Carbohydrate-mediated targeting of antigen to dendritic cells leads to enhanced presentation of antigen to T cells

Abstract

The unique therapeutic value of dendritic cells (DCs) for the treatment of allergy, autoimmunity and transplant rejection is predicated upon our ability to selectively deliver antigens, drugs or nucleic acids to DCs in vivo. Here we describe a method for delivering whole protein antigens to DCs based on carbohydrate-mediated targeting of DC-expressed lectins. A series of synthetic carbohydrates was chemically-coupled to a model antigen, ovalbumin (OVA), and each conjugate was evaluated for its ability to increase the efficiency of antigen presentation by murine DCs to OVA-specific T cells (CD4(+) and CD8(+)). In vitro data are presented that demonstrate that carbohydrate modification of OVA leads to a 50-fold enhancement of presentation of antigenic peptide to CD4(+) T cells. A tenfold enhancement is observed for CD8(+) T cells; this indicates that the targeted lectin(s) can mediate cross-presentation of antigens on MHC class I. Our data indicate that the observed enhancements in antigen presentation are unique to OVA that is conjugated to complex oligosaccharides, such as a high-mannose nonasaccharide, but not to monosaccharides. Taken together, our data suggest that a DC targeting strategy that is based upon carbohydrate-lectin interactions is a promising approach for enhancing antigen presentation via class I and class II molecules.

Scheme 1

Synthetic analogues of the high-mannose oligosaccharide (Man)₉(GlcNAc)₂ that were used in the preparation of ovalbumin conjugates for dendritic cell targeting. These structures were chosen for DC targeting on the basis of their recognition by the DC lectin, DC-SIGN. The panel of structures consists of three branched oligosaccharides (1−3), a linear trisaccharide 4 that is derived from the D1 arm of the high-mannose nonasaccharide and two monosaccharides, mannose 5 and galactose 6. Conjugation of each structure was made possible by the incorporation of a thiol-bearing linker (shown as “R” here) The stereochemistry at the reducing end is as shown; it was β for structures 1−3 and 6, and it was α for structures 4 and 5.

Figure 1

Carbohydrate modification of OVA leads to enhanced presentation to antigen-specific T cells. A) OVA, SMCC-activated OVA and carbohydrate modified OVA were resolved by SDS-PAGE electrophoresis and immunoblotted with anti-OVA polyclonal antibody to reveal changes in molecular weight as a function of carbohydrate addition. B) Oligosaccharide modification of OVA elicits stronger presentation to OT-II T cells than unmodified OVA. Unfractionated OT-II splenocytes (3 × 10⁵ per well) were incubated with graded doses of OVA or oligosaccharide-modified OVA for 84 h with [³H]thymidine (1 μCi) added for the last 12 h. Thereafter [³H]thymidine incorporation levels were measured and plotted. C) Observed enhancements in antigen presentation of (OVA)-1 to T cells is carbohydrate dependent. Splenocytes were incubated with antigen as in (B) ±(BSA)-1 at various doses; incubations were performed for 72 h, and [³H]thymidine (1 μCi) was added for the last 12 h. (D). Nonasaccharide 1 does not directly stimulate T cell proliferation. A mixed leukocyte reaction (MLR) was carried out with purified C57BL/6 CD11c⁺ DC and purified Balb/c CD4⁺ T cells at a 1:5 DC:T cell ratio (OVA)-1 (50 μg mL⁻¹) for 72 h; [³H]thymidine (1 μCi) was added for the last 12 h. Control incubations were DCs + (OVA)-1 (50 μg mL⁻¹) and T cells (–DCs) + (OVA)-1 (50 μg mL⁻¹).

Figure 2

Nonasaccharide 1-promoted presentation via MHC class I pathway for presentation to CD8⁺ T cells. A) (OVA)-1 enhances presentation of antigenic peptides to CD8⁺ T cells in a dose and carbohydrate-dependent fashion. Unfractionated OTI splenocytes (3 × 10⁵ per well) were incubated with graded doses of OVA ±(OVA)-1 (50 ng ml⁻¹) or (OVA)-1 ±(BSA)-1 (10 μg mL⁻¹). Incubations were performed for 72 h, and [³H]thymidine (1 μCi) was added for the last 12 h. B) The monosaccharide mannose 5 targets a different receptor than 1. OT-II splenocytes were incubated with graded doses of monosaccharide-modified OVA ±(BSA)-1 and T cell proliferation was measured as in (A). C) The receptor-mediated uptake of (OVA)-1 has a different binding profile than that which was observed for DC-SIGN. Unfractionated splenocytes were incubated with OVA or (OVA)-1 ±mBSA or Lewis^x–BSA (each at 100 μg mL⁻¹). Incubations were performed for 72 h, and [³H]thymidine (1 μCi) was added for the last 12 h. All values reported are the mean of triplicate measurements; Ag: antigen.

Figure 3

CD11c⁺ DCs are the main APC-presenting nonasaccharide 1-targeted antigen. A) Purified CD11c⁺ DCs efficiently present (OVA)-1 to purified CD4⁺ T cells. 1.5 × 10⁴ Splenic CD11c⁺ DCs that were purified from C57BL/6 mice and 3.0 × 10⁴ purified OT-II T cells were incubated with OVA (starting concentration 300 μg mL⁻¹) or (OVA)-1 (starting concentration 25 μg mL⁻¹) in graded doses for 84 h, [³H]thymidine (1 μCi) was added for the last 12 h. B) The receptor-mediating uptake of (OVA)-1 preferentially binds complex mannans. Purified CD11c⁺ DCs and OT-II T cells were incubated with (OVA)-1 (25 μg mL⁻¹) ±each potential inhibitor (100 μg mL⁻¹). T cell proliferation was determined as in (A). Note: OVA incubation alone gave cpm counts of 30 000. C) Toll-like-receptor-induced DC maturation significantly decreases uptake and presentation of (OVA)-1 to T cells. CD11c⁺ DCs and OT-II T cells were incubated with OVA or (OVA)-1 ±lipopolysaccharide (1 μg mL⁻¹). Also included are all “non-DCs” (macrophages and B cells) that were obtained during the purification of the DCs. T cell proliferation was determined as in (A). D) Targeting (OVA)-1 to DCs leads to a more vigorous T cell response to pro-inflammatory conditions. Supernatants from (C) were collected at 48 and 72 h and were measured for IFN-γ by ELISA; the values presented are from the 72 h time point. All measurements were performed in triplicate.

Figure 4

A) Both DC subsets can present (OVA)-1. CD11c⁺ DCs were stained with antibody against CD8α and I-A^b MHC class II molecules and sorted into their respective CD8α⁺ and CD8α⁻ populations. 2 × 10⁴ Cells of each subtype were incubated with 1 × 10⁵ purified OT-II T cells with graded doses of OVA or (OVA)-1 for 84 h; [³H]thymidine (1 μCi) was added for the last 12 h. All data points were performed in triplicate. B) Nonsaccharide 1 does not inhibit or potentiate pro-inflammatory signalling networks relative to unmodified OVA. 10⁵ Purified CD11c⁺ splenic DCs were incubated with media, OVA or (OVA)-1 at two different doses ±various TLR stimuli for 24 h. Supernatants were collected and analyzed for IL-10. LPS was used at 1 μg mL⁻¹; poly(IC), 25 μg mL⁻¹; Sendai Virus, 40 hemagglutination units (H.U.) mL⁻¹; CpG1826, 2 mm. C) IL-6, as in (B). D) IFN-γ, as in (B). Values represent the mean of duplicate measurements.