Effective stimulation of invariant natural killer T cells by oligomannose-coated liposomes

Highlights

• The response of iNKT cells to αGalCer-containing oligomannose-coated liposomes was examined.

• Splenocytes stimulated with αGC-OMLs produced higher levels of IFN-γ.

• Systemic administration of αGC-OMLs led to more rapid and continuous IFN-γ release in sera.

• iNKT cells proliferated and expanded upon activation of the cells with αGC-OMLs more extensively.

Abstract

vThe in vitro and in vivo response of invariant natural killer T (iNKT) cells to alpha-galactosylceramide (αGC)-containing oligomannose-coated liposomes (αGC-OMLs) was examined to determine whether selective delivery of αGC to dendritic cells (DCs) and subsequent activation of iNKT cells could be achieved. Splenocytes stimulated with αGC-OMLs produced higher levels of IFN-γ compared to those stimulated with bare liposomes without an oligomannose coating (αGC-BLs). The ratio of IFN-γ/IL-4 produced from αGC-OML-treated splenocytes was higher than those produced from αGC-BL- and soluble αGC-treated cells. Depletion of CD3⁺-, DX5⁺- or CD11c⁺-cells from splenocytes almost completely abolished the αGC-OML-stimulated cytokine production, suggesting that both NKT cells and DCs were involved in the response to αGC-OML stimulation. In addition, αGC-OMLs were incorporated into both splenic and bone marrow-derived DCs more effectively than αGC-BLs. iNKT cells stimulated with DCs with ingested αGC-OMLs produced much higher levels of IFN-γ than those stimulated with DCs containing αGC-BLs or soluble αGC. Systemic administration of αGC-OMLs led to modification of the kinetics of IFN-γ production in vivo and also resulted in predominant production of IFN-γ from splenocytes over IL-4. In addition, iNKT cells proliferated and expanded upon in vivo activation of the cells with αGC-OMLs much more extensively than with αGC-BLs or soluble αGC. Collectively, our results suggest that αGC-OMLs can be used as a preferential delivery system for lipid antigens to DCs to activate iNKT cells in vivo and ex vivo.

Introduction

A subpopulation of T cells referred to as invariant natural killer T (iNKT) cells recognize glycolipid antigens presented by the MHC class l-like molecule CD1d [1], [2]. The glycolipid antigen, alpha-galactosylceramide (αGC), which was originally derived from a marine sponge, has been used as an exogenous ligand for CD1d to stimulate mouse Vα14 NKT and human Vα24 NKT cells [3]. The antitumor and anti-infectious properties of αGC have also attracted attention, since αGC-reactive iNKT cells rapidly produce significant amounts of Th1 (e.g., IFN-γ) and Th2 (e.g., IL-4) cytokines in response to stimulation, which can subsequently activate other immune cells. Studies in animals have shown that intravascular administration of soluble αGC produces antitumor effects against various cancers in metastasis models [4], [5], [6] and can lead to elimination of several pathogens [7], [8]. However, intravascular or intraperitoneal administration of the soluble form of αGC also induces long-term anergy in iNKT cells, since it is taken up preferentially by circulating, CD1d-expressing B cells, rather than dendritic cells (DCs) [9], [10], [11]. Therefore, a method for delivery of αGC specifically to DCs is required, although it has been demonstrated that iNKT cell anergy is attenuated by subcutaneous and intradermal routes [12], [13]. Particles of 500–2000 nm are superior carriers of vaccines due to their preferable uptake by DCs [14], and thus formulation of αGC into particles such as liposomes may achieve selective delivery to DCs and modify the bioavailability of αGC. Formulation approaches to delivery of αGC have received relatively little attention, but αGC formulation in particles has been shown to promote iNKT cell-mediated immune responses [14], [15], [16], [17], [18].

DCs express numerous C-type lectin receptors (CLRs) that act as phagocytic receptors [19], [20]. Among the CLRs, the family of DC-SIGNs and langerin are of particular interest, since expression of these CLRs is restricted to professional antigen presenting cells (APCs) such as DCs [21], [22], [23], [24]. Several laboratories have demonstrated that targeting DC-SIGN by anti-DC-SIGN antibody affords the best strategy to deliver antigens or drug into DCs [25], [26], [27]. DC-SIGNs and langerin recognize carbohydrate ligands with terminal mannose, fucose and N-acetylglucosamine residues, such as high-mannose type glycans [28], [29]. Therefore, mannose residues attached to antigens or coated on antigen-containing particles have the potential to deliver antigens to DCs [30].

We have shown that liposomes coated with a mannotriose-containing neoglycolipid (Man3-DPPE) can deliver encapsulated protein antigens to DCs [31], promote presentation of the encapsulated antigen on MHC class I and II molecules [32], and lead to maturation of DCs [33], with resulting induction of antigen-specific immune responses against protozoan infectious diseases, cancer, and allergy [34], [35], [36]. Based on these results, we hypothesized that Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) could also be used as a vehicle for preferential delivery of lipid antigens such as αGC to DCs to activate iNKT cells via presentation of lipids on CD1d. In this study, we compared the in vitro and in vivo responses of iNKT cells to αGC-containing liposomes coated with Man3-DPPE (αGC-OMLs) and to those without a Man3-DPPE coating (αGC-BLs).