Carbohydrate-Lectin Interactions Reprogram Dendritic Cells to Promote Type 1 Anti-Tumor Immunity

Abstract

Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) is thought to shape immunity by directing T cell function. Controlling downstream responses, however, remains a major challenge. A critical goal in advancing vaccine development involves the identification of receptors that drive type 1 cellular immunity. The immune system monitors cells for aberrant glycosylation (a sign of a foreign entity), but potent activation occurs when a second signal, such as single-stranded RNA or lipopolysaccharide, is present to activate TLR signaling. To exploit dual signaling, we engineered a glycan-costumed virus-like particle (VLP) vaccine that displays a DC-SIGN-selective aryl mannose ligand and encapsulates TLR7 agonists. These VLPs deliver programmable peptide antigens to induce robust DC activation and type 1 cellular immunity. In contrast, VLPs lacking this critical DC-SIGN ligand promoted DC-mediated humoral immunity, offering limited tumor control. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4⁺ and CD8⁺ T cells that infiltrated solid tumors, significantly inhibiting tumor growth in a murine melanoma model. The tailored VLPs also afforded protection against the reintroduction of tumor cells. Thus, DC lectin-driven immune reprogramming, combined with the modular programmability of VLP platforms, provides a promising framework for directing cellular immunity to advance cancer immunotherapies and vaccines.

Keywords: antitumor; dendritic cell; immunotherapy; lectin; toll-like receptor; virus-like particles.

Figure 1.. Aryl mannoside-substituted VLPs as a platform to induce cellular immunity.

(a) Schematic of VLP-ArMan-OvaI/II engagement of DC-SIGN and TLR7 on DCs for antigen presentation and T cell priming. VLP decoration with ArMan facilitates DC-SIGN binding and antigen internalization. Following endocytosis, ssRNAs engage TLR7 to activate DCs and facilitate endosomal MHC loading to induce tumor antigen-specific T cells. (b) Design of VLPs decorated with tumor antigens OvaI and OvaII, control pentaerythritol (PE) or ArMan ligands, and encapsulated ssRNAs. (c) Surface-accessible lysine residues were functionalized to incorporate a fluorophore and an azide handle and the resulting VLP was subjected to copper(I)-catalyzed Huisgen azide-alkyne cycloaddition (CuAAC), a type of click chemistry to append tumor antigens and ligands. Steps (i,ii): 0.1 M potassium phosphate buffer, pH 7.4; room temperature, 2 h; step (iii): tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), CuSO₄, sodium ascorbate, aminoguanidine, 0.1 M potassium phosphate, pH 7.0, 50 °C, 1 h.

Figure 2.. VLP-ArMan-OvaI/II co-engages DC-SIGN and TLR7 for DC activation.

(a) Representative flow cytometry plots of VLP uptake in CD11c⁺ moDCs after 30 min. (b) moDCs exposed to 8 nM Alexa Fluor 647 (AF647)-labeled VLPs were assessed for VLP uptake using flow cytometry. (c) Mean fluorescence intensity (MFI) of AF647-labeled VLPs in CD11c⁺ moDCs at 30 min. (d) moDCs were pre-treated with blocking antibodies against lectins for 20 min and then 8 nM VLP-ArMan-OvaI/II for 15 min. VLP uptake was assessed by flow cytometry. Relative uptake was calculated by normalization against a control sample without blocking antibodies. (e) Confocal microscopy images showing VLP localization in moDCs after 1 h exposure to 8 nM VLPs. Green: AF405 anti-DC-SIGN; Pink: AF488 anti-TLR7; Cyan: AF647-VLP. Scale bar, 5 μm. (f,g) Pearson’s correlation coefficient is reported for the colocalization of VLPs with DC-SIGN (f) or TLR7 (g) after 1 h incubation of moDCs with 8 nM VLPs (40 cells per treatment). (h) Representative flow cytometry plots of moDC activation, demonstrated by expression level of surface marker CD40, following treatment with 8 nM VLPs for 24 h. (i) Activation of moDCs by VLPs was determined by expression of costimulatory signals. moDCs were incubated with 8 nM VLPs for 24 h, and the expression levels of activation surface markers CD40, CD83, CD86, and CD80 were measured by flow cytometry. Data represent mean +/− standard error of the mean (s.e.m.) from a representative experiment (n=3). Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparisons test. *P<0.1, **P<0.01, ***P<0.001, ****P<0.0001. Each experiment was repeated at least twice with similar results.

Figure 3.. VLP-ArMan-OvaI/II induces type 1-associated responses.

(a) Altered gene expression profiles between VLP-ArMan-OvaI/II- and VLP-PE-OvaI/II-treated moDCs. moDCs were treated with 8 nM VLPs for 6 h. (b) TLR7 downstream gene signature module score for unstimulated, VLP-PE-OvaI/II-treated, and VLP-ArMan-OvaI/II-treated moDCs. (c) GSEA analysis of upregulated and downregulated pathways in DCs treated with VLP-ArMan-OvaI/II or VLP-PE-OvaI/II. (d) The moDCs were treated with VLPs for 32 h and supernatants were collected for cytokine analysis. (e) The moDCs were pre-treated with blocking antibodies against DC-SIGN sfor 20 min followed by stimulation with VLP-ArMan-OvaI/II for 32 h, and TNF-α secretion was measured. Data represent mean +/− s.e.m. from a representative experiment (n=3). Statistical analysis was performed by one-way ANOVA with Bonferroni’s multiple comparisons test. *P<0.1, **P<0.01, ***P<0.001, ****P<0.0001. For (d) and (e), experiments were repeated at least twice with similar results.

Figure 4.. VLP-ArMan-OvaI/II induces tumor antigen-specific T cells.

(a) C57/BL6 mice were inoculated with 5×10⁵ B16F10-OVA cells subcutaneously (s.c.) on day 0 and treated with VLPs and 2’3’-cGAMP (s.c.) and anti-PD-1 intraperitoneally (i.p.) on days 3, 9, and 15. Splenocytes were isolated on day 21 for immunophenotyping. (b,c) Representative flow cytometry plots of CD4⁺ (b) and CD8⁺ (c) T cells in the spleens. (d) Percentages of CD4⁺ and CD8⁺ T cells in the spleens. (e-g) IFN-γ ELISpot analysis of splenocytes restimulated with OVA(323–339) (e) or OVA(257–264) (f) or co-cultured with irradiated B16F10-OVA cells (g). (h) Representative flow cytometry plots of IFN-γ and TNF-α-secreting CD8⁺ T cells in the spleens following restimulation with OVA antigens. (i) IFN-γ and TNF-α-secreting CD4⁺ T cells in the spleen were measured following restimulation with OVA(323–339). (j) IFN-γ and TNF-α-secreting CD8⁺ T cells in the spleen were measured following restimulation with OVA(257–264). (k) Representative flow cytometry plots of T cells in the tumors. (l) Percentages of CD4⁺ and CD8⁺ T cells in the tumors. (m) Percentages of CD4⁺ and CD8⁺ among CD3⁺ T cells within the tumor. Data represent mean +/− s.e.m. from a representative experiment (n=5). Statistical analysis was performed by one-way ANOVA with Bonferroni’s multiple comparisons test. *P<0.1, **P<0.01, ***P<0.001, ****P<0.0001. Each experiment was repeated twice with similar results.

Figure 5.. VLP-ArMan-OvaI/II elicits therapeutic and prophylactic anti-tumor responses.

(a) C57/BL6 mice were inoculated with 5×10⁵ B16F10-OVA cells (s.c.) on day 0 and treated with VLPs and 2’3’-cGAMP (s.c.) and anti-PD-1 (i.p.) on days 3, 9, and 15. (b) Mean tumor growth curves. (c) Tumor volumes on day 21. (d) Survival curves. (e) Weight change measurements. (f) Surviving mice were re-challenged with 5×10⁴ B16F10-OVA cells on day 28. (g) Tumor growth curves. (h,i) OVA(323–339)-specific antibody profiles were analyzed by ELISA measurements from serum of mice immunized with VLP-PE-OVAI/II (h) or VLP-ArMan-OvaI/II (i). (j) IgG2c:IgG1 antibody ratio for mice immunized with VLPs. Line represents IgG2c:IgG1 ratio of 1. Data represent mean +/− s.e.m. from a representative experiment (n=8 (a-e) and n=4 (f-g) and n=5 (h-j)). Statistical analysis was performed by one-way ANOVA with Bonferroni’s multiple comparisons test or by log-rank (Mantel-Cox) test for the survival analysis. *P<0.1, **P<0.01, ***P<0.001, ****P<0.0001. Each experiment was repeated twice with similar results.

Figure 6.. ArMan VLPs demonstrate self-adjuvanting capacity.

(a) C57/BL6 mice were inoculated with 5×105 B16F10-OVA cells (s.c.) on day 0 and treated with VLPs and 2’3’-cGAMP (s.c.) and anti-PD-1 (i.p.) on days 3, 9, and 15. (b) Mean tumor growth curves. (c) Tumor volumes on day 21. (d) Survival curves. Differences with or without 2’3’-cGAMP were not significant. (e) Representative flow cytometry plots of IFN-γ and TNF-α-secreting CD8+ T cells in the spleens following restimulation with OVA antigens. (f) IFN-γ and TNF-α-secreting CD4+ T cells in the spleen were measured following restimulation with OVA(323–339). (g) IFN-γ and TNF-α-secreting CD8+ T cells in the spleen were measured following restimulation with OVA(257–264). (h) Representative flow cytometry plots of T cells in the tumors. (i) Percentages of CD4+ and CD8+ T cells in the tumors were analyzed on day 21. Data represent mean +/− s.e.m. from a representative experiment (n=8 (b-d) and n=5 (e-g)). Statistical analysis was performed by one-way ANOVA with Bonferroni’s multiple comparisons test or by log-rank (Mantel-Cox) test for the survival analysis. *P<0.1, **P<0.01.