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International Immunology Advance Access originally published online on September 11, 2006
International Immunology 2006 18(11):1563-1573; doi:10.1093/intimm/dxl089
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Dendritic cell activating peptides induce distinct cytokine profiles

Gloria Telusma1, Sandip Datta2, Ivan Mihajlov2, Wenxue Ma3, Jianhua Li1,4, Huan Yang1,4, Walter Newman5, Bradley T. Messmer1,6, Boris Minev3, Ingo G. H. Schmidt-Wolf7, Kevin J. Tracey1,4, Nicholas Chiorazzi1,6 and Davorka Messmer1,6,8

1 The Feinstein Institute for Medical Research, North Shore—LIJ Health System, Manhasset, NY 11030, USA
2 Department of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
3 University of California San Diego, Rebecca and John Moores UCSD Cancer Center, 3855 Health Science Drive, La Jolla, CA 92093, USA
4 Department of Surgery, North Shore University Hospital and Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
5 Critical Therapeutics Inc., Lexington, MA 04241-3108, USA
6 Department of Medicine, North Shore University Hospital and NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA
7 Department of Internal Medicine, Rheinische Friedrich-Wilhelm Universitaet, Sigmund Freud Strasse 25, Bonn, Germany
8 Present address: Moorse UCSD Cancer Center, 3855 Health Science Drive, La Jolla, CA 92093-0820, USA

Correspondence to: D. Messmer; E-mail: dmessmer{at}ucsd.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
High-mobility group box 1 protein (HMGB1), a DNA-binding nuclear and cytosolic protein, is a pro-inflammatory cytokine released by monocytes and macrophages. HMGB1 as well as its B box domain induce maturation of human dendritic cells (DCs). This report demonstrates that the B box domain induces phenotypic maturation of murine bone marrow-derived dendritic cells (BM-DCs) as evidenced by increased CD86, CD40 and MHC-II expression. The B box domain enhanced secretion of pro-inflammatory cytokines and chemokines: IL-1ß, IL-2, IL-5, IL-8, IL-12 and tumor necrosis factor (TNF)-{alpha}, but not IL-6 and IL-10. Furthermore, four peptides whose sequences correspond to different regions of HMGB1 induced production of IL-1ß, IL-2 and IL-12 (p70), but not IL-10 and IL-6 in mouse BM-DCs. Interestingly, these peptides differed in their capacity to induce TNF-{alpha}, IL-5, IL-18 and IL-8. B box domain as well as peptide-activated DCs acted as potent stimulators of allogeneic T cells in a mixed leukocyte reaction. DCs exposed to HMGB1 peptides induced proliferation of ovalbumin-specific syngeneic T cells. These DC-activating peptides could serve as an adjuvant in immunotherapeutic or vaccine context and the selective activity of these different peptides suggests a means to customize the functional properties of DCs.

Keywords: cytokines, immune adjuvant, inflammation, necrosis


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs) play a central role in the initiation and control of an adaptive immune response. DCs detect evolutionarily conserved molecular structures unique to foreign pathogens, such as LPS (1) or DNA molecules containing unmethylated CpG motifs (2) and also respond to endogenous signals of cellular distress or damage (3–6). Interaction with these agents stimulates DCs to mature. It has been proposed that endogenous stress factors if released in the context of infection can activate/mature DCs and contribute to the initiation and/or perpetuation of an immune response against pathogens. Alternately, if these factors are released constitutively and/or in the absence of infection, they could play a role in the development of autoimmunity (3, 4).

High-mobility group box 1 protein (HMGB1), a nuclear and cytosolic protein, was originally identified as an intra-nuclear factor with an important structural function in chromatin organization (7). HMGB1 has since been described as a pro-inflammatory cytokine that mediates endotoxin lethality, local inflammation and macrophage activation (8–12). HMGB1 is actively secreted by activated macrophages and monocytes following exposure to LPS, tumor necrosis factor (TNF)-{alpha} or IL-1ß and as a result of tissue damage (13, 14). It also acts as a potent stimulus/signal to monocytes by inducing the synthesis of pro-inflammatory cytokines (9). Furthermore, HMGB1 also enhances IFN-{gamma} release from macrophage-stimulated NK cells (15). Receptor for advanced glycation end-products (RAGE) (16) as well as Toll-like receptor (TLR)-2 and TLR4 (17) have been reported as HMGB1 receptors. HMGB1 is secreted from necrotic but not apoptotic cells and acts as a signal of tissue damage (13) and as immune adjuvant (18). It has recently been shown that human DCs secrete HMGB1 upon LPS stimulation and that HMGB1 acts back on DCs via RAGE inducing DC maturation. Furthermore, HMGB1 secreted from DCs is required for DC-mediated T cell activation (19). It has also been shown that HMGB1 through RAGE causes activation of human plasmacytoid DCs (20). HMGB1 contains two homologous DNA-binding motifs termed HMG A and HMG B boxes (21, 22). The pro-inflammatory domain of HMGB1 is located in the B box domain (HMGB1-Bx), which alone is sufficient to recapitulate the cytokine-stimulating effect of full-length HMGB1 in vivo (23). The intracellular abundance of HMGB1 and its pro-inflammatory activities suggest that its release at sites of cell injury or damage may play a role in the initiation and/or perpetuation of an immune response. This model is strengthened by in vivo results showing that HMGB1 induces arthritis when injected into murine joints (24) and acute lung injury when administered intra-articularly (25, 26). Furthermore, HMGB1 is found in the serum of patients with acute (sepsis) and chronic (rheumatoid arthritis) inflammatory conditions (10, 20).

We have previously shown that HMGB1 and HMGB1-Bx are potent stimuli for maturation of human monocyte-derived DCs (27). HMGB1 is a crucial component of necrotic lysates and it has adjuvant activity in vivo (18). However, it has not been investigated to what extent HMGB1 alone induces maturation of murine DCs. While the potential for whole HMGB1 protein as adjuvant has been demonstrated, there are limitations for using large recombinant proteins as adjuvants and it would be more practical and desirable to use synthetic small protein fragments or preferably peptides as adjuvants due to the uncomplicated production and purity obtained. We have investigated the DC stimulatory capacity of smaller HMGB1 fragments.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals
Female C57BL/6 and BALB/c mice, 6–8 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed at the North Shore–LIJ Research Institute's animal facility. All animal studies were approved by the Institutional Animal Care and Use Committee and the Biosafety Committee of the North Shore–LIJ Research Institute and were performed in accordance with the institutional guidelines. OT-I (OVA TCR, MHC-I restricted) mice on the C57BL/6 background, expressing a transgenic TCR that recognizes ovalbumin (OVA)-derived peptide in the context of MHC-I (H-2Kb) (28), were a gift from M. Bevan (University of Washington, Seattle, WA, USA) to S. Datta and were subsequently crossed onto the C57BL/6-RAG1–/– background and bred at our animal facilities in San Diego. Male OT-II (OVA TCR, MHC-II restricted) mice on the C57BL/6 background, expressing a transgenic TCR that recognizes OVA-derived peptide in the context of MHC-II (I-Ab) (29), were a gift from W. Heath (Walter and Eliza Hall Institute, Melbourne, Australia) to S. Datta and were subsequently bred at our animal facilities in San Diego.

Reagents and cell lines
Recombinant HMGB1-Bx was expressed in Escherichia coli and purified as described (30). Purified HMGB1-Bx contained trace amounts of LPS (19 pg LPS µg–1 B box) as measured by the chromogenic Limulus amebocyte lysate assay (BioWhittacker Inc., Walkersville, MD, USA). Therefore, all experiments using HMGB1-Bx as well as the peptides were performed in the presence of 200 U ml–1 of polymyxin B sufficient to neutralize >10-fold the amount of contaminating LPS in HMGB1-Bx preparations. We have previously shown that the DC stimulatory capacity of HMGB1-Bx requires an intact tertiary structure and is not due to contaminating amounts of LPS, as trypsinization abolished HMGB1-Bx activity (27). The tumor infiltrating lymphocytes (TIL771) was kindly provided by John R. Wunderlich (National Institutes of Health (NIH)/National Cancer Institute, Bethesda, MD, USA) to B. Minev.

Peptides
All peptides, except the ones labeled non-bio, were synthesized with an N-terminal biotin (SynPep Corp., Dublin, CA, USA and GenScript Corp., Piscataway, NJ, USA). The peptides are named by their first amino acid in the HMGB1 sequence. The peptide sequences are listed in Table 1.


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  		Table 1 Biochemical properties of HMGB1-spanning peptides

 
T cell isolation
T cells were isolated by negative selection using the mouse SpinSep antibody cocktail from StemCell Technologies (Vancouver, CA, USA) according to the manufacturer's instructions. The purity of isolated T cells was routinely ~99%.

Generation of human DCs
PBMCs were isolated from the blood of normal volunteers (Long Island Blood Services, Melville, NY, USA) over a Ficoll–Hypaque (Amersham Biosciences, Uppsala, Sweden) density gradient. CD14+ monocytes were isolated from PBMCs by positive selection using anti-CD14 beads (Miltenyi Biotech, Auburn, CA, USA) following the manufacturer's instructions. To generate DCs, CD14+ cells were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine (GIBCO-BRL Life Technologies; Grand Island, NY, USA), 50 µM 2-mercaptoethanol (Sigma, St Louis, MO, USA), 10 mM HEPES (GIBCO-BRL), penicillin (100 U ml–1)–streptomycin (100 µg ml–1) (GIBCO-BRL) and 5% human AB serum (Gemini Bio-Products, Woodland, CA, USA). Cultures were maintained for 7 days in six-well trays (3 x 106 cells per well) supplemented with 1000 U granulocyte macrophage colony-stimulating factor (GM-CSF) per milliliter (Immunex, Seattle, WA, USA) and 200 U IL-4 per milliliter (R&D Systems; Minneapolis, MN, USA) at days 0, 2, 4 and 6.

Generation of mouse DCs
BM-DCs were generated using modifications of the original method described by Inaba et al. (31). In brief, bone marrow (BM) suspensions were incubated with red cell lysis buffer (PUREGENETM RBC Lysis Solution, Gentra Systems, Minneapolis, MN, USA) to remove RBCs. After washing in media, lymphocytes and Ia-positive cells were killed with a cocktail of mAbs and rabbit complement for 60 min at 37°C. The mAbs were GK1.5 anti-CD4, TIB 211 anti-CD8, TIB 120 anti-Ia and TIB 146 anti B220 (kindly provided by Ralph Steinman). The cells were subsequently cultured in media containing 5% FCS and 10 ng ml–1 recombinant mouse GM-CSF (R&D Systems) for 7 days. For some experiments, the cells were further purified at day 7 using CD11c+ microbeads (Miltenyi Biotech) according to the manufacturer's instructions. For Figs 2(C) and 5, mouse BM-DCs were cultured as previously described (32, 33). Briefly, BM from femurs and tibia of C57BL/6 mice were plated on day 0 into bacterial Petri dishes (Fisher Scientific) at 5 x 105 cells ml–1 in DC medium, which consisted of RPMI (Irvine Scientific, Irvine, CA, USA) supplemented with 10% heat-inactivated FCS (Life Technologies, Gaithersburg, MD, USA), 2 mM L-glutamine (Cellgro, Natham, VA, USA), 100 U ml–1 penicillin/100 µg ml–1 streptomycin (Pen/Strep; Cellgro) and 10 ng ml–1 recombinant murine GM-CSF (BD PharMingen, La Jolla, CA, USA). On day 3, an equal volume of DC medium was added. The non-adherent cells were harvested on day 6.


Figure 2
Figure 2
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  		Fig. 2 HMGB1-Bx and HMGB1 peptides enhance the secretion of cytokines and chemokines in mouse BM-DCs. Secreted cytokine levels were measured by ELISA 48 h after addition of the various stimuli. Polymyxin B (200 U ml–1) was added to all cultures except to the ones with LPS before addition of the peptides. (A) Immature BM-DCs were cultured in the presence of HMGB1 peptides (200 µg ml–1), HMGB1-Bx (50 µg ml–1), LPS (100 ng ml–1) or left untreated (medium). Results represent mean ± SEM of three independent experiments. (B) Immature BM-DCs were cultured either with HMGB1 peptides (200 µg ml–1) or left untreated (medium). All peptides except Hp-106 (non-bio) were N-terminally biotinylated. Results represent mean ± SEM of three independent experiments. (C) Immature BM-DCs were cultured with HMGB1 peptides (200 µg ml–1), LPS (10 ng ml–1), biotin (20 µg ml–1) or left untreated (medium). All peptides except Hp-16 (non-bio) were N-terminally biotinylated. Results represent means ± SEM of two independent experiments.

 

Figure 5
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  		Fig. 5 BM-DCs were incubated overnight with media, H-2Kb-restricted OVA peptide (10 pM) and Hp-peptides (100 µg ml–1) as indicated. The DCs were then washed and incubated with CFSE-labeled OT-I CD8+ T cells. After 3 days, T cell proliferation was assessed by flow cytometry (CFSE dilution). The percentage of cells undergoing at least one round of cell division is indicated within each histogram. The x-axis indicates fluorescence intensity for CFSE and the y-axis depicts the number of cells.

 
Stimulation of DCs
At day 7 of culture, immature DCs were either left untreated or were stimulated with indicated doses of HMGB1-Bx, HMGB1 peptides or LPS (E. coli serotype 026:B6, Sigma). In all experiments, DCs were analyzed 48 h after stimulation.

Analysis of DC phenotype
DCs (1 x 104) were reacted for at least 20 min at 4°C in 100 µl of PBS/5% FCS/0.1% sodium azide (staining buffer) with FITC-conjugated IgG mAb specific for CD86, CD40 and MHC-II (eBioscience). Cells were then washed four times with staining buffer, fixed in 3.7% formaldehyde in PBS (pH 7.2–7.4) and examined by flow cytometry using a FACScan (BD). In all experiments, isotype controls were included using FITC-conjugated irrelevant mAb of the same Ig class.

Measurement of cytokines and chemokines
After 48 h of activation, the production of cytokines and chemokines in cell culture supernatants was measured by ELISA (Pierce Boston Technology Center, SearchLightTM Proteome Arrays Multiplex Sample Testing Services, Woburn, MA, USA). For Fig. 2(C), TNF-{alpha} (eBioscience, San Diego, CA), IL-2, IL-5 and IL-12 p70 (BD PharMingen) production was assessed by routine ELISA techniques according to the manufacturer's instructions.

Mixed leukocyte reaction
To assess levels of T cell activation and proliferation, cells were plated at 105 cells per well in a round-bottomed 96-well tray at DC:T cell ratios of 1:120 for 5 days. The microcultures were pulsed with [3H]thymidine (1 µCi per well) for the final 8 h of culture. Cell cultures were harvested onto glass fiber filters with an automated multiple sample harvester and the amount of isotope incorporation was determined by liquid scintillation ß-emission. Responses are reported as mean counts per minute of thymidine incorporation by triplicate cultures (±SEM).

Enzyme-linked immunospot assay
On day 5 of culture, immature DCs were stimulated with Hp-peptide or controls for 48 h. Subsequently DCs were washed and re-suspended in serum-free media. DCs (2 x 104 per well) were distributed to triplicate or quadruplicate wells of a nitrocellulose bottom enzyme-linked immunospot (ELISPOT) plate (Millipore, Billerica, MA, USA) that had previously been coated with 10 µg ml–1 monoclonal human anti-IFN-{gamma} antibody (Mabtech, Stockholm, Sweden) overnight. Gp100 peptide, residue 154–162 KTWGQYWQV, of the human melanocytic protein gp100 (synthesized by GenScript Corp.) was added to the wells at a final concentration of 500 ng ml–1. The peptide corresponds to HLA-A*0201-restricted CTL epitope. Subsequently, responder cells, TIL771, were added to the DC cultures at a DC to responder ratio of 1:2. Cells were cultured for 24 h at 37°C. ELISPOT plates were developed using biotinylated anti-human IFN-{gamma} (2 µg ml–1), avidin-bound biotinylated HRP (Vector Laboratories, Burlingame, CA, USA) and AEC substrate for peroxidase (Vector Laboratories). Plate was scanned and the spots were counted automatically using the image analysis system ELISpot reader (CTL Analyzers LLC, Cleveland, OH, USA).

In vitro T cell proliferation assay
T cell proliferation was assessed by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution using the previously described DECOT assay (32). Briefly, BM-derived C57BL/6 DCs were incubated overnight with H-2-Kb-restricted OVA peptide (SIINFEKL, PeptidoGenic Research, Livermore, CA, USA) or I-Ab-restricted OVA peptide (ISQAVHAAHAEINEAGR, PeptidoGenic Research) in the absence or presence of Hp-peptides. CD8+ or CD4+ T cells from sex-matched OT-I or OT-II mice, respectively, were purified from splenocytes using anti-CD8 or anti-CD4 magnetic beads (Miltenyi Biotec) according to the manufacturer's instructions. The purified T cells were stained in PBS containing 1 µM CFSE (Molecular Probes, Eugene, OR, USA). The non-adherent DCs were then washed and incubated with an equal number of the CFSE-labeled T cells in supplemented RPMI for 3 days. Flow cytometry was then done on the gated CD4+ or CD8+ T cell population to assess T cell proliferation, reflected by halving of CFSE fluorescence intensity in daughter cells produced with each round of proliferation.

Statistical analysis
Data are represented as mean ± SEM. Data were analyzed for statistical significance using Student's t-test. P-values <0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
HMGB1 peptides induce cytokine secretion in human DCs
We have previously shown that an 18-amino acid long peptide whose sequence corresponds to a part of HMGB1-Bx induced IL-6 secretion in human monocyte-derived DCs (27). The search for DC-activating peptides was extended by testing 18-amino acid long peptides that span the whole HMGB1 molecule (Table 1). When immature human monocyte-derived DCs were exposed to these different peptides for 48 h, peptide Hp-31 in addition to the previously described peptide Hp-106, enhanced secretion of IL-6 by DCs (Fig. 1A). Subsequently, peptides that overlap by three amino acids either N- or C-terminal of these two peptides were tested (Fig. 1B). We found that the C-terminal flanking peptide of Hp-106, Hp-91 also enhanced the IL-6 secretion. This peptide shares only three amino acids (CSE) with Hp-106. The two peptides flanking Hp-31 showed no activity. Furthermore, the N-terminal biotin was required for the DC stimulatory effect of the active peptides. Hp-106 without the N-terminal biotin (Fig. 1B, Hp-106 non-bio) did not enhance IL-6 secretion. However, the activity was not due to the biotin moiety, since peptides with different sequences that were also N-terminally biotinylated (Hp-16, Hp-46, Hp-121, etc), did not enhance IL-6 secretion by DCs (Fig. 1A and B).


Figure 1
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  		Fig. 1 HMGB1-derived peptides enhance cytokine secretion in human DCs. Secreted cytokine levels were measured by ELISA 48 h after addition of the various stimuli. Polymyxin B (200 U ml–1) was added to all cultures except to the ones exposed to LPS before addition of the stimuli. (A) Immature DCs were cultured in the presence of peptides (200 µg ml–1), whose sequence maps different regions of the HMGB1 molecule (Table 1.). Results represent mean ± SEM of two independent experiments using DCs generated from different donors. (B) Immature DCs were cultured in the presence of selected peptides (200 µg ml–1), HMGB1-Bx (50 µg ml–1) or left untreated (medium). All peptides are N-terminally biotinylated except Hp-106 (non-bio). Results represent mean ± SEM of three independent experiments using DCs generated from different donors. (C) Immature DCs were cultured in the presence of selected peptides (200 µg ml–1), biotin (20 µg ml–1), LPS (100 ng ml–1) or left untreated (medium). Results represent mean ± SEM of two independent experiments using DCs generated from different donors. (D) Immature DCs were cultured in the presence of Hp-91 or Hp-46 (200 µg ml–1), biotin (20 µg ml–1) or left untreated (medium) for 48 h. DCs were washed and cultured with gp100 peptide (500 ng ml–1) and responder cells, TILs, at a DC to responder ratio of 1:2. The number of IFN-{gamma}-secreting cells was determined 24 h later. The plate was scanned and the spots were counted automatically using the image analysis system ELISpot reader. The data shown, number of IFN-{gamma} spot-forming cells per well, are means (±SEM) of two experiments using DCs from different donors.

 
Next, the cytokine profile induced by the active peptides was investigated (overview in Table 2). Only HMGB1-Bx increased secretion of IL-8. Neither HMGB1-Bx nor the peptide-treated DCs showed enhanced secretion of IL-10 and IL-1ß (Table 2). Hp-91 significantly increased secretion of IL-2, IL-5, IL-12 (p70), IL-15 and IFN-{alpha}. Hp-31 and Hp-106 caused a very low but statistically significant increase in IL-2 secretion (Fig. 1C). Hp-31 was the only peptide that induced IL-18 secretion (data not shown). No additional increase or synergistic effect was observed in cytokine secretion when the peptides were added together with low-dose LPS (data not shown). Biotin only, when tested at equimolar amounts as present in the peptide preparations, did not cause secretion of any of the tested cytokines (Fig. 1C). Since Hp-91 appeared to be the most potent cytokine-inducing peptide for human DCs, Hp-91-treated DCs were assessed for their ability to induce antigen-specific T cell responses as measured by INF-{gamma} secretion in an ELISPOT assay (Fig. 1D). DCs generated from HLA-A*0201-positive donors were exposed to Hp-91, Hp-46 (inactive peptide), biotin or medium only for 48 h. DCs were then washed and cultured with gp100 peptide and HLA-A*0201-restricted gp100-specific TILs overnight. DCs prior exposed to Hp-91, but not the control peptide Hp-46, or biotin, induced a significant increase in gp100-specific IFN-{gamma} secretion by the responder CD8+ T cells (Fig. 1D).


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  		Table 2 Summary of cytokine secretion induced by HMGB-1 peptides

 
HMGB1-Bx and HMGB1 peptides cause secretion of pro-inflammatory cytokines and chemokines in murine BM-DCs
To determine whether HMGB1-Bx and HMGB1 peptides also enhance cytokine and chemokine secretion in mouse DCs, immature BM-DCs were exposed to HMGB1-Bx, HMGB1 peptides or LPS for 48 h. HMGB1-Bx-stimulated DCs showed significantly enhanced secretion of IL-5, TNF-{alpha} and IL-8 as well as a small but significant increase in IL-12 (p70) (Fig. 2A and Table 2). In contrast to human DCs, mouse BM-DCs exposed to HMGB1-Bx or Hp-peptides did not increase IL-6 secretion, but did increase IL-1ß secretion (Table 2). Furthermore, all three peptides that showed activity in human DCs (Hp-106, Hp-91 and Hp-31) were also active on mouse BM-DCs. Overall, the peptides induced a more prominent cytokine profile in mouse DCs. Hp-16 which was inactive on human DCs induced cytokines secretion in mouse BM-DCs. Hp-91 induced a similar cytokine profile in mouse BM-DCs as observed in human DCs, except that it did not induce IL-6 secretion in mouse DCs, but did induce IL-1ß (Table 2). The other peptides showed a very different profile in mouse BM-DCs compared with human DCs. All four peptides (Hp-16, Hp-31, Hp-91 and Hp-106) enhanced secretion of IL-1ß, IL-2 and IL-12 (p70) (Fig. 2 and Table 2). Hp-16 induced only a very small but statistically significant increase in IL-12 (p70) secretion and we did not observe increased IL-12 (p70) secretion in combination with low-dose LPS. No synergistic effects were observed (data not shown). Hp-31, Hp-91 and Hp-106 also enhanced secretion of IL-5 (Fig. 1A and C and Table 2). Only Hp-31 and Hp-106 enhanced secretion of TNF-{alpha} (Fig. 2A and C) and none of the peptides enhanced IL-10 secretion (Table 2). Although Hp-91 and Hp-106 overlap by 3 amino acids and are both representative of regions within the HMGB1-Bx, only Hp-106 enhances TNF-{alpha}, IL-8 and IL-18 secretion (Table 2). Apart from this difference, the remaining cytokine profile is the same.

Furthermore, as observed in the human system, N-terminal biotinylation of the peptides was required for their activity. The non-biotinylated peptide Hp-106 non-bio that has the same sequence as Hp-106 did not enhance IL-12 secretion (Fig. 2B) neither did the non-biotinylated Hp-16 non-bio that has the same sequence as Hp-16 (Fig. 2C). Again, although the N-terminal biotin was critical, the DC stimulatory capacity of the peptides was sequence dependent, since biotinylated peptides with different sequences (Hp-46 and Hp-133) did not enhance IL-12 secretion (Fig. 2B). Furthermore, when biotin only was tested at equimolar amounts as present in the peptide preparations, no enhanced secretion of any of the tested cytokines was observed (Fig. 2C).

HMGB1 peptides induce phenotypic maturation of murine BM-DCs
Previous work has linked the pro-inflammatory activity of HMGB1 to HMGB1-Bx (30). To determine whether HMGB1-Bx and HMGB1 peptides can induce phenotypic maturation of murine DCs, immature BM-DCs were exposed to HMGB1-Bx, HMGB1 peptides or LPS for 48 h (Fig. 3). Exposure to HMGB1-Bx elicited only a small increase in CD86 expression and no changes in CD40 and MHC-II expression. Hp-16 induced a strong up-regulation of CD86, MHC-II and CD40 to levels comparable to or higher than those generated by LPS. Interestingly, although Hp-106 induced high levels of cytokine secretion in BM-DCs, it did not significantly enhance the surface expression of the analyzed maturation markers. No altered expression in MHC-II, CD86 or CD40 was detected using the control peptide Hp-121.


Figure 3
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  		Fig. 3 Phenotypic maturation of mouse BM-DCs is induced by a HMGB1 peptide. FACS analysis of immature DCs cultured in the presence of HMGB1-Bx (50 µg ml–1), HMGB1 peptides (200 µg ml–1), LPS (100 ng ml–1) or left untreated (medium) for 48 h. DCs were gated on CD11c+ cells and analyzed for expression of the indicated markers by surface membrane immunofluorescence techniques using FITC-conjugated mAbs. Results represent mean fluorescence intensity (MFI) ± SEM of three independent experiments.

 
HMGB1-Bx and HMGB1 peptides induce functional maturation of BM-DCs
Mature, cytokine-producing DCs induce T cell activation and proliferation, leading to the development of adaptive immunity (34, 35). To assess whether HMGB1-Bx and HMGB1 peptides induce functional maturation of BM-DCs, immature BM-DCs generated from C57/BL6 mice were exposed to HMGB1-Bx or HMGB1 peptides for 48 h and subsequently co-cultured with allogeneic T cells for 5 days. BM-DCs that were exposed to HMGB1-Bx, Hp-16 or Hp-106 activated resting allogeneic T cells in a mixed lymphocyte reaction (MLR) (Fig. 4A), whereas DCs exposed to control peptides Hp-46 or Hp-121 did not. To investigate whether the functional maturation of DCs by HMGB1-Bx was strain specific, BM-DCs were generated from BALB/c mice. HMGB1-Bx-treated BM-DCs showed a strong capacity to induce T cell proliferation (Fig. 4B) similar to that observed in C57BL/6 mice. Next, we assessed the capacity of HMGB1 peptide-stimulated DCs to induce antigen-specific proliferation of syngeneic T cells. BM-DCs were exposed to OVA peptide and different HMGB1 peptides to induce maturation overnight. Subsequently, DCs were washed and mixed with TCR transgenic OVA-specific OT-I CD8+ T cells. BM-DCs exposed to Hp-31, Hp-91 and Hp-106 induced strong proliferation of CD8+ T cells, whereas Hp-16 only had a small effect (Fig. 5). The control peptide Hp-46 showed no effect and neither did Hp-106 when added to OT-I cells in the absence of DCs.


Figure 4
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  		Fig. 4 HMGB1-Bx- and HMGB1 peptide-stimulated mouse BM-DCs enhance the proliferation of allogeneic T cells. (A) Immature BM-DCs generated from C57BL/6 mice were incubated for 48 h with HMGB1-Bx (50 µg ml–1), HMGB1 peptides (200 µg ml–1) or left untreated (medium). DCs were then co-cultured with 105 allogeneic T cells at a DC:T cell ratio of 1:100. T cell proliferation was assessed by measuring the amount of [3H]thymidine incorporated during the last 8 h of a 5-day culture period. A representative example of three independent experiments is shown as mean counts per minute (c.p.m.) ± SEM, from triplicate cultures. (B) Immature BM-DCs generated from BALB/c mice were incubated for 48 h with HMGB1-Bx (50 µg ml–1), LPS (100 ng ml–1) or left untreated (medium). Their T cell stimulatory capacity was assessed as above. Data shown are mean c.p.m. ± SEM, from triplicate cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
These results demonstrate that HMGB1-Bx and select HMGB1 peptides are potent activation stimuli for mouse and human DCs, and as such represent endogenous immunostimulatory molecules. Endogenous factors that stimulate DC are intriguing because they may represent a class of well-tolerated natural adjuvants (3). Hp-106, Hp-31, Hp-91 and Hp-16 act as Th1 stimuli by enhancing the production of IL-12 (p70) and IL-2. Hp-16 and Hp-106 also increase secretion of IL-18 in mouse BM-DCs. The different cytokine spectra induced by these peptides in DCs (Table 2) might allow us to use peptides to custom tailor DCs with special features for therapeutic use. For example, the IL-18-inducing capacity of certain peptides is very attractive for cancer vaccine design as IL-18 together with IL-12 promotes anti-tumor immune responses (36, 37). Hp-31, Hp-91 and Hp-16 also induced strong antigen-specific CD8+ T cell proliferation by mouse DCs.

Biotinylation of the active peptides was necessary for the observed effects in both human and mouse DCs. It is possible that biotin stabilizes the peptides. In this regard, it has been shown that introduction of biotin to the N-terminus of the insulin-like peptide promoted conformational stability which, in turn, facilitated better receptor activation (38). Biotin-binding IgM has been detected in healthy subjects (39) and a biotin-binding protein has been detected in sera of female rats (40). It is conceivable that biotin-binding proteins are present in the serum added to our culture medium. Binding to these proteins could promote multimerization of the peptides and lead to receptor cross-linking, whereas non-biotinylated peptides might not be able to bind to the receptor due to their monomeric nature.

Interestingly, Hp-106, as well as Hp-31 and Hp-91 (data not shown) did not induce phenotypic changes in BM-DCs but did cause high levels of cytokine secretion and similar level of T cell activation as Hp-16 in an allogeneic MLR, which showed strong increase in MHC-II, CD86 and CD40 expression. This suggests that cytokines can compensate for the lack of up-regulation of co-stimulatory molecules and MHC-II in BM-DCs. A similar phenomenon has been observed in macaque and human DCs that expressed the HIV or SIV Nef protein. The DCs secreted high levels of cytokines and chemokines without any phenotypic changes but showed an increased capacity to activate T cells (41). In fact, we show that Hp-31, Hp-91 and Hp-106 despite the lack of phenotypic changes induce strong antigen-specific T cell activation by mouse DCs.

It has been shown that the A box domain of HMGB1 can inhibit HMGB1 activity and reverse established sepsis (42). Interestingly, two peptides whose sequence maps to the A box domain of HMGB1 (Hp-16 and Hp-31) have a stimulatory effect on mouse DCs. Hp-16 peptide induced lower levels of the cytokines but was the only one capable of inducing phenotypic maturation. The difference between phenotypic maturation and the induction of cytokine secretion between different peptides could be due to different receptor usage and or the activation of signaling pathways. It was tested whether peptides whose sequence maps to other regions within the A box domain could inhibit the cytokine-inducing capacity of Hp-31 by mixing the flanking peptides with Hp-31. None of the peptides tested affected the stimulatory capacity of Hp-31 (data not shown). Hp-31 is not predicted to be on the surface of HMGB1 and therefore might not be accessible when the whole A box domain was tested in the sepsis model (42), but when used as peptide shows strong stimulatory activity. Hp-16 is the only peptide that was found to induce cytokine production in the mouse but not human DCs. This could be due to different receptor or adaptor molecule expression in the two species.

HMGB1 has been shown to contribute to LPS-mediated DC maturation via RAGE (19). The C-terminal motif of HMGB1 (150–183 amino acids) is responsible for RAGE binding (43). The HMGB1-Bx and the ‘active’ peptides do not contain the C-terminal motif; therefore, it is unlikely that DC maturation induced by HMGB1-Bx or the peptides occurs through RAGE. Since TLRs have been shown to be involved in HMGB1-induced activation of nuclear factor-kappa B (17), we tested whether the peptides induce DC activation through a TLR-dependent mechanism. The peptides were tested for their DC stimulatory capacity using BM-DCs from TLR2, TLR4, TLR9 and Myd88 knockout mice. No differences with respect to phenotypic changes or cytokine secretion induced by the peptides was observed in comparison with DCs from wild-type mice (data not shown), suggesting that the peptides activate mouse BM-DCs through a TLR2, -4 and -9 and Myd88-independent mechanism. Peptides of this length are not necessarily expected to have a structure and it is difficult to imagine how a single peptide could bind a receptor. Therefore, we favor the model that the N-terminal biotin allows for multimerization of the peptides. However, another possibility is that biotin transporters shuttle the peptides into the cell where they interact with intracellular targets.

Hp-91 induces high levels of IL-2 and IL-15 in human DCs and also increases secretion of IL-12 (p70) and IFN-{alpha}. Previously, LPS has been shown to induce IL-2 secretion from BM-DCs (44). Here we show that a peptide derived from an endogenous danger molecule can increase IL-2 secretion in both human and mouse DCs. Interestingly, Hp-91 also increased secretion of IFN-{alpha} in human DCs. So far, only plasmacytoid DCs have been identified as IFN-producing cells. However, the levels we observed are lower compared with what has been reported for plasmacytoid DCs. IL-15 shares many properties with IL-2, like T cell proliferation (45, 46) and it plays a crucial role in the development, survival and activation of NK cells (47). Overall, the properties of Hp-91, the induced cytokine profile and augmented T cell activation by human and mouse DCs make it a potentially potent immune adjuvant that could be used to induce anti-tumor and anti-viral immune responses.

We show in this study that HMGB1-Bx, which is about one-third the size of HMGB1, is sufficient to induce phenotypic and functional maturation of mouse BM-DCs. In addition, several HMGB1-derived peptides were identified that can activate both human and mouse DCs. Those are attractive candidates for vaccine adjuvants. The peptides not only represent attractive tools to study the contribution of different DC-derived cytokines/chemokines in various DC functions, but the selective activity of the peptides suggests a means to customize the functional properties of DCs in immunotherapeutic or vaccine context.


   Acknowledgements
 
We thank Catherine Rapelje for FACS analyses. We particularly thank Sylvie Beaulieu and Marie Larsson for critical reading of the manuscript. Supported in part by NIH grant AI 052046 to (S.D.) and NIGMS and the NCRR (to K.J.T.) and in part by the The Peter J. Sharp Foundation, the Marks Family Foundation, the Tebil Foundation and the Horace W. Goldsmith Foundation.


   Abbreviations
 
BM, bone marrow
BM-DC, bone marrow-derived dendritic cell
CFSE, 5,6-carboxyfluorescein diacetate succinimidyl ester
DC, dendritic cell
ELISPOT, enzyme-linked immunospot
GM-CSF, granulocyte macrophage colony-stimulating factor
HMGB1, high-mobility group box 1 protein
HMGB1-Bx, HMGB1 B box domain
MLR, mixed lymphocyte reaction
NIH, National Institutes of Health
OVA, ovalbumin
RAGE, receptor for advanced glycation end-products
TIL, tumor infiltrating lymphocytes
TLR, Toll-like receptor
TNF, tumor necrosis factor

   Notes
 
Transmitting editor: R. A. Flavell

Received 26 July 2005, accepted 14 August 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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