The Adjuvanticity of a Mannosylated Antigen Reveals TLR4 Functionality Essential for Subset Specialization and Functional Maturation of Mouse Dendritic Cells

Kuo-Ching Sheng, Martha Kalkanidis, Dodie S. Pouniotis, Mark D. Wright, Geoffrey A. Pietersz and Vasso Apostolopoulos
J Immunol August 15, 2008, 181 (4) 2455-2464; DOI: https://doi.org/10.4049/jimmunol.181.4.2455

Abstract

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24high (conventional CD8+ equivalent) and CD11bhigh (CD8− equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA+ plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-β, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.

Dendritic cells (DCs)4 are immune sentinels that express an extensive repertoire of pattern recognition receptors to detect pathogen-associated molecular patterns (PAMPs). One class of pattern recognition receptors is the TLR family, which recognizes a wide range of microbial and endogenous structures (1). PAMP recognition by TLRs induces DC activation exemplified by production of proinflammatory cytokines and phenotypic maturation, which are critical in control of polarization and magnitude of T cell responses (2, 3). Additionally, emerging evidence suggests that Ag processing/presentation is also largely dependent on concomitant TLR signaling, due to its role in phagosome maturation and Ag routing (4, 5, 6). TLR stimulation can also significantly lower the threshold of Ag concentration required to stimulate a T cell response (6).

DCs are comprised of heterogeneous subsets (7). DC functionality in Ag presentation is closely associated with its phenotype. For instance, while both CD8+ and CD8− conventional DCs present endogenous viral Ags on MHC class I and II molecules, only CD8+ DCs cross-present exogenous Ags. This intrinsic feature of CD8+ and CD8− DCs is dictated by Ag processing, but not capture (8, 9). CD11c+B220+ plasmacytoid DCs are inefficient in stimulating naive T cells, in contrast to CD11c+B220− DCs (10). Such a poor capability in T cell stimulation results from the immature phenotype and ineffective Ag presentation. However, after activation with appropriate stimuli, plasmacytoid DCs become efficient T cell stimulators (11, 12).

In contrast to human DCs, TLRs are less differentially expressed in mouse DC subsets (13, 14, 15, 16). Nonetheless, there is evidence to suggest that TLRs play a role in DC subset specialization, on the basis of cytokine (e.g., type I IFN or IL) induction (3, 11, 12, 16, 17). Their role in Ag presentation by DC subsets has, however, not yet been thoroughly investigated. Given the influence of both TLR stimulation and intrinsic divergence on Ag presentation capabilities of DCs, it is open to question as to whether a PAMP-associated Ag induces differential functional maturation (both Ag processing/presentation and phenotypic activation) among DC subsets in correspondence to TLR expression and how the TLR is involved in this process. This is particularly important to resolve how DC subsets selectively present specific Ags and distinguish foreign from self Ags to initiate T cell responses when exposed to various PAMPs in the course of microbial infection.

We previously synthesized a mannosylated Ag that was linked with a dendrimer (mannosylated dendrimer OVA (MDO)). Its immunogenicity was demonstrated by the strong OVA-specific immune responses induced in vitro and in vivo and by its efficacy in induction of tumor protection in MDO-immunized mice (18). In the present study, using MDO as a PAMP-associated Ag, we further investigate its adjuvanticity in two heterogeneous DC populations, bone marrow DCs (BMDCs) composed of CD24high and CD11bhigh subsets and fms-like tyrosine kinase 3 ligand (Flt3-L) DCs, which contain CD24high, CD11bhigh, and B220+ DCs (functional equivalents of mouse CD8+, CD8−, and plasmacytoid subsets in vivo) (15, 19). The advantage of using in vitro DCs is that they do not constitutively mature, unlike freshly isolated mouse DCs. In this study, we show that, although MDO binds all in vitro DCs, DC subsets display differential capabilities in processing and presenting MDO. MDO-loaded BMDCs competently stimulate both CD4+ and CD8+ T cells. In comparison, Flt3-L CD24high DCs are efficient in cross-presenting MDO to CD8+ T cells, while the CD11bhigh subset is potent in activating CD4+ T cells. Plasmacytoid DCs present MDO poorly to either CD4+ or CD8+ T cells. MDO induces maturation of BM, CD24high, and CD11bhigh Flt3-L DCs, but not plasmacytoid B220+ DCs. This preferential maturation effect is largely mediated by TLR4. Interestingly, defective TLR4 signaling does not affect the binding and internalization of MDO, but it does affect cellular and subcellular maturation of DCs, despite indirect interaction between MDO and TLR4. TLR4-defective DCs are unable to process MDO, due to the failure to localize the Ag in lysosomes. Moreover, as a TLR signaling adaptor, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-β (TRIF), but not MyD88, regulates MDO-induced DC maturation and Ag presentation. We conclude that TLR functionality plays a critical role in efficient localization and processing of a PAMP-associated Ag, as well as functional maturation and specialization of DC subsets.

Materials and Methods

Animals

C57BL/6, C3H/He, C3H/HeJ, OTI, OTII, and MyD88-deficient mice (aged 6–10 wk) used throughout this study were purchased from the animal facilities of the Walter and Eliza Hall Institute (Melbourne, Australia). C57BL/6 mice were used as wild-type mice. C3H/HeJ mice were donors of TLR4-defective DCs due to an inactivating point mutation in the Lpsd/Ran gene, while C3H/He DCs were TLR4-competent. OTI and OTII mice were donors of OVA peptide-specific CD8+ and CD4+ T cells, respectively. MyD88-deficient mice were of the C57BL/6 background.

Preparation of MDO

Before MDO synthesis, endotoxin (>6 endotoxin units (E.U.)/ml (1 ng/ml)) (EML) was removed from OVA (Sigma-Aldrich) with Triton X-114 (BDH). The OVA solution containing 1% (v/v) Triton X-114 was gently mixed on a rotating wheel at 4°C for 30 min, then incubated at 30°C for 10 min and centrifuged at 2500 rpm for 10 min. The upper layer of the solution was collected. The above procedure was repeated three times, followed by overnight dialysis into PBS. Following this protocol, the final OVA product contained an undetectable level of LPS (<0.06 E.U./ml (0.01 ng/ml)) (EML). To synthesize MDO (18), mannosylated dendrimer (MD) was prepared. Eighty-four microliters of α-d -mannopyranosylphenyl isothiocyanate (Sigma-Aldrich) in DMSO (31.33 mg/ml) was slowly added into a solution containing 44 μl of 10% (w/v in methanol) generation 4 PAMAM dendrimer (Dendritic Nanotechnologies) and 372 μl of 0.2 M sodium bicarbonate buffer (pH 9.0). The solution was mixed on the rotating wheel for 16 h at room temperature (RT) and then dialyzed overnight into PBS at 4°C to remove unconjugated α-d -mannopyranosylphenyl isothiocyanate. Mannose residues present in dendrimer were quantitated by a colorimetric assay as previously described (20). To prepare MDO, 100 μl of 0.2 M tris(2-carboxyethyl)phosphine (TCEP) (Pierce) in PBS was added to the MD solution and left for 30 min at RT to generate the reduced form of MD. The solution was dialyzed overnight into PBS containing 2 mM EDTA at 4°C to remove excess TCEP. The sulfhydryl groups were quantitated as described previously (21, 22). OVA was modified by addition of 18 μl of 20 mg/ml N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Pierce) in DMSO to 500 μl of 10 mg/ml OVA in PBS, and mixed for 16 h at RT to generate OVA-SPDP. The solution was then dialyzed overnight at 4°C to remove unconjugated SPDP. The number of activated disulfide groups present in OVA-SPDP was quantitated as described previously (23). OVA-SPDP was subsequently added to the reduced MD solution (OVA-SPDP/MD, 1/12) to form MDO. The number of MD residues incorporated onto OVA was determined by increased OD at 343 nm. The sample was concentrated using an Amicon centrifugal filtration device (Millipore). Excess MD and OVA was removed by gel filtration chromatography using a Superdex-75 column (0.5 × 30 cm) (Pharmacia Biotech). The amount of OVA of MDO was quantitated by reduction of MDO with 2-ME, followed by densitometry analysis after SDS-PAGE electrophoresis. Therefore, the concentration of MDO was calculated based on OVA throughout this study. Following these procedures, MDO was tested for the endotoxin level and contained lower than <0.06 E.U./ml (0.01 ng/ml) of LPS (EML). This concentration of LPS was not sufficient to stimulate DC activation (24). To prepare MDO-FITC, 0.0226 mg FITC dissolved in 0.0226 ml DMSO was added to 0.5 mg MDO dissolved in 0.2 M, 0.399 ml sodium bicarbonate. The solution was rotated for 16 h at RT and dialyzed overnight in PBS at 4°C.

Generation of BMDC and Flt3-L DC subpopulations

BM cells from femurs and tibias of C57BL/6, C3H/He, C3H/HeJ, or MyD88-deficient mice were collected and treated with ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA) to lyse erythrocytes. Cells were washed and cultured with complete RPMI 1640 media (2% HEPES buffer, 0.1 mM 2-ME, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 10% FCS) in 24-well plates. To generate BMDCs, 5 × 105 cells were seeded in 1 ml complete medium supplemented with 10 ng/ml GM-CSF and IL-4 (BD Pharmingen) in each well and cultured for 6–7 days. For Flt3-L DC generation, 106 cells were seeded in 500 μl complete medium supplemented with 300 ng/ml Flt3-L (Sigma-Aldrich) in each well and cultured for 8–9 days. By these culture protocols, cells were consistently >85% and >96% CD11c+ in GM-CSF/IL-4 and Flt3-L cultures, respectively. To purify Flt3-L DC subpopulations, cells were labeled with fluorochrome-conjugated anti-CD11c, anti-CD11b, anti-CD24, and anti-CD45RA (BD Pharmingen) and separated by the FACSAria cell sorter (BD Biosciences). Alternatively, cells were labeled with biotinylated anti-CD45RA, biotinylated anti-CD24, or anti-CD11b (BD Pharmingen) and serially purified with anti-biotin or anti-CD11b magnetic beads through the autoMACS cell-sorting system (Miltenyi Biotec). With these methods, the purities of Flt3-L DC subsets were at least 93%.

MDO binding and internalization

To examine the binding of MDO and OVA to DCs, 5 × 105 pelleted DCs were incubated with 100 μl of 40 μg/ml MDO-FITC or OVA-FITC at 4°C for 30 min. Alternatively, cells were stained with 40 μg/ml MDO at 4°C for 30 min, washed, and resuspended with 100 μl rabbit polyclonal anti-OVA Ab (Bethyl Laboratories) at 4°C for 30 min. These cells were washed and stained with the FITC-conjugated sheep anti-rabbit Ig Ab (Silenus) and anti-CD11c-APC (BD Pharmingen) at 4°C for 30 min. The binding of MDO or OVA was demonstrated by FITC fluorescence intensity of CD11c+ BMDC (CD24high and CD11bhigh) subsets and Flt3-L DC (CD24high, CD11bhigh, and CD24−CD11b−) subsets. To study MDO internalization, C3H/He and C3H/HeJ BMDCs (5 × 105) were incubated with 40 μg/ml MDO in 37°C culture and harvested at 0 min, 5 min, 30 min, 2 h, and 4 h after pulsing. In some experiments, before MDO treatment, cells were fixed by 4% (w/v) paraformaldehyde in PBS for 10 min and permeablized by 0.5% (w/v) saponin in PBS for 10 min. Cells treated with MDO or PBS (background control) were labeled with rabbit anti-OVA Ab, followed by FITC-conjugated sheep anti-rabbit Ig Ab.

T cell purification

Splenocytes from OTI or OTII mice were collected, washed, and incubated in ACK lysis buffer at 37°C for 5 min to lyse erythrocytes. Splenocytes were washed, counted, and incubated with Ab cocktail containing rat anti-mouse Gr-1 (RB6–8C5), anti-B220 (RA3–6B2), anti-CD11b (M1/70.15), anti-erythrocyte (TER-119), and anti-MHC class II (M5/114) at 4°C for 30 min. Cells were washed and unwanted cells were depleted with two rounds of bead separation. In each round, cells were incubated with BioMag goat anti-rat magnetic beads (Qiagen) (8 beads/cell) at 4°C for 25 min. Cells bound to the beads were removed by magnets. The purity of T cells was consistently >95%.

T cell proliferation

To evaluate peptide-specific T cell proliferative responses, DCs were incubated with specified concentrations of control peptides (H-2Kb-restricted SIINFEKL and I-Ab-restricted ISQAVHAAHAEINEAGR (OVA323–339)), OVA, or MDO for 18 h. DCs were irradiated, washed, and seeded at specified cell numbers (0–4 × 103) into the 96-well plates containing 2 × 104 OTI or OTII T cells in quadruplicates with a final volume of 200 μl per well. In some experiments, seeded BMDCs were pretreated with either 0–50 μg/ml anti-TLR4/MD2 Ab (MTS510) (BioLegend) at RT for 20 min or 80 μM (−)-epigallocatechin-3-gallate (EGCG) (Sigma-Aldrich) as a TRIF signaling inhibitor at 37°C for 1 h, before incubation with peptides or Ags. Proliferation of T cells was monitored between days 1 and 5 by addition of 1 μCi [3H]thymidine (Amersham Biosciences). The radioactivity was measured in cpm by the Packard TopCount scintillation counter (PerkinElmer). Peak proliferation of OTI (on day 2) and OTII (on day 3) between various stimulants was compared.

Induction of DC maturation

PBS, MDO (40 μg/ml), LPS (1 μg/ml), poly(I:C) (100 μg/ml), or CpG1668 (GeneWorks) (10 μg/ml) were added into DC cultures. In some experiments, DCs were pretreated with monoclonal anti-TLR4/MD2 Ab MTS510 or EGCG. After 18 h incubation, 5 × 105 cells collected from each stimulant condition were pelleted and stained individually with standardized in-house FITC-conjugated anti-CD40, CD80, CD86, and MHC class II (IAb), together with anti-CD11c-APC and/or anti-CD11b-PE, anti-CD24-PECy7. The FITC fluorescence intensity of each DC population was examined by flow cytometry to determine maturation states. To investigate the effect of stimulation on DC morphology, MDO or LPS was added into C3H/He and C3H/HeJ DC cultures. After 18 h stimulation, DCs were visualized by the Leitz DMX light microscope (Leica).

TLR4 knockdown

TLR4 expression in the DC culture was silenced using a TLR4 siRNA kit (Santa Cruz Biotechnology) with a modified protocol from the manufacturer. Briefly, for each tranfection, 8 μl of control or TLR4 siRNA duplex was added into 200 μl of transfection medium, which contained 6 μl of the transfection reagent. The solution was incubated at RT for 15–45 min and mixed with 800 μl of transfection medium. Freshly isolated BM cells (5 × 105) prewashed with 2 ml transfection medium were resuspended with the solution prepared previously and incubated at 37°C for 7 h in a 24-well plate, followed by addition of 2 ml complete RPMI 1640 media that contained 2× concentrations of the normal serum and antibiotics. After 24 h incubation, the medium was replaced with normal complete RPMI 1640 and cells were cultured for an additional 48 h. To examine TLR4 expression of DCs, siRNA-treated cells were pelleted and treated with 50 μg/ml anti-TLR4 Ab at 4°C for 30 min. Cells were washed and stained with FITC-conjugated goat polyclonal anti-rat Ab and anti-CD11c-APC, followed by flow cytometry analysis. TLR4-deficient DCs as well as control DCs were further tested for MDO-FITC binding.

Confocal microscopy studies

BM cells (2 × 104) were seeded onto sterile poly-l -lysine (Sigma-Aldrich) coated round coverslips in 24-well plates (BD Biosciences) in complete media containing 10 ng/ml of recombinant GM-CSF and IL-4. At day 5, culture media was aspirated and replaced with 200 μl of serum-free media. Cells were incubated with MDO-FITC (25 μg/ml) for 24 h at 37°C. They were then washed and resuspended in serum-free media with or without LysoTracker Red (100 nM) (Molecular Probes) for 30 min at 37°C. Cells were washed twice with Ca2+- and Mg2+-free HBSS buffer (Invitrogen) and resuspended with −20°C acetone for 2 min. Coverslips were removed, air-dried, and mounted on frosted glass slides with the anti-fade reagent (Citifluor). Cells were visualized by the Krypton Leica TCS NT laser scanning confocal microscope at Monash University (Melbourne, Australia).

Results

MDO binds to DC subpopulations

Both GM-CSF/IL-4-cultured BMDCs and Flt3-L DCs were characterized for their binding to MDO and OVA. As shown in Fig. 1⇓, CD11c+ BMDCs were divided into CD11c+CD24high and CD11c+CD11bhigh subsets, previously characterized as inflammatory DCs, while Flt3-L DCs were separated into CD11c+CD24high, CD11c+CD11bhigh, and CD11c+CD24lowCD11blow (also CD45RA+B220+, not shown) subsets, which were equivalent to splenic CD8+, CD8−, and plasmacytoid DCs, respectively (10, 15, 19). While MDO bound to all DCs, OVA showed limited or no binding to BMDCs and Flt3-L DCs (Fig. 1⇓).

FIGURE 1.
FIGURE 1.

MDO, but not OVA, binds to DCs. BMDCs (5 × 105) or Flt3-L DCs (5 × 105) were incubated with 40 μg/ml MDO-FITC or OVA-FITC at 4°C and stained with fluorochrome-conjugated Abs (anti-CD11c, anti-CD11b, and/or anti-CD24) and analyzed by FACS. CD11c+ BMDC (CD24high and CD11bhigh) subsets and Flt3-L DC (CD24high, CD11bhigh, and CD11blowCD24low) subsets were gated based on the dot plots. The relative binding of MDO (solid line) and OVA (dotted line) in comparison to the untreated cell control (gray area) was determined by FITC fluorescence intensity.

Both BMDCs and Flt3-L DCs present MDO efficiently

BMDCs or Flt3-L DCs pulsed with MDO (40 μg/ml) were highly stimulatory to OVA-specific CD8+ (OTI) and CD4+ (OTII) T cells, in comparison to DCs loaded with OVA (40 μg/ml). DCs loaded with peptides (SIINFEKL (1 μg/ml) and OVA323–339 (10 μg/ml)) were used as internal positive controls (Fig. 2⇓). T cell proliferative responses to MDO-loaded DCs were further increased with elevated DC numbers. Therefore, MDO was internalized, processed, and presented effectively by DCs following either MHC class I or MHC class II Ag presentation pathways.

FIGURE 2.
FIGURE 2.

MDO-pulsed BMDCs and Flt3-L DCs induce OVA-specific CD4+ and CD8+ T cell proliferation. Titrated (1–4 × 103) BMDCs (A) and Flt3-L DCs (B) pulsed with MDO (40 μg/ml), OVA (40 μg/ml), or control peptides (SIINFEKL (1 μg/ml) and OVA323–339 (10 μg/ml)) were seeded with 2 × 104 OTI or OTII T cells. T cell proliferation was monitored for 5 days with [3H[thymidine incorporation. Peak proliferation on day 2 and day 3 for OTI and OTII T cells was compared. The data shown are representative of three experiments.

Flt3-L DC subpopulations differentially present MDO

Sorted Flt3-L DCs were compared for their capabilities in processing and presenting MDO (Fig. 3⇓). The MDO-loaded CD24high DCs were more effective in stimulating CD8+ T cells, while CD11bhigh subset was a more efficient CD4+ T cell stimulator. MDO-pulsed plasmacytoid DCs stimulated little OTI and a low level of OTII T cell proliferation. All peptide-loaded control DCs were relatively efficient in stimulating CD4+ and CD8+ T cells. Given the comparable levels of maturation markers expressed on CD24high and CD11bhigh DCs after MDO stimulation (Fig. 4⇓A), these results suggested that, after uptake of exogenous MDO, CD24high DCs were efficient in MDO delivery into MHC class I compartments for processing and cross-presentation, whereas CD11bhigh DCs primarily processed the Ag via the endocytic pathway for MHC class II presentation. Plasmacytoid DCs failed to process and present MDO effectively to either OTI or OTII T cells.

FIGURE 3.
FIGURE 3.

Flt3-L DC subsets exhibit differential capabilities in presenting MDO. Sorted Flt3-L CD45RA+, CD24high, and CD11bhigh DCs (2–4 × 103) pulsed with 40 μg/ml MDO or control peptides (SIINFEKL (1 μg/ml) and OVA323–339 (10 μg/ml), denoted with S and H, respectively) were seeded with 2 × 104 OTI or OTII T cells. Peak T cell proliferation was monitored and compared as described. The data shown are representative of five experiments.

FIGURE 4.
FIGURE 4.

MDO induces differential levels of maturation among DC subsets. BMDC and Flt3-L DC subsets were incubated with various stimulants including PBS (negative control, thin line), MDO (thick line), LPS (dotted line), and CpG (broken line) for 18 h in culture and analyzed for activation marker CD40, CD80, CD86, and MHC class II expression. CD24high and CD11bhigh subsets derived from either BMDC and Flt3-L DC cultures were both stimulated by MDO, LPS, and CpG (not shown) (A), while the B220high/CD45RA+ plasmacytoid subset derived from the Flt3-L DC culture was only responsive to CpG stimulation (B).

MDO induces maturation of all DCs, except the plasmacytoid subset

In the steady-state, BMDCs generally expressed higher levels of CD40, CD80, and CD86 than did Flt3-L DCs and were less responsive to MDO and LPS stimulation (indicated by only slight up-regulation of activation markers) (Fig. 4⇑A). Nevertheless, MDO intermediately stimulated up-regulation of CD40, CD80, CD86, and MHC class II of CD24high and CD11bhigh DC subpopulations derived from both cultures in comparison to LPS (Fig. 4⇑A). The maturation patterns of CD24high and CD11bhigh DC subsets of each culture condition were somewhat similar. By contrast, B220high/CD45RA+ plasmacytoid DCs appeared to be least mature among all DCs examined, expressing low levels of CD40, CD80, CD86, and MHC class II in the steady-state (Fig. 4⇑). Both MDO and LPS barely induced CD40, CD80, CD86, and MHC class II up-regulation, while CpG induced strong activation of this DC subpopulation (Fig. 4⇑B).

MDO-induced DC maturation is largely dependent on TLR4

Before stimulation, C3H/He and C3H/HeJ DCs expressed similar levels of CD40, CD80, and CD86 (Fig. 5⇓A). In a manner similar to LPS, MDO failed to induce up-regulation of CD40, CD80, and CD86 in TLR4-defective C3H/HeJ BMDCs, while it induced maturation of the wild-type C3H/He DCs (Fig. 5⇓A). Both C3H/He and C3H/HeJ DCs were equally responsive to TLR9-mediated CpG stimulation by upregulating CD40, CD80, and CD86 (Fig. 5⇓A). Morphologically, C3H/He DCs and C3H/HeJ DCs appeared similar (Fig. 5⇓B, i and ii). After MDO, however, C3H/He DCs developed distinct intracellular granular structures (Fig. 5⇓Biii), which were not observed in MDO-stimulated C3H/HeJ DCs (Fig. 5⇓Biv). The high granularity was also demonstrated by an elevated level of the side scatter in flow cytometry (not shown). In comparison to MDO, LPS-stimulated C3H/He DCs also developed distinct intracellular granules that were less glittery (Fig. 5⇓Bv). Notably, C3H/He BMDCs were more responsive to MDO stimulation than were C57BL/6 counterparts (Figs. 4⇑A and 5⇓A).

FIGURE 5.
FIGURE 5.

MDO-induced DC maturation is largely dependent on functional TLR4. C3H/He (TLR4-competent, solid line) and C3H/HeJ (TLR4-defective, dotted line) BMDCs that were not stimulated (NS) or stimulated with MDO, LPS, and CpG for 18 h were analyzed for activation marker CD40, CD80, and CD86 expression by FACS (A). The morphology of C3H/He and C3H/HeJ DCs, which were not stimulated (i and ii) or stimulated with MDO (iii and iv) or LPS (v and vi), was compared by direct light microscopy (B). The inset (v) represents a LPS-stimulated cell from another field.

TLR4 is required for competent MDO processing and presentation, but not uptake by DCs

As shown in Fig. 6⇓A, surface binding of MDO to DCs was effective within 5 min, while MDO was continuously internalized by DCs within 4 h. No difference in surface binding or internalization of MDO was observed, indicating that impaired TLR4 signaling did not affect MDO uptake (Fig. 6⇓A). However, MDO failed to localize at the lysosomal compartments in C3H/HeJ DCs, in contrast to C3H/He DCs, which showed colocalization of MDO with lysosomes (Fig. 6⇓B), indicating the role of TLR4 in endolysosomal reorganization. Moreover, DCs that were pretreated with titrated TLR4/MD2-blocking Ab exhibited an increasingly reduced capability in processing MDO (Fig. 6⇓C). The maturation effect of MDO (up-regulation of CD40, CD80, and CD86) was also abolished when DCs were pretreated with 50 μg/ml anti-TLR4 (Fig. 6⇓D, right panel). Anti-TLR4 Ab treatment did not alter the expression of costimulatory markers in control DCs (Fig. 6⇓D, left panel). These results suggest that TLR4 functionality is critical in Ag processing and presentation, due to its role in cellular and subcellular maturation of DCs.

FIGURE 6.
FIGURE 6.

Competent TLR4 is required for successful Ag processing and DC functional maturation. C3H/He and C3H/HeJ BMDCs incubated with 40 μg/ml MDO at 37°C were harvested at 0 min, 5 min, 30 min, 2 h, and 4 h and analyzed for MDO binding and internalization by surface staining (SS) and intracellular staining (IS), respectively (A). The localization of MDO-FITC and acidic lysosomal compartments in C3H/He (a and b) and C3H/HeJ (c and d) DCs was visualized with confocal imaging after 16 h incubation (B). C57BL/6 BMDCs pretreated with titrated (0–50 μg/ml) anti-TLR4/MD2 Ab (MTS510) were loaded with MDO, OVA323–339, and PBS (control). DCs were seeded with OTII T cells in the proliferation assay, and peak proliferation for each anti-TLR4 Ab titration was compared at day 3 (C). In the absence (left panel) or presence (right panel) of MDO, maturation states of BMDCs, which were pretreated with (shaded area) or without (solid line) TLR4 blocking Ab, were determined by CD40, CD80, and CD86 expression (D).

MDO indirectly interacts with the TLR4/MD2 complex and fails to synergize with LPS

TLR4 expression in the DC population was greatly reduced by TLR4 siRNA (>95%), in contrast to control siRNA (Fig. 7⇓A). TLR4-deficient DCs bound to MDO-FITC at a level comparable to TLR4-expressing control DCs (Fig. 7⇓A). TLR4-expressing DCs that were pretreated with 100 μg/ml or 300 μg/ml (not shown) TLR4/MD2-blocking Ab MTS510 did not show decreased MDO-FITC binding (Fig. 7⇓B). These results indicated that TLR4 did not bind directly to MDO. Interestingly, in contrast to anti-TLR4, LPS partially blocked the binding of MDO to DCs, suggesting that certain DC surface receptor(s) that recognized LPS also bound to MDO (Fig. 7⇓B). Since both MDO and LPS activated DCs through TLR4, the synergy of MDO with substimulatory doses of LPS should indicate whether such a dependence was interconnected. As shown in Fig. 7⇓C, trace amounts of LPS (≤10 ng/ml) did not enhance the MDO effect on DC maturation, while high concentrations of LPS overrode the MDO effect. This result indicates that the respective pathways used by LPS and MDO to initialize upstream TLR4 signaling are divergent and not interconnected.

FIGURE 7.
FIGURE 7.

MDO does not directly interact with the TLR4/MD2 complex or synergize with LPS to induce DC maturation. On day 3, BM cultures treated with siRNA (solid line) or control siRNA (dotted line) were labeled with the anti-TLR4/MD2 Ab MTS510, followed by the secondary FITC-conjugated anti-rat Ab, at 4°C for 30 min. siRNA-treated CD11c+ cells were compared for TLR4 expression with non-siRNA treated CD11c+ cells stained with the secondary Ab (shaded area) by flow cytometry (A, left). TLR4-silenced and control cells were treated with MDO-FITC at 4°C for 30 min, in comparison to nontreated cells (A, right). BMDCs pretreated with 100 μg/ml MTS510 or 80 μg/ml LPS at 4°C for 30 min (solid line) or without (dotted line) were stained with MDO-FITC, in comparison to nonstained cells (shaded area) (B). BMDCs were incubated with or without 40 μg/ml MDO in the presence of titrated LPS (0–1 μg/ml) in separated wells for 18 h at 37°C. Cells were harvested and stained for CD40 and CD80 expression in the CD11c+ DC population. Mean fluorescence intensity values of CD40 and CD80 expression derived from DCs pulsed with titrated LPS in the absence (open bars) or presence (gray bars) of MDO were compared (C).

TRIF, but not MyD88, is a critical TLR signaling modulator for MDO-induced DC maturation and Ag presentation

To investigate whether MyD88 was crucial for MDO-induced TLR4 functionality, MyD88-deficient BMDCs were tested for phenotypic activation and Ag presentation after MDO stimulation. MyD88-deficient Flt3-L CD24high and CD11bhigh DCs were responsive to LPS (not shown) and MDO stimulation by up-regulating CD40, CD80, CD86, and MHC class II expression, while they were not upon CpG stimulation (Fig. 8⇓A). MDO-loaded MyD88-deficient BMDCs were also capable of efficiently processing and presenting MDO, leading to both CD4+ and CD8+ T cell proliferation, suggesting that MyD88 was not required for effective MDO processing and presentation (Fig. 8⇓B). To investigate the role of TRIF in MDO adjuvanticity, EGCG as a potential TRIF and MyD88 signaling pathway inhibitor was used (25). As shown in Fig. 9⇓A, EGCG diminished up-regulation of CD40, CD80, and CD86 induced with poly(I:C) or MDO to a much greater extent than did CpG, suggesting that EGCG served primarily as a TRIF signaling inhibitor. To examine the role of TRIF in MDO presentation by DCs, cross-presentation induced by MDO and CpG/OVA mix (6) was investigated. When pulsed with MDO, EGCG-pretreated BMDCs failed to efficiently stimulate CD8+ T cells, while presentation of CpG/OVA mix, surface-bound SIINFEKL, or OVA alone by these DCs remained unaffected (Fig. 9⇓B). This finding suggests that the TRIF pathway largely regulates MDO-mediated DC activation.

FIGURE 8.
FIGURE 8.

MyD88 is not a critical signaling modulator for MDO-induced DC maturation and Ag presentation. MyD88-deficient CD24high and CD11bhigh Flt3-L DCs were stimulated with PBS (control, shaded area), MDO (dotted line), LPS (not shown), and CpG (solid line) and surface expression of CD40, CD86, CD86, and MHC class II was evaluated by flow cytometry analysis (A). Titrated MyD88-deficient BMDCs pretreated with MDO, OVA, and control peptides (SIINFEKL and OVA323–339) for 18 h were seeded with OTI or OTII T cells. Peak proliferation for each DC titration at day 2 and day 3 for OTI and OTII T cells was compared (B).

FIGURE 9.
FIGURE 9.

The TRIF pathway regulates MDO-induced DC maturation and Ag presentation. BMDCs pretreated with EGCG (80 μM) at 37°C for 1 h were pulsed with PBS (denoted as NS, nonstimulant), MDO (40 μg/ml), poly(I:C) (100 μg/ml), and CpG (10 μg/ml) for an additional 18 h. DCs were harvested and examined for CD40, CD80, and CD86 expression by flow cytometry. The shaded area represents the isotype control (A). EGCG-pretreated BMDCs (2–8 × 103) pulsed with OVA (40 μg/ml), MDO (40 μg/ml), CpG (10 μg/ml)/OVA (40 μg/ml) mix, or SIINFEKL (1 μg/ml) for 18 h were seeded with 2 × 104 OTI T cells. T cell proliferation was monitored and peak proliferation on day 2 was compared (B). The data shown are representative of at least two experiments.

Discussion

BMDCs and Flt3-L DCs are two diverse DC populations that have been extensively characterized in recent years. Both DC populations have been shown to represent specific in vivo DCs based on the criteria generated with phenotyping and functional characterization. BMDCs are considered as in vivo monocyte-derived DC equivalents during inflammation. The delineation of their subset specialties, however, is not yet clear. In contrast, Flt3-L CD24high, CD11bhigh, and B220/CD45RA+ DC subsets as representatives of conventional splenic CD8+, CD8−, and plasmacytoid B220+ DCs are well defined (10, 15, 19, 26, 27). In the present study, we show that both BMDCs and Flt3-L DCs process the PAMP-associated Ag MDO efficiently by both MHC class I and II Ag presentation pathways. Interestingly, Flt3-L DC subsets are distinct in their capabilities in processing and presenting exogenous MDO. Plasmacytoid DCs process and present MDO poorly, while both CD24high and CD11bhigh DCs are competent presenters. CD11bhigh DCs primarily process MDO following the MHC class II presentation pathway. CD24high DCs appear to be proficient in cross-presenting MDO to CD8+ T cells. This finding is consistent with previous studies (8, 15, 26, 28). All DC subsets are highly stimulatory to T cells when loaded with control peptides.

Through characterizing the adjuvanticity of MDO as a PAMP-associated Ag in DC subsets, TLR4 functionality in Ag presentation is fully demonstrated. MDO induces Ag presentation and DC maturation only in the presence of competent TLR4, while MDO internalization can be mediated by receptors including C-type lectins (18). MDO fails to elicit morphological/phenotypic maturation of C3H/HeJ DCs, in which no lysosomal localization is observed, given that it is bound and internalized at levels equivalent to wild-type C3H/He DCs. BMDCs pretreated with anti-TLR4 blocking Ab do not optimally process and present MDO nor display phenotypic activation. Flt3-L-derived plasmacytoid DCs do not express TLR4 and barely respond to MDO or LPS stimulation, in contrast to conventional CD24high and CD11bhigh DCs and BMDCs (15). They do, however, respond robustly to stimuli such as viral nucleic acid patterns (e.g., CpG) (10). To this end, the poor response of plasmacytoid DCs to MDO is reasoned by the lack of corresponding TLR4 expression.

The variability of DC subsets in response to MDO suggests that DC functionality toward a specific pathogen can be significantly influenced by the expression of the targeted TLR (5). It is possible that PAMP stimulation, on the one hand, results in specific proinflammatory cytokines to polarize T cell responses (3); on the other hand, it induces phenotypic maturation and enhances Ag processing/presentation, leading to optimal T cell activation (4, 29). Apart from TLR expression, the intrinsic variance of DC subsets plays a role in determining how DCs process a pathogen-derived Ag. CD24high DCs, after MDO stimulation, still maintain a strong capability to cross-present the exogenous Ag to prime CD8+ T cell proliferation, while CD11bhigh cells are particularly efficient in presenting MDO following the MHC class II presentation pathway. These results may indicate a cognate role of two conventional DC subsets in establishing immunity against pathogen-associated mannosylated Ags such as yeast mannoproteins (30, 31).

In our study, concomitant TLR4 stimulation is critical for successful MDO processing and presentation in TLR4-expressing DC subsets, suggesting that coupling TLR signaling with Ag delivery can greatly enhance the Ag processing capacity of DCs. This finding is consistent with previous studies (4, 5). Activation of DCs with TLR agonists such as LPS has been shown to transform MHC class II compartments from intracellular endosomes into endolysosomal tubules that move toward the cell surface in a microfilament-dependent manner (32, 33). Maturation of DCs enhances the lysosomal function and Ag proteolysis (5, 21). We have previously demonstrated that intracellular routing of MDO into endolysosomal compartments is dependent on actin microfilaments (18). Hence, it is possible that, without TLR4 signaling, internalized MDO fails to be localized and processed at early endolysosomal or cytosol compartments in the absence of microtubular modulation, leading to poor Ag presentation. In line with this, TLR4-defective C3H/HeJ DCs fail to exhibit a morphological change after MDO stimulation.

The functionality of TLR4 in MDO presentation may also elucidate the adjuvant mechanism of oxidized mannan-conjugated fusion mucin 1 protein (MFP), of which the adjuvanticity is also TLR4-mediated (24, 34). MFP has been used for immunotherapy in phase III cancer clinical trails (35). The immunogenicity of yeast-derived mannoproteins may be also due to enhanced Ag presentation mediated by TLR2 and/or TLR4 (30, 31, 36).

Although MDO induces DC activation through TLR4, unlike LPS, it does not directly interact with the TLR4/MD2 complex, since no difference in MDO-FITC binding between TLR4-silenced or MTS510-treated DCs and control DCs was observed. Interestingly, LPS can partially block the binding of MDO to DCs. LPS has been shown to interact not only with TLR4/MD2, but also with the prominent mouse DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN) homolog, SIGNR1, which is also associated with TLR4/MD2 to facilitate signal transduction (37). In line with this, it has been shown that mannosylated dendrimer induces clustered organization of DC-SIGN due to a high level of binding affinity (38). It is therefore possible that MDO activates TLR4 signaling indirectly through SIGNR1. The lack of synergy with LPS in induction of DC maturation further suggests that the pathway used by MDO to initialize upstream TLR4 signaling is divergent from LPS and is not interconnected. In comparison to MDO and LPS, zymosan also binds to the C-type lectin, dectin-1, in cooperation with TLR2, to induce maturation of DCs, which subsequently become regulatory and induce immune tolerance (39). Mannosylated lipoarabinomannan, another DC-SIGN ligand, induces intermediate or little DC maturation, resulting in a tolerogenic DC phenotype (40, 41). Hence, depending on the binding property of the ligand, the interactions between C-type lectins and TLRs can determine how the receptors are engaged to regulate innate cellular responses.

In our studies, because the concentration of MDO is calculated based on OVA, it is clearly demonstrated that the conjugation of mannosylated dendrimer to OVA promotes its immunogenicity (18). While OVA is glycosylated and contains three to seven branched mannose residues, at 40 μg/ml, it binds very weakly to DCs unless a high dose is used (18, 42). Due to the recognition by the mannose receptor, OVA in a high dose can also be processed in early endosomes and cross-presented on MHC class I (18, 42). In this way, mannose receptor has been shown to be indispensable for cross-presentation of OVA to induce T cell responses both in vitro and in vivo (42, 43). In comparison to mannose receptor, DC-SIGN recognizes high-mannose oligosaccharide (44) and, instead of targeting endosomal compartments, it delivers mannosylated Ags into late endolysosomes (45). In line with this, MDO is colocalized with acidic lysosomal compartments after internalization by DCs (18). Similar to mannose receptor-mediated uptake of OVA, DC-SIGN-mediated uptake of MDO may also be cross-presented on MHC class I. However, because both dendrimer and mannose residues contribute significantly to MDO binding to DCs, it is not known whether or how dendrimer is involved in Ag shuttling (18). Nonetheless, given the evidence of receptor-mediated uptake and intracellular compartment targeting, under low Ag dosage, TLR-aided functional maturation of DCs facilitates Ag processing and presentation (6, 18).

Since TLR4 functionality is critical for MDO presentation among DC subsets, how does TLR4 signaling regulate MDO processing and presentation? TLR4 engages two adaptor protein pathways upon ligation: MyD88 and TRIF. MyD88 recruits IL-1 receptor-associated kinase 4 (IRAK-4) and induces phosphorylation of IRAK1, resulting in activation of TNF receptor-associated factor 6 (TRAF6) and subsequent IKKα/β/γ for NF-κB activation or the MAPK pathway for AP-1 production. TRIF activates the downstream kinases TBK1 and IKKε, leading to activation of IFN regulatory factor 3 (IRF3) (25, 46). While MyD88-mediated signaling is critical for DC generation of several inflammatory cytokines, the TRIF-mediated pathway accounts primarily for DC maturation and type I IFN/Th1 cytokine production (46, 47, 48). Accordingly, MDO induces maturation of MyD88-deficient DCs, while CpG, of which signaling is solely dependent on MyD88, fails to do so. Moreover, MDO-induced Ag presentation in MyD88-deficient DCs appears robust. MyD88 is not required for TLR4-induced formation of endolysosomal tubules (32).

When EGCG is used to treat BMDCs, the function of the TRIF pathway in DC activation is apparent. EGCG has been shown to inhibit the activity of IKKβ important for NF-κB activation, while EGCG also suppresses the primary downstream kinase TBK1, which regulates IRF3. Therefore, EGCG has been thought to be inhibitory for both MyD88 and TRIF-dependent signaling pathways (25). Interestingly, in our DC maturation study where MDO, CpG, and poly(I:C) as TLR stimulants are investigated, EGCG acts primarily as a TRIF, but not MyD88, pathway inhibitor, indicating that inhibition of IKKβ alone does not hinder MyD88-mediated DC activation and that the MAPK signaling pathway is more important in induction of DC maturation (49). EGCG-treated BMDCs fail to efficiently cross-present MDO, in contrast to CpG/OVA mix (6), suggesting that the TRIF pathway is indeed important for MDO processing and presentation.

The negative effects of EGCG on cell physiology have been reported. EGCG is found to be inhibitory to the proteasome in cancer cell lines (50). With the dose of EGCG used throughout the study, our findings suggest that it has little impact on the capability of DCs to present exogenous Ags with or without TLR9 mediation. EGCG has also been shown to induce apoptosis and suppress growth of cancer cells (51). In our study, EGCG-treated BMDCs remained healthy after 18 h treatment when examined by cell staining. Therefore, we consider that these effects associated with EGCG on maturation and Ag presentation of BMDCs are inconsequential.

Note that while DC maturation induced by poly(I:C), similar to MDO, is dependent on the TRIF pathway, poly(I:C)/OVA mix remains efficiently cross-presented by EGCG-treated DCs (data not shown). This is in line with a previous study that demonstrated that poly(I:C)-mediated cross-presentation is MyD88-dependent (6). It is possible that there is an unknown signaling pathway connected with MyD88 that governs TLR3-induced cross-presentation. Hence, the role of TRIF or MyD88 in TLR-mediated Ag cross-presentation is complex and cannot be generalized only by DC maturation or cytokine production studies. In terms of CpG, it has been shown that TLR9 is not required for the CpG-aided cross-presentation of a conjugated Ag, while it is essential for CD8+ T cell cross-priming (52). In our studies, OVA is not conjugated to CpG. The doses of CpG and OVA used clearly demonstrate CpG enhances cross-presentation of the OVA Ag, resulting in increased CD8+ T cell proliferation. Moreover, CpG-mediated soluble OVA cross-presentation is MyD88-dependent (6).

In this study, we have clarified not only the connection between TLR4 stimulation and functional maturation of DCs, but also differential responses of DC subsets representative of major in vivo DCs to a mannosylated Ag. Although DC subsets vary in their capabilities in Ag presentation due to intrinsic variances, TLR functionality is critical for not only cellular maturation, which provides T cell costimulation, but also for subcellular maturation, which enhances presentation of the PAMP-associated Ag in DCs. Hence, DCs possess an ability to select and present a PAMP Ag like MDO through recognition of a specific TLR expressed by DC subsets, which takes part in the decision of immune induction. The use of a PAMP Ag to target specific DC subsets should be of particular significance in immunotherapeutic applications.

Disclosures

The authors have no financial conflicts 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 advertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported a National Health and Medical Research Council project grants (266818 to G.A.P.) (Medical Bioinformatics Genomics and Proteomics Program grant to M.D.W. (406660)) and an American Institute for Cancer Research grant (to M.D.W.). V.A. was a National Health and Medical Research Council R. Douglas Wright Fellow (223316).

  • ↵2 G.A.P. and V.A. contributed equally to this paper.

  • ↵3 Address correspondence and reprint requests to Dr. Vasso Apostolopoulos, Burnet Institute (Austin Campus), Kronheimer Building, Studley Road, Heidelberg, 3084, Victoria, Australia. E-mail address: vasso{at}burnet.edu.au

  • ↵4 Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; BMDC, bone marrow dendritic cell; EGCG, (−)-epigallocatechin-3-gallate; E.U., endotoxin unit; Flt3-L, fms-like tyrosine kinase 3 ligand; MD, mannosylated dendrimer; MDO, mannosylated dendrimer OVA; PAMP, pathogen-associated molecular pattern; RT, room temperature; TRIF, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-β.

  • Received December 10, 2007.
  • Accepted May 18, 2008.

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