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The Journal of Immunology, 2005, 175: 2244-2251.
Copyright © 2005 by The American Association of Immunologists

Enhancement of Dendritic Cell Antigen Cross-Presentation by CpG DNA Involves Type I IFN and Stabilization of Class I MHC mRNA1

John Kuchtey*,§, Peter J. Chefalo*, Reginald C. Gray*,{dagger}, Lakshmi Ramachandra*,{ddagger} and Clifford V. Harding2,*,§

* Department of Pathology, {dagger} Department of Pharmacology, and {ddagger} Department of Pediatrics and § Center for AIDS Research, Case Western Reserve University, Cleveland, OH 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs) internalize exogenous Ags and process them for cross-presentation by class I MHC (MHC-I) to CD8+ T cells. This processing can occur by transporter for Ag presentation (TAP)-dependent or TAP-independent mechanisms. We observed that CpG DNA enhanced cross-presentation of Ags by Flt-3L-cultured bone marrow-derived murine DCs by a type I IFN (IFN-{alpha}{beta})-dependent mechanism. Myeloid DCs provided cross-presentation function in this system. Both TAP1 knockout and wild-type DCs showed enhanced cross-presentation when treated with CpG DNA at 26°C, demonstrating that TAP is not essential to this regulatory mechanism, although TAP is an important determinant of MHC-I expression. Enhancement of cross-processing by CpG DNA did not involve increased Ag uptake or proteolysis but did correlate with IFN-{alpha}{beta}-dependent increases in expression of MHC-I mRNA and protein. Increased MHC-I mRNA levels resulted in part from stabilization of MHC-I mRNA, a novel posttranscriptional mechanism for regulation of MHC-I expression. Thus, a major mechanism by which CpG oligodeoxynucleotide increase cross presentation by DCs appears to be an IFN-{alpha}{beta}-mediated increase in MHC-I synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Class I MHC (MHC-I)3-restricted CD8+ effector CTLs recognize viruses and other intracellular pathogens that synthesize or deposit foreign Ags in the cytosol of a wide variety of host cell types, where they are accessible to conventional cytosolic MHC-I Ag-processing mechanisms. Cytosolic Ags are processed by proteasomes into peptides that are conveyed by the transporter for Ag presentation (TAP) into the endoplasmic reticulum (ER), where they bind to MHC-I. In contrast, priming of naive CD8+ T cells occurs in secondary lymphoid organs and requires MHC-I Ag presentation by mature DCs that express costimulator molecules. Many pathogens do not infect DCs, and priming of CD8+ T cell responses to these pathogens requires "cross-priming", wherein DCs internalize and process Ags derived from other cells or the extracellular space (i.e., the Ags are exogenous to the DCs). Processing of exogenous Ags for MHC-I "cross" presentation occurs by mechanisms that are termed alternate MHC-I processing or cross-processing (1, 2), resulting in cross-priming of CD8+ T cell responses.

Cross-processing is more efficient with particulate Ag (bacteria, apoptotic cells, or Ag-conjugated beads) than soluble Ag (3, 4, 5, 6, 7). Phagocytosed particulate Ag is proteolyzed subsequent to phagosome-lysosome fusion and, through both TAP-dependent and TAP-independent mechanisms, processed into peptides and loaded onto MHC-I for cross-presentation (1, 2). TAP-dependent processing involves transit of Ags from the phagosome to the cytosol (8) for proteasome-mediated proteolytic processing and transport into a MHC-I loading compartment (either the ER or ER-associated phagosomes that contain TAP (9, 10, 11)). TAP-independent cross-presentation mechanisms have been reported, e.g., the vacuolar pathway in which phagosomal processing generates peptides that bind phagosomal MHC-I without transit to the cytosol (1, 3, 12, 13, 14, 15).

In murine vaccination models, efficient cross-priming of CD8+ T cells to injected Ags requires coadministration of an immune adjuvant. CpG oligodeoxynucleotides (ODNs) contain an unmethylated CpG dinucleotide in a specific sequence context that is recognized by TLR9 (16). CpG ODNs mimic the effects of bacterial DNA on the vertebrate immune system (16, 17). CpG ODNs are effective adjuvants that promote cross-priming of CD8+ T cell responses (18, 19, 20, 21). Enhancement of cross-priming by CpG ODN is partially dependent on IFN-{alpha}{beta}, and the effect is attenuated in mice lacking IFN-{alpha}{beta}R (22, 23).

CpG ODNs may promote cross-priming by enhancing Ag cross-processing and presentation. Treatment of DCs with CpG ODN results in IFN-{alpha}{beta}-dependent increases in expression of both TAP and MHC-I (23). TAP is required for efficient cross-presentation in vitro (21) or cross-priming in vivo (23). Increased expression of TAP could regulate cross-presentation by enhancing transport of Ag-derived peptides from the cytosol into the ER for binding to MHC-I. Increased expression of TAP could also enhance cross-presentation indirectly by increasing endogenous peptide loading to stabilize and increase expression of post-Golgi MHC-I (that may bind antigenic peptides by peptide exchange in phagosomes) (12, 13). Mechanisms other than regulation of TAP could also increase MHC-I expression, directly increasing MHC-I availability for peptide binding and subsequent peptide presentation.

The effect of CpG ODNs on MHC-I expression has not been studied in detail, but published reports have shown that CpG ODNs increase MHC-I expression by DCs (14, 20, 23). CpG ODNs cause DCs (especially plasmacytoid DCs (pDCs)) to produce IFN-{alpha}{beta} (16), which is known to increase MHC-I expression (24). Thus, paracrine or autocrine IFN-{alpha}{beta} signaling may mediate the effects of CpG ODNs on DC MHC-I expression.

CpG-induced increases in MHC-I expression may result from increased transcription of MHC-I mRNA because 5' regulatory sequences of MHC-I genes bind NF-{kappa}B (which is activated by CpG) and IFN regulatory factor-1 (which is activated by IFN-{alpha}{beta}) (24, 25). Protein stabilization is another possible means of controlling MHC-I expression because MHC-I protein half-life is increased on DCs by viral or bacterial infection (7, 26) or LPS (27), another TLR agonist that induces IFN-{alpha}{beta} (28). Our studies have addressed the mechanisms whereby CpG ODNs increase MHC-I expression and cross-processing.

To investigate enhancement of cross-processing by CpG ODN, we measured cross-presentation of particulate Ag by DCs to T hybridoma cells. CpG ODN enhanced cross-presentation by wild-type but not IFN-{alpha}{beta}R–/– DCs. CpG ODN also increased MHC-I mRNA and surface protein by wild-type but not IFN-{alpha}{beta}R–/– DCs. Expression of TAP was not required for CpG ODN to increase cross-presentation or surface expression of MHC-I, although it influenced baseline MHC-I expression and may contribute to regulation of MHC-I expression by CpG ODN. We observed a novel posttranscriptional mechanism for enhancement of MHC-I expression involving stabilization of MHC-I mRNA. Thus, the main mechanism by which CpG ODN enhances cross-presentation by DCs appears to be an IFN-{alpha}{beta}-mediated increase in MHC-I synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and animals

Mice were housed under specific pathogen-free conditions. C57BL/6 and TAP1–/– mice were purchased from The Jackson Laboratory, and 129S6/SvEv mice (formerly designated as 129/SvEv Tac) were purchased from Taconic Farms. IFN-{alpha}{beta}R–/– A129 mice (29) on a 129/SvEv background were purchased from B&K Universal. Homozygous deletions were confirmed by PCR using primers 5'-AAGATGTGCTGTTCCCTTCCTCTGCTCTGA-3' and 5'-ATTATTAAAAGAAAAGACGAGGCGAAGTGG-3'. Unmethylated phosphorothioate-modified CpG ODN 1826 (5'-TCCATGACGTTCCTGACGTT-3') and non-CpG ODN 1982 (5'-TCCAGGACTTCTCTCAGGTT-3') were provided by Coley Pharmaceutical Group. OVA beads were prepared by passive adsorption of chicken egg OVA onto 2 µM polystyrene beads (Polysciences). Beads (0.5 ml) were resuspended in 10 mg/ml OVA in 0.5 ml of citrate buffer (pH 4.2), incubated overnight at 4°C with agitation, and washed and resuspended in culture medium, yielding 6 x 109 beads/ml and 100 µg/ml bead-associated OVA.

DC culture

After lysis of erythrocytes, mouse femur bone marrow cells were suspended at 106 cells/ml in standard medium (RPMI 1640 with 10% FBS, 50 µM 2-ME, and antibiotics) containing 100 ng/ml Flt-3L (R&D Systems) and cultured in 6-well plates (10 ml/well) (30, 31). On days 3 and 6, half of the volume was removed and replaced with medium containing 200 ng/ml Flt-3L. On day 8 of culture, nonadherent cells were resuspended in medium without Flt-3L (1–2 x 106 cells/ml). For some experiments (Fig. 2, D and E), Flt-3L-cultured DCs were separated using Ab-coated magnetic microbeads (Miltenyi Biotec) and the manufacturer’s protocols to produce preparations containing myeloid DCs (mDCs) or pDCs; mDCs were prepared by positive selection with anti-CD11b microbeads, whereas pDCs were prepared by negative selection with anti-CD11b microbeads followed by positive selection with anti-B220 microbeads.



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  		FIGURE 2. CpG ODN 1826 induces processing and cross-presentation of particulate Ag by DCs. C57BL/6 DCs were harvested from Flt-3L cultures (A–C). In some experiments, mDCs or a population enriched in pDCs were separated (D and E). Cells were then incubated with or without CpG ODN 1826 or non-CpG ODN 1982 for 12 h (A), 16 h (B), or 24 h (C). The cells were then incubated for 1 h with bead-conjugated OVA, fixed with paraformaldehyde, and incubated with CD8OVA1.3 T hybridoma cells to detect presentation of SIINFEKL:Kb complexes. Production of IL-2 was assessed with a colorimetric IL-2 bioassay. Error bars represent SD of triplicate wells and where not shown are smaller than symbol size. Values of p from a two-tailed t test comparing treated and untreated values are shown (#, p < 0.05; +, p < 0.01; *, p < 0.001). Data are representative of eight experiments (A–C) or three experiments (D and E), all of which showed statistically significant increases (p < 0.05) in T cell responses to 300, 1000, and 3000 ng/ml bead-bound OVA presented by unfractionated Flt-3L DCs after 16 and 24 h of treatment with CpG ODN 1826 or mDCs after 16 h of treatment with CpG ODN.

 
Ag processing and presentation assays

DCs were plated in round-bottom 96-well culture plates (50 µl at 2 x 106 cells/ml) and incubated with CpG ODN 1826 or non-CpG ODN 1982 for 18 h at 37°C (or as otherwise indicated). Particulate Ag was added, plates were centrifuged at room temperature for 5 min at 450 x g, and cells were incubated with Ags for 1 h at 37°C (18 h at 26°C for experiments with TAP1–/– cells), fixed with 1% paraformaldehyde, washed, and incubated with CD8OVA1.3 T hybridoma cells (specific for OVA-derived peptide, SIINFEKL, presented by H-2Kb) (3). Release of IL-2 by CD8OVA1.3 cells was determined by colorimetric (OD550 nm–OD595 nm) bioassay based on metabolism of Alamar blue by IL-2-dependent CTLL-2 cells (12). Assays were performed in triplicate and statistical significance was evaluated by Student’s two-tailed t test.

Flow cytometry

DCs were plated (0.5 ml/well at 2 x 106 cells/ml) in 48-well plates (nontissue culture treated) and incubated with or without CpG ODN 1826 or non-CpG ODN 1982 for 18–24 h at 37°C. For experiments comparing MHC-I expression on TAP1–/– and wild-type cells, incubations were at 37°C with or without addition of 5 µM SIINFEKL peptide or at 26°C without exogenous peptide. Cells were stained with fluorochrome- or biotin-conjugated mAbs recognizing CD11c (HL3), CD11b (M1/70), or MHC-I (28-8-6) (BD Biosciences) and an appropriate secondary reagent. Data were collected with a BD LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

MHC-I protein stability

Stability of peptide:MHC-I complexes was determined after overnight incubation of DCs with or without 30 nM CpG ODN 1826 or 10 ng/ml LPS and with or without 5 µM SIINFEKL at 37°C. Cells were washed to remove peptide and incubated for various intervals in continued presence or absence of CpG ODN. Surface expression of SIINFEKL:Kb complexes was determined by flow cytometry after staining with mAb 25-D1.16 (32). Data are presented as SIINFEKL:Kb-specific fluorescence, defined as geometric mean fluorescence value (MFV) of cells incubated with peptide minus MFV of cells incubated without peptide. Alternatively, stability of total MHC-I was determined on DCs after overnight incubation with or without 30 nM CpG ODN 1826 or 10 ng/ml LPS. Cells were incubated with brefeldin A for various periods, and surface expression of Kb and Db was determined by flow cytometry after staining with mAb 28-8-6.

ELISA, RT-PCR, and determination of mRNA half-life

To measure IFN-{alpha} protein production, DCs were plated in flat-bottom 96-well plates at a density of 2 x 106 cells/ml with or without CpG ODN 1826 for 16 h. Cell-free supernatants were analyzed for IFN-{alpha} using an ELISA kit (PBL Biomedical). For RT-PCR studies, DCs were cultured for 18 h with or without CpG ODN 1826 (30 nM) or non-CpG ODN 1982 (30 nM) and with or without 5 µM SB203580, a p38 MAPK inhibitor (Calbiochem). Total RNA was extracted using the RNeasy kit (Qiagen) with DNase digestion. Oligo(dT)-primed reverse transcription of RNA was performed with Superscript II First-Strand Synthesis kit from Invitrogen Life Technologies, and 5% of the product was used for each of triplicate real-time quantitative PCR using PCR buffer with hot start Taq polymerase and SYBR green (iQ SYBR Green Supermix; Bio-Rad) performed with an iCycler (Bio-Rad). Primer pairs for IFN-{beta} and all forms of IFN-{alpha} were made using published sequences (33). OLIGO software (Molecular Biology Insights) was used to design primer pairs for TAP1 (5'-CAGCGGCTCCTGTATGAGA-3' and 5'-CAGTCCAGAGGCCTTGTCAGT-3'), TAP2 (5'-GCCTGTGCTGTTCTCGGGTTC-3' and 5'-TGCTGTACCAGGTGGGCGTAG-3'), Kb (5'-GCCCTCAGTTCTCTTTAGTCA-3' and 5'-GCCCTAGGTCAAGATGATAAC-3'), Db (5'-TCCGAGATTGTAAAGCGTGAA-3' and 5'-TGTGGTTGCTGGGATTTGA-3'), and GAPDH (34). GAPDH mRNA/cell was unaffected by CpG and served as an internal control. For Figs. 5 and 9, expression was normalized to GAPDH. Each PCR data point for TAP1, TAP2, Kb, and Db was divided by mean GAPDH expression. Mean and SD were calculated with normalized values, and statistical significance was evaluated by Student two-tailed t test.



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  		FIGURE 5. TAP mRNA is increased by CpG ODN. DCs from wild-type 129S6/SvEv (A) or IFN-{alpha}{beta}R–/– (B) mice were treated for various periods with or without 30 nM CpG ODN 1826. Quantitative RT-PCR was performed using primers specific for the TAP1 and TAP2 subunits of TAP. TAP1 and TAP2 were normalized to GAPDH (GAPDH expression was not affected by CpG treatment). At 3 h, CpG ODN induced significant increases in TAP1 and TAP2 (+, p < 0.01). Data are representative of three experiments that each showed statistically significant (p < 0.05) CpG-induced increases in TAP1 and TAP2 mRNA. Error bars represent SD of triplicate PCR samples and where not shown are smaller than the symbol size.

 


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  		FIGURE 9. MHC-I mRNA is increased by CpG ODN in an IFN-{alpha}{beta}-dependent manner. DCs from wild-type 129S6/SvEv mice (A and C) or IFN-{alpha}{beta}R–/– mice (B and D) were cultured for various intervals with or without 30 nM CpG ODN 1826. MHC-I mRNA was quantified by quantitative RT-PCR using primers specific for Db (A and B) or Kb (C and D). RT-PCR for GAPDH provided an internal control for normalization. Error bars represent SD of triplicate PCR samples and where not shown are smaller than the symbol size. Symbols indicate p values for difference in MHC-I expression with CpG ODN vs no ODN (#, p < 0.05; +, p < 0.01). Data are representative of three experiments that each showed significant (p < 0.05) increases in MHC-I expression after treatment of wild-type cells with CpG ODN 1826. Data from the three experiments showed that the CpG-induced mean fold increase in both Kb and Db expression (normalized copy number) was significantly (p < 0.05) greater for wild-type than IFN-{alpha}{beta}R–/– cells; at 6–12 h, the mean fold increase (± SD) was 2.10 ± 0.02 for Kb on wild-type cells, 1.30 ± 0.28 for Kb on IFN-{alpha}{beta}R–/– cells, 3.12 ± 0.7 for Db on wild-type cells, and 1.09 ± 0.02 for Db on IFN-{alpha}{beta}R–/– cells.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CpG ODN induces IFN-{alpha}{beta} and enhances cross presentation of particulate Ag by DCs

Murine bone marrow cells were cultured for 8 days with Flt-3L (30, 31) to produce DCs (~80% CD11c+ DCs). Of CD11c+ cells, 30–40% were CD11b– pDCs (known to produce IFN-{alpha}{beta} in response to CpG ODN), and 60–70% were CD11b+ mDCs (in mice both pDCs and mDCs express TLR9 and respond to CpG ODN). Incubation of DCs with CpG ODN 1826 for 18 h induced IFN-{alpha} and IFN-{beta} (Fig. 1). IFN-{alpha} mRNA, IFN-{beta} mRNA, and IFN-{alpha} protein were optimally induced by 30 nM CpG ODN 1826, and this concentration was used for subsequent experiments. For six experiments in which DCs were stimulated with 30 nM CpG ODN 1826 for 12–18 h, CpG ODN increased IFN-{alpha} mRNA 26- to 106-fold (mean = 49.5-fold) with statistical significance (p < 0.01) in each experiment. Likewise, CpG ODN increased IFN-{beta} mRNA 11- to 426-fold (mean = 121-fold, p < 0.01 for each experiment). Four of these experiments assessed the effects of control non-CpG ODN 1982, which did not produce statistically significant changes in IFN mRNA expression in any experiment (0.82- to 1.54-fold change for IFN-{alpha} mRNA and 1.0- to 1.7-fold change IFN-{beta} mRNA), indicating that the induction of IFN-{alpha}{beta} mRNA is specific for CpG ODN. All of three experiments with ELISA showed that CpG ODN 1826 induced statistically significant increases (p < 0.05) in IFN-{alpha} protein with fold induction ranging from 11- to 82-fold.



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  		FIGURE 1. Induction of IFN-{alpha} mRNA, IFN-{beta} mRNA, and IFN-{alpha} protein by CpG ODN 1826. C57BL/6 DCs were incubated for 18 h with or without varying concentrations of ODN 1826 (A and C) or with no ODN, 30 nM control non-CpG ODN 1982, or 30 nM CpG ODN 1826 (B). IFN-{alpha}, IFN-{beta}, and GAPDH mRNA levels were determined by quantitative RT-PCR (A and B). For detection of IFN-{alpha} mRNA, primers common to all forms of IFN-{alpha} were used. Fold change was calculated after normalization to GAPDH. IFN-{alpha} protein released by DCs stimulated with varying concentrations of ODN 1826 was measured by ELISA (C). C, ELISA readings below the limit of detection for the assay are indicated as "B" on the y-axis. Data are representative of three experiments. Error bars represent SD of triplicate PCR samples (A and B) or duplicate ELISA wells (C) and where not shown are smaller than symbol size. Values of p from a two-tailed t test comparing treated and untreated values are shown (+, p < 0.01; *, p < 0.001).

 
To assess MHC-I Ag cross-processing and presentation, DCs were replated, cultured with or without CpG ODN 1826 or non-CpG ODN 1982 for 12–24 h, incubated for 1 h with OVA beads, and fixed with paraformaldehyde. Cross-presentation was assessed by incubating fixed DCs with a T hybridoma (CD8OVA1.3) that produces IL-2 upon recognition of OVA (257-264):Kb complexes (3) and determining IL-2 production by CTLL-2 bioassay with an Alamar blue colorimetric readout (OD550 nm–OD595 nm). Cross-presentation was not detected with DCs cultured in control medium, but CpG-treated DCs produced significant cross-presentation (Fig. 2). This effect required exposure to CpG ODN for at least 12 h, and strong cross-presentation occurred after exposure to CpG ODN for 16–24 h (Fig. 2). Analysis of pooled data from eight independent experiments showed that statistically significant increases in T cell responses (mean OD550 nm–OD595 values) were produced by CpG ODN relative to no ODN at 16 h (0.45 ± 0.05 vs 0.05 ± 0.08, p < 0.001 at 1000 ng/ml OVA; 0.54 ± 0.08 vs 0.18 ± 0.13, p < 0.01 at 3000 ng/ml OVA) and at 24 h (0.40 ± 0.08 vs –0.02 ± 0.01, p < 0.001 at 1000 ng/ml OVA; 0.56 ± 0.05 vs 0.10 ± 0.06, p < 0.001 at 3000 ng/ml OVA). Statistically significant increases were not seen consistently in pooled data from cells treated 12 h with CpG ODN 1826 or from cells treated 16–24 h with control non-CpG ODN 1982. CpG ODN did not change uptake of fluorescent OVA beads, as determined by flow cytometry, or the rate of degradation of OVA within phagosomes, as determined by flow organellometry (35, 36) (data not shown). We conclude that CpG ODN increased MHC-I cross-presentation of exogenous Ags by DCs.

DCs were separated using CD11b- and B220-coated microbeads to produce CD11b+CD11c+B220– mDCs (>89% purity) and a population enriched in CD11b–CD11c+B220+ pDCs (>47% purity). Incubation of isolated mDCs with CpG ODN induced MHC-I cross-processing and presentation of exogenous Ags (Fig. 2D). In contrast, pDCs had little or no cross-presentation activity with or without CpG ODN (Fig. 2E). Phagocytosis of polystyrene beads was similar for pDCs and mDCs (data not shown), suggesting that Ag uptake was not the limiting factor in pDCs. MHC-I expression was ~2-fold lower on pDCs than mDCs (data not shown), which may have contributed partially to the lower cross-presentation function of pDCs, but other factors may also contribute. Thus, MHC-I cross-processing and presentation in Flt-3L-cultured DCs is mediated by mDCs, and pDCs function poorly at cross-presentation.

Enhancement of cross-presentation by CpG ODN is dependent on IFN-{alpha}{beta} signaling

To determine the role of IFN-{alpha}{beta} in modulation of cross-presentation, DCs were produced from IFN-{alpha}{beta}R–/– and wild-type mice (both on 129/SvEv background). IFN-{alpha}{beta}R–/– mice lack the subunit 1 chain of the heterodimeric IFN-{alpha}{beta}R, rendering the receptor nonfunctional (29). Because this is the only receptor for the multiple IFN-{alpha} subtypes and IFN-{beta}, IFN-{alpha}{beta}R–/– mice lack responsiveness to IFN-{alpha}{beta} (29). CpG ODN was not able to enhance cross-presentation by IFN-{alpha}{beta}R–/– DCs but did induce significant cross-presentation by wild-type DCs (Fig. 3). Thus, enhancement of cross-presentation by CpG ODN requires IFN-{alpha}{beta}R, indicating a requirement for paracrine or autocrine signaling by IFN-{alpha}{beta}.



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  		FIGURE 3. CpG-stimulated cross-presentation by DCs is dependent on IFN-{alpha}{beta}. DCs were prepared from wild-type 129S6/SvEv (A) or IFN-{alpha}{beta}R–/– (B) mice, incubated with or without CpG ODN 1826 for 18 h, incubated with bead-conjugated OVA for 1 h, and fixed. Presentation of Ag-derived peptide was assessed with CD8OVA1.3 T hybridoma cells as in Fig. 1. Error bars represent SD of triplicate wells and where not shown were smaller than the symbol size. Values of p from a two-tailed t test comparing treated and untreated values are shown (*, p < 0.001). Data are representative of five experiments, all of which showed statistically significant (p < 0.05) CpG-induced increases in T cell responses to 300, 1000, and 3000 ng/ml bead-bound OVA for wild type but not for IFN-{alpha}{beta}R–/– cells.

 
CpG ODN induces an IFN-{alpha}{beta}-dependent increase in MHC-I expression by DCs

To determine whether CpG ODN regulates MHC-I expression, wild-type and IFN-{alpha}{beta}R–/– DCs were incubated overnight with or without CpG ODN 1826 and analyzed by flow cytometry. CpG ODN increased MHC-I expression on wild-type but not IFN-{alpha}{beta}R–/– cells (Fig. 4). The similar IFN-{alpha}{beta}R dependence of CpG-induced increases in MHC-I expression and cross-presentation suggests that modulation of MHC-I expression is a mechanism by which CpG ODN regulate cross-presentation.



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  		FIGURE 4. Treatment with CpG ODN increases surface expression of MHC-I in an IFN-{alpha}{beta}-dependent manner. DCs from wild-type 129S6/SvEv mice (A) or IFN-{alpha}{beta}R–/– mice (B) were cultured for 24 h with or without 30 nM CpG ODN 1826 and analyzed by flow cytometry. MHC-I expression on CD11c+ cells is shown. Staining with isotype-matched control Ab is shown in shaded histograms. Data are representative of three experiments.

 
CpG ODN induces a transient increase in TAP expression that is dependent on IFN-{alpha}{beta} signaling, but CpG ODN enhances MHC-I expression and cross-presentation even in the absence of TAP

TAP is a determinant of cross-presentation, and treatment of DCs with CpG ODN was reported to increase expression of TAP mRNA in wild-type but not IFN-{alpha}{beta}R–/– cells (23). Treatment of mice with CpG ODN was also found to increase TAP mRNA expression in splenocytes from wild-type but not IFN-{alpha}{beta}R–/– animals (23). In theory, increased TAP expression could enhance cross-presentation either by increasing transport of peptides derived from Ags by cytosolic processing into the ER for MHC-I loading or by increasing loading and stabilization of MHC-I with endogenous peptides to increase post-Golgi MHC-I that may subsequently bind peptides derived by vacuolar Ag cross-processing mechanisms (12, 13). Despite these considerations, the regulatory role of TAP in CpG-induced regulation of cross-presentation remains unclear, although it is clearly necessary for full physiological levels of MHC-I expression and cross-processing, which are decreased in its absence.

Quantitative RT-PCR demonstrated that treatment of DCs with CpG ODN increased expression of mRNA encoding both TAP subunits (TAP1 and TAP2) by ~6-fold within 3 h (p < 0.01; Fig. 5A). CpG ODN did not increase TAP mRNA in IFN-{alpha}{beta}R–/– DCs (Fig. 5B), indicating that IFN-{alpha}{beta} signaling was required for this effect. The CpG-induced increase in TAP mRNA was transient, and expression of TAP1 and TAP2 returned to near resting levels within 12 h (Fig. 5A), although cross-processing was enhanced for much longer periods (Fig. 2). Thus, CpG induces TAP expression, but the degree to which this contributes to modulation of cross-processing and presentation by CpG ODN is unclear.

To test the role of TAP in CpG-induced regulation of MHC-I, we compared the effect of CpG ODN on surface expression of MHC-I by TAP1–/– and wild-type DCs (Fig. 6). Because MHC-I expression is reduced in TAP1–/– cells at 37°C due to less efficient peptide loading and decreased MHC-I stability (37), some cells were incubated at reduced temperature (26°C) or with SIINFEKL peptide at 37°C to stabilize MHC-I on TAP1–/– cells and increase the signal for MHC-I by flow cytometry. To confirm that similar results were produced under physiological conditions, some cells were incubated at 37°C without exogenous peptide. Under all experimental conditions, CpG ODN increased MHC-I expression on both wild-type and TAP1–/– cells (Fig. 6). Control non-CpG ODN produced no change in MHC-I expression, indicating specificity of the effect for CpG ODN. Because CpG ODN increased MHC-I expression in the absence of TAP1, a CpG-induced increase in TAP expression was not necessary for CpG ODN to increase MHC-I expression. Although TAP may be required for normal expression of MHC-I under physiological conditions, CpG ODN can modulate MHC-I expression independent of TAP.



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  		FIGURE 6. CpG ODN can enhance MHC-I expression by mechanisms independent of TAP. DCs isolated from C57BL/6 wild-type (A, C, and E) and TAP1–/– (B, D, and F) mice were incubated with or without 30 nM CpG ODN or 30 nM control non-CpG ODN for 18 h at 37°C in the absence (A and B) or presence (C and D) of SIINFEKL peptide or at 26°C in the absence of added peptide (E and F). MHC-I expression was determined by staining with mAb 28-8-6, which recognizes both Kb and Db, followed by flow cytometry. Shaded histograms, no ODN; dashed lines, control non-CpG ODN; thick lines, CpG-ODN. Thin lines show staining with isotype control Ab, which did not vary substantially with ODN condition. The following values represent CpG ODN-induced mean fold increase (± SD) in MHC-I-specific MFV (MFV with 28-8-6 minus MFV with isotype control Ab). At 37°C without exogenous peptide, fold increase was 4.2 ± 1.4 for wild-type cells (n = 10 independent experiments) and 4.5 ± 0.9 for TAP1–/– cells (n = 3 independent experiments). At 37°C with peptide, fold increase was 3.7 ± 0.4 for wild-type cells (n = 2 independent experiments) and 5.3 ± 1.5 for TAP1–/– cells (n = 2 independent experiments). At 26°C without peptide, fold increase was 2.3 ± 0.8 for wild-type cells (n = 3 independent experiments) and 3.5 ± 1.6 for TAP1–/– cells (n = 3 independent experiments). The average fold increase in response to control non-CpG ODN was 1.1 ± 0.1 for eight independent experiments (two to three independent experiments for each condition).

 
To test the role of TAP in modulation of cross-processing, we assessed cross-presentation with TAP1–/– and wild-type DCs. Incubations were done at 26°C to allow sufficient MHC-I expression on TAP1–/– cells for analysis of cross-presentation and its regulation by CpG ODN. DCs were incubated with or without CpG ODN for 24 h at 26°C and then with OVA beads for 18 h at 26°C. The cells were fixed, and cross-presentation was determined with CD8OVA1.3 cells. Without CpG ODN, cross-presentation was very low or absent with both TAP1–/– and wild-type DCs, but both TAP1–/– and wild-type DCs processed Ags efficiently after stimulation with CpG ODN (Fig. 7). Under these conditions, cross-processing occurs by TAP-independent mechanisms that are induced by CpG ODN. This does not exclude physiological contribution of TAP-dependent mechanisms (e.g., at 37°C). Although TAP expression may be required for efficient MHC-I expression and cross-processing under physiological conditions, TAP is not required for CpG ODN to modulate MHC-I expression and cross-processing activity. This implies that CpG ODN and IFN-{alpha}{beta} modulate MHC-I by another, perhaps more direct, mechanism.



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  		FIGURE 7. TAP is not required for CpG-dependent induction of cross-presentation at 26°C. DCs from C57BL/6 wild-type (A) or TAP1–/– (B) mice were cultured for 24 h at 26°C with or without 30 nM CpG ODN 1826. Cells were then incubated with bead-conjugated OVA for 18 h at 26°C, fixed, and incubated with CD8OVA1.3 T hybridoma cells at 37°C to assess SIINFEKL:Kb presentation. Error bars represent SD of triplicate samples and where not shown are smaller than the symbol size. Values of p from a two-tailed t test comparing treated and untreated values are shown (#, p < 0.05; +, p < 0.01; *, p < 0.001). Data are representative of three experiments, all of which showed statistically significant increases (p < 0.05) in T cell responses at 300, 1000, and 3000 ng/ml bead-bound OVA for wild-type but not for TAP1–/– cells.

 
CpG ODN induces an IFN-{alpha}{beta}-dependent increase in MHC-I protein but does not increase MHC-I protein stability

Enhancement of MHC-I expression by CpG ODN could be due to increased expression of MHC-I mRNA or posttranslational enhancement of MHC-I protein stability. Exposure of DCs to LPS or bacteria increases the half-life of MHC-I protein (7, 27). Expression of class II MHC is also increased by enhanced protein half-life following treatment of DCs with CpG ODN (38, 39).

To determine the half-life of peptide:MHC-I complexes, DCs were treated overnight with or without CpG ODN in the presence or absence of SIINFEKL peptide. Peptide was removed, and cells were incubated for additional periods with or without CpG ODN. Peptide:MHC-I complexes were detected by flow cytometry with mAb 25-D1.16, which recognizes SIINFEKL:Kb complexes. The log2 of SIINFEKL:Kb-specific fluorescence (MFV of cells incubated with SIINFEKL minus MFV of cells incubated without SIINFEKL) was plotted vs time, a best-fit line was determined, and the half-life of SIINFEKL:Kb complexes was determined as the negative reciprocal of the slope. Exposure to CpG ODN did not increase the half-life of peptide:MHC-I complexes (Fig. 8A). In contrast, LPS increased half-life of SIINFEKL:Kb complexes (Fig. 8A), consistent with previously reported data (27).



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  		FIGURE 8. CpG ODN does not affect stability of MHC-I protein on DCs. C57BL/6 DCs were incubated overnight with or without 30 nM CpG ODN 1826 or 10 ng/ml LPS and with or without 5 µM SIINFEKL peptide. SIINFEKL was removed, cells were incubated for various chase periods, and SIINFEKL:Kb complexes were detected by staining with mAb 25-D1.16 and flow cytometry (A). SIINFEKL:Kb-specific fluorescence was determined as MFV of cells with peptide minus MFV of cells without peptide. Data are presented as log2(SIINFEKL:Kb-specific fluorescence) vs time after removal of peptide. The half-life of SIINFEKL:Kb complexes was 23 h with no stimulus, 20 h after treatment with CpG ODN and 40 h after treatment with LPS. To determine the half-life of total Kb/Db MHC-I (B), DCs were cultured overnight with or without 30 nM CpG ODN 1826 and then for various periods with brefeldin A. Cell surface expression of MHC-I (Kb + Db) was determined by staining with mAb 28-8-6 and flow cytometry. Data are presented as log2(specific MFV) vs time (specific MFV = MFV with 28-8-6 minus MFV with isotype control Ab). MHC-I half-life was 7.3 h for unstimulated cells and 7.6 h for CpG-treated cells. A is representative of two experiments, and B is representative of four experiments.

 
To investigate the half-life of total surface MHC-I, DCs were incubated overnight with or without CpG ODN. Brefeldin A was added to block egress of nascent MHC-I from the Golgi apparatus (thereby defining a population of post-Golgi MHC-I for study of its stability without addition of new molecules), and cells were incubated for various chase intervals to determine decay of MHC-I expression, as assessed by staining with anti-MHC-I mAb (recognizing H-2Kb and H-2Db). Specific MFV was defined as MFV with anti-MHC-I minus MFV with isotype control Ab. Specific MFV was plotted vs time of chase incubation. The slope of the best-fit line was used to determine MHC-I half-life, as described above. MHC-I protein stability was not increased by CpG ODN (Fig. 8B). We conclude that CpG ODN, in contrast to LPS, does not increase the half-life of MHC-I protein on DCs. These observations suggest that CpG ODN regulates MHC-I by transcriptional or posttranscriptional regulation of mRNA expression.

CpG ODN increases DC expression of MHC-I mRNA by mechanisms that include mRNA stabilization

To explore modulation of MHC-I mRNA by CpG ODN, DCs were treated with CpG ODN for various times, and MHC-I mRNA expression was determined by quantitative RT-PCR. CpG ODN produced a 2- to 3-fold-increase in mRNA for both Kb and Db that occurred within 6 h and was sustained for at least 24 h (Fig. 9). Enhancement of MHC-I mRNA was dependent on expression of IFN-{alpha}{beta}R (Fig. 9). The increase in MHC-I mRNA may explain much of the increase in MHC-I protein because the 2- to 3-fold increase in mRNA closely matches the fold-change in MHC-I protein (Figs. 4A and 6).

Increased mRNA expression may result from increased mRNA stability, as well as enhanced transcription. To assess MHC-I mRNA stability, DCs were cultured for 16 h with or without CpG ODN. Actinomycin D was added to block nascent mRNA synthesis, and cells were incubated for various intervals to allow decay of mRNA. Quantitative RT-PCR was performed using primers specific for Kb, Db, and GAPDH. GAPDH was used as a control because CpG ODN did not change GAPDH mRNA expression (Fig. 10B and data not shown), and GAPDH mRNA stability was unaltered (Fig. 10C). Log2 of specific RNA/cell was plotted vs time (Fig. 10, A and B). The slope of the best-fit line was used to calculate MHC-I mRNA half-life. Treatment with CpG ODN produced statistically significant (p < 0.05) increases in MHC-I mRNA half-life in all of six experiments. Analysis of data from six experiments revealed a mean half-life (± SD) for Kb mRNA of 2.2 ± 0.7 h without CpG ODN and 6.5 ± 2.0 h with CpG ODN (p < 0.001) and a mean half-life for Db mRNA of 2.6 ± 1.1 h without CpG ODN and 9.4 ± 7.3 h with CpG ODN (p < 0.05). In two experiments, non-CpG ODN did not change mRNA half-life (as shown in Fig. 10B). For some genes, regulation of mRNA stability requires activation of p38 MAPK (40), which is known to be activated by CpG ODN, but SB203580, an inhibitor specific for p38, did not affect MHC-I mRNA decay (Fig. 10A). Therefore, MHC-I stability may be controlled through a p38-independent mechanism.



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  		FIGURE 10. MHC- I mRNA is stabilized by treatment of DCs with CpG ODN. C57BL/6 DCs were incubated for 18 h with or without 30 nM CpG ODN 1826. Some CpG-treated cells were also treated with the p38 MAPK inhibitor, SB203580, at the time of CpG addition. After 18 h, 5 µM actinomycin D (Act D) were added to block mRNA synthesis. The amount of mRNA remaining as a function of time after addition of Act D is shown for Kb (A), Db (B), and GAPDH (C). Data from six separate experiments are summarized in D, showing mRNA half-life for Kb (D, left side) and Db (D, right side) with and without CpG ODN treatment. Each line represents results from a single experiment. Error bars represent SD of triplicate PCR samples (A–C) and where not shown were smaller than the symbol size.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Cross-priming of naive CD8+ T cells requires that DCs take up extracellular Ags in the periphery and migrate to T cell areas of secondary lymphoid organs for MHC-I cross-presentation of peptides with expression of costimulatory molecules. CpG ODNs affect each of these essential steps. CpG ODNs induce migration and expression of costimulatory molecules by DCs (16, 17). Treatment of DCs with CpG ODNs enhances Ag-specific activation of naive CD8+ T cells (23). Although previous observations indicate that CpG ODNs enhance cross-presentation of Ags, the mechanisms of this enhancement remain unclear.

CpG ODNs modulate many aspects of DC Ag presentation, including expression of costimulators, but our experiments with fixed DCs and T hybridoma assays were insensitive to costimulators, which are destroyed by fixation and not required by T hybridoma cells. Thus, our studies focused on the ability of CpG ODN to enhance MHC-I cross-processing, i.e., the production of peptide:MHC-I complexes from exogenous Ags. CpG ODN dramatically enhanced MHC-I cross-processing activity of DCs. Furthermore, CpG ODN enhanced cross-processing by an IFN-{alpha}{beta}-dependent mechanism because there was no cross-presentation by DCs lacking expression of IFN-{alpha}{beta}R.

MHC-I cross-processing and presentation by Flt-3L-cultured DCs was dependent on IFN-{alpha}{beta}, and cross-presentation function was attributed to mDCs. In fact, isolated mDCs responded to CpG ODN by increasing cross-presentation. This suggests that mDCs may directly respond to CpG ODN, although we cannot exclude the possible contribution of some CpG-induced IFN-{alpha}{beta} production by pDCs that may contaminate the mDC preparation. It is often assumed that CpG-induced IFN-{alpha}{beta} is produced by pDCs, not mDCs, but CpG DNA increases expression of MHC-I, TAP, and costimulatory molecules by murine mDCs from GM-CSF bone marrow cultures in an IFN-{alpha}{beta}-dependent manner (23). Thus, murine mDCs may respond to CpG ODN and produce IFN-{alpha}{beta}, although at lower levels than pDCs. However, physiologically, pDCs appear to be the main source of IFN-{alpha}{beta}, and pDC-derived IFN-{alpha}{beta} may modulate mDC cross-presentation function in vivo. This may be particularly important in humans, which (unlike mice) do not have TLR9 expression on mDCs or macrophages.

Our studies further dissected mechanisms whereby CpG ODNs enhance cross-processing. Uptake and proteolytic processing of bead-OVA were unaffected by CpG ODN 1826 (data not shown), consistent with recent reports that CpG ODNs do not enhance uptake of inert microspheres, whereas uptake of bacteria is enhanced by CpG ODNs (41). Although studies of bead-OVA processing may not recapitulate all regulatory mechanisms of phagocytic uptake and processing seen with bacteria, the simplicity of the bead-OVA system allowed us to isolate and identify other mechanisms whereby CpG ODNs regulate MHC-I cross-processing. Because uptake and proteolysis of Ags were unaffected, we tested whether cross-processing was modulated by changes in expression of TAP or MHC-I. CpG ODN 1826 increased expression of MHC-I protein by IFN-{alpha}{beta}-dependent mechanisms, and our subsequent experiments assessed the roles of TAP and altered MHC-I synthesis in this effect.

Increased expression of TAP may contribute to enhanced cross-presentation by DCs undergoing maturation, and treatment of DCs with CpG ODN increases expression of TAP mRNA (23). However, our data suggest that increased TAP expression cannot fully explain CpG enhancement of cross-processing. CpG ODN enhanced cross-presentation in the absence of TAP (Fig. 7), demonstrating that regulation of TAP expression is not necessary for CpG ODN to regulate cross-presentation.

Our experiments assessed several possible mechanisms to explain enhancement of MHC-I expression by CpG ODN. MHC-I expression could be controlled by changes in the half-life of protein or mRNA. Unlike LPS (27), CpG ODN did not increase the half-life of MHC-I protein (Fig. 8). Instead, an increase in the amount of MHC-I mRNA was found (Fig. 9), the magnitude of which was similar to the magnitude of the increase in surface protein expression. One possible mechanism for increasing MHC-I mRNA that has not been described previously would be to increase its stability, which we observed in DCs stimulated with CpG ODN (Fig. 10). Increased MHC-I transcription may also contribute, but the magnitude of message stabilization is sufficient to explain most or all of the increase in MHC-I mRNA. Thus, CpG ODNs may control cross-processing by regulation of MHC-I mRNA stability, a novel mechanism for regulation of MHC-I expression.


   Acknowledgments
 
We appreciate helpful advice from Dr. Arthur Krieg and the gift of CpG ODN from Coley Pharmaceutical Group.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI34343, AI47255, and AI055793 (to C.V.H.) and AI36219 to the Case Center for AIDS Research. Quantitative real-time PCR was performed in a core facility of the Case Comprehensive Cancer Center (supported by National Institutes of Health Grant P30 CA43703). Back

2 Address correspondence and reprint requests to Dr. Clifford Harding, Department of Pathology, WRB 5534, Case Western Reserve University, 2103 Cornell Road, Cleveland, OH 44106-7288. E-mail address: cvh3{at}cwru.edu Back

3 Abbreviations used in this paper: MHC-I, class I MHC; TAP, transporter for Ag presentation; ER, endoplasmic reticulum; ODN, oligodeoxynucleotide; IFN-{alpha}{beta}, type I IFN; DC, dendritic cell; pDC, plasmacytoid DC; mDC, myeloid DC; MFV, geometric mean fluorescence value; OVA, ovalbumin. Back

Received for publication October 12, 2004. Accepted for publication June 6, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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