 h with either unsupplemented media, CpG ODN (6 μg/ml), control CpG ODN (6 μg/ml), OVA (5 mg/ml), or SIINFEKL (100 ng/ml) as indicated. T cells were obtained from spleens of OT-1 mice that have TCRs that recognize SIINFEKL-H-2Kb (OT-1 CD8 T cells), from mice infected with an adenovirus that expresses OVA (Ad-OVA CD4 T cells), and from wild-type C57BL/6 mice (WT CD4 T cells), separated into CD8+ or CD4+ fractions using magnetic bead separation, and cultured with washed DCs in a T:DC ratio of 100:1. Where indicated, ligation of CD40 via anti-CD40 Abs was used to provide T cell help. After 48 h of incubation, supernatant was harvested and IFN-γ production was measured by a standard ELISA. Proliferation was measured by [3H]thymidine incorporation. A, IFN-γ production when OVA was used as Ag. B, IFN-γ production when SIINFEKL was used as Ag. C, Proliferation was measured by a 6-h [3H]thymidine incorporation. All samples were run in triplicate. WT, wild type. Error bars represent SEM.

FIGURE 5.

Although CpG ODN treatment of Ag-pulsed DCs can result in activation of class I-restricted T cells in the absence of T cell help, the presence of T cell help will further enhance activation. DCs were incubated for 24 h with either unsupplemented media, CpG ODN (6 μg/ml), control CpG ODN (6 μg/ml), OVA (5 mg/ml), or SIINFEKL (100 ng/ml) as indicated. T cells were obtained from spleens of OT-1 mice that have TCRs that recognize SIINFEKL-H-2Kb (OT-1 CD8 T cells), from mice infected with an adenovirus that expresses OVA (Ad-OVA CD4 T cells), and from wild-type C57BL/6 mice (WT CD4 T cells), separated into CD8+ or CD4+ fractions using magnetic bead separation, and cultured with washed DCs in a T:DC ratio of 100:1. Where indicated, ligation of CD40 via anti-CD40 Abs was used to provide T cell help. After 48 h of incubation, supernatant was harvested and IFN-γ production was measured by a standard ELISA. Proliferation was measured by [3H]thymidine incorporation. A, IFN-γ production when OVA was used as Ag. B, IFN-γ production when SIINFEKL was used as Ag. C, Proliferation was measured by a 6-h [3H]thymidine incorporation. All samples were run in triplicate. WT, wild type. Error bars represent SEM.

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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-γ 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.

FIGURE 6.

Fixation of CpG ODN-treated DCs blocks their ability to induce enhanced activation of MHC class I-restricted T cells. DCs were treated with CpG ODN (5 μg/ml) for 12 h and pulsed with SIINFEKL (20 ng/ml) for 12 h. Some DCs were then fixed in 1% paraformaldehyde and washed extensively. DCs (5 × 103) were cultured for 48 h with T cells (5 × 105) obtained from spleens of OT-1 mice that have TCRs that recognize SIINFEKL-H2kb. IFN-γ production was measured in culture supernatants by a standard ELISA. All samples were run in triplicate. Error bars represent SEM.

FIGURE 6.

Fixation of CpG ODN-treated DCs blocks their ability to induce enhanced activation of MHC class I-restricted T cells. DCs were treated with CpG ODN (5 μg/ml) for 12 h and pulsed with SIINFEKL (20 ng/ml) for 12 h. Some DCs were then fixed in 1% paraformaldehyde and washed extensively. DCs (5 × 103) were cultured for 48 h with T cells (5 × 105) obtained from spleens of OT-1 mice that have TCRs that recognize SIINFEKL-H2kb. IFN-γ production was measured in culture supernatants by a standard ELISA. All samples were run in triplicate. Error bars represent SEM.

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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.

FIGURE 7.

IL-12 production contributes to the ability of CpG ODN-treated DCs to activate Ag-specific T cells. DCs obtained from wild-type C57BL/6 mice or IL-12−/− knockout mice were treated with CpG ODN (5 μg/ml) for 12 h and pulsed with soluble OVA (100 μg/ml) for 12 h. Washed DCs (5 × 103) were then cultured for 48 h with T cells (5 × 105) obtained from spleens of OT-1 mice that have TCRs that recognize SIINFEKL-H2kb. A, Proliferative response was measured by a 6-h pulse with [3H]thymidine. B, IFN-γ production was measured in culture supernatants by a standard ELISA. All samples were run in triplicate. Error bars represent SEM.

FIGURE 7.

IL-12 production contributes to the ability of CpG ODN-treated DCs to activate Ag-specific T cells. DCs obtained from wild-type C57BL/6 mice or IL-12−/− knockout mice were treated with CpG ODN (5 μg/ml) for 12 h and pulsed with soluble OVA (100 μg/ml) for 12 h. Washed DCs (5 × 103) were then cultured for 48 h with T cells (5 × 105) obtained from spleens of OT-1 mice that have TCRs that recognize SIINFEKL-H2kb. A, Proliferative response was measured by a 6-h pulse with [3H]thymidine. B, IFN-γ production was measured in culture supernatants by a standard ELISA. All samples were run in triplicate. Error bars represent SEM.

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Classic immunologic teaching states that intracellular proteins are processed and presented in class I MHC, and this results in a cellular response. In contrast, extracellular Ags are presented in class II MHC and lead to a humoral response. Increasing evidence suggests there is cross-talk between these two pathways and that exogenous proteins are processed for presentation via the same pathway described for conventional MHC class I-restricted cytosolic Ags (44, 45). The immune signals that regulate this cross-priming are not well understood. Understanding approaches that can enhance cross-priming could have a significant impact on other immunization strategies, including the development of cancer vaccines.

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-γ (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.

1

This work was supported by Grants R01CA74542 (to G.J.W. and C.E.D.) and T32 HL07344 (to T.L.W. and S.K.B.) from the National Institutes of Health.

4

Abbreviations used in this paper: CpG ODN, cytosine-phosphorothioate-guanine oligodeoxynucleotide; DC, dendritic cell; CM, culture medium.

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