2-Hydroxypropyl-β-cyclodextrin was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Ficoll-Paque PLUS was obtained from GE Healthcare (Uppsala, Sweden). Fetal bovine serum (FBS) was purchased from GIBCO (Grand Island, NY, USA). RPMI-1640 medium and HyClone™ penicillin–streptomycin solution were from HyClone (Logan, UT, USA). Anti-human CD14 magnetic beads (clone: MΦP9) and anti-human CD3 magnetic beads (clone: HIT3a) were purchased from BD Biosciences (San Diego, CA, USA). Recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4 were purchased from R&D Systems (Minneapolis, MN, USA) and CreaGene (Sungnam, Korea), respectively. Recombinant murine GM-CSF was obtained from CreaGene. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate and enzyme-linked immunosorbent assay (ELISA) kits for the quantification of human TNF-α, IL-6, IL-12p70, and IL-10, and mouse TNF-α and IL-6 were purchased from BioLegend (San Diego, CA, USA). 5,6-Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and the Vybrant^® Alexa Fluor^® 594 lipid raft-labeling kit were obtained from Molecular Probes (Eugene, OR, USA). Filipin, bovine serum albumin (BSA), 2,2,2-tribromoethanol, 2-methyl-2-butanol, ovalbumin (OVA), and red blood cell (RBC)-lysis buffer were purchased from Sigma-Aldrich. Luria Bertani broth and Bacto™ Agar were purchased from BD Biosciences. FITC-labeled anti-human CD80 (clone: 2D10), PE-labeled anti-human CD83 (clone: HB15e), APC-labeled anti-human CD86 (clone: IT2.2), APC-labeled anti-PD-L1 (clone: 29E.2A3), PE-labeled anti-human PD-L2 (clone: 24F.10C12), APC-labeled anti-human CD25 (clone: BC96), PE-labeled anti-human CD4 (clone: RPA-T4), and PE-labeled anti-human CD8 (clone: RPA-T8) antibodies were purchased from BioLegend. FITC-labeled anti-human HLA-DR, DP, DQ (clone: Tu39) antibody for MHC class II, FITC-labeled anti-mouse CD86 (clone: GL-1) antibody, PE-labeled anti-mouse CD80 (clone: 16-10A1) antibody, PerCP-labeled anti-mouse CD11c (clone: N418) antibody, and FITC-labeled anti-mouse I-A^b (clone: 25-9-17) antibody for MHC class II were obtained from BD Biosciences. All isotype-matched antibodies were purchased from BD Biosciences or BioLegend. Horseradish peroxidase (HRP)-conjugated anti-mouse total IgG, anti-mouse IgG1, and anti-mouse IgG2a were purchased from Southern Biotech (Birmingham, AL, USA).

Human peripheral blood samples donated by healthy adult male subjects (n = 15) were provided from the Korean Red Cross (Seoul, Korea) after obtaining informed consent. All experiments using human blood were conducted under the approval of the Institutional Review Board of Seoul National University. The peripheral blood was diluted in phosphate-buffered saline (PBS) and overlaid on the Ficoll-Paque PLUS, and peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation as previously described (16, 17). PBMCs were washed with PBS three times to remove platelets and the remaining Ficoll. To isolate CD14⁺ monocytes, PBMCs were incubated with anti-human CD14 magnetic beads for 30 min at room temperature. The cells were separated on a magnetic field, and then CD14⁺ cells were enriched by positive selection. CD14⁺ monocytes were suspended at a concentration of 2 × 10⁶ cells/ml in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin solution. The isolated monocytes were differentiated into immature DCs in the presence of human recombinant GM-CSF (5 ng/ml) and IL-4 (10 ng/ml) for 6 days. Cell culture medium supplemented with GM-CSF and IL-4 was added every 3 days.

Escherichia coli BL21 (DE3) strain obtained from Stratagene (La Jolla, CA, USA) was cultured in Luria Bertani broth at 37°C until reaching mid-log phase and then harvested by centrifugation. The harvested bacterial cells were resuspended in PBS and killed by heating at 60°C for 1 h. To confirm the complete killing, the heat-treated E. coli was plated onto Luria Bertani agar plates and cultured overnight at 37°C. No bacterial colonies were observed.

Mouse BM-DCs (5 × 10⁵ cells/ml) were stimulated with either HP-β-CD (0, 0.1, or 1 mg/ml) or LPS (100 ng/ml) in the presence of murine GM-CSF (10 ng/ml) for 24 h. Human monocyte-derived DCs (2.5 × 10⁵ cells/ml) were stimulated with either HP-β-CD (0, 0.1, 0.3, or 1 mg/ml) or HKEC (1 × 10⁷ CFU/ml) in the presence of GM-CSF (2.5 ng/ml) and IL-4 (5 ng/ml) for 24 h. The unstimulated or stimulated DCs were then stained with fluorochrome-conjugated monoclonal antibodies specific to CD80, CD83, CD86, MHC class II, PD-L1, or PD-L2 for 30 min on ice, and then the cells were washed once with PBS. The geometric mean fluorescence intensity (MFI) of each group of DCs was obtained by flow cytometric analysis. More than 8,500 events were acquired for each group, and cell debris and dead cells were gated out. Phenotypes of DCs were analyzed using flow cytometry with FACSCalibur (BD Biosciences) and FlowJo software (TreeStar, San Carlos, CA, USA).

The levels of cytokines produced by DCs were quantified by ELISA, as previously described (18). Briefly, mouse BM-DCs (5 × 10⁵ cells/ml) were stimulated with either HP-β-CD (0, 0.1, or 1 mg/ml) or LPS (100 ng/ml) in the presence of murine GM-CSF (10 ng/ml) for 24 h. Human monocyte-derived DCs (2.5 × 10⁵ cells/ml) were stimulated with either HP-β-CD (0, 0.1, 0.3, or 1 mg/ml) or HKEC (1 × 10⁷ CFU/ml) in the presence of GM-CSF (2.5 ng/ml) and IL-4 (5 ng/ml) for 24 h. The levels of TNF-α, IL-6, IL-12p70, and IL-10 in the culture supernatants were measured by ELISA kits according to the manufacturers’ instructions.

To isolate CD3⁺ T lymphocytes, CD14⁺ monocyte-depleted PBMCs were incubated with anti-human CD3 magnetic beads for 30 min, and then CD3⁺ T lymphocytes were enriched by positive selection. The isolated CD3⁺ T lymphocytes were labeled with 10 μM CFDA-SE for 15 min at 37°C and washed with PBS. Immature DCs (2.5 × 10⁵ cells/ml) were stimulated with either HP-β-CD (0, 0.1, 0.3, or 1 mg/ml) or HKEC (1 × 10⁷ CFU/ml) in the presence of GM-CSF (2.5 ng/ml) and IL-4 (5 ng/ml) for 16 h. After removal of the culture supernatant, the DCs (5 × 10⁴ cells) were cocultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous CD3⁺ T lymphocytes (5 × 10⁴ cells) for 4–5 days. To analyze DC-mediated proliferation and activation of T lymphocyte subsets, the cells were stained with anti-human CD4, anti-human CD8, or anti-human CD25 antibodies and then analyzed by flow cytometry.

Dendritic cells (2.5 × 10⁵ cells/ml) were stimulated with HP-β-CD (1 mg/ml) in the presence or absence of filipin (30 μg/ml) for 45 min. Cell staining was performed with the lipid raft-labeling kit according to the manufacturer’s instructions. Briefly, unstimulated or HP-β-CD-stimulated DCs were washed once with ice-cold PBS and stained with Alexa Fluor^® 594-conjugated CTB conjugate for 10 min on ice. The DCs were washed once with ice-cold PBS and incubated with rabbit serum containing anti-CTB antibodies for 10 min on ice to crosslink lipid rafts on the surface of the DCs. Formation of lipid raft on the DCs was analyzed by confocal laser scanning microscopy (Carl Zeiss MicroImaging GmbH, Jena, Germany). Fluorescence intensity of DCs was analyzed by ZEN software, Lite Edition (Carl Zeiss, Oberkochen, Germany).

Seven-week-old male C57BL/6 mice were purchased from Orient Bio (Sungnam, Korea) and maintained in a specific pathogen-free animal facility. All experiments using animals were conducted under the approval of the Institutional Animal Care and Use Committee of Seoul National University. Care and treatment of the animals were carried out in accordance with the approved guidelines. Mice were anesthetized by intraperitoneal injection of Avertin (2,2,2-tribromoethanol and 2-methyl-2-butanol) and administered with 20 μg OVA with or without 3 mg HP-β-CD through a hind footpad. The mice were maintained for 24 h or 7 days and sacrificed to obtain the draining lymph nodes and the blood, respectively.

Twenty-four hours after the immunization with OVA in the presence or absence of HP-β-CD, draining lymph nodes, including popliteal and inguinal lymph nodes, were harvested and dissociated into a single cell suspension on a cell strainer (BD Biosciences). To analyze DC populations in the lymph nodes, the cells were stained with fluorochrome-conjugated antibodies specific to CD11c, MHC class II, CD86, and CD80 at 4°C for 30 min. CD11c⁺ MHC class II⁺ cells in the lymph nodes and their phenotypic changes upon the administration of OVA in the presence or absence of HP-β-CD were analyzed by flow cytometry using FACSVerse (BD Biosciences).

Bone marrow-derived DCs (BM-DCs) were generated as previously described (19), with minor modifications. Briefly, BM cells were isolated from mouse femurs and tibias, and the RBCs were lysed with the RBC lysis buffer. Isolated BM cells were suspended in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin solution and plated in petri dishes at 4 × 10⁵ cells/ml. The BM cells were differentiated into immature DCs in the presence of murine recombinant GM-CSF (20 ng/ml) for 7 days. Cell culture medium supplemented with GM-CSF was added every 4 days.

The statistical significance of differences between the experimental groups and the control group was analyzed using Student’s t-test. P-values less than 0.05 were considered statistically significant. Results are presented as mean value ± SD or SEM.

Maturation of DCs is an essential process for activating antigen-specific adaptive immunity and includes the upregulation of costimulatory molecules, MHC class I/II, and certain cytokines (12). Thus, we first examined the effects of HP-β-CD on the phenotypic maturation and cytokine production in human monocyte-derived DCs. Notably, HP-β-CD was not cytotoxic to DCs at concentrations up to 1 mg/ml (Figure S1A in Supplementary Material). Stimulation with HP-β-CD remarkably augmented the expression of costimulatory molecules, such as CD80, CD83, and CD86 (Figures 1A,D), together with MHC class II (Figures 1B,E). Additionally, HP-β-CD-treated DCs exhibited increased expression of inhibitory molecules, such as PD-L1 and PD-L2 (Figures 1C,F). Moreover, HP-β-CD weakly increased the expression of proinflammatory cytokines, including TNF-α and IL-6, in a dose-dependent manner (Figures 1G,H). Furthermore, HP-β-CD treatment slightly increased IL-10 expression in DCs (Figure 1I), and IL-12p70 was not detected in the culture supernatant of HP-β-CD-treated DCs (data not shown). These results suggest that HP-β-CD upregulates the expression of maturation markers coincident with weak induction of cytokines in human monocyte-derived DCs.

To examine whether HP-β-CD potentiates the T lymphocyte-activating ability of DCs, unstimulated or HP-β-CD-stimulated DCs were cocultured with autologous T lymphocytes, and the extents of T lymphocyte proliferation and activation were analyzed. HP-β-CD-sensitized DCs significantly induced T lymphocyte proliferation and CD25 expression, an activation marker of T lymphocytes (Figures 2A,B). The enhancement of proliferation and CD25 expression was observed in CD4⁺ T lymphocytes but not in CD8⁺ T lymphocytes (Figures 2C,D). However, HP-β-CD did not directly enhance the proliferative activity or CD25 expression of the T lymphocytes, indicating that HP-β-CD requires the help of DCs to activate T lymphocytes (Figures S2A,B in Supplementary Material). Therefore, the results suggest that HP-β-CD potentiates the ability of DCs to induce CD4⁺ cells.

Lipid rafts are hydrophobic microstructures that play an important role as signal transduction platforms in many eukaryotic cells (20). Since HP-β-CD interacts with cellular cholesterol (21), an essential component of lipid rafts, we examined whether HP-β-CD induces lipid raft formation on the plasma membrane based on the hypothesis that lipid rafts are involved in the HP-β-CD-induced DC maturation. DCs were stimulated with HP-β-CD in the presence or absence of filipin, which disrupts a lipid raft formation, and fluorochrome-conjugated CTB was used to detect the formation of lipid rafts. As shown in Figure 3, HP-β-CD triggered lipid raft formation on the surface of DCs, whereas such effect was blocked by preexposure to the lipid raft inhibitor filipin.

Next, we further determined the role of lipid rafts in HP-β-CD-mediated DC maturation by blocking the lipid raft formation using filipin. Of note, treatment with filipin and/or HP-β-CD was not cytotoxic to DCs (Figure S1B in Supplementary Material). As shown in Figures 4A–C, treatment with filipin remarkably attenuated HP-β-CD-elicited expression of surface costimulatory molecules, PD-L1/L2, but not MHC class II. In addition, HP-β-CD-mediated induction of TNF-α, IL-6, and IL-10 in DCs was significantly reduced upon the inhibition of lipid raft (Figure 4D). Furthermore, lipid raft inhibition in DCs stimulated with HP-β-CD abrogated their ability to activate T lymphocytes (Figure 4E). These results suggest that HP-β-CD requires the formation of lipid rafts to trigger DC maturation and further to activate T lymphocytes.

Next, we determined whether HP-β-CD has an adjuvanticity with a mouse footpad immunization model (22). To immunize mice, OVA with or without HP-β-CD was injected through a hind footpad, and titers of serum antibodies specific to OVA were determined. Coadministration with HP-β-CD and OVA efficiently increased OVA-specific total IgG in the blood (Figure S3A in Supplementary Material). IgG1 was the major antibody subtype induced following immunization (Figure S3B in Supplementary Material), and no IgG2a antibodies were detected (data not shown). As DCs are crucial in the mediation of naive T cell responses, we subsequently analyzed DC populations in the draining lymph nodes, including popliteal and inguinal lymph nodes upon administration of OVA with or without HP-β-CD. Mice administered with OVA with HP-β-CD showed an increase in the size and cell number of the draining lymph nodes (Figure 5A). OVA administration with HP-β-CD markedly augmented the number of CD11c⁺ MHC class II⁺ cells in the popliteal and inguinal lymph nodes (Figures 5B,C). In addition, the DCs showed an upregulation in the expression of costimulatory molecules, including CD80 and CD86 (Figures 5D,E). To determine whether DC activation in the draining lymph nodes of mice administered with OVA and HP-β-CD is directly attributed to stimulatory functions of HP-β-CD, we examined HP-β-CD effects on the maturation of BM-DCs generated in vitro. HP-β-CD markedly upregulated the expression of surface costimulatory molecules, such as CD80, CD83, and CD86, and MHC class II (Figure 5F). In addition, HP-β-CD-treated BM-DCs modestly increased the production of proinflammatory cytokines including TNF-α and IL-6 in BM-DCs compared to LPS-stimulated BM-DCs (Figure 5G). Collectively, these results suggest that HP-β-CD-induced DC maturation and activation in vivo that might be necessary for the adjuvanticity.