Differential pathways regulating innate and adaptive antitumor immune responses by particulate and soluble yeast-derived β-glucans

Abstract

β-glucans have been reported to function as a potent adjuvant to stimulate innate and adaptive immune responses. However, β-glucans from different sources are differential in their structure, conformation, and thus biologic activity. Different preparations of β-glucans, soluble versus particulate, further complicate their mechanism of action. Here we show that yeast-derived particulate β-glucan activated dendritic cells (DCs) and macrophages via a C-type lectin receptor dectin-1 pathway. Activated DCs by particulate β-glucan promoted Th1 and cytotoxic T-lymphocyte priming and differentiation in vitro. Treatment of orally administered yeast-derived particulate β-glucan elicited potent antitumor immune responses and drastically down-regulated immunosuppressive cells, leading to the delayed tumor progression. Deficiency of the dectin-1 receptor completely abrogated particulate β-glucan-mediated antitumor effects. In contrast, yeast-derived soluble β-glucan bound to DCs and macrophages independent of the dectin-1 receptor and did not activate DCs. Soluble β-glucan alone had no therapeutic effect but significantly augmented antitumor monoclonal antibody-mediated therapeutic efficacy via a complement activation pathway but independent of dectin-1 receptor. These findings reveal the importance of different preparations of β-glucans in the adjuvant therapy and allow for the rational design of immunotherapeutic protocols usable in clinical trials.

Figure 1

β-glucan phagocytosis and binding on DCs and macrophages. BMDCs (A) and macrophages (B) were incubated with DTAF-WGP at 37°C and phagocytosis of particulate β-glucan was assessed by flow cytometry and ImageStream. Percent of particulate β-glucan positive cells were summarized. One representative dot plot from 3 independent experiments with similar results. (C) BMDCs and macrophages were incubated with PGG-DTAF β-glucan on ice and β-glucan binding was assessed by flow cytometry. The data indicate that soluble β-glucan binding on DCs and macrophages is independent of dectin-1 or CD11b receptors. (D) BMDCs and macrophages were incubated with soluble PGG β-glucan or dextran and then mixed with DTAF-WGP on ice. Particulate β-glucan binding on DCs or macrophages was measured by flow cytometry. Cells were gated on CD11c⁺ or F4/80⁺ cells.

Figure 2

Surface marker expression and cytokine secretion by DCs stimulated with β-glucans. (A) BMDCs from WT, CD11b^−/−, or dectin-1^−/− mice were stimulated with particulate β-glucan (100 μg/mL) and surface marker expression was assessed by flow cytometry. One representative histogram from 3 independent experiments with similar results. (B) BMDCs were stimulated with soluble PGG β-glucan and surface marker expression was measured by flow cytometry. (C) Supernatants from particulate β-glucan–stimulated DCs were collected and assayed for TNF-α, IL-12, IL-6, and IL-10 (**P < .01; ***P < .001).

Figure 3

CD4 T-cell differentiation and CD8 priming and differentiation stimulated by β-glucan–activated DCs. (A) OVA Tg CD4⁺ T cells were cocultured with BMDCs from WT, CD11b^−/− or dectin-1^−/− mice in the presence or absence of OVA and β-glucans. CD4⁺ T cells cultured with β-glucans were used as controls. Percent of CD4⁺IFN-γ⁺ cells and CD4⁺ IL-17⁺ cells is shown (n = 4). (B) CFSE-labeled OVA Tg CD8⁺ T cells were cultured together with BMDCs in the presence of OVA and β-glucan. CD8⁺ T cells cultured with β-glucans were used as controls. Graphs show CFSE dilution versus intracellular IFN-γ on day 3 of culture. Percent of CD8⁺IFN-γ⁺ cells is shown (n = 4).

Figure 4

Particulate β-glucan treatment significantly reduces tumor burden with enhanced antitumor immunity. (A) Groups of WT mice were implanted subcutaneously with EO771/OVA tumor cells. After palpable tumors formed, mice were treated daily with or without particulate β-glucan for 2 weeks. Tumor diameter was recorded at the indicated time. Tumor mass was weighted when mice were killed. (B) Single cell suspensions prepared from tumor samples or spleen as indicated were stained with fluorochrome labeled mAbs. Summarized data are shown. (C-D) Single cell suspensions from tumors were stimulated with PMA plus ionomycin and stained intracellular IFN-γ and IL-17. Cells were gated on CD4⁺ (C) or CD8⁺ (D) T cells. (E) Single cell suspensions from tumors or tumor DLNs were stimulated with ovalbumin and intracellular IFN-γ staining was performed. (F) Splenocytes were labeled with CFSE and then stimulated with ovalbumin. Graphs show CFSE dilution versus intracellular IFN-γ production. (G). RNAs from tumor specimens were extracted and qRT-PCR was performed for the indicated cytokines and arginase.

Figure 5

Dectin-1–deficient completely abrogates particulate β-glucan–mediated antitumor therapeutic efficacy. (A) Groups of dectin-1^−/− mice were implanted subcutaneously with EO771/OVA tumor cells. After palpable tumors formed, mice were treated daily with or without particulate β-glucan for 2 weeks. Tumor diameter was recorded at the indicated time. (B) Single cell suspensions prepared from tumor samples or spleen as indicated were stained with fluorochrome labeled mAbs. Summarized data are shown. (C) Single cell suspensions from tumors were also stimulated with PMA plus ionomycin and stained intracellular IFN-γ and IL-17. Cells were gated on CD8⁺ T cells. (D) RNAs from tumor specimens were extracted and qRT-PCR was performed for the indicated cytokines and arginase.

Figure 6

Soluble β-glucan augments antitumor monoclonal antibody-elicited therapeutic efficacy. (A) Groups of WT mice were implanted subcutaneously with RMA-MUC1, after 7 days, to allow tumor formation, were treated with different regimens. Both tumor progression and tumor-free survival were monitored. (B) Dectin-1^−/− or (C) C3^−/− mice were implanted with RMA-MUC1 tumor cells. After tumors were formed, mice were subject to different treatment as indicated. Tumor diameter was recorded at the indicated time. (D) Neutrophils were marginated from WT and CD11b^−/− mice treated with soluble PGG β-glucan for 1 week. iC3b-opsonized tumor cells and un-opsonized tumor cells were labeled with a different intensity of CFSE and mixed at a 1:1 ratio and incubated with neutrophils. Percent of cytotoxicity is shown. (E) WT mice treated with soluble PGG β-glucan in combination with antitumor mAbs were preinjected with anti–Gr-1 monoclonal antibody to deplete neutrophils or isotype the control monoclonal antibody. Tumor diameter and tumor-free survival were monitored.