Significance Sialic acids are terminal glycan structures present on cellular glycoproteins and often overexpressed on certain pathogens and tumors. Sialic acids interact with sialic acid-binding Ig-type lectin (siglec) receptors, suggesting a potential regulatory role in homeostasis or pathology-mediated immune modulation. Here, we show that modification of antigens with sialic acids alters their immunogenicity. Sialylated antigens impose a regulatory program on dendritic cells (DCs) via Siglec-E. DCs loaded with sialylated antigens induce de novo regulatory T (Treg) cells and inhibit the generation of new effector T cells as well as the function of existing ones. This dual tolerogenic DC function is maintained under inflammatory conditions and, therefore, sialylation of antigens could provide a novel way to induce antigen-specific immune tolerance to treat patients who suffer from autoimmunity and allergies.

Abstract Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigen-specific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4+ and CD8+ T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen–loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E–mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro–established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelin-reactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance.

Antigen-presenting cells (APCs), and predominantly dendritic cells (DCs), are crucial to maintain immune homeostasis, because these cells determine whether or not an immune reaction is mounted against pathogens or self or innocuous foreign antigens. In healthy individuals, encounter with innocuous (self-)antigens will lead to induction of regulatory T (Treg) cells, which in turn dampen excessive immune responses, a process that fails in autoimmune patients (1).

Ways to promote this immune-inhibitory signature of DCs may therefore provide new therapeutic strategies to fight autoimmune disorders. Although different methods are described to push DCs into an immune-inhibitory mode, none of these methods is antigen-specific and may therefore exert off-target effects. Additional evidence suggests that merely generating a large pool of Treg cells is not sufficient to control aberrant immunity: the inflammatory milieu in the affected areas promotes a strong APC-mediated activation of antigen-specific autoreactive T cells, which makes these autoreactive T cells refractory to suppression by Treg cells (2⇓⇓–5). These findings suggest that strategies that aim at controlling autoimmunity should not only involve inducing Treg-cell populations but also dampening autoreactive T cells, thereby creating a milieu that allows Treg cells to exert their suppressive function. Certain pathogenic infections and tumors are marked by the presence of tolerogenic DCs and T cells (6⇓⇓–9). An often overlooked feature shared by these pathological conditions is the presence of aberrant glycosylation patterns (7, 10⇓–12). In particular, increased levels of sialic acids have been demonstrated in these situations. On tumors, enhanced sialylation driven by enhanced expression and activity of β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1) and/or α-N-acetylgalactosaminide α2,6-sialyltransferase 1 (ST6GalNAc-I) often correlates with tumor invasion and poor prognosis (13⇓–15). Additionally, the hypersialylated pathogens Campylobacter jejuni and Neisseria meningitides were shown to negatively affect human APC function and consequently subvert immune responses (7, 16⇓⇓–19).

Sialic acids are the outermost monosaccharides on glycan chains of glycoproteins and glycolipids, attached to the underlying glycans with α2,3, α2,6, or α2,8 linkage (14) and as such form the recognition elements for sialic acid-binding Ig-like lectins (siglecs) (14, 20). Siglecs are predominantly expressed by innate immune cells, such as DCs, macrophages, and B cells (20). On these cells, siglecs function as endocytic receptors as well as can regulate activation status and cytokine secretion. Most siglecs are characterized by the presence of one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their intracellular domain (21) and, thus, siglec triggering often counteracts activatory signals elicited by receptors containing immunoreceptor tyrosine-based activatory motifs (ITAMs) (20). Although engagement of the hCD33rSiglecs on innate cells by sialylated antigens has been shown to negatively modulate the proinflammatory functions of APCs, effects on T-cell responses have not yet been investigated in detail.

Because the immune-inhibitory effects induced by sialylated pathogens and tumors may be attributed to different configurations of sialic acid-containing glycoproteins or glycolipids, we set out to characterize the effects of sialic acids on DCs and T-cell responses using a well-characterized neoglycoconjugate approach based on the model antigens ovalbumin (OVA) or the encephalitogenic peptide derived from myelin oligodendrocyte glycoprotein (MOG 35–55 ) that we modified with either α2,3- or α2,6-linked sialyl-lactose (hereafter named Sia-antigens). Our data reveal that internalization of Sia-antigen by DCs endows them with the ability to promote the differentiation of naive CD4+ T cells into Treg cells at the expense of functional CD4+ and CD8+ effector T cells, both in vitro and in vivo. We provide evidence that this feature is antigen-specific and even effective under inflammatory conditions. Moreover, our findings demonstrate that Sia-antigen–loaded DCs also dampen the function of established effector T cells, suggesting that sialylation of antigens provides a means to dampen excessive T-cell pathologies.

Discussion Here, we show, for the first time to our knowledge, that DCs become tolerogenic upon uptake of soluble sialylated antigens. Moreover, we provide evidence for induction of antigen-specific immune tolerance under inflammatory conditions using sialylated antigens. We observed that under inflammatory conditions, effector T-cell functions were also dampened by Sia-antigen–loaded DCs. Importantly, under these circumstances Sia-antigen–loaded DCs retained the capacity to polarize naive CD4+ T cells toward Treg cells. To the best of our knowledge, this tolerizing effect under inflammatory conditions has not been demonstrated for other tolerizing compounds. The possibility of inducing tolerance in vivo in an antigen-specific manner via this new strategy of sialylation of antigens may open up new avenues for treating patients who suffer from unwanted immune reactions such as autoimmunity and allergies. Tolerogenic DCs, either occurring in vivo or generated in vitro, are characterized by an antiinflammatory phenotype attributable to low expression of costimulatory molecules and the ability to promote Treg-cell induction. Although uptake of Sia-antigen by DCs in vitro did not result in expression of surface markers associated with “classic” tolerogenic DCs, DCs are clearly tolerogenic in function upon internalization of sialylated antigens. We previously observed a similar absence of clear effects on the phenotype of human monocyte-derived DCs after incubation with the highly sialylated pathogen Neisseria gonorrhea, although T-cell polarizing capacity was affected (16, 27). Despite producing lower amounts of the proinflammatory cytokines IL-6 and TNF, we did not observe significant differences in the quantity of IL-10 or TGF-β produced by Sia-OVA–loaded DCs compared with OVA-loaded DCs. Therefore, it is possible that Sia-OVA–pulsed DCs induce Treg-cell induction and prevent effector T-cell generation via cell surface receptor–ligand interactions, rather than secretion of antiinflammatory cytokines. This possibility is further substantiated by our findings that Sia-OVA–loaded DCs did not confer tolerogenic capacity onto CD4+ T cells when physically separated or when the supernatant of Sia-OVA–loaded DCs was used to treat OVA-loaded DCs. Our findings indicate that the differentiation of naive CD4+CD25− T cells into Treg cells and reduced generation of effector IFN-γ+ T cells by Sia-OVA–pulsed DCs were not dependent on the mouse strain, because DCs and T cells from both C57BL/6 and BALB/c mice underwent equivalent responses. Additionally, we even observed de novo Treg-cell generation using naive CD4+ T cells from DO11.10×Rag−/− mice, which lack natural Treg (nTreg) cells. However, we anticipate that DCs tolerized by the uptake of Sia-antigen in vivo may also propagate nTreg cells, because the detection of Foxp3+CD4+ T cells does not discriminate between induced Treg (iTreg) cells and nTreg cells. Nevertheless, the net result of Sia-antigen internalization by DCs is the formation of increased numbers of T cells with suppressive capacity that can inhibit detrimental effector T-cell responses. In this process, Siglec-E plays an important role. In fact, our data indicate that DCs are endowed with tolerogenic characteristics via Siglec-E signaling upon internalization of sialylated antigens. This finding is underscored by our observations that intracellular trafficking of antigens is not altered by sialylation of antigens and that the presence of Siglec-E is required on DCs to induce Treg cells in response to Sia-antigens. Besides affecting CD4+ T-cell responses, DCs loaded with antigens modified with sialic acids also strongly restrained CD8+ T-cell responses. Both the expansion of CD8+ T cells as well as the acquisition of cytotoxic effector functions was significantly dampened, even under inflammatory conditions. Similarly, reduced CTL generation was observed in vivo when pretreating mice with Sia-antigen or Sia-antigen–loaded DCs. How CD8+ T-cell responses are regulated in vivo by Sia-antigen pretreatment is unclear. The CD8+ T-cell responses can either be controlled by Sia-antigen–induced Treg cells or via direct effects of the Sia-antigen–tolerized DCs, as shown in vitro. Because autoreactive CTLs have been shown to contribute to the pathogenesis in type 1 diabetes and multiple sclerosis (28⇓⇓–31), treatment with sialylated antigens could thus also be effective to dampen these pathologies. CTLs also play crucial functions in the immune response against viral infections. However, certain viruses such as herpes simplex virus (HSV)-1 and -2 adopt latency as a strategy to evade immune defense and persist in the host (32). Infections caused by these viruses are characterized by low antigen expression and a low level of CD8+ T-cell activation (33). Because high levels of sialylated antigens have been found on the envelope glycoproteins of HSV-1/-2, it can be hypothesized that sialic acids on HSV contribute to HSV immune escape by tolerizing DCs. Although the focus of the present study was to examine the effect of sialylated antigens on DCs and T-cell responses, B-cell responses may also be modulated. Liposomes coated with high-affinity sialic acid derivatives that target CD22 have been shown to induce B-cell tolerance (34). Many siglecs act as negative regulators of immune responses via the expression of ITIM motifs in their cytoplasmic tails. Therefore, it is likely that the observed tolerogenic effect of Sia-antigen results from interaction of Sia-antigen with siglec receptors on DCs. Our studies showed a significant reduction in the binding and uptake of Sia-OVA by Siglec-E−/− DCs and, concomitant, drastic inhibition of Treg-cell generation. This finding suggests a relevant role for the Siglec-E receptor in the interaction of Sia-antigens with DCs. Moreover, in view of these findings, Sia-antigen–mediated Treg-cell induction and T-effector inhibition may occur via two different modes, with Siglec-E mainly implicated in Treg-cell polarization. Loading WT DCs with low concentrations of Sia-OVA antigen did not lead to enhanced generation of Treg cells but still dampened effector T-cell generation. It can be speculated that low antigen concentrations do not evoke clustering of Siglec-E, which is necessary to relay intracellular ITIM signaling (21). In healthy individuals, the most important role of Treg cells is to maintain immune tolerance to self- and innocuous exogenous antigens as well as the intestinal microflora to prevent the development of autoimmune and allergic diseases (35). Defects in Treg-cell number and/or function have been shown to contribute to autoimmune diseases (4). Thus, therapies directed at resolving Treg-cell defects have the potential to prevent and also cure such diseases. Modification of specific antigens using sialic acids has a number of advantages over current therapeutic strategies aimed at establishing large cohorts of Treg cells: this method provides DCs with a dual-tolerogenic function, because in addition to induction of Treg cells, these DCs simultaneously inhibit the generation of IFN-γ–producing T cells. Thus, using sialylated antigens as a therapy may directly dampen excessive inflammation, a process that occurs during autoimmunity. Furthermore, in contrast to current adjuvant approaches such as administration of retinoic acid or TGF-β, nonspecific suppression is minimized, because the specific antigen is directly modified with sialic acids. Moreover, we provide evidence for induction of antigen-specific immunotolerance under inflammatory conditions using sialylated antigens. Importantly, under these circumstances Sia-antigen–loaded DCs retained the capacity to polarize naive CD4+ T cells toward Treg cells. To the best of our knowledge, this sustained antigen-specific tolerizing effect has not been demonstrated for other tolerizing compounds. In conclusion, our data demonstrate that sialylation alters the immunogenicity of an antigen and provides a novel way to induce tolerogenic DCs for the treatment of autoimmune diseases and allergies.

Experimental Procedures Mice. C57BL/6 mice were used at 8–12 wk of age. OT-II, DO11.10, DO11.10×Rag2−/−, and 2D2 TCR transgenic mice were bred in the animal facilities of VU Medical Center (VUmc), Erasmus University Medical Center (Erasmus MC), and the TWINCORE Institute under specific pathogen-free conditions. All experiments were approved by the Animal Experiments Committee of the Erasmus MC and VUmc and performed in accordance with national and international guidelines and regulations. Dendritic Cells. Spleens from C57BL/6 mice were enriched for CD11c+ cells by magnetic separation according to the manufacturer’s protocol (Miltenyi Biotec). BMDCs were cultured from bone marrow of WT or Siglec-E−/− mice as described by Lutz et al. (36) with minor modifications (25). Antibodies. The phycoerythrin (PE)-labeled antibodies were anti-CD8b (H35-17.2) and anti-CD4 (GK1.5); the APC-labeled antibodies were anti-CD62L (MEL-14), anti-Foxp3 (FJK-169), and anti-IFN-γ (XMG1.2). Anti-CD62L and –IFN-γ antibodies were purchased from BD Pharmingen (BD Biosciences); others were obtained from eBiosciences. Modification of Antigens with Sialylated Glycans. To obtain Sia-OVA and Sia-MOG, maleimide-activated 6′-sialyl-N-acetyllactosamine (SLN306; Neu5Acα2,6Galβ1,4Glc; DEXTRA Labs) and 3′-sialyl-N-acetyllactosamine (SLN302; Neu5Acα2,3Galβ1,4Glc) were conjugated to thio-activated OVA (Calbiochem) and to MOG 35–55 peptide, which is described in detail in SI Materials and Methods. Th-Cell Differentiation and Testing of Suppressive Capacity. Naive CD4+CD62LhiCD25− T cells were purified from spleen and LN cell suspensions using the Dynal mouse CD4+CD62L+ T-cell isolation kit II mouse (Miltenyi Biotec) or by sorting on a MoFlo (DakoCytomation). Naive CD4+CD62L+CD25− T cells (5 × 104) were added to wells containing DCs (1 × 104) that were pulsed with the indicated concentrations of Sia-antigen or native antigen 3 h prior. After 2 d, 10 U/mL recombinant mouse IL-2 (Invitrogen) was added, and T-cell polarization was evaluated on day 6 by intracellular staining for Foxp3 and IFN-γ following 5 h of restimulation with phorbol 12-myristate 13-acetate (PMA) (30 μg/mL)/ionomycin (500 ng/mL; Sigma) in the presence of Brefeldin A (5 µg/mL; Sigma). To determine whether Sia-OVA–loaded DCs induce Treg-cell differentiation via a soluble factor, the cells were cultured in 0.4-μm pore size Transwell plates (Millipore). Suppressive capacity of Sia-OVA-DC–primed T cells was determined using 4-d cocultures with 1 × 104 OVA-loaded DCs and CFSE-labeled responder OT-II T cells. Cytokine Analysis. Cytokines were determined by cytometric bead arrays (CBA) using the CBA Th1/2/17 kit and mouse inflammation kit (BD Biosciences) or by ELISA using specific antibody pairs (eBiosciences) following the manufacturers’ instructions. In Vivo Treatment. For modulation of endogenous DCs, C57BL/6 mice were injected i.v. with 50 µg of Sia-OVA or OVA and primed 1 wk later by s.c. injection of 200 µg of OVA/25 µg of polyinosinic–polycytidylic acid [poly(I:C)]/25 µg of anti-CD40. Seven days after priming, the mice were killed, and spleens were analyzed for the frequency of Treg cells and effector T cells after restimulation with either 2 µg/mL SIINFEKL or 200 µg/mL EKLTEWTSSNMEER OVA peptides. Confocal Microscopy and Imaging Flow Cytometry. Intracellular routing of Sia-OVA or native OVA in DCs was analyzed confocal laser-scanning microscopy and ImageStream X (Amnis) imaging flow cytometer as described in SI Materials and Methods. Statistical Analysis. Prism 5.0 software (GraphPad) was used for statistical analysis. The Student’s t test and one-way ANOVA with Bonferroni correction were used to determine statistical significance. Statistical significance was defined as P < 0.05.

SI Materials and Methods Modification of Antigens with Sialylated Glycans. To obtain Sia-OVA and Sia-MOG, maleimide-activated 6′-sialyl-N-acetyllactosamine (SLN306; Neu5Acα2,6Galβ1,4Glc; DEXTRA Labs) and 3′-sialyl-N-acetyllactosamine (SLN302; Neu5Acα2,3Galβ1,4Glc) were conjugated to thio-activated OVA (Calbiochem) and to MOG 35–55 peptide with an added cysteine to the N terminus, through a thiol-ene reaction. MOG 35–55 peptide was produced by solid-phase peptide synthesis using fluorenylmethyloxycarbonyl chemistry with a Symphony peptide synthesizer (Protein Technologies). The glycans were activated with the bifunctional cross linker 4-N-maleimidophenyl butyric acid hydrazide (MPBH) (Pierce) and OVA was activated with the linker N-succinimidyl S-acetylthioacetate (SATA) (Pierce). The hydrazide moiety of MPBH was covalently linked to the reducing end of the carbohydrate via reductive amination at a 3:1 molar ratio, OVA was reacted with SATA via the amino groups on its surface at a 6:1 molar ratio, and the final reaction of OVA-SATA with the derivatized carbohydrate was performed at a molar ratio of 1: 10. The final reaction of MOG 35–55 with the derivatized carbohydrates was performed at a molar ratio of 1:1.5. Briefly, SATA, dissolved in DMSO, was added to a filtered solution of OVA in phosphate buffer (pH 8.2; filtered to 200 nm). After vigorous stirring, the resulting OVA-SATAAc solution was purified using a PD10 column (GE Healthcare) and diluted with 10% vol/vol of 0.5 M NH 2 OH⋅HCl solution (pH 7.2). After another 40 min of vigorous stirring and a second purification using a disposable size-exclusion cartridge (PD10 column; GE Healthcare Life Sciences), OVA-SATASH was ready for coupling with the MPBH-glycans. A mixture of MPBH (3 eq), 3′-sialyl-N-acetyllactosamine (or 6′-sialyl-N-acetyllactosamine) (1 eq), and picoline borane complex (10 eq; Sigma-Aldrich) dissolved in DMSO/AcOH (8:2) was incubated for 2 h at 65 °C, cooled to room temperature (RT), 1.4 mL of ice-cold isopropanol (anhydrous; Sigma-Aldrich) was added, and then incubated at –20 °C for 1 h. Subsequently, the precipitated MPBH–carbohydrates were pelleted, washed twice with cold isopropanol, and dissolved in 50 µL of PBS. The derivatization was confirmed by electrospray ionization mass spectrometry. The obtained MPBH–3′sialyl-N-acetyllactosamine and MPBH–6′-sialyl-N-acetyllactosamine were used immediately for coupling to OVA-SATASH and to MOG 35 – 55 peptide. Conjugation of OVA-SATASH and MOG 35 – 55 to the activated glycans was performed o/n at 4 °C, and the neoglycoconjugates were purified by size exclusion chromatography. The concentrations of OVA and MOG were determined using the bicinchoninic acid assay (Pierce). The presence of α2,6- or α2,3-linked sialic acids on the antigens was confirmed by matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF), HPLC, and ELISA (Fig. S1). In brief, 10 µg/mL Sia-antigen was coated directly onto ELISA plates (Nunc Maxisorb; Nunc), followed by incubation with the biotinylated plant lectins from Maackia amurensis Lectin-I (MAL-I) and Sambucus nigra (SNA) (Vector Labs) and peroxidase-labeled streptavidin (Sigma-Aldrich). Confocal Microscopy and Imaging Flow Cytometry. BMDCs were incubated with 30 μg/mL atto633-labeled Sia-OVA or native OVA for 2 h at 37 °C, fixed and permeabilized for 20 min on ice, and stained with primary and secondary antibodies. Colocalization was analyzed using a Leica AOBS SP2 CLSM system containing a DM-IRE2 microscope with glycerol objective lens (PL APO 63×/NA1.30); images were acquired using Leica confocal software (version 2.61). For imaging flow cytometry, ∼1 × 106 BMDCs were incubated with Sia-OVA or OVA and after 2 h cells were washed twice, fixed in ice-cold 4% (wt/vol) PFA/PBS for 20 min, and permeabilized in 0.1% saponin (Sigma) in PBS for 30 min at RT. Stainings were performed at RT in PBS/0.1% saponin/2% BSA. After staining, cells were washed twice, resuspended in PBS/1% BSA/0.02% NaN 3 and kept at 4 °C until analysis. Cells were acquired on the ImageStream X (Amnis) imaging flow cytometer. Analysis was performed using the IDEAS version 6.1 software (Amnis). Cells were first gated based on the Gradient RMS (bright field) feature and then based on area vs. aspect ratio intensity (both on bright field). The first gating identified the cells that appeared in focus, whereas the second excluded doublets and cells other than BMDCs. Three-color colocalization was calculated using the bright detail colocalization 3 feature.

Acknowledgments We thank our biotechnicians for excellent caretaking of the animals. This work was supported by the Seventh Framework Program of Marie Curie Actions (7th Framework Programme for Research and Technological Development-Carmusys), Koningin Wilhelmina Fonds (Dutch Cancer Foundation) Grant VU2009-2598, Senternovem Grant SII071030, and European Research Council Grants ERCAdvanced339977 and ESTAR14104 “SIAGEN-MS.”

Footnotes Author contributions: J.M.M.d.H., Y.v.K., and W.W.J.U. designed research; M.P., J.M.I., M.I.V., L.A.M.C., S.T.T.S., S.E., M.A., H.K., H.V., L.A.v.B., and J.J.G.-V. performed research; J.N.S., P.R.C., T.S., and L.B. contributed new reagents/analytic tools; M.P., J.M.I., M.I.V., L.A.M.C., S.T.T.S., S.E., M.A., H.V., J.J.G.-V., and W.W.J.U. analyzed data; and M.P., J.M.I., J.J.G.-V., Y.v.K., and W.W.J.U. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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