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Immunology. Mar 2010; 129(3): 406–417.
PMCID: PMC2826685

Dendritic cells activated by an anti-inflammatory agent induce CD4+ T helper type 2 responses without impairing CD8+ memory and effector cytotoxic T-lymphocyte responses


Prevalence of pro-inflammatory diseases is rising in developed country populations. The increase in these diseases has fuelled the search for new, immune suppressive, anti-inflammatory therapies, which do not impact, or minimally impact, CD4+ and/or CD8+ T-cell-mediated immunity. The goal of this study was to determine if antigen-presenting cells (APCs) activated by the anti-inflammatory oligosaccharide, lacto-N-fucopentaose III (LNFPIII), would have an impaired ability to drive CD4+ T helper (Th) or CD8+ memory and effector T-cell responses. To investigate this we activated splenic dendritic cells (SDCs) with LNFPIII and examined their ability to drive antigen-specific CD4+ Th, and CD8+ memory and cytotoxic T-cell (CTL) responses compared with lipopolysaccharide (LPS) -stimulated SDCs. The LNFPIII-activated SDCs had altered co-stimulatory molecule expression compared with LPS-stimulated SDCs, while the levels of SDC chemokines following activation by either compound were similar. LNFPIII-activated SDCs produced significantly lower levels of interleukin-12 but surprisingly higher levels of interleukin-6 than LPS-activated SDCs. Similar to previous studies using bone-marrow-derived DCs, LNFPIII-activated SDCs induced strong Th2 responses in vivo and ex vivo. LNFPIII activation of APCs was independent of the Toll-interleukin-1 receptor adaptor myeloid differentiating factor 88. Importantly, LNFPIII-matured DCs induced CD8+ memory and effector CTL responses similar to those driven by LPS-matured DCs, including the frequency of interferon-γ-producing CD8+ T cells and induction of CTL effectors. Treatment of APCs by the anti-inflammatory glycan LNFPIII did not impair their ability to drive CD8+ effector and memory cell-mediated immunity.

Keywords: anti-inflammatory, dendritic cells, lacto-N-fucopentaose III, myeloid differentiating factor 88


Prevalence of pro-inflammatory-based autoimmune diseases and metabolic disorders has increased steadily over the past two decades in so-called ‘developed’ country populations.13 Medications used to bring patients into remission have severe side-effects associated with dramatic suppression of immune responses.4 Therefore, identifying new anti-inflammatory or immune suppressive agents that have minimal impact on overall systemic CD4+ and CD8+ T-cell responses is a high priority. In this regard, the pentasaccharide lacto-N-fucopentaose III (LNFPIII) functions in vivo to drive anti-inflammatory immune responses, and it has been used to prevent or treat psoriasis in a mouse model of the disease.5 The protective, anti-inflammatory activity of LNFPIII appears to be a result of its ability to activate antigen-presenting cells (APCs) which are anti-inflammatory in vivo.6 Because of the potential use of LNFPIII as a therapeutic agent to prevent or treat pro-inflammatory diseases, we wanted to determine if LNFPIII-treated APCs would negatively impact the development of CD4+ and CD8+ T-cell responses.

Classically, APCs are spoken of as the first line of defence against microbial infection.7 However, APCs are also immune regulatory, can be anti-inflammatory and increasing evidence points to these cells having roles in the resolution of inflammation-based diseases.810 In addition to instructing the differentiation and polarization of T helper (Th) responses, APCs are essential for the activation and proliferation of cytotoxic T cells (CTLs).

The LNFPIII is a biologically conserved pentasaccharide that contains the LewisX trisaccharide.11 It has been shown to induce otherwise naive peritoneal cells to produce the anti-inflammatory mediators, interleukin-10 (IL-10) and prostaglandin E2.12 LNFPIII is found in high concentrations in human milk at all times post-partum and LewisX is found on immune suppressive helminth parasites.1315 Multivalent conjugates of LNFPIII induce the maturation of alternatively activated APCs, including bone-marrow-derived dendritic cells type 2 (DC2s) that have potent Th2-driving properties.1618 Activation of murine and human DCs by LNFPIII/LewisX occurs via a process that involves/antagonizes Toll-like receptor 4 (TLR4), and preferentially activates extracellular signal-regulated kinase among the mitogen-activated protein kinase family.1820 For example, Helicobacter pylori lipopolysaccharide (LPS) expressing LewisX antagonizes TLR4 via TLR2,20 and activation of human DCs by schistosome soluble egg antigens which contain LewisX occurs via C-type lectin ligation in a process that subsequently antagonizes TLR4.20

The LNFPIII has been shown to drive production of the anti-inflammatory mediators IL-10 and prostaglandin E2 in murine peritoneal cells, and IL-10 from human peripheral blood mononuclear cells.12,21,22 In addition, injection of LNFPIII-conjugates into otherwise naive mice, expands alternatively activated macrophages in vivo, which can adoptively transfer the ability of recipient splenic T cells to produce significantly increased levels of IL-10 and IL-13.6,23 Lastly, the anti-inflammatory action of LNFPIII-conjugates was shown in a study where injection of LNFPIII-conjugates prevented the development of psoriasis-like lesions in Fsn/Fsn mice, including reduction of skin thickness and restoration of CD4/CD8 T-cell balance.5 These studies, taken together, demonstrate the therapeutic potential of LNFPIII-conjugates for pro-inflammatory diseases. Because of the aforementioned properties, we wanted to determine if LNFPIII-activated splenic dendritic cells (SDCs) have an impairment or alteration in driving cellular immunity in vitro and in vivo.15,19 Therefore the goals of the present study were to determine if activation and maturation of SDCs by LNFPIII would lead to a defect in the ability of these APCs to drive CD4+ Th, CD8+ memory and effector CTL responses compared with LPS-activated SDCs. To determine the impact of LNFPIII activation on APCs, we focused on phenotype and properties of the SDCs as well as the functional competence of the SDCs in driving various parameters of T-cell immune responses. Furthermore, as LNFPIII/LewisX uses/antagonizes TLR4, we performed experiments to determine if LNFPIII-induced signalling in APCs required the Toll interleukin-1 receptor (TIR) adaptor myeloid differentiating factor 88 (MyD88).24,25

We report that LNFPIII treatment induced maturation of SDCs which were fully functional in terms of their ability to drive Th2 responses in C57BL/6 mice, a prototypical Th1-type mouse strain. Surprisingly, as LNFPIII/LewisX has been shown to antagonize TLR4,20,21 we found that maturation of DC2s by LNFPIII and the subsequent induction of Th2 and CTL responses were MyD88-independent. Though LNFPIII-activated SDCs drove Th2-type CD4+ T-cell responses, these SDCs did not suppress, or alter the generation or frequency of antigen-specific CD8+ CTLs in vitro or in vivo. These results, taken together with earlier studies, suggest that utilization of LNFPIII as an anti-inflammatory agent as a therapy for pro-inflammatory diseases is unlikely to lead to defects in CD4+ or CD8+ T-cell compartments, and so represents an anti-inflammatory agent that could be administered long-term.

Materials and methods


Eight- to 12-week-old female C57BL/6 mice, T-cell receptor transgenic OT-I and OT-II mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MyD88 gene knockout (MyD88−/−) mice were obtained from Dr D.T. Golenbock (University of Massachusetts Medical School, Worcester, MA). Transgenic mice were on the C57BL/6 background, and donors and recipients were matched for sex and age. Mice were maintained under pathogen-free conditions at the animal housing facilities of the Harvard School of Public Health (HSPH). Mice were used following the HSPH guidelines and Institutional Animal Care and Use Committee-approved protocols.

Cell culture

Cells were cultured at 37° in humidified air/5% CO2 atmosphere in Dulbecco’s modified Eagle’s minimal essential medium supplemented with 10% fetal bovine serum (FBS), 2 mm l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich, St Louis, MO) and 0·05 mm 2-mercaptoethanol.

Reagents and peptides

The LNFPIII, conjugated to human serum albumin (LNFPIII-HSA), was provided by Neose Technologies (Horsham, PA) or by Dr Thomas Norberg, Uppsala, Sweden.17 LNFPIII has a molecular weight of 853·7 and the LNFPIII-HSA conjugates used in this study had on average six or nine LNFPIII molecules conjugated to each HSA. Lipopolysaccharide from Escherichia coli 055:B5 was obtained from Sigma-Aldrich. Ovalbumin 257–264 (OVA257–264; SIINFEKL) and OVA323–339 (ISQAVHAAHAEINEAGR) peptides were purchased from Sigma-Genosys (Woodlands, TX), and stock solutions of 0·5 mg/ml were prepared in dimethylsulphoxide.

Flow cytometry

Fluorescence-activated cell sorting (FACS) analysis was performed using standard methodology.23 Briefly, after Fc blocking, 1 × 106 cells were stained with appropriate antibodies for 30 min at 4°. Cells were washed with phosphate-buffered saline (PBS)/2% FBS. 7-Amino-actinomycin D (BD Biosciences, San Jose, CA) was used to exclude dead cells. Fluorescent staining of 1 × 105 to 5 × 105 live cells was analysed on a FACScalibur flow cytometer using CellQuest software (BD Biosciences).

Enzyme-linked immunosorbent assay

Levels of cytokines and chemokines in culture supernatants were determined by enzyme-linked immunosorbent assays (ELISA) using BD OptEIA cytokine ELISA Sets (BD Biosciences) and Quantikine chemokine ELISA kits (R&D Systems, Minneapolis, MN), respectively, according to the manufacturers’ instructions. Tetramethylbenzidine/hydrogen peroxide was used as a substrate and reactions were stopped by the addition of 5% phosphoric acid to all wells. Optical densities at 450 nm were measured using a Spectra Max 190 ELISA plate reader (Molecular Devices, Sunnyvale, CA). Concentrations of cytokines and chemokines were calculated by using standard curves created on each plate.

Preparation and pulse of DCs

Dendritic cells were isolated from murine spleens, as described by Smith et al.26 with modifications. Briefly, spleen fragments were digested for 30 min at room temperature with collagenase (1 mg/ml collagenase type II; Worthington Biochemicals, Lakewood, NJ) and DNAse I (1 μg/ml grade II bovine pancreatic DNAse I; Roche Applied Science, Indianapolis, IN), and washed with PBS/5 mm ethylenediaminetetraacetic acid/5% FBS. The DCs were enriched by density gradient centrifugation using NycoPrep 1.077A (Axis-shield PoC AS., Oslo, Norway) and then purified with CD11c (N418) MicroBeads (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer’s instructions. The purity of the CD11c-positive cell fraction was routinely > 90%. Enriched SDCs were stimulated with LNFPIII-HSA (50 μg/ml, which is equal to approximately 5 μg/ml LNFPIII for the nine-mer LNFPIII-HSA conjugates), HSA (50 μg/ml), LPS (0·1 μg/ml) or medium, and simultaneously pulsed with the appropriate OVA peptide (500 nm) in 12-well plates (106 cells/ml; 2 ml/well). After 16–24 hr of stimulation, DCs were harvested, washed three times with medium, and used in co-culture with T cells or for in vivo administration. For the analysis of cytokine production, DC culture supernatants were harvested at 12, 24 and 48 hr post stimulation.

In vivo administration of pulsed SDCs

Enriched SDCs were pulsed with OVA257–264 for 24 hr in the presence of LPS, LNFPIII-HSA, HSA or medium. The cells were then washed extensively in PBS, and 2 × 106 to 2·5 × 106 SDCs were injected intravenously into the lateral tail vein of recipient mice.

In vitro analysis of T-cell responses

For in vitro T-cell response studies, naive OVA peptide-specific CD4+/CD8+ T cells were isolated by negative selection from the spleens of OT-II/OT-I mice respectively, using magnetic antibody cell sorting (MACS) T Cell Isolation Kits (Miltenyi Biotec). The purity of the selected cell fraction was > 95%. Purified CD4+ CD8+ T cells were incubated with 2·5 μm 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) at 37° for 7 min, and then washed extensively. For T-cell response analysis, CFSE-stained cells were mixed with peptide-pulsed SDCs at ratios of 1 : 1, 2 : 1, 4 : 1 or 8 : 1 (T : DC), respectively (results at 4 : 1, the optimal condition, are shown). At the indicated time-points, cells were washed with PBS/2% FBS, stained with the appropriate antibodies, and analysed by flow cytometry, as described above. Culture supernatants were harvested at 24, 48 and 72 hr post stimulation and used for cytokine detection.

In vivo CTL assay

This assay was performed as described elsewhere with modifications.27 Briefly, target cell suspensions were prepared from the spleens of naive C57BL/6 mice. Cells were pulsed with 100 nm OVA257–264 peptide for 1 hr at 37°, or left untreated. The pulsed fraction was then labelled with a high concentration (2·5 μm) of CFSE (CFSEhigh cells), and the non-treated control fraction was labelled with a low concentration (0·25 μm) of CFSE (CFSElow cells) (Fig. 3a). Equal numbers of cells from each population (107) were mixed and adoptively transferred via the tail vein into mice immunized 7 days before with peptide-pulsed DCs as described. At the indicated time-points, mice were killed and spleens were removed. Cell suspensions were analysed by flow cytometry; 5 × 105 CFSE-positive cells were collected for analysis. Peptide-pulsed and untreated target cells were distinguished by their different fluorescent intensities. Percentage of specific lysis was calculated by loss of the peptide-pulsed population compared with control population using the formula described previously.27

Figure 3
Lacto-N-fucopentaose III (LNFPIII) matured splenic dendritic cells (SDCs) induce cytotoxic CD8+ T cells. SDCs from C57BL/6 mice were treated with lipopolysaccharide (LPS), LNFPIII, human serum albumin (HSA), or culture medium, loaded with the ovalbumin ...

To investigate the effect of LNFPIII-matured SDCs on memory cytotoxic CD8+ T cells, C57BL/6 mice were immunized twice (8 weeks apart) with SDCs treated with LPS, LNFPIII-HSA, HSA, or culture medium, loaded with the OVA257–264 peptide. CFSE-labelled target cells were administered on day 4 after the secondary immunization, and in vivo cytotoxicity was detected at the indicated time-points.

Statistical analysis

Student’s t-test was used for statistical analysis. Results are expressed as the means ± SD. Each experiment was repeated at least twice.


LNFPIII-matured SDCs are distinct from LPS-matured cells

Dendritic cell maturation of naive T cells is partly dependent on the DC surface expression of co-stimulatory molecules including CD40, CD80 (B7.1) and CD86 (B7.2);28 for this reason we compared expression of these molecules and major histocompatibility complex (MHC) class II on the surfaces of LNFPIII-matured versus LPS-matured SDCs (Fig. 1a). Freshly isolated SDCs from naive C57BL/6 mice expressed co-stimulatory molecules and MHC class II at low levels. In comparison, DCs activated by treatment with either LPS or LNFPIII consistently up-regulated expression of these cell surface molecules associated with T-cell activation. CD40 was significantly elevated at 48 hr to similar levels in LPS-matured or LNFPIII-treated DCs. The LPS promoted a marked increase in CD80/CD86 expression on SDCs, whereas LNFPIII-stimulated DCs had increased expression of CD86 and a slight increase in CD80 expression. Examining the kinetics of co-stimulatory molecule up-regulation, we observed that CD80 expression was significantly increased at 24 hr on LPS-stimulated DCs, whereas the mean fluorescence intensity (MFI) of CD80 expression for LNFPIII-activated SDCs remained at baseline. By 48 hr LPS-activated SDCs had dramatically up-regulated CD80 expression, with significantly higher MFI (P< 0·05) compared with LNFPIII-activated SDCs, which exhibited slightly, but significantly, increased levels of CD80. Conversely, LNFPIII stimulation induced a markedly higher level of CD86 expression compared with LPS at both 24 and 48 hr. The MHC class II expression on SDCs was up-regulated to similar levels on LPS-stimulated and LNFPIII-stimulated cells, being significantly increased by 12 hr, and > 2·5-fold higher than control treated SDCs at 48 hr (Fig. 1a).

Figure 1
Lacto-N-fucopentaose III (LNFPIII) stimulation induces splenic dendritic cell (SDC) maturation. SDCs were treated with lipopolysaccharide (LPS), LNFPIII-human serum albumin (HSA), HSA, or culture medium. (a) Expression levels of CD40, CD80, CD86 and major ...

LNFPIII-activated SDCs produce inverse levels of IL-6 and IL-2 compared with LPS-activated SDCs

We next examined production of IL-6, IL-12, regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein 1a (MIP-1a) and monocyte chemotactic protein 1 (MCP-1) from LNFPIII-HSA or LPS-activated SDCs at several different time-points. Amounts of cytokines/chemokines were determined by ELISA (Fig. 1b,c). As expected, LPS-treated SDCs produced high levels of IL-12. In contrast, LNFPIII-treated SDCs produced IL-12 only at baseline levels. Surprisingly, LNFPIII-stimulated SDCs produced significantly more IL-6 than LPS-stimulated cells, and the patterns for chemokine production between LPS-treated and LNFPIII-treated SDCs were similar. High levels of RANTES and MIP-1α were detected as early as 12 hr and for MCP-1 at 48 hr. As a control and to eliminate the possibility of endotoxin contamination, we added polymyxin B to SDC cultures and observed that as expected, polymyxin B significantly reduced chemokine production by LPS-activated SDCs but did not significantly impair LNFPIII-driven chemokine production (Fig. 1d). Collectively, these data show that like LPS, LNFPIII clearly activates SDCs, maturing them such that they express distinct co-stimulatory molecules and cytokine profile from LPS-activated SDCs. These observations suggest that the direction of adaptive T-cell responses may be regulated by the combined pattern of soluble and cell-associated signals that pro-inflammatory or anti-inflammatory activated SDCs express.

LNFPIII-matured SDCs induce Th2 maturation

As we expected, LNFPIII-treated SDCs produced little or no IL-12, a potent Th1 promoting cytokine (for review see ref. 29). This raised the question of whether the adaptive CD4+ Th responses are directly triggered by different types of SDCs. In an earlier study, we demonstrated that treatment of bone-marrow-derived DCs from the Th2-type BALB/c mouse with LNFPIII were able to induce Th2-type responses from naive CD4+ T cells.18 Here we wanted to extend this observation by using splenic DCs from the prototypical Th1-type C57BL/6 mouse. To investigate whether LNFPIII-treated SDCs were capable of directly inducing a Th2 response, enriched SDCs were stimulated with LNFPIII, pulsed with OVA323–339 peptide, and co-cultured with CD4+ T cells isolated from T-cell receptor transgenic OT-II mice. SDCs treated with LPS, HSA or medium were used as controls. CD4+ T cells were labelled with CFSE to permit discrimination between the two cell populations, and for measuring cell proliferation. As shown in Fig. 2, there was a dramatic increase in interferon-γ (IFN-γ) production from CD4+ T cells co-cultured with LPS-treated SDCs but not from CD4+ T cells cultured with LNFPIII-stimulated SDCs (Fig. 2a). Conversely, we observed a significant increase in levels of IL-4 in supernatants of CD4+ T cells co-cultured with LNFPIII-treated SDCs, compared with CD4+ cells co-cultured with LPS or control SDCs. Consistent with up-regulated levels of cytokines in culture supernatants, we determined that OT-II transgenic CD4+ T cells cultured with OVA323–339 peptide-pulsed LPS-matured or LNFPIII-matured SDCs had significantly increased levels of proliferation compared with T cells co-cultured with control (HSA-treated or media-treated) SDCs (Fig. 2b). Hence, LNFPIII-activated SDCs do not have T-cell suppressive activity similar to LNFPIII-activated macrophages.23 Overall, LNFPIII-treated SDCs from a Th1-type mouse induced potent type 2 cytokine (IL-4) production from CD4+ T cells, extending earlier observations made using bone-marrow-derived DCs from a Th2-type mouse.18

Figure 2
Lacto-N-fucopentaose III (LNFPIII) -activated splenic dendritic cells (SDCs) drive T helper type 2 (Th2) maturation. (a) DCs were isolated from the spleens of C57BL/6 mice, treated with lipopolysaccharide (LPS), LNFPIII-human serum albumin (HSA), HSA, ...

LNFPIII-matured SDCs induce functional CD8+ T cells

To further determine if LNFPIII activation of SDCs negatively impacts adaptive immunity, we investigated the ability of LNFPIII-activated SDCs to generate CTLs by performing an in vivo CTL assay. Naive SDCs were treated with LPS, LNFPIII, HSA or culture medium, and pulsed with OVA257–264 peptide. Naive C57BL/6 mice were immunized with OVA-peptide-pulsed SDCs and the in vivo CTL assay was performed on day 6 post-immunization as described in the Materials and methods. As depicted in Fig. 3(b), mice that received LPS-treated or LNFPIII-treated SDCs showed profound antigen-specific CTL activity 6–7 days after priming, compared with mice that received HSA-treated or non-treated SDCs. No apparent differences in CTL activity were observed at any time-points between mice that received LPS-treated or LNFPIII-treated SDCs, indicating that LNFPIII-activated and -matured SDCs are fully capable of inducing CTL responses. We performed additional experiments to detect memory immune responses by giving mice a secondary intravenous administration of OVA257–264-pulsed SDCs (106 cells) 8 weeks after the primary challenge. Similar to primary in vivo CTL generation, we observed no differences in secondary (memory) cytotoxic responses between mice receiving LPS-treated or LNFPIII-treated SDCs (Fig. 4). As a consequence, treatment of SDCs with the anti-inflammatory glycan LNFPIII did not negatively impact the ability of these SDCs to drive robust CTL effector and memory T cells.

Figure 4
Lacto-N-fucopentaose III (LNFPIII) matured splenic dendritic cells (SDCs) induce memory cytotoxic CD8+ T cells. C57BL/6 mice were immunized twice (with an 8-week interval) with SDCs treated with lipopolysaccharide (LPS), LNFPIII-human serum albumin (HSA), ...

The activation of effector CD8+ T cells is a critical step in the initiation of an efficient CTL response. To determine whether there is a difference in regulating CD8+ T-cell activation between LPS-treated and LNFPIII-treated SDCs, as well as the role of different phenotypes of CD4+ T cells induced by SDCs, antigen-specific IFN-γ-producing CD8+ T cells were counted. The SDCs from naive C57BL/6 mice were treated with LPS, LNFPIII, or HSA or were left untreated, and then pulsed with OVA257–264 peptide. They were co-cultured with OT-I transgenic CD8+ T cells plus or minus the addition of CD4+ T cells from C57BL/6 mice. Cells were collected and stained for CD8+ and intracellular IFN-γ at the indicated time-points. Stimulator cells were OVA257–264 peptide-pulsed or non-pulsed and mitomycin-C-treated splenocytes from naive C57BL/6 mice. The profile of specific CD8+ T-cell activation by SDCs with the help of CD4+ T cells are shown in Fig. 5. No obvious activation for CD8+ T cells could be detected at 12 hr. However, we did find a significantly higher percentage of IFN-γ production among CD8+ T cells cultured with LPS-treated or LNFPIII-treated SDCs at 24 and 48 hr, compared with CD8+ T cells cultured with HSA-treated or non-treated SDCs (37% versus 10% at 48 hr, P< 0·001). Consistent with the Th response and yields of IFN-γ induced in CD4+ T cells by LNFPIII versus LPS, LPS-treated SDCs induced a significantly higher level of IFN-γ-producing cells among the CD8-negative cell population (most of them were CD4+ T cells from C57BL/6 mice), whereas LNFPIII-treated SDCs only induced baseline levels of IFN-γ-producing cells among the CD8-negative cells. These studies also demonstrated that activation of CD8+ T cells was significantly impaired without CD4+ T-cell help (data not shown).

Figure 5
Specific activation of CD8+ T cells induced by mature splenic dendritic cells (SDCs). SDCs from C57BL/6 mice were treated with lipopolysaccharide (LPS), lacto-N-fucopentaose III (LNFPIII), human serum albumin (has), or culture medium and pulsed with the ...

To determine the ability of LNFPIII-activated SDCs to induce antigen-specific CD8+ T-cell proliferation, an in vitro proliferation assay was performed. OT-I transgenic CD8+ T cells were purified and labelled with CFSE, and co-cultured with LNFPIII or LPS-treated SDCs with or without CD4+ T cells (as described above). The results of these experiments showed that LPS-matured or LNFPIII-matured SDCs induced identical kinetics of CD8+ T-cell proliferation in vitro (Table 1). In those experiments where CD4+ T cells were not added, we observed that specific expansion of CD8+ T cells was significantly delayed, again indicating the important role of CD4+ Th cells in this response. Interestingly, coincident with the results of CTL activation, both Th1 and Th2 CD4+ T cells showed similar function in their ability to help SDCs drive antigen-specific proliferation of cytotoxic CD8+ T cells.

Table 1
Kinetics of CD8+ cell proliferation induced by different splenic dendritic cells

LNFPIII-induced SDC maturation is independent of MyD88

Myeloid differentiating factor 88 is considered a common adaptor protein recruited by the activation of all TLRs except TLR3.30 Previous studies have shown that LNFPIII/LewisX-induced DC maturation is a process that involves/antagonizes TLR4.18 This observation suggested that MyD88 might play a role in LNFPIII-induced DC maturation. To test this hypothesis, SDCs were isolated from MyD88−/− mice and treated with LPS, LNFPIII, HSA or medium in combination with OVA peptide as described above. Expression of co-stimulatory molecules and MHC II were detected by flow cytometry at 24 hr. For LNFPIII-activated SDCs similar levels of expression of each of the co-stimulatory molecules and MHC II were seen on SDCs from MyD88−/− or wild-type C57BL/6 mice (Fig. 6a). In contrast, LPS-activated SDCs from MyD88−/− mice had decreased levels of expression of CD80, CD86 and MHC II (Fig. 6a). We next examined cytokine and chemokine production at 24 hr from MyD88−/− and wild-type SDCs. As expected the pronounced up-regulation in both IL-12 and IL-6 production from LPS-stimulated wild-type SDCs was reduced to baseline in MyD88−/− SDCs (Fig. 6b). In contrast, absence of MyD88 did not alter the production of IL-6 from LNFPIII-treated SDCs. Similar results were seen for the chemokines RANTES and MIP-1α. Levels of both chemokines were significantly reduced in LPS-treated MyD88−/− SDCs whereas chemokine production by LNFPIII-treated MyD88−/− SDCs remained significantly higher than levels from control-treated SDCs (data not shown).

Figure 6
Maturation of splenic dendritic cells (SDCs) by lacto-N-fucopentaose III (LNFPIII) is myeloid differentiating factor 88 (MyD88) independent. The SDCs from MyD88−/− or wild-type C57BL/6 mice were treated with lipopolysaccharide (LPS), LNFPIII-human ...

LNFPIII induces Th2 activity and specific T-cell proliferation independently of MyD88

To further investigate the role of MyD88 in the differentiation of Th responses, enriched CD4+ T cells from OT-II transgenic mice were co-cultured with SDCs from MyD88−/− or wild-type mice previously stimulated with LPS, LNFPIII, HSA or medium simultaneously with a pulse of OVA323–339 peptide. Levels of cytokines (IFN-γ, IL-4 and transforming growth factor-β) were measured at 48 and 72 hr by ELISA (Fig. 7a). Cytokine levels were similar between LNFPIII-treated MyD88−/− or wild-type SDCs, showing that LNFPIII-activated SDCs are able to drive naive CD4+ T cells to produce Th2 responses in the absence of MyD88. In contrast and as expected, MyD88 was required for the induction of Th1 responses by LPS-treated SDCs. Together with the observation that maturation of LNFPIII-treated SDCs was unimpaired in the absence of MyD88, these results further suggest that LNFPIII induces Th2 responses in the host via a MyD88-independent mechanism.

Figure 7
Lacto-N-fucopentaose III (LNFPIII) matured myeloid differentiating factor 88 double-negative (MyD88−/−) splenic dendritic cells (SDCs) induce T helper type 2 (Th2) responses and specific T-cell proliferation. (a) Cytokine production of ...

To further investigate the role of MyD88 in activating T-cell function, the division profiles of OVA-transgenic CD4 and CD8 T cells in the context of OVA peptide stimulation were compared. The SDCs from MyD88−/− or wild-type mice were treated with LPS or LNFPIII plus appropriate OVA peptide, and co-cultured with CFSE-labelled OT-II CD4+ T cells, or mixed with CD4+ T cells from naive C57BL/6 mice and co-cultured with CFSE-labelled OT-I CD8+ T cells. CD4 and CD8 T-cell proliferation was analysed at different time-points (Fig. 7b). As expected, fractions of LPS-matured or LNFPIII-matured SDCs from C57BL/6 mice showed similar kinetics in inducing antigen-specific proliferation of either CD4 or CD8 T cells. Similar to other findings in this study, we observed that LNFPIII-matured SDCs from MyD88−/− mice were fully capable of inducing antigen-specific proliferation of both CD4+ and CD8+ T cells to the same extent as those induced by LNFPIII-matured SDCs from C57BL/6 mice. Consistent with the impaired function of LPS-treated MyD88−/− SDCs, we observed a significant reduction in the extent of division in both CD4 and CD8 T cells co-cultured with LPS-treated MyD88−/− SDCs. As a consequence, LNFPIII-activation and maturation of fully competent SDCs is via a MyD88-independent activation pathway, rather than the MyD88-dependent pathway seen with LPS-matured SDCs.


The prevalence of pro-inflammatory-based diseases continues to rise, necessitating the search for new anti-inflammatory agents capable of placing patients in remission or for maintenance. Whether treatment with anti-inflammatory agents will dampen or suppress CD4+ or CD8+ T-cell responses is a major concern, and likely to be agent specific. In this study, we demonstrated that treatment of SDCs with the anti-inflammatory glycoconjugate LNFPIII-HSA, did not impair their ability to drive CD4+ T helper, or CD8+ memory and effector T-cell responses in vivo or in vitro. These are important observations as previously we demonstrated the anti-inflammatory therapeutic potential of LNFPIII in a murine model of psoriasis.5 Further, in earlier studies we demonstrated that LNFPIII conjugates activated naive bone-marrow-derived DCs to mature to DC2s, driving Th2 cytokine production from CD4+ T cells, and adoptive transfer of LNFPIII-conjugate-activated macrophages induced significant increases in antigen-specific IL-10 and IL-13 in recipient T cells.6,18

The results presented in this study provide several insights into the mechanism of DCs in directing adaptive immune responses. We demonstrated that stimulation of naive SDCs with LNFPIII-HSA conjugates induced full maturation of naive SDCs, including increased antigen-presenting function, up-regulation of MHC expression, co-stimulatory molecule expression and cytokine/chemokine production. However, LNFPIII-HSA-conjugate-matured SDCs showed distinct expression profiles of co-stimulatory molecules in comparison with LPS-matured type 1 SDCs. First, they had similar expression of CD40 compared with LPS-matured SDCs. CD40/CD40 ligand interactions have been shown to be critical in driving Th1 and Th2 response development in a mouse model of Leishmania major infection.31,32 The expression patterns of CD80/86 on LNFPIII-HSA-matured SDCs was reversed from that measured on LPS-matured SDCs. The LPS-activated SDCs had high levels of expression of CD80 at 24 and 48 hr post-stimulation whereas LNFPIII-HSA-stimulated SDCs had no increase in CD80 at 24 hr and a slight increase at 48 hr post-stimulation. For CD86, LNFPIII-HSA-treated SDCs expressed higher levels than LPS-treated SDCs at 24 hr post-stimulation, and this increased level of CD86 expression remained at 48 hr post-stimulation for LNFPIII-HSA-treated SDCs, but dropped to near background for LPS-treated SDCs. As a consequence, there was differential expression of these two co-stimulatory molecules, with CD80 expression increased more for LPS-treated cells than for LNFPIII-HSA-treated cells and these levels of expression were reversed for CD86. This observation is interesting with regards to Th1 and Th2 biasing of CD4+ T cells as several studies have suggested that CD80 is linked with Th1-type maturation and CD86 with Th2-type.3739 As expected, LNFPIII-matured SDCs produced no IL-12, in contrast to the high levels from LPS-activated SDCs. Surprisingly, LNFPIII-conjugate-matured SDCs produced more IL-6 than LPS-matured SDCs. This is the only instance where our laboratory has seen an increase in IL-6 from murine APCs. Examination of LPS-conjugate-activated peritoneal macrophages, RAW cell line macrophages or bone-marrow-derived macrophages has routinely shown decreases in levels of IL-6 (personal observations, data not shown). In this study we used unfractionated SDCs, it may be that the unusual increase in IL-6 was from one SDC subset and was not indicative of the total population. Functionally, LNFPIII-matured SDCs showed the ability to induce potent Th2 responses in a Th1-biased mouse strain, indicating the role of these cells as type 2 DCs in driving adaptive immune responses.

The ability of schistosome infection or schistosome antigens to drive Th2 type and anti-inflammatory responses has been well documented (for review see ref. 33). Few studies have examined the impact of schistosome infection or schistosome antigens on the generation of CTL responses. Actor et al.,34 showed that in BALB/c mice, vaccinia-virus-specific CTL responses were impaired in mice co-infected with Schistosoma mansoni. However, a study by Ekkens et al.35 demonstrated that Th2 cells induced by schistosome soluble egg antigen are able to provide help to primary and memory CTL responses, comparable to the responses driven by Th1 cells. Our data suggest that, similar to LPS-induced Th1 cells, Th2 cells matured by LNFPIII-treated DC2s provided intact help to CTLs in terms of specific activation and proliferation. Furthermore, both Th1 and Th2 cells helped to develop memory cytotoxic responses to the same extent.

How various pathogens or pathogen-associated molecular patterns differentially activate and mature DCs is of great interest with regard to developing therapies for inflammatory diseases or adjuvants for vaccines. Among APC receptors that initiate activation and maturation, TLRs have been widely studied and shown to induce distinct adaptive responses through differential use of adaptor molecules.36 For TLR4, LPS stimulation of APCs drives type 1 and pro-inflammatory responses in wild-type cells, but in the absence of MyD88, LPS-matured APCs drive type 2 responses.38 Studies from our laboratory and others have shown that LNFPIII/LewisX activation of APCs involves and/or antagonizes TLR4.1820 For example, Lepper et al.20 showed that H. pylori LPS expressing LewisX antagonizes TLR4 in a TLR2-dependent process. Examining how schistosome soluble egg antigens activate human DCs, Van Liempt et al.19 showed that schistosoma egg glycans activate human DCs via C-type lectins and endosome formation, which subsequently antagonizes TLR4. These studies confirm our earlier observations on a role for TLR4 in LNFPIII/LewisX activation of APCs, and suggest that receptor cross-talk is responsible for LNFPIII/LewisX-induced alternative activation of APCs. Currently we know that LNFPIII/LewisX binds to DC-SIGN, Mgl-1 the macrophage mannose receptor and depending on the cell type, some of the MSIGNRs.37 How LNFPIII/Lewis X-induced signalling via these C-type lectins impacts TLR4, and possibly other TLR-induced signalling cascades in APCs is a new area of investigation that will begin to tell us how APCs combine different receptor-induced signalling cascades to modify APC maturation.

Because each of these studies suggests a role for TLR4 in LNFPIII/LewisX activation of APCs, and the study by Kaisho et al.38 showed that MyD88-deficient APCs mature to type 2 APCs following LPS stimulation, we asked if the TIR adaptor MyD88 was required for LNFPIII-HSA-induced responses in SDCs. In this study we observed that the absence of MyD88 had a profound impact on the ability of LPS to activate and mature APCs, from their inability to up-regulate co-stimulatory molecule expression and the production of IL-12 and IL-6. In contrast, MyD88−/− SDCs behaved in an identical manner to wild-type SDCs following LNFPIII-HSA stimulation. These changes in maturation of LPS-activated MyD88−/− SDCs largely account for why these cells were unable to drive Th1-type CD4+ responses as measured by the production of IFN-γ, whereas LNFPIII-HSA-stimulated MyD88−/− SDCs functioned similarly to wild-type SDCs. Hence, LPS requires MyD88 to mount type 1 pro-inflammatory responses whereas LNFPIII-HSA activation and subsequent maturation of SDCs is MyD88 independent. These observations fit with the study by Kaisho et al.38 demonstrating that APCs from MyD88−/−-deficient mice drive DC2 maturation through TLR4 signalling.

The use of LNFPIII/Lewis X conjugates as therapeutic agents for pro-inflammatory diseases may have other risks in addition to altering cellular immunity. As LNFPIII drives Th2-type responses depending on the carrier molecule used for conjugation, continued, repeated injection of the LNFPIII conjugate could lead to antibody production. In an earlier study we demonstrated that LNFPIII functioned as an adjuvant to enhance Th2-type antibody responses to the carrier molecule human serum albumin (HSA).39 Consequently, if LNFPIII conjugates were to be employed therapeutically, the use of more inert carrier molecules such as dextran, dendrimers or nanoparticles would be suggested. In the event that LNFPIII conjugates cannot be used as therapeutic agents, then elucidation of how LNFPIII conjugates activate APCs via the various C-type lectin and TLR signalling pathways to drive alternative activation of APCs represents an approach to define the signalling pathways and molecules that are specific to LNFPIII activation of APCs. We are currently employing bioinformatics approaches to identify signalling pathways unique to LNFPIII-conjugate activation in an attempt to identify signalling pathway(s) and molecules which may be required for induction of anti-inflammatory responses.


We would like to thank Ms Michele Carter for technical assistance with this study. This work was supported by National Institutes of Health Grant (5RO1AI056484) to D.A.H. Y.W. was a CJ Martin Fellow supported by the Australian National Health and Medical Research Council (Grant 316978).


The authors have no conflict of interest.



antigen-presenting cell
5- (and 6-) carboxyfluorescein diacetate succinimidyl ester
cytotoxic T lymphocyte
enzyme-linked immunosorbent assay
fluorescence-activated cell sorting
fetal bovine serum
human serum albumin
lacto-N-fucopentaose III
monocyte chemotactic protein 1
major histocompatibility complex
macrophage inflammatory protein 1a
myeloid differentiating factor 88
pathogen-associated molecular pattern
phosphate-buffered saline
regulated on activation normal T cell expressed and secreted
splenic dendritic cell
schistosome soluble egg antigen
T helper
Toll-interleukin-1 receptor
Toll-like receptor


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