Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-αβ and -γ, TNF-α, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8+ and CD4+ lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-αβ pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8+ CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4+ Th cells by ISS-based vaccines.

Immunostimulatory sequence (ISS)3 DNA, also known as CpG motifs, is a family of unmethylated CpG dinucleotides within a consensus sequence of 5′-pur-(pur or T)-CpG-pyr-pyr-3′ (1, 2). These sequences, which are common in certain bacterial and viral genomes but suppressed in mammalian genomes, have a broad range of stimulatory effects on NK cells, B cells, and APC such as dendritic cells (DC) (3, 4, 5). Stimulation results in the secretion of cytokines, typically type I IFNs (IFN-αβ), IFN-γ, TNF-α, IL-6, and IL-12, and up-regulation of surface molecules such as ICAM-1, CD40, B7-1 and -2, and MHC classes I and II (5, 6, 7). By these criteria, ISS DNA have very little direct effect on T cells. However, immunization with ISS-based vaccines results in the priming of two T cell functions: Ag-specific CTL activity and a Th1-biased immune response (6, 8). Immunization of wild-type (wt), CD4−/−, or MHC class II−/− mice with ISS oligodeoxynucleotide (ODN)-based vaccines results in similar levels of CTL activation (9), indicating that these vaccines efficiently prime CTL against exogenous Ags (cross-priming) (10) independently of Th cells. ISS-based vaccines appear to act upon APC to facilitate cross-presentation and promote the expression of costimulatory molecules, allowing them to directly prime naive CD8+ lymphocytes.

It is likely that the priming interaction between ISS-stimulated APC and CD4+ or CD8+ lymphocytes is mediated by the cytokines and surface molecules that are induced by ISS DNA. However, it is unclear whether there are differential costimulatory requirements for CTL vs Th1 priming, or whether there is a temporal sequence to these signals. It is possible that an early event is the lynchpin in the transition from innate to Ag-specific immunity. One key class of mediators that may anchor the adaptive response to ISS-based vaccines is IFN-αβ, which have a broad range of activities in both innate and adaptive immunities (11). Like ISS DNA, IFN-αβ promote maturation of APC as measured by increased expression of costimulatory molecules (12). In addition, proinflammatory cytokines, including IFN-γ and TNF-α, have been shown to modulate the expression of TAP in many tissues, including macrophages (13). TAP plays a critical role in the presentation of exogenous Ags (cross-presentation) in the context of MHC class I (14). Although a role for IFN-αβ in regulating TAP in APC has not been reported to date, IFN-β stimulation has been shown to increase the expression of TAP in human pulmonary epithelial cells (15). These findings suggest a link between IFN-αβ and the regulation of both costimulation and cross-presentation by APC, two essential functions for generating adaptive T cell activity against pathogens. ISS DNA promotes the secretion of IFN-αβ in both murine and human innate immunity (16, 17, 18) and appears to facilitate both costimulation and cross-presentation in priming T cell activity (9). This suggests that ISS DNA promotes priming interactions between APC and Ag-specific T cells via IFN-αβ-dependent mechanisms.

In this report, we demonstrate that ISS-ODN stimulation of APC regulates costimulatory molecules and TAP expression via IFN-αβ activity. The costimulation and cross-presentation mediated by this mechanism result in the efficient priming of both CTL and Th cell activity by ISS-based vaccines.

wt C57BL/6 and 129 mice, and CD28−/−, TAP1−/−, and IL-12p40−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). IFN-αβR−/− mice were purchased from B & K Universal (East Yorkshire, U.K.). CD40−/− mice were the gift of N. Phillips (University of Massachusetts, Amherst, MA) and H. Kikutani (Osaka University, Osaka, Japan). Animal protocols were approved by the University of California-San Diego Office of Animal Resources (San Diego, CA).

Single-stranded phosphorothioate ISS-ODN (ISS motif underlined) (sequence 5′-TGA CTG TGA ACG TTC GAG ATG A-3′) and mutated ODN (mODN; sequence 5′-TGA CTG TGA AGG TTG GAG ATG A-3′) were purchased from Tri-Link Biotechnology (San Diego, CA).

H-2b MHC class I-restricted peptides were purchased from PeptidoGenic (Fullerton, CA): OVA peptide, NH2-SIINFEKL-COOH; influenza virus nucleoprotein peptide (target control), NH2-ASNENMETM-COOH.

Murine thymoma EL4 cells were cultivated in RPMI (Irvine Scientific, Santa Ana, CA) supplemented with 10% (v/v) heat-inactivated FCS (Life Technologies, Gaithersburg, MD), 50 mM 2-ME (Sigma-Aldrich, St. Louis, MO), 2 mM l-glutamine (Cellgro, Herndon, VA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Pen/Strep; Cellgro) (RP10). Cells were grown in 250-ml tissue culture flasks (Falcon, Franklin Lakes, NJ) at 37°C in 5% CO2.

Mouse BM-DC were cultured as previously described (19). Briefly, bone marrow from IFN-αβR−/− or wt 129/SvEv mice was plated in petri dishes (Fisher, Pittsburgh, PA) at 2 × 105 cells/ml in RP10, a tissue culture medium preparation consisting of RPMI (Irvine Scientific) supplemented with 10% (v/v) FCS, supplemented with 5 ng/ml recombinant murine GM-CSF (BD PharMingen, La Jolla, CA). On day 3 an equal volume of RP10 and GM-CSF was added. On day 6 half the volume of RP10 and GM-CSF was replaced. The nonadherent cells were harvested on day 7. The resultant population consisted of ∼70–80% CD11c+ DC.

Day 7 BM-DC from wt or IFN-αβ R−/− mice were incubated with medium or ISS (1 μg/ml) for 48 h. All Abs were from BD PharMingen. The DC were resuspended in FACS buffer (RPMI medium without phenol red, supplemented with 3% FCS and 0.02% sodium azide) and Fc block (BD PharMingen) for 10 min before staining for 30 min with FITC-labeled anti-CD11c (clone HL3), PE-labeled anti-B7-1 (clone 16-10A1), PE-labeled anti-B7-2 (clone GL1), PE-labeled anti-CD40 (clone 3/23), PE-labeled anti-H-2Kb (clone AF6-88.5), PE-labeled anti-I-Ab (clone M5114.15.2), or isotype control Abs. The cells were then washed and analyzed on a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). Data analysis was performed using FlowJo 3.4 software (Treestar, San Carlos, CA).

T cell subsets were quantified by the following method. Splenocytes were recovered, and RBC were lysed by incubation in 1.0 ml ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, and 0.1 mM Na2EDTA (pH 7.2)) for 5 min at room temperature. Splenocytes (∼106 cells/sample) were washed and resuspended in FACS buffer containing Fc block (BD PharMingen). After 10 min at 4°C anti-CD4-PE (clone RM25043; Caltag Laboratories, San Francisco, CA), anti-CD8 FITC (clone RM2201; Caltag Laboratories), or isotype control Abs were added. After a 30-min incubation at 4°C, dead cells were stained by addition of propidium iodide at 5 μg/ml for 5 min. The cells were then washed and analyzed on a FACSCalibur flow cytometer (BD Biosciences).

Bone marrow from wt C57BL/6 mice was washed and resuspended at 2 × 105/ml in DMEM high glucose (Irvine Scientific) with 10% (v/v) heat-inactivated FCS (Life Technologies), 2 mM l-glutamine (Cellgro), 100 U/ml penicillin, and 100 μg/ml streptomycin (Pen/Strep; Cellgro), and 30% (v/v) L cell-conditioned medium. These were cultivated for 6–8 days in petri dishes (Fisher) at 37°C in 5% CO2. BM-DM were harvested, replated at 5 × 105 cells/ml in six-well tissue culture plates (Costar, Cambridge, MA), and stimulated 10 μg/ml ISS-ODN or mODN. Stimulated BM-DM were harvested, and total RNA was recovered for FACS analysis or RT-PCR.

Female 129 mice were injected i.v. with 20 μg ISS-ODN or mODN in sterile saline. Mice were sacrificed at the indicated time intervals, and total splenocytes were recovered for RNA isolation and RT-PCR.

The following primers were used: TAP1: sense, 5′-CGG ACT CCA ACC ATG GAG GAA ATC ACA G-3′; antisense, 5′-TCA GTC TGC AGG AGC CGC AAG AGC C-3′; TAP2: sense, 5′-CAG GCG GCC TGT GCA GAC GAC TTC AT-3′; antisense, 5′-TCA TGC CTC CAG CCG CTG CTG TAC CAG GT-3′; and G3PDH: sense, 5′-ACC ACA GTC CAT GCC ATC AC-3′; antisense, 5′-TCC ACC ACC CTG TTG CTGTA-3′ (Integrated DNA Technologies, Coralville, IA). Total RNA isolated from BM-DM or splenocytes was analyzed by RT-PCR using a Superscript preamplification system (Life Technologies) and Advantaq Plus DNA polymerase (Clontech Laboratories, Palo Alto, CA) according to the manufacturer’s instructions. Amplification was conducted at 94°C for 30 s and at 68°C for 1 min in a GeneAmp 9600 thermal cycler (PerkinElmer/Cetus, Norwalk, CT; 18 cycles for G3PDH and 24 cycles for TAP1 and -2).

Total bone marrow was harvested from TAP1−/− and wt C57BL/6 donors and washed. BM cells were depleted of mature T cells by resuspension at 30 × 106/ml in RP2 containing anti-Thy1 mAb (clone YTS 154), anti-CD4 mAb (clone RL172), and anti-CD8 mAb (clone 3.155) on ice and then lysed with batch-tested guinea pig complement (Pel-Freez Biologicals, Rogers, AR) at a 1/10 dilution and 10 μg/ml DNase I (Sigma) at 37°C as previously described (8). Cells were washed and resuspended at 107/100 μl (108/ml) in RPMI, and 107 cells were injected i.v. into lethally irradiated recipient C57BL/6 mice. The mice were rested for 8 wk, then engraftment was verified by FACS analysis of peripheral blood. The chimeric mice were then vaccinated as described in Vaccination protocol.

The test animals were vaccinated according to a previously described protocol (9). Briefly, wt animals received mAb blockade (no azide, low endotoxin formulation) i.p. 6 h before vaccination. Doses were 200 μg for anti-CD40 ligand (anti-CD40L; clone MR1, BD PharMingen) and 100 μg each for anti-CD80 (clone 16-10A1; BD PharMingen) and CD86 (clone PO3; BD PharMingen). Ab-treated and gene-deficient mice received 50 μg OVA (Sigma-Aldrich) and 50 μg ISS-ODN (Tri-Link) in sterile saline intradermally at the tail base on days 0 and 14. Control mice received 50 μg OVA alone intradermally or no treatment. Three days before sacrifice, mice received an i.v. boost of 50 μg OVA (Sigma-Aldrich) in sterile saline. Animals were sacrificed at 6 wk, and total splenocytes were recovered for FACS analysis, secondary CTL, and cytokine restimulation assays.

CTL activity was assayed as previously described (9). Briefly, 2 × 106 effector splenocytes were restimulated in culture for 5 days with 1.8 × 107 OVA peptide-pulsed stimulator splenocytes and 50 U/ml recombinant human IL-2 (BD PharMingen) in RP10. After restimulation, viable lymphocytes were recovered by centrifugation over Ficoll Lympholyte M (Cedarlane Laboratories, Hornby, Canada) and washed, then serially diluted to several E:T cell ratios in 96-well U-bottom culture plates (Costar) in colorless RPMI (Irvine Scientific) supplemented with 2% BSA (Sigma-Aldrich), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Target EL4 cells were pulsed with OVA or nucleoprotein peptide at 37°C for 1 h, washed, and added to effector cells. Plates were incubated for 4 h, and supernatants were recovered. Specific lysis was assayed with the CytoTox 96 kit (Promega, Madison, WI) according to the manufacturer’s instructions.

Cytokine restimulation was assayed as previously described (9). Purified rat anti-mouse IFN-γ capture Ab and purified, biotinylated rat anti-mouse IFN-γ-detecting Ab were purchased from BD PharMingen. Briefly, splenocytes were isolated as described and incubated for 3 days with or without 50 μg/ml OVA (Sigma-Aldrich) restimulation in 96-well plates (Costar). Aliquots of tissue culture supernatant were removed for cytokine ELISA. Half-area 96-well plates (Costar) were coated with capture Ab diluted 1/1000 in carbonate buffer (15 mM Na2CO3 and 35 mM NaHCO3 (pH 9.6)) overnight at 4°C. Plates were washed with 1× BBS (160 mM NaCl, 40 mM NaOH, and 200 mM boric acid (pH 8.0)) and then blocked for 2 h at 37°C with blocking buffer (1% BSA in BBS). Plates were washed and incubated with tissue culture supernatants diluted 1/2 in blocking buffer overnight at 4°C. Plates were washed and incubated with detecting Ab diluted 1/1000 in blocking buffer at room temperature for 1 h. Plates were washed and incubated with streptavidin-HRP conjugate (Zymed Laboratories, South San Francisco, CA) diluted 1/2000 in blocking buffer at room temperature for 1 h. Plates were washed and incubated with TMB substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The reaction was stopped with 1 M phosphoric acid (Sigma-Aldrich), and the plates were read at 450 nm on a ThermoMax microplate reader (Molecular Devices, Menlo Park, CA).

To examine the role of IFN-αβ in regulating costimulatory molecules, BM-DC from wt and IFN-αβR−/− mice were stimulated with ISS-ODN in vitro and analyzed by FACS. wt BM-DC stimulated with ISS-ODN increased surface expression of CD40, B7-1 and -2, and MHC class I (Fig. 1,A, top row). In contrast, only a subpopulation of BM-DC from IFN-αβR−/− mice was capable of up-regulating CD40 and B7 in response to ISS-ODN stimulation (Fig. 1 A, bottom row). IFN-αβR−/− BM-DC did not up-regulate MHC class I under these conditions. This result indicated that ISS-ODN signaling via IFN-αβ promoted expression of these costimulatory molecules on the majority of BM-DC.

FIGURE 1.

ISS-ODN regulates the expression of costimulatory molecules and TAP via IFN-αβ. A, BM-DC from wt 129 and IFN-αβR−/− mice were prepared and stimulated as described in Materials andMethods. Expressions of B7-1 and -2, CD40, MHC class I (H-2Kb), and MHC class II (I-Ab) were quantified by FACS gated on CD11c+ cells. The mean fluorescence of the unstimulated (dashed lines) and stimulated (solid lines) cells are depicted in each histogram. Isotype control staining of unstimulated cells is depicted in the shaded curve of the upper left panel (wt BM-DC, anti-B7-1) and is representative of control staining in all groups depicted. Results are quantified in each histogram as the mean fluorescence intensity ratio (MFIR), defined as MFIR = MF (cells + Ag-specific mAb)/MF (cells + isotype control Ab) (7 ). Results are displayed in each histogram as MFIR (unstimulated cells)/MFIR (ISS-stimulated cells). B, BM-DC from wt and IFN-αβR−/− mice were stimulated with ISS-ODN and mODN in vitro as described in Materials and Methods, and total RNA was analyzed by RT-PCR (leftcolumn). RT-PCR with primers specific for G3PDH is shown for comparison. wt 129 and IFN-αβR−/− mice were stimulated in vivo with ISS-ODN or mODN (right column). These mice were sacrificed, and total splenocytes were recovered as described in Materials and Methods. RT-PCR was performed with primers specific for TAP1 and G3PDH. Results shown are representative of three experiments each.

FIGURE 1.

ISS-ODN regulates the expression of costimulatory molecules and TAP via IFN-αβ. A, BM-DC from wt 129 and IFN-αβR−/− mice were prepared and stimulated as described in Materials andMethods. Expressions of B7-1 and -2, CD40, MHC class I (H-2Kb), and MHC class II (I-Ab) were quantified by FACS gated on CD11c+ cells. The mean fluorescence of the unstimulated (dashed lines) and stimulated (solid lines) cells are depicted in each histogram. Isotype control staining of unstimulated cells is depicted in the shaded curve of the upper left panel (wt BM-DC, anti-B7-1) and is representative of control staining in all groups depicted. Results are quantified in each histogram as the mean fluorescence intensity ratio (MFIR), defined as MFIR = MF (cells + Ag-specific mAb)/MF (cells + isotype control Ab) (7 ). Results are displayed in each histogram as MFIR (unstimulated cells)/MFIR (ISS-stimulated cells). B, BM-DC from wt and IFN-αβR−/− mice were stimulated with ISS-ODN and mODN in vitro as described in Materials and Methods, and total RNA was analyzed by RT-PCR (leftcolumn). RT-PCR with primers specific for G3PDH is shown for comparison. wt 129 and IFN-αβR−/− mice were stimulated in vivo with ISS-ODN or mODN (right column). These mice were sacrificed, and total splenocytes were recovered as described in Materials and Methods. RT-PCR was performed with primers specific for TAP1 and G3PDH. Results shown are representative of three experiments each.

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IFN-β has been shown to modulate the expression of TAP (15). To investigate the effect of ISS-ODN on TAP expression in APC and the potential role of IFN-αβ in this activity, BM-DC from wt and IFN-αβR−/− mice were treated in vitro with ISS-ODN or mODN that did not contain the CpG motif, and total RNA was isolated at several time points. RT-PCR demonstrated that ISS-ODN treatment increased the transcription of TAP1 mRNA ∼4 h after stimulation, whereas mODN treatment did not (Fig. 1,B, first column). Real-time semiquantitative RT-PCR demonstrated a 1.5-fold increase in TAP1 transcription at 4 h in ISS-ODN-stimulated BM-DC, escalating to a 1.9-fold increase at 6 h (data not shown). TAP1 transcription did not increase in response to ISS-ODN in BM-DC from IFN-αβR−/− mice, suggesting that IFN-αβ mediated this activity as well. This result is consistent with the absence of augmented MHC class I expression in IFN-αβR−/− BM-DC following ISS-ODN stimulation (Fig. 1 A).

ISS-ODN and IFN-αβ also regulated TAP expression in vivo. wt and IFN-αβR−/− mice were treated by i.v. injection of ISS-ODN or mODN and were sacrificed at subsequent time points. Splenocytes were isolated, and total RNA was analyzed by RT-PCR. mRNA for TAP1 increased at 2 h in response to ISS-ODN, but not mODN, and this effect was sustained up to 6 h (Fig. 1 B, second column). In contrast, TAP1 was not up-regulated in splenocytes from IFN-αβR−/− mice.

ISS-ODN appeared to regulate the expression of TAP and costimulatory molecules via IFN-αβ. To examine the role of IFN-αβ in priming of CTL by ISS-based vaccines, wt 129 and IFN-αβR−/− mice were immunized with OVA and ISS-ODN, and OVA-specific CTL activity was measured. wt mice demonstrated efficient priming of OVA-specific CTL activity (Fig. 2,A). IFN-αβR−/− mice exhibited a 41–88% reduction in CTL activity between 50:1 and 2:1 E:T cell ratios. IFN-αβR−/− mice treated with mAb blockade against B7-1 and -2 before immunization showed a similar reduction in CTL priming, indicating that the observed reduction was due to the absence of IFN-αβ signaling. The IFN-αβR−/− and wt mice had similar CD4+ and CD8+ T cell subsets, as determined by FACS analysis (Table I); therefore, the observed differences were not an artifact due to reduction of the CD8+ population. These data demonstrated that IFN-αβ played a critical role and acted upstream of B7 costimulation in priming of CTL by ISS-based vaccines.

FIGURE 2.

Cross-priming of CTL activity by ISS-based vaccines is dependent upon B7 costimulation and TAP-dependent cross-presentation. wt and gene-deficient mice (with or without mAb blockade) were immunized, and total splenocytes were recovered for secondary CTL assay as described in Materials and Methods. A, IFN-αβR−/− (•) mice pretreated with isotype control Ab showed a significant decrease in priming OVA-specific CTL activity compared with 129 wt mice (▴). This decrease was not affected by addition of anti-B7-1 and -2 mAb blockade (▪). Untreated wt 129 mice did not exhibit OVA-specific CTL activity (♦). Target cells loaded with an irrelevant H-2b-restricted peptide derived from influenza nucleoprotein were not lysed, indicating that CTL activity was Ag specific (data not shown). B, C57BL6 wt→wt bone marrow chimeras (▪) primed similar levels of OVA-specific CTL activity as cells from wt mice (▴). In contrast, TAP−/− → wt bone marrow chimeras (•) did not prime detectable CTL activity, indicating that TAP activity is an absolute requirement for cross-priming by ISS-based vaccines. CD4 and CD8 T cell subsets were similar in the chimeric groups, indicating that the observed difference was not due to a failure of CD8 development (Table I). Untreated wt C57BL6 mice did not exhibit OVA-specific CTL activity (♦). C, CD40−/− (•) and wt mice treated with mAb blockade against CD40L (▪) primed similar levels of OVA activity compared with wt mice (▴), indicating that cross-priming by ISS-based vaccines bypassed CD40-dependent Th cell activity. wt animals immunized with OVA alone (▾) did not exhibit OVA-specific CTL activity, nor did untreated negative control mice (♦). D, Wild-type mice treated with mAb blockade against B7-1 and -2 (•) and CD28−/− mice (▪) primed similar levels of OVA-specific CTL activity that were significantly lower compared with wt animals (▴). Addition of anti-CD40L mAb blockade to either group (○ and □) did not further decrease CTL activity, indicating that B7/CD28 mediated the dominant costimulatory interaction between APC and naive CTL. E, IL-12p40−/− mice (•) exhibited an intermediate reduction of OVA-specific CTL activity compared with wt controls (▴). However, addition of mAb blockade against B7 and CD40L (▪) resulted in CTL activity comparable to that observed in wt mice treated with mAb blockade of B7 alone or CD28−/− mice. Results are shown as the average specific lysis ± SEM and represent four to six mice per group.

FIGURE 2.

Cross-priming of CTL activity by ISS-based vaccines is dependent upon B7 costimulation and TAP-dependent cross-presentation. wt and gene-deficient mice (with or without mAb blockade) were immunized, and total splenocytes were recovered for secondary CTL assay as described in Materials and Methods. A, IFN-αβR−/− (•) mice pretreated with isotype control Ab showed a significant decrease in priming OVA-specific CTL activity compared with 129 wt mice (▴). This decrease was not affected by addition of anti-B7-1 and -2 mAb blockade (▪). Untreated wt 129 mice did not exhibit OVA-specific CTL activity (♦). Target cells loaded with an irrelevant H-2b-restricted peptide derived from influenza nucleoprotein were not lysed, indicating that CTL activity was Ag specific (data not shown). B, C57BL6 wt→wt bone marrow chimeras (▪) primed similar levels of OVA-specific CTL activity as cells from wt mice (▴). In contrast, TAP−/− → wt bone marrow chimeras (•) did not prime detectable CTL activity, indicating that TAP activity is an absolute requirement for cross-priming by ISS-based vaccines. CD4 and CD8 T cell subsets were similar in the chimeric groups, indicating that the observed difference was not due to a failure of CD8 development (Table I). Untreated wt C57BL6 mice did not exhibit OVA-specific CTL activity (♦). C, CD40−/− (•) and wt mice treated with mAb blockade against CD40L (▪) primed similar levels of OVA activity compared with wt mice (▴), indicating that cross-priming by ISS-based vaccines bypassed CD40-dependent Th cell activity. wt animals immunized with OVA alone (▾) did not exhibit OVA-specific CTL activity, nor did untreated negative control mice (♦). D, Wild-type mice treated with mAb blockade against B7-1 and -2 (•) and CD28−/− mice (▪) primed similar levels of OVA-specific CTL activity that were significantly lower compared with wt animals (▴). Addition of anti-CD40L mAb blockade to either group (○ and □) did not further decrease CTL activity, indicating that B7/CD28 mediated the dominant costimulatory interaction between APC and naive CTL. E, IL-12p40−/− mice (•) exhibited an intermediate reduction of OVA-specific CTL activity compared with wt controls (▴). However, addition of mAb blockade against B7 and CD40L (▪) resulted in CTL activity comparable to that observed in wt mice treated with mAb blockade of B7 alone or CD28−/− mice. Results are shown as the average specific lysis ± SEM and represent four to six mice per group.

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Table I.

T cell subsets after immunization with ISS-based vaccinesa

GroupCD4+ (%)CD8+ (%)
wt, no treatment 17.4 ± 1.2 11.3 ± 0.5 
TAP1−/−→wt chimera 17.6 10.3 
wt→wt chimera 18.1 11.1 
CD40−/− 22.9 ± 1.7 12.9 ± 0.4 
wt, anti-CD40L mAb 17.5 ± 0.4 11.9 ± 0.6 
wt, anti-B7 mAbs 19.2 ± 0.6 10.4 ± 0.1 
wt, isotype control 19.5 ± 0.7 11.7 ± 0.6 
IL-12−/− 18.5 ± 1.0 11.9 ± 1.0 
IL-12−/−, anti-B7 and anti-CD40L mAbs 19.8 ± 0.5 12.0 ± 0.4 
IFN-α/βR−/− 25.6 ± 0.8 12.5 ± 0.3 
IFN-α/βR−/−+ anti-B7 mAb 23.5 ± 0.6 11.4 ± 0.4 
wt, OVA+ ISS-ODN 21.2 ± 1.1 12.6 ± 0.3 
GroupCD4+ (%)CD8+ (%)
wt, no treatment 17.4 ± 1.2 11.3 ± 0.5 
TAP1−/−→wt chimera 17.6 10.3 
wt→wt chimera 18.1 11.1 
CD40−/− 22.9 ± 1.7 12.9 ± 0.4 
wt, anti-CD40L mAb 17.5 ± 0.4 11.9 ± 0.6 
wt, anti-B7 mAbs 19.2 ± 0.6 10.4 ± 0.1 
wt, isotype control 19.5 ± 0.7 11.7 ± 0.6 
IL-12−/− 18.5 ± 1.0 11.9 ± 1.0 
IL-12−/−, anti-B7 and anti-CD40L mAbs 19.8 ± 0.5 12.0 ± 0.4 
IFN-α/βR−/− 25.6 ± 0.8 12.5 ± 0.3 
IFN-α/βR−/−+ anti-B7 mAb 23.5 ± 0.6 11.4 ± 0.4 
wt, OVA+ ISS-ODN 21.2 ± 1.1 12.6 ± 0.3 
a

CD4+ and CD8+ subsets of total splenocytes for test animals were determined by FACS analysis as described in Materials and Methods. Percentages of total splenocytes are expressed as the average ± SEM for four to six mice per group, except for TAP1−/−→wt and wt→wt chimeras, in which the percentages were derived from a single representative mouse from each group.

TAP activity is essential in certain animal models of cross-priming against pathogen-derived Ags (14, 20). To examine the role of TAP in cross-presentation by ISS vaccines, TAP1−/− into wt (TAP−/−→wt) and wt→wt bone marrow chimeras were generated. The chimeras were necessary because TAP1−/− mice do not support the native development of CD8+ CTL (21). TAP1−/−→wt chimeras did not prime CTL activity in response to OVA and ISS-ODN vaccination, whereas wt→wt chimeras did, demonstrating that TAP was essential for ISS-induced cross-presentation (Fig. 2,B). The chimeric and wt mice also had similar T cell subsets (Table I).

Current models of cross-priming CTL activity against exogenous protein Ags posit a “licensing” step between APC and CD4+ Th cells, followed by an activation step between licensed APC and naive CD8+ lymphocytes. The licensing step between CD4+ T cells and APC requires CD40/CD40L signaling (22, 23, 24), but the costimulatory requirements for the activation interaction between licensed APC and naive CD8+ lymphocytes are unknown. As noted previously, ISS-based vaccines can bypass CD4+ Th activity in priming CTL (9), allowing evaluation of the activation interaction independent of the licensing step. To examine the role of CD40, wt and CD40−/− mice were immunized with OVA and ISS-ODN. wt mice were also treated with blocking mAb to CD40L or isotype control before immunization. wt mice immunized with OVA and ISS-ODN demonstrated efficient priming of OVA-specific CTL activity (Fig. 2,C). Lytic activity from splenocytes of CD40−/− mice and wt mice treated with anti-CD40L mAb did not differ significantly from that of wt mice. Treatment with an isotype control mAb did not affect CTL activation (data not shown). The T cell subsets were also similar among these groups (Table I). These data indicate that CD40/CD40L signaling did not make a significant contribution to the priming interaction between APC and CD8+ lymphocytes in response to ISS-based vaccines.

B7-1 and -2 have been shown to play a critical role in priming of CTL activity in vivo (25). To examine the role of these molecules in ISS-based vaccination, wt mice were treated with mAb blockade to B7-1 and -2 before immunization with OVA and ISS-ODN. Both B7 molecules were blocked, because previous studies demonstrated that either signal alone can support the priming of CTL activity (25). Mice deficient in CD28, the positive signaling receptor for the B7 molecules (26), were also immunized. Treatment with anti-B7 mAb or immunization of CD28−/− mice resulted in a 52–80% reduction (between 25:1 and 1:1 E:T ratios) in CTL activity compared with that in wt mice, strongly suggesting that B7 signaling through CD28 provided critical costimulation for CTL priming (Fig. 2 D). Addition of anti-CD40L mAb did not further reduce CTL activity, supporting the findings in the previous section.

IL-12 is a proinflammatory cytokine that has been shown to promote CTL proliferation (27). Both ISS DNA and IFN-α have also been shown to directly up-regulate IL-12 expression in APC (6). Production of IL-12 by APC can also be stimulated by CD40/CD40L and B7/CD28 interactions (28, 29). To determine the role of IL-12 signaling in priming of CTL activity, wt and IL-12p40−/− animals were immunized with OVA and ISS-ODN, with one group of IL-12p40−/− mice receiving anti-B7 and anti-CD40L mAb blockade. IL-12p40−/− mice exhibited a 35% reduction in CTL activation compared with wt mice at a 25:1 E:T cell ratio (Fig. 2,E). The addition of blocking mAb against B7 and CD40L resulted in an additional 46% reduction in CTL activation. This CTL activity was comparable to mAb blockade of B7 alone (Fig. 2 D), suggesting that B7 was the dominant costimulation in priming of CTL by ISS-based vaccines. In this setting, IL-12 appeared to make a nonsynergistic contribution to CTL priming in response to ISS-based vaccines.

Priming of Th1-biased CD4+ helper activity also exhibited dependence upon autocrine IFN-αβ. Mice were immunized as described in the previous sections, and splenocytes were isolated and restimulated in culture with OVA. IFN-γ secretion was quantified by ELISA as a measure of Th1-biased immune responses. Splenocytes from immunized IFN-αβR−/− mice secreted 62% less IFN-γ (Fig. 3, bar B) in restimulation assays compared with wt controls (Fig. 3, 129 (+) control). IFN-αβR−/− mice treated with mAb blockade against B7 molecules exhibited similarly reduced levels of IFN-γ (Fig. 3, bar A), suggesting that IFN-αβ also acted upstream of B7 costimulation in CD4+ priming. Thus, priming of Th1-biased CD4+ lymphocyte activity by ISS-based vaccines appeared to be partially dependent upon IFN-αβ, consistent with its regulation of CD40 and B7 expression (Fig. 1). Splenocytes from TAP→wt and wt→wt bone marrow chimeras produced similar levels of IFN-γ in restimulation assays (Fig. 3, bars C and D, respectively), indicating that TAP did not make a significant contribution to Th1 priming. It is likely that total body irradiation, adoptive transfer of bone marrow, and older age of the chimeric animals contributed to the lower overall IFN-γ secretion compared with wt (Fig. 3, B6 (+) control).

FIGURE 3.

Priming of Th1-biased CD4+ lymphocyte activity by ISS-based vaccines via IFN-αβ. Total splenocytes from wt, chimeric, gene-deficient, and mAb blockade-treated mice were isolated as described in the text, and IFN-γ secretion was analyzed by cytokine restimulation assay. 129 (+) control, wt 129 immunized with ISS-ODN, and OVA. A, IFN-αβR−/− and anti-B7 mAb blockade. B, IFN-αβR−/− and isotype control Ab. B6 (+) control, wt C57BL/6 immunized with ISS-ODN and OVA. C, TAP−/−→wt BM chimera. D, wt→wt BM chimera. E, CD40−/−. F, wt and anti-CD40L mAb blockade. G, wt and anti-B7 mAb blockade. H, wt and anti-B7 and anti-CD40L mAb blockade. I, CD28−/−. J, CD28−/− and anti-CD40L mAb blockade. K, IL-12p40−/−. L, IL-12 p40−/− and anti-B7 mAb blockade. M, wt immunized with OVA alone. (−) control, untreated wt C57BL6. Results are shown as the average IFN-γ concentration ± SEM and represent four to six mice per group.

FIGURE 3.

Priming of Th1-biased CD4+ lymphocyte activity by ISS-based vaccines via IFN-αβ. Total splenocytes from wt, chimeric, gene-deficient, and mAb blockade-treated mice were isolated as described in the text, and IFN-γ secretion was analyzed by cytokine restimulation assay. 129 (+) control, wt 129 immunized with ISS-ODN, and OVA. A, IFN-αβR−/− and anti-B7 mAb blockade. B, IFN-αβR−/− and isotype control Ab. B6 (+) control, wt C57BL/6 immunized with ISS-ODN and OVA. C, TAP−/−→wt BM chimera. D, wt→wt BM chimera. E, CD40−/−. F, wt and anti-CD40L mAb blockade. G, wt and anti-B7 mAb blockade. H, wt and anti-B7 and anti-CD40L mAb blockade. I, CD28−/−. J, CD28−/− and anti-CD40L mAb blockade. K, IL-12p40−/−. L, IL-12 p40−/− and anti-B7 mAb blockade. M, wt immunized with OVA alone. (−) control, untreated wt C57BL6. Results are shown as the average IFN-γ concentration ± SEM and represent four to six mice per group.

Close modal

In contrast to cross-priming of CTL activity, priming of Th1-biased CD4+ lymphocytes was not preserved if CD40/CD40L signaling was disrupted. Splenocytes from CD40−/− mice demonstrated an 83% reduction in IFN-γ secretion (Fig. 3, bar E), and splenocytes from wt mice treated with anti-CD40L mAb blockade demonstrated a 59% reduction (Fig. 3, bar F) compared with wt controls (Fig. 3, B6 (+) control). mAb blockade of B7 resulted in an 89% reduction in IFN-γ secretion (Fig. 3, bar G), and CD28−/− splenocytes had a 92% reduction (Fig. 3, bar I). Splenocytes from CD28−/− mice treated with anti-CD40L mAb demonstrated complete inhibition of IFN-γ secretion (Fig. 3, bar J). Splenocytes from IL-12p40−/− mice secreted ∼2% IFN-γ compared with wt (bar K), and this was not affected by mAb blockade of B7 (Fig. 3, bar L).

Interestingly, mAb blockade of CD40L on CD4+ lymphocytes was not equivalent to the absence of CD40 on APC (Fig. 3, bars E and F). The addition of anti-CD40L mAb blockade to either anti-B7 mAb blockade (Fig. 3, bar H) or IL-12p40−/− mice (Fig. 3, bar L) resulted in higher levels of IFN-γ secretion than either condition alone, but the addition of anti-CD40L to CD28−/− mice (Fig. 3, bar J) reduced IFN-γ secretion from 8% of the control value to zero. These data suggest that engagement of the blocking mAb to CD40L may transduce partial positive signaling to the CD4+ cell, and this stimulation is dependent upon the presence of intact CD28.

In this report, we demonstrate that ISS-based vaccines act upon APC via a novel IFN-αβ mechanism to regulate the expression of costimulatory molecules and TAP. These molecules then mediate the priming interactions between APC and naive CD4+ and CD8+ lymphocytes via cell-to-cell interactions and cross-presentation of Ag in the context of MHC class I. Priming of CD8+ CTL by ISS-based vaccines requires TAP activity and B7, and costimulation is partially dependent upon IL-12 and is independent of CD40. In contrast, priming of CD4+ Th1 lymphocytes requires CD40, B7, and IL-12 costimulation and is independent of TAP activity.

Priming of Ag-specific CTL by ISS-based vaccines is independent of CD4+ T helper activity (9). The data presented in this study suggest the following sequence of events in this activity (depicted in the right half of Fig. 4). First, ISS-ODN and Ag are acquired by the APC. ISS-ODN promotes autocrine signaling via IFN-αβ (Fig. 4, arrow 1), which up-regulates TAP (Fig. 4, arrow 2) and B7 expression (Fig. 4, arrow 3). TAP-dependent cross-presentation of exogenous Ag in the context of MHC class I (Fig. 4, arrow 4) in conjunction with B7 costimulation results in priming of naive CTL. Although IL-12 appears to make a contribution to CTL priming (Fig. 4, arrow 5), it is unclear whether this is a direct effect on CTL. Because the experiments reported in this study were conducted in animals with an intact CD4+ compartment, it is possible that IL-12 acts via priming of Th cells to promote B7 up-regulation rather than by directly affecting CTL. The enhanced inhibition of CTL priming observed in IL-12p40−/− mice treated with anti-B7 mAb blockade supports this hypothesis. Further investigation will be required to discern among these possibilities.

FIGURE 4.

Proposed mechanism of CD4+ and CD8+ T cell priming by ISS-based vaccines. Cross-priming CD8+ CTL activity (right half). 1, ISS-ODN and Ag are acquired by APC and ISS stimulates autocrine IFN-αβ. 2, Autocrine IFN-αβ up-regulates TAP, which promotes cross-presentation of exogenous Ag in the context of MHC class I. 3, Autocrine IFN-αβ also up-regulates B7 costimulation. 4, Cross-presentation of exogenous Ag and B7 costimulation primes naive CD8+ cells, resulting in Ag-specific CTL activity. 5, ISS-ODN also promotes IL-12 secretion, either directly or via IFN-αβ. IL-12 appears to contribute to CTL priming, although it is unclear whether this is a direct effect or an indirect one via CD4+ T cell help. Priming of Th1-biased CD4+ T helper activity (left half). 1, ISS-ODN and Ag are acquired by APC and ISS stimulates autocrine IFN-αβ. 6, Autocrine IFN-αβ up-regulates CD40 and B7 costimulatory molecules on APC. 7, Engagement of costimulatory molecules promotes secretion of IL-12, which acts upon CD4+ Th cells in a paracrine fashion to mature Th1-biased helper activity. Differences in the requirements and observed responses between CD4+ and CD8+ cell priming suggest that different APC populations may mediate these activities.

FIGURE 4.

Proposed mechanism of CD4+ and CD8+ T cell priming by ISS-based vaccines. Cross-priming CD8+ CTL activity (right half). 1, ISS-ODN and Ag are acquired by APC and ISS stimulates autocrine IFN-αβ. 2, Autocrine IFN-αβ up-regulates TAP, which promotes cross-presentation of exogenous Ag in the context of MHC class I. 3, Autocrine IFN-αβ also up-regulates B7 costimulation. 4, Cross-presentation of exogenous Ag and B7 costimulation primes naive CD8+ cells, resulting in Ag-specific CTL activity. 5, ISS-ODN also promotes IL-12 secretion, either directly or via IFN-αβ. IL-12 appears to contribute to CTL priming, although it is unclear whether this is a direct effect or an indirect one via CD4+ T cell help. Priming of Th1-biased CD4+ T helper activity (left half). 1, ISS-ODN and Ag are acquired by APC and ISS stimulates autocrine IFN-αβ. 6, Autocrine IFN-αβ up-regulates CD40 and B7 costimulatory molecules on APC. 7, Engagement of costimulatory molecules promotes secretion of IL-12, which acts upon CD4+ Th cells in a paracrine fashion to mature Th1-biased helper activity. Differences in the requirements and observed responses between CD4+ and CD8+ cell priming suggest that different APC populations may mediate these activities.

Close modal

In contrast, the priming of CD4+ T helper activity appears to have different costimulatory requirements. ISS-ODN stimulates autocrine IFN-αβ (Fig. 4, arrow 1), which, in turn, up-regulates CD40 and B7 costimulatory molecules (Fig. 4, arrow 6). ISS-ODN also up-regulates IL-12 expression (Fig. 4, arrow 7), and this may be a direct effect of ISS-ODN or may also be mediated by autocrine IFN-αβ (6). Priming of Th1-type responses (depicted in the left half of Fig. 4) is achieved by cognate interaction between the TCR of naive CD4+ Th cells and Ag in the context of MHC class II and receptor-ligand interactions between CD40/CD40L, B7/CD28, and IL-12/IL-12R. CD40- and B7-dependent signaling is known to enhance IL-12 production, which may provide amplification of the stimulatory signal (29, 30). These costimulatory paths act upon CD4+ lymphocytes to promote Th1-type priming. As expected, CD4+ lymphocyte priming is not dependent upon TAP.

TAP activity is required for cross-presentation and CTL priming by ISS-based vaccines. The data presented in this study appear to be the first report of regulation of TAP expression in APC by IFN-αβ, suggesting a potential mechanism for facilitation of cross-presentation of exogenous Ags by ISS-based vaccines. Cytokine-induced increases in TAP expression have been shown to promote Ag presentation and stabilization of MHC class I complexes (31, 32). These findings suggest that ISS-ODN facilitates cross-presentation at least in part by regulation of TAP expression via IFN-αβ-dependent signals.

It has been reported that IFN-αβ secreted by ISS-stimulated APC can act directly upon T cells to induce partial activation (33). However, our data show that costimulatory molecules and TAP-dependent cross-presentation act downstream of IFN-αβ, because interruption of these activities in animals with intact IFN-αβ signaling prevents CTL and Th1 priming. The addition of anti-B7 mAb blockade does not enhance the reduction of CTL priming in IFN-αβR−/− mice, also indicating that costimulation acts downstream of IFN-αβ. Interestingly, IFN-αβ have been shown to act directly upon activated T cells to promote their survival (34). Thus, ISS-based vaccines may prime T cells via indirect IFN-αβ-dependent mechanisms and maintain T cell activity by a direct mechanism. The in vitro model presented demonstrates that autocrine IFN-αβ signaling can support the regulation of costimulatory molecules and TAP. However, ISS-ODN also stimulate the secretion of IFN-αβ by other cellular components of innate immunity. The more rapid induction of TAP in vivo strongly suggests that other sources of IFN-αβ may contribute to this activity. Further investigation is needed to determine whether autocrine or paracrine signaling predominates in vivo.

ISS-ODN is an ideal molecular probe for examining the cross-priming interaction between APC and naive CD8+ lymphocytes, because it achieves this activity independently of CD4+ T cell help. The results reported in this study provide further refinement of the licensing model of cross-priming, which invokes a three-cell interaction between Th cells, APC, and naive CD8+ lymphocytes (22, 23, 24). By directly up-regulating B7 and TAP, ISS-based vaccines bypass CD40-dependent T cell help. This model implies that a major function of Th-dependent licensing in cross-priming is the up-regulation of B7 expression on APC via CD40/CD40L interaction (35). Although this model explains many aspects of cross-priming, there are still unresolved issues that require further investigation. First, it is apparent that a subpopulation of IFN-αβR−/− BM-DC is capable of up-regulating costimulatory molecules in response to ISS-ODN stimulation. What are the signaling pathways that mediate this redundant activity? Second, do novel members of the CD40/CD40L (36, 37) and B7/CD28 (38) families of signaling molecules play roles in this system? Third, what are the interactions between IFN-αβ and the pathways critical to ISS DNA signaling, Toll-like receptor 9 (39), and DNA-PK (40)? Finally, do ISS-ODN have different activities on different subsets of DC, and how does this affect T cell priming?

ISS-based vaccines are uniquely suited to efficiently prime CTL activity due to their coordinated regulation of cross-presentation and costimulation. CTL priming by ISS-based immunization has been demonstrated for experimental Ags OVA and β-galactosidase (41) and for viral Ags HIV gp120 (41, 42) and hepatitis B virus surface Ag (43). This immunization strategy has already demonstrated efficacy in an animal model of cancer (9). Although the efficient priming of Ag-specific tumor immunity has been an elusive goal, ISS-based vaccines represent a promising new approach to this problem. Further investigation of the formulation of vaccines with naturally occurring tumor-associated Ags could result in effective cancer immunotherapy.

We thank Nancy Phillips and Hitoshi Kikutani for the generous gift of CD40−/− mice; Nina Bhardwaj, Carl Nathan, and Roy Silverstein for critical reading of the manuscript; Nancy Noon and Jane Uhle for their editorial assistance; and Minh-Duc Nguyen, Yucan Chiu, Abe Chang, Samantha Kim, Megan Anderson, and Nadya Cinman for their expert technical assistance.

1

This work was supported by National Institutes of Health Grants AI40682, AI47078, and AR44850 and by a grant from Dynavax Technologies (Berkeley, CA). H.J.C. was supported by a Research Resident Fellowship from The Sam and Rose Stein Institute for Research on Aging.

3

Abbreviations used in this paper: ISS, immunostimulatory sequence; DC, dendritic cell; BM-DC, bone marrow-derived DC; ODN, oligodeoxynucleotide; CD40L, CD40 ligand; mODN, mutated ODN; wt, wild type.

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