Cross-presentation through langerin and DC-SIGN targeting requires different formulations of glycan-modified antigens

Abstract

Dendritic cells (DCs) and Langerhans cells (LC) are professional antigen presenting cells (APCs) that initiate humoral and cellular immune responses. Targeted delivery of antigen towards DC- or LC-specific receptors enhances vaccine efficacy. In this study, we compared the efficiency of glycan-based antigen targeting to both the human DC-specific C-type lectin receptor (CLR) DC-SIGN and the LC-specific CLR langerin. Since DC-SIGN and langerin are able to recognize the difucosylated oligosaccharide Lewis Y (Le^Y), we prepared neoglycoconjugates bearing this glycan epitope to allow targeting of both lectins. Le^Y-modified liposomes, with an approximate diameter of 200 nm, were significantly endocytosed by DC-SIGN⁺ DCs and mediated efficient antigen presentation to CD4⁺ and CD8⁺ T cells. Surprisingly, although langerin bound to Le^Y-modified liposomes, LCs exposed to Le^Y-modified liposomes could not endocytose liposomes nor mediate antigen presentation to T cells. However, LCs mediated an enhanced cross-presentation when antigen was delivered through langerin using Le^Y-modified synthetic long peptides. In contrast, Le^Y-modified synthetic long peptides were recognized by DC-SIGN, but did not trigger antigen internalization nor antigen cross-presentation. These data demonstrate that langerin and DC-SIGN have different size requirements for antigen uptake. Although using glycans remains an interesting option in the design of anti-cancer vaccines targeting multiple CLRs, aspects such as molecule size and conformation need to be taken in consideration.

Introduction

Dendritic cells (DCs), the most efficient antigen presenting cells (APCs) of the immune system, continuously sample their environment for pathogens in order to endocytose, process and, ultimately, present antigens on MHC molecules to T cells. To facilitate antigen recognition, DCs are equipped with a variety of pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs) and NOD-like receptors (NLRs) [1]. Although the specificity and function of these receptors is rather diverse, the CLR family members detect carbohydrate structures in a Ca^2 +-dependent fashion and mediate antigen uptake to facilitate antigen processing and presentation, whereas TLRs and NLRs are signaling receptors that recognize pathogen-associated molecular patterns and elicit signals that result in proper DC maturation and cytokine production.

Some CLRs are selectively expressed by specific APC subsets and can be used to define APC subpopulations, such as DC-SIGN, expressed on human dermal DCs (dDCs) and other APCs of the myeloid lineage [2]; and langerin, which is highly expressed on Langerhans cells (LCs) [3] and lower expression levels were recently also detected on intestinal lamina propria DCs [4] and on CD1c⁺ myeloid DCs [5]. The glycan binding profiles of DC-SIGN and langerin show some overlap: both receptors are known to bind mannosylated glycans, but have a different specificity towards fucose-containing glycans [6], [7], [8]. For example, DC-SIGN shows specificity for all Lewis blood group antigens (Le^a, Le^b, Le^X and Le^Y), whereas langerin only interacts with the difucosylated glycans Le^b and Le^Y [8].

Additionally, differences in molecular orientation between langerin and DC-SIGN have been described. Langerin forms trimers through a coiled-coil structure in the extracellular neck-region, leading to a rather rigid position in the membrane compared to DC-SIGN, which is organized in tetramers [9]. DC-SIGN forms oligomers via its stem region, which allows for a higher level of flexibility to the carbohydrate recognition domains (CRDs) that facilitate interaction with its ligands [10]. An interesting feature of langerin is its association with Birbeck granules (BGs), which are rod-shaped structures and subdomains of the endosomal recycling compartment uniquely present in LCs [3], [11]. The presence of langerin is crucial for BG formation [12]. Antibodies directed against langerin are internalized in BGs, providing access of antigen to a LC-specific non-classical antigen-processing pathway [3], but how BGs influence the processing and presentation of antigens in MHC class I and II is still not fully understood. Although langerin and DC-SIGN show significant overlap in ligand specificity, they are expressed on distinct DC subsets, have a distinctive structural organization, and target to different intracellular processing machineries and organelles, altogether suggesting that these CLRs mediate different biological responses.

Many CLRs facilitate the internalization of antigens after binding to the receptor, leading to antigen processing and presentation on MHC-II molecules to activate CD4⁺ T cells. CLR-mediated uptake of exogenous antigens has also been shown to result in cross-presentation of antigen on MHC-I molecules for the activation of CD8⁺ T cells [13], [14], [15]. In most of these studies, monoclonal antibodies (moabs) against CLRs were used as targeting agents. For both DC-SIGN and langerin, internalization of the receptors after moab targeting and induction of T cell responses have been described, suggesting that DC-SIGN and langerin targeting routes antigen to MHC class I and II loading compartments [16], [17], [18]. However, the Kd of antibodies is several orders of magnitude lower than that of natural CLR ligands (glycans), which might certainly affect the antigen routing and processing. Therefore, it would be interesting to analyze and compare langerin and DC-SIGN internalization and induction of T cell responses when both CLRs are targeted with antigens conjugated to glycan structures.

The internalization route and T cell stimulating capacity of several CLRs upon targeting with their ligands have already been described. For instance, mannose receptor (MR) targeting using the glycans, 3-sulfo-Lewis^a and chitotriose conjugated to OVA, resulted in MR-dependent cross-presentation and Th1 polarization in vivo [19]. Similar results have been obtained using Le^b- or Le^X-modified OVA that target transgenic human DC-SIGN⁺ murine DCs [20]. Although most of these experiments have been performed with bone marrow-derived DC or in vivo mouse models, little is known about the potency of CLR-targeting vaccines in human skin, the primary vaccination site. The complexity of targeting and mobilizing skin APC subsets for the improvement of antigen-specific CD4⁺ and CD8⁺ T cell responses directed against tumors or viruses are major questions to be addressed [19], [21], [22], [23].

Although both DC-SIGN and langerin are able to internalize antigen, but little is known on the preferences of these receptors for any form of glycosylated antigen, if there are restrictions related to glycan valency or the size of the vaccine formulation. For DC-SIGN it has been described that glycan multivalency favors the strength of binding, since targeting of DC-SIGN using glycan-modified dendrimers, or glycan-modified liposomes facilitated DC-SIGN-mediated internalization and resulted in the induction of strong CD4⁺ and CD8⁺ T cell responses [24], [25]. On the other hand, less is known about the ligand preferences of langerin. Similar to DC-SIGN, langerin recognizes pathogens such as HIV, Candida, Saccharomyces and measles virus (MV) in a glycan-dependent manner [26], [27], [28]. Although the targeting of antigens to langerin using moabs leads to the development of antigen-specific Th1 and CD8⁺ T-cell responses [13], langerin-mediated internalization of MV only induced MV-specific CD4⁺ T cell responses, but no antigen cross-presentation occurred [27]. It is currently unclear whether langerin facilitates internalization and induction of T cell responses of glycan-modified antigens of any formulation.

Therefore, we set out to study the preferences of DC-SIGN and langerin for glycan-based vaccine formulations using glycan-modified peptides or liposomes as a model for small sized soluble-based molecular platforms versus large multivalent particulate antigenic carriers.