Elsevier

Human Immunology

Volume 76, Issues 2–3, March 2015, Pages 146-154
Human Immunology

β-Glucan enhances cytotoxic T lymphocyte responses by activation of human monocyte-derived dendritic cells via the PI3K/AKT pathway

https://doi.org/10.1016/j.humimm.2015.01.009Get rights and content

Abstract

Purpose

To investigate the effects of β-(1,3/1,6)-d-glucan on dendritic cells (DCs) maturation, cytotoxic T lymphocyte responses and the molecular mechanisms of its transition.

Methods and results

Human monocyte-derived DCs were matured using yeast-derived particulate β-glucan (WGP) or a mix of TNF-α, IL-1β and IL-6 (“Conv mix”). Multicolor flow cytometry was used to study the DCs phenotype and cytotoxic T-lymphocyte priming and differentiation. ELISA and RT-PCR assays were used to evaluate cytokine production. Western blot was used to investigate the signal pathways. WGP-matured DCs functions were compared with those of Conv mix-matured DCs. WGP-matured DCs expressed higher levels of CD11c, CD86, CD40 and HLA-DR; produced higher levels of pro-inflammatory cytokines; and elicited more CTL priming and differentiation than Conv mix-matured DCs. The PI3K/AKT signaling pathway was involved in WGP-induced dendritic cell maturation. Furthermore, WGP-matured DCs significantly increased tumor-specific CTL responses.

Conclusion

Excellent ability of yeast-derived particulate β-glucan to induce DCs maturation and tumor-specific CTL responses explains, in part, its clinical benefits and emphasizes its utility in ex vivo maturation of DCs generated for therapy.

Introduction

Recently, new approaches in immunotherapy offer hope of prolonged survival in cancer patients [1]. There is great interest, in this regard, in the use of anti-tumor vaccines, in particular DC-based vaccines [2]. Vaccines operate through the activation of DCs that eventually stimulate antigen-specific T and B lymphocytes. Adjuvants promote and enhance immune responses to vaccine components. Adjuvants derived from microorganisms stimulate DCs directly, leading to the up-regulation of cytokines, MHC class II, and co-stimulatory molecules and to their migration to the T cell area of lymph nodes. Among all adjuvants, toll-like receptor (TLR) agonists and cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF) have been tested in both preclinical and clinical settings for cancer treatment. In addition to TLR agonists and cytokine adjuvants, ligands for the RIG-I-like, the NOD and the dectin-1 receptors have been demonstrated to activate DCs and to promote innate and adaptive immune responses. β-Glucan has been recognized as a major fungal pathogen-associated molecular pattern (PAMP), which can strongly influence natural and adaptive host immune responses, mostly through engagement of the C-type lectin receptor dectin-1 [3]. β-Glucan, as a biological response modifier, was first reported 45 years ago and has been extensively investigated for both its anti-tumor and anti-infective activity [4], [5], [6]. Recent studies also demonstrate that β-glucan can function as a potent adjuvant to stimulate innate and adaptive immune responses [7], [8]. Most β-glucans are derived from yeast, bacteria, barely or fungi and have a backbone structure of linear β-1,3-linked d-glucose molecules (β-1,3-d-glucan). They also have β-1,6-linked side chains of β-1,3-d-glucan of varying sizes that occur at different intervals along the backbone [9]. There are at least four β-glucan receptors that have been identified: complement receptor 3 (CR3, CD11b/CD18, αMβ2-integrin, Mac-1) [10], lactosylceramide (LacCer) [11], selected scavenger receptors (SRs) including SR CD36 [12], and dectin-1 [8], [13].

Dectin-1 was originally reported as a DC-specific molecule with a T cell co-stimulatory capacity, but later, its expression was found more strongly in monocytes, macrophages and neutrophils and weakly in a subset of T cells, and in humans, B cells and eosinophils. The receptor dectin-1 expressed on polymorphonuclear leukocytes, mononuclear cells, macrophages, and DCs mediates the biological effects of β-glucan, including glucan-dependent anti-cancer immune responses. In murine tumor models, we found that orally administered β-glucan elicits potent anti-cancer immune responses, leading to delayed tumor progression [8], [14]. Stimulation with β-glucan leads to DC maturation and cytokine secretion, enabling DCs to elicit T lymphocyte activation. Dectin-1 signaling in human monocyte-derived DCs triggers NF-κB activation through two independent signaling pathways: one through Syk [15], [16] and the second through the kinase Raf-1 [17], [18]. Both signaling pathways converge at the level of NF-κB activation and regulation to control antifungal adaptive immunity. Engagement of dectin-1 by β-glucan can trigger a series of intracellular signal transduction pathways through Syk kinase and Raf-1 signaling pathways, activating the cells and inducing a variety of cellular responses, such as cytokine production.

In this study, we demonstrated that whole β-glucan particles (WGPs) could activate and maturate human monocyte-derived DCs, up-regulate co-stimulatory molecules, increase pro-inflammatory cytokine production, and lead to augment tumor-specific cytotoxic T lymphocyte (CTL) responses. Furthermore, we showed that the PI3K/AKT pathway was involved in activation of human monocyte-derived dendritic cells by β-glucan.

Section snippets

Preparation of β-glucans

WGP β-glucan (kindly provided by Prof Jun Yan, University of Louisville) was purified from the cell walls of Saccharomyces cerevisiae. A series of alkaline and acid extractions yielded hollow yeast cell wall “ghosts” composed primarily of long β-1,3 glucose polymers with 3–6% of the backbone glucose units possessing a β(1,6) branch (β-(1,3/1,6)-d-glucan). WGPs were hydrated in distilled water and sonicated to produce a single-particle suspension. To remove any trace amounts of LPS

Distinct phenotype of DCs matured by WGP vs. a conventional maturation cocktail

A widely used conventional combination of cytokines for human monocyte-derived DC maturation consists of TNF-α, IL-1β and IL-6. We compared this mixture with WGPs after 48 h of maturation, which results in maximal effects as determined in preliminary studies (data not shown). Both procedures resulted in a significant upregulation of all DC surface markers, including the maturation markers CD11c, CD40, CD80, CD83, CD86 and HLA-DR. However, several differences were observed in the phenotype of

Discussion

Over the last decade, the convergence of several lines of research has bolstered interest in the use of dendritic cells (DCs) as the prime platform for immune therapy of cancers [22]. One of the critical developments in the field is related to gaining a better understanding of the central role of DCs for the initiation of anti-tumor immunity. Ample evidence now indicates the occurrence of an immune response against cancer involving DC recruitment and activation within the tumor tissue. Thus,

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (81272323 to C.Q.), the Natural Science Foundation of Jiangsu Province (BK2011244 to C.Q. and BK2012590 to Y.W.), the Key Project of Changzhou Health Bureau (ZD201201 to C.Q.), and the Science and Technology Development Project of Changzhou (CE20125019 to C.Q. and CE20125024 to K.Q.).

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    J.D. and T.F. contributed equally to this work.

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