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The Journal of Immunology, 2003, 170: 2802-2805.
Copyright © 2003 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Toll-Like Receptor 9 Expression Is Not Required for CpG DNA-Aided Cross-Presentation of DNA-Conjugated Antigens but Essential for Cross-Priming of CD8 T Cells1

Antje Heit, Tobias Maurer, Hubertus Hochrein, Stefan Bauer, Katharina M. Huster, Dirk H. Busch and Hermann Wagner2

Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Covalent linkage of immunostimulatory CpG DNA to OVA results in CpG DNA-aided cross-presentation of OVA by dendritic cells (DCs). In vivo, cross-presentation is conditional for cross-priming of OVA-specific CD8 T cells. In this study, we investigated the involvement of the CpG DNA receptor Toll-like receptor (TLR)9 in CpG DNA-aided cross-presentation and cross-priming. Although CpG DNA-aided cross-presentation is not altered in TLR9-deficient cells, TLR9 is required for maturation of APC allowing cross-priming, as resulting in CTL function. These findings imply that TLR9 does not trigger endocytosis of CpG-OVA conjugates, but activates DCs downstream of endocytosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Tcells recognize Ags complexed to MHC molecules. In general, MHC class I molecules are complexed with peptides (CD8 T cell epitopes) derived from cytosolic Ag, and MHC class II molecules are complexed with peptides from internalized Ag (CD4 T cell epitopes) (1, 2). However, when APC such as dendritic cells (DCs)3 internalize high amounts of Ag, they appear to be able to route internalized Ag into the MHC class I presentation pathway, a process termed cross-presentation (3). Cross-presentation of Ag via MHC class I molecules can lead to activation/priming of naive CD8 T cells, a process referred to as cross-priming (4), to paralysis/deletion, or to cross-tolerance (5).

Immature DCs lack costimulatory signals required for productive T cell activation but are well equipped to sample Ag, for example via receptor-mediated endocytosis or fluid phase pinocytosis (6). Because of its selectivity for the Ag in question, the efficacy of Ag internalization via receptor-mediated endocytosis is high, examples being C-type lectins that bear mannose specificity (6), Ag/Ab immune complexes that bind FcRs on DCs (7), or Ag delivered via apoptotic cells (8).

CpG oligonucleotides covalently linked to soluble Ags result in enhanced receptor-like endocytosis of Ag, Ag cross-presentation by DCs, and activation of DCs into professional APC (9). Immunostimulatory CpG DNA motifs activate immature DCs via Toll-like receptor (TLR)9 (10, 11, 12) at late (lysosome-associated membrane protein 1+) endosomal organelles (13). This raises the fundamental question of whether TLR9 effects receptor-mediated endocytosis of covalently linked Ag in addition to its known ability to activate the Toll-IL-1 signaling pathway. To address this issue, we analyzed Ag uptake, cross-presentation, and cross-priming of CpG DNA-linked OVA in TLR9-deficient DCs in vitro and in vivo. We now report that TLR9 does not impact receptor-mediated endocytosis but is essential for cross-priming.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
C57BL76 mice were purchased from Harlan Winkelmann (Borchen, Germany). TLR9-/- mice were a gift from Dr. S. Akira (Osaka, Japan). All animals were kept under specific pathogen-free conditions and were used at 8–12 wk of age.

Cell lines and in vitro culture medium

EL-4 (H-2b) thymoma cells were purchased from the American Type Culture Collection (Manassas, VA). B3Z, a somatic T cell hybrid generated by fusing the OVA/Kb-specific cytotoxic clone B3 with a lacZ-inducible derivative of BW5147 fusion partner (14), was kindly provided by Dr. B. Kelsall (National institutes of Health, Bethesda, MD). Cells were cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FBS, 100 IU/ml penicillin G, 100 IU/ml streptomycin sulfate (all from Biochrom, Berlin, Germany) and 50 µmol/L 2-ME (Life Technologies, Karlsruhe, Germany) at 37°C and 5% CO2.

Generation of GM-CSF- and Flt3 ligand-cultured DC from bone marrow

Flt3 ligand-supplemented bone marrow cell cultures were generated as described (9), and cells were used at 10 days of culture. Generation of GM-CSF-induced, bone marrow-derived DC cultures was performed as previously described (15). Cells were used at days 5–7.

Reagents

Chicken egg albumin (OVA) was from Sigma-Aldrich (Taufkirchen, Germany). The peptide SIINFEKL (OVA peptide 257–264) was custom synthesized by Research Genetics (Huntsville, AL). FITC-labeled OVA was purchased from Molecular Probes (Leiden, The Netherlands).

Phosphothioate-modified immunostimulatory CpG DNA was custom synthesized by MWG (Ebersberg, Germany). The phosphothioated sulfhydryl-modified oligodeoxynucleotides (ODN; TriLink Biotechnologies, La Jolla, CA) used throughout this study consisted of 20 bases and contained a CpG motif (1668: 5'-S-TCCATGACGTTCCTGATGCT-3'). OVA was incubated with the cross-linker sulfo-maleimidobenzoyl-N-hydroxysuccinimide ester (S-MBS; Pierce, Bonn, Germany) in a 50 mM EDTA-PBS buffer (pH 7.0) at a molar ratio of 1:10 for 1 h at room temperature. The sulfhydryl-modified ODN were reduced in a 50 mM 1,4-DTT-PBS solution. Subsequently unbound S-MBS and 1,4-DTT were removed by chromatography on a Bio-Rad P-6 gel column (Bio-Rad, Munich, Germany). The activated ODN were incubated with the linker-modified OVA at a molar ratio of 5:1 for 2.5 h at room temperature and thereafter L-cysteine was added to quench reactive S-MBS. Free ODN were removed by chromatography on a Superdex 75HR column (Amersham Biosciences, Freiburg, Germany). Purified conjugates were analyzed on a 6–20% gradient SDS-PAGE and consecutively silver-stained. To determine the ratio of bound ODN on OVA, a 4–15% gradient nondenaturing, nonreducing PAGE was run and silver-stained or visualized using ethidium bromide staining. Protein concentration was determined by the Lowry method (Pierce). The batches of CpG-OVA conjugates used in this study had a ratio of 2.5 CpG DNA molecules linked to one OVA molecule.

Escherichia coli-derived LPS was purchased from Sigma-Aldrich.

Immunization of chromium release assay

For induction of CTL, OVA and adjuvant were injected into both hind footpads of mice. Four days later, draining lymph nodes (LN) were removed and a single-cell suspension was prepared. LN cells (3 x 106 cells/ml) were cultured for an additional 4 days in medium conditioned with 10 IU/ml rIL-2. The chromium-51 release assay was performed as described (13). SIINFEKL peptide-untreated cells served for specificity control. Specific lysis was calculated according to the formula: percent specific lysis = (cpmsample - cpmspontaneous release/maximum release - cpmspontaneous release) x 100.

Uptake and activation analysis, mAbs

To examine uptake of FITC-labeled CpG-OVA in vitro by bone marrow derived Flt3 DC, cells were exposed to FITC-labeled OVA, mixed with CpG DNA 1668 or 1668-OVA-FITC conjugates (0.5 h at 37°C), washed with ice-cold 2% FCS-PBS containing 2 mM EDTA, and stained with the allophycocyanin-labeled anti-CD11c (clone HL3).

To analyze activation of DC, GM-CSF-cultured bone marrow-derived DC (15) were incubated with 10 µg/ml OVA conjugated with 1.14 µM CpG DNA. Cells were cultured for 24 h, and thereafter washed and stained with allophycocyanin-labeled anti-CD11c (clone HL3), FITC-labeled anti-CD4 (clone 3/23), anti-CD80 (clone 16-10A1), and anti-CD86 (clone GL1). FACS analysis was performed using a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany) acquiring at least 30,000 events per sample. FACS data were analyzed using CellQuest software (BD Biosciences).

mAbs and corresponding isotype controls were purchased from BD PharMingen (Heidelberg, Germany).

Presentation assay

Presentation of SIINFEKL after CpG DNA-aided uptake of OVA by DCs was assayed as previously described (13) by measuring induction of lacZ activity in SIINFEKL/Kb-specific T cell hybridoma B3Z (14). To this, 1 x 105 GM-CSF-cultured DC were incubated with the indicated reagents for 18 h at 37°C. Plates were washed, and 1 x 105 B3Z cells were added to each well. After additional incubation overnight at 37°C, cells were fixed with 0.5% glutaraldehyde for 10 min and incubated with X-Gal solution (Taufkirchen, Germany) (16) at 37°C. After 6–8 h, blue B3Z cells were counted under the microscope.

Detection of cytokines

For determination of cytokines, the supernatant of stimulated cells was collected. Cytokine release (IL-12p40 and TNF-{alpha}; R&D Systems, Wiesbaden, Germany) was assayed by ELISA as described by the manufacturer.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Previously, we (9) and others (17, 18) have shown that immunostimulatory CpG DNA motifs linked to OVA fulfill a dual role: enhancement of OVA uptake yielding efficient Ag cross-presentation by DCs and activation of DC into professional APCs. To analyze the involvement of TLR9 during CpG DNA-aided cellular uptake of OVA, we conjugated CpG DNA to FITC-labeled OVA (CpG-OVA conjugate), exposed wild-type (wt) as well as TLR9-deficient bone marrow-derived DCs to these reagents, and measured cellular uptake by flow cytometry. As shown in Fig. 1, Flt3 ligand-cultured CD11c+ DCs exposed to either OVA-FITC (0.5 mg/ml) or OVA-FITC (0.5 mg/ml) mixed with stimulatory CpG DNA yielded 0.2% (wt) or 0.4% (TLR9-/-) FITC-positive DCs. In contrast, CpG-OVA conjugates enhanced OVA uptake by DCs >30-fold, both in wt and TLR9-/- DCs. Additionally, cross-presentation of Ag, as determined by activation of B3Z hybridoma cells expressing a TCR specific for the Kb-restricted OVA-derived CD8 T cell peptide SIINFEKL, functioned equally well in TLR9-/- or wt DCs (Fig. 2). We conclude that CpG DNA aided enhanced OVA uptake and processing, and SIINFEKL loading to MHC class I molecules functioned in the absence of TLR9.



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  		FIGURE 1. TLR9 is not required for uptake of OVA-CpG DNA conjugates in primary DC. Flt3 ligand-cultured bone marrow-derived DC from wt C57/B6 or TLR9-/- C57/B6 mice were incubated with 0.5 µg/ml OVA-FITC alone, mixed or conjugated with the CpG DNA 1668 (22.5 nM) for 30 min at 37°C, stained with allophycocyanin-labeled anti-CD11c, and analyzed by FACS. Percentages of FITC and allophycocyanin double-positive, Ag-bearing CD11c+ DC are shown. The data are representative of three independent experiments.

 


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  		FIGURE 2. Ag presentation by DC’s is not altered by the absence of TLR9. GM-CSF-derived bone marrow-derived DCs were incubated with 5 µg of OVA alone (Ova), mixed with CpG DNA 1668 (1668+Ova), or conjugated with 0.28 µM CpG DNA 1668 (1668-Ova) for 18 h. SIINFEKL presentation was assayed via B3Z hybridoma cells expressing an OVA-specific TCR via induction of {beta}-galactosidase upon TCR engagement. Mean and SD of three wells are shown. The data are representative of three independent experiments.

 
Next, we addressed the issue of whether immunostimulatory CpG DNA covalently linked to OVA is effective in activating cross-presenting TLR9-deficient DCs into professional APCs. As shown in Fig. 3a, wt but not TLR9-deficient DCs up-regulated CD40 and CD80 and secreted IL-12 p40 and TNF-{alpha} (Fig. 3b) upon in vitro uptake of immunostimulatory CpG-OVA conjugates. Of note, the DC activation power of CpG DNA linked to protein (Fig. 3) was somehow reduced compared with free CpG DNA (9). Whether processing of OVA when linked to CpG DNA is still TAP-dependent has not yet been analyzed. However, in general, DCs of TLR9-/- were fully able to up-regulate CD40 and CD80 and secrete cytokine as shown by stimulation via TLR4 with LPS (Fig. 3).



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  		FIGURE 3. a, Up-regulation of costimulatory molecules is dependent on TLR9. Bone marrow-derived DCs from either wt or TLR9-/- mice were stimulated with CpG DNA 1668 conjugated to OVA (1668-Ova) (black line), LPS (dotted line), or medium only (filled) for 24 h at 37°C. Cells were washed two times, stained with anti-CD40 or anti-CD86 mAbs, and subjected to FACS analysis. b, TLR9 presence is essential for IL12 p40 and TNF-{alpha} secretion. Supernatants of stimulated cells (a) with CpG DNA 1668 conjugated to OVA (1668-Ova), LPS, or medium only, were collected and determined in triplicate by commercially available (IL12 p40; TNF-{alpha}) ELISA kits. Mean and SD are shown. Similar results were obtained in two additional experiments.

 
To investigate whether primary CTL responses were induced in vivo by CpG-OVA conjugates, TLR9-deficient and wt mice were challenged s.c. in the footpad with immunostimulatory CpG-OVA conjugates or OVA. After 4 days, cells of the draining LNs were harvested, cultured for an additional 72 h, and tested for CTL activity. As shown in Fig. 4, wt but not TLR9-deficient mice generated SIINFEKL-specific CTL in response to CpG-OVA conjugates. However, Ag-specific CTL were generated in both TLR9-/- and wt mice when OVA was administered with IFA.



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  		FIGURE 4. CTL priming induced by CpG-OVA-conjugates in wt and TLR9-/- mice. TLR9-/- and wt mice (two mice per group) were challenged in hind footpads with 0.56 µM CpG DNA 1668 conjugated to 10 µg of OVA (1668-Ova) or 10 µg of OVA mixed with 50 µl of IFA, or 10 µg of OVA only. After 4 days, draining LN were harvested, and cells were isolated and cultured in IL-2-conditioned medium for an additional 4 days before a chromium release assay was performed. {blacksquare}, SIINFEKL peptide-pulsed EL-4 target cells; {square}, unpulsed EL-4 cells. A representative experiment of three is shown.

 
In conclusion, these data provide compelling evidence that, following administration of CpG-Ag conjugates, the function of TLR9 is restricted to activation of OVA cross-presenting DCs in professional APCs. CpG DNA-aided cellular uptake and cross-presentation of linked OVA does not involve TLR9. The enhanced Ag loading of DC with OVA seen by conjugation with CpG DNA (9, 17) (18) must be mediated by a yet-undefined DNA-binding receptor lacking nucleic acid sequence specificity. Following endocytosis, immunostimulatory CpG DNA meets TLR9 in late endosomes where the adaptor molecule MyD88 is recruited (13, 19). This contrasts with LPS, which activates TLR4 expressed at the cell membrane (13, 20). Of note, cross-presentation of conjugated Ag by TLR9-/- DCs is qualitatively similar to the pattern seen in wt DCs independently of the CpG sequence used (9). In contrast, activation of cross-presenting DC to allow cross-priming requires the presence of TLR9 and immunostimulatory CpG DNA (Fig. 3) (9). Thus, the use of DNA-OVA conjugates provides opportunities to influence the subsequent adaptive immune response, first by enhancement of Ag uptake, and second by DC activation through TLR9. Future studies can address the question of whether CpG-Ag conjugates are primarily cross-presented by CD8+ DCs (3, 21) and whether CpG-Ag conjugates in TLR9-/- mice or nonstimulatory DNA-Ag conjugates in wt mice (9) efficiently induce cross-tolerance.


   Acknowledgments
 
With gratitude, we acknowledge the assistance of M. Hammel. Thanks to Dr. S. Akira of Japan for providing a TLR9 knockout breeding stock.


   Footnotes
 
1 This work was supported by grants from the Sonderforschungsbereich 456 and 576 and Coley Pharmaceutical Group. Back

2 Address correspondence and reprint requests to Dr. Hermann Wagner, Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität München, Trogerstrasse 9, D-81675 Munich, Germany. E-mail address: h.wagner{at}lrz.tu-muenchen.de Back

3 Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; ODN, oligodeoxynucleotide; S-MBS, sulfo-maleimidobenzoyl-N-hydroxysuccinimide ester; LN, lymph node; wt, wild type. Back

Received for publication December 20, 2002. Accepted for publication January 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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F. K. Stevenson, C. H. Ottensmeier, P. Johnson, D. Zhu, S. L. Buchan, K. J. McCann, J. S. Roddick, A. T. King, F. McNicholl, N. Savelyeva, et al.
DNA vaccines to attack cancer
PNAS, October 5, 2004; 101(suppl_2): 14646 - 14652.
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 BloodHome page
T. R. Kollmann, S. S. Way, H. L. Harowicz, A. M. Hajjar, and C. B. Wilson
Deficient MHC class I cross-presentation of soluble antigen by murine neonatal dendritic cells
Blood, June 1, 2004; 103(11): 4240 - 4242.
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S. L. McCoy, S. E. Kurtz, F. A. Hausman, D. R. Trune, R. M. Bennett, and S. H. Hefeneider
Activation of RAW264.7 Macrophages by Bacterial DNA and Lipopolysaccharide Increases Cell Surface DNA Binding and Internalization
J. Biol. Chem., April 23, 2004; 279(17): 17217 - 17223.
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A. Heit, K. M. Huster, F. Schmitz, M. Schiemann, D. H. Busch, and H. Wagner
CpG-DNA Aided Cross-Priming by Cross-Presenting B Cells
J. Immunol., February 1, 2004; 172(3): 1501 - 1507.
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