Orally administered particulate beta-glucan modulates tumor-capturing dendritic cells and improves antitumor T-cell responses in cancer

Abstract

Purpose: The beneficial properties of β-glucans have been recognized for centuries. Their proposed mechanisms of action in cancer therapy occur via stimulation of macrophages and priming of innate neutrophil complement receptor 3 for eliciting complement receptor 3-dependent cellular cytotoxicity of iC3b-opsonized tumor cells. The current study is to investigate whether β-glucan therapy has any effect on antitumor adaptive T-cell responses.

Experimental design: We first examined the trafficking of orally administered particulate yeast-derived β-glucan and its interaction with dendritic cells (DC) that captured tumor materials. Antigen-specific T cells were adoptively transferred into recipient mice to determine whether oral β-glucan therapy induces augmented T-cell responses. Lewis lung carcinoma and RAM-S lymphoma models were used to test oral β-glucan therapeutic effect. Further mechanistic studies including tumor-infiltrating T cells and cytokine profiles within the tumor milieu were determined.

Results: Orally administered particulate β-glucan trafficked into spleen and lymph nodes and activated DCs that captured dying tumor cells in vivo, leading to the expansion and activation of antigen-specific CD4 and CD8 T cells. In addition, IFN-γ production of tumor-infiltrating T cells and CTL responses were significantly enhanced on β-glucan treatment, which ultimately resulted in significantly reduced tumor burden. Moreover, β-glucan-treated tumors had significantly more DC infiltration with the activated phenotype and significant levels of Th1-biased cytokines within the tumor microenvironment.

Conclusions: These data highlight the ability of yeast-derived β-glucan to bridge innate and adaptive antitumor immunity and suggest that it can be used as an adjuvant for tumor immunotherapy.

Figure 1. Oral WGPs migrate to the spleen and lymph node and interact with DCs

(A) Mice were fed orally with DTAF-WGPs (green). Spleen (Sp) and inguinal lymph nodes (LN) were cryosectioned and stained with anti-CD11c mAb (red). (B) Mice were injected with 2 ×10⁷ CFSE-labeled apoptotic LLC/OVA tumor cells or PBS. At 2 h after injection, spleen cells were harvested and stained with mAbs against CD11c and CD8α. Data show that DCs uptaking CFSE-positive apoptotic tumor cells are CD8α⁺. Cells were gated on CD11c⁺ population. (C) Groups of mice (n=5) were treated with WGPs daily for 7 days (400 μg/mouse). Fluorescein dye PKH26-labeled apoptotic LLC/OVA tumor cells (2 × 10⁷/mouse) were injected i.v. into C57Bl/6 mice. Five hours later, mice were sacrificed and spleen cells were isolated and stained with mAbs against CD11c, CD40, CD80, CD86, MHC class II, and relevant isotype controls. Cells were gated on CD11c⁺populations or CD11c⁺ and PKH26⁺ populations. Data show that co-stimulatory CD40, CD80, CD86, and MHC class II molecules are significantly upregulated in DCs capturing tumor materials after WGP treatment. Numbers represent mean fluorescent intensity (MFI).

Figure 2. WGPs significantly increase Ag-specific T cell proliferation in vitro but do not enhance DC-mediated apoptotic tumor cell phagocytotosis

(A) BMDCs were co-cultured with apoptotic LLC/OVA tumor cells and purified CD4 OVA Tg T cells in the presence or absence of varying amounts of WGPs. DCs with CD4 Tg T cells in the presence of OVA Ag (50 μg/ml) with or without WGPs were used as positive controls. (B) CFSE-labeled apoptotic LLC/OVA cells were co-cultured with BMDCs in the presence or absence WGPs in vitro for 24 hrs. Cells were then analyzed by flow cytometry.

Figure 3. Enhanced Ag-specific T cell responses upon oral WGP in vivo treatment

(A) Groups of mice (n=3) treated with or without WGPs (400 μg/daily/mouse for 7 days) were injected i.v. with or without apoptotic LLC/OVA tumor cells. After 24 h, mice were adoptively transferred with CFSE-labeled OT-II CD4 T cells. Mice were continuously treated with or without WGPs for 5 days and sacrificed. Splenocytes were examined by flow cytometry. Cells were gated on CFSE positive cells. Percentage indicates proliferated cells. (B) Splenocytes were stained with mAbs against CD44 and CD62L. Cells were gated on CFSE positive populations. (C) Groups of mice (n=3) treated with or without WGPs were injected with or without apoptotic LLC/OVA tumor cells. After 24 h, mice were adoptively transferred with CFSE-labeled OT-I CD8 T cells. Mice were continuously treated with or without WGPs for 5 days and sacrificed. Splenocytes were examined by flow cytometry. Cells were gated on CFSE positive cells. Percentage indicates proliferated cells. (D) Splenocytes were restimulated with OVA (50 μg/ml) for 24 h and then stained for intracellular IFN-γ production. Cells were gated on CD8⁺ T cells.

Figure 4. WGP treatment significantly reduces tumor burden and prolongs survival with increased CD8 T cell killing activity

(A) Groups of mice (n=10) were implanted s.c. with RMA-S-MUC1, and after 11 days, to allow tumor formation, were treated with oral WGPs at different does (100 μg, 200 μg, 400 μg, daily) for 3 weeks. Tumor-free survival was also monitored. Points, mean; bars, SE. *p<0.05, **p<0.01. (B) Groups of mice (n=12) were implanted s.c with LLC/OVA tumor cells. After palpable tumors formed, mice were treated with or without orally administered WGPs for three weeks. Tumor diameters were recorded at the indicated time. *p<0.05, **p<0.01. Points, mean; bars, SE. (B) Tumor-bearing mice with LLC/OVA (n=3) treated with or without WGPs for three wks were adoptively transferred with OVA Class I peptide loaded CFSE^high splenocytes with unloaded CFSE^low splenocytes. Mice were sacrificed after 24 hrs and CFSE⁺ cells were gated and analyzed by flow cytometry. Data show that tumor-bearing mice treated with WGPs have the highest cytotoxicity against target cells compared to PBS treated or naïve mice.

Figure 5. WGP treatment significantly increases IFN-producing T cells and DC infiltration within the tumors and drives Th1 cytokine production in the tumor microenvironment

(A) LLC/OVA tumor specimens (n=5) from WGP-treated or untreated mice were prepared for single cell suspensions. Tumor infiltrating cells were assessed by flow cytometry. Data indicate that oral WGP treatment in vivo significantly increases DC and macrophage infiltration within the tumors. Tumor samples were cryosectioned for immunohistochemistry staining with anti-CD11c mAb, which shows brown staining. Magnification ×200. The sections shown in this figure are representative tumor sections of 10 total tumor specimens. (B) Single cells from tumors treated with or without WGPs were re-stimulated with OVA (50 μg/ml) for overnight and then performed surface staining with mAbs against CD4 or CD8 and intracellular IFN-γ staining. Cells were gated on CD4⁺ or CD8⁺ T cell populations. (C) Single cell suspensions were stained with mAbs against CD11c, CD40, CD80, CD86, and MHC class II. Cells were gated on a CD11c⁺ population. *P<0.05. (D) RNAs from tumor specimens treated with or without WGPs (5 tumors per group) were extracted and reverse-transcribed for RT-PCR for the indicated cytokines and transcriptional factor FoxP3. *p<0.05; **p<0.01.