β-Glucan Enhances Anti-Tumor Immune Responses By Regulating Suppressor And Effector Cells In Melanoma Mouse Model

Purpose:(1) Recent findings have demonstrated that β-glucans are capable of functioning as a potent adjuvant to stimulate innate and adaptive immune responses and are capable of stimulating dendritic cells (Dendritic cells, DCs). Dectin-1(DC-associated C-type lectin) receptors on the cell surface is commonly expressed in macrophages, neutrophil lineages, DC, and some T-cells. Glucocorticoid-induced tumor necrosis factor receptor family-related ligand (glucocorticoid-induced TNFR-related protein ligand, GITRL) is predominantly expressed by antigen presenting cells (APC) including dendritic cells (DCs) and macrophages. In this study, β-glucan-treated DC stimulated Th17responses in established Melanoma mice model. The β-glucan treatments affected tumor development, and also enhanced the influence of Th17response in vivo.(2) In this study, the use of Curdlan in stimulating MDSC cells was employed to observe changes in the number and associated effector molecules of MDSC cells. Curdlan inhibition of cellular immune function of MDSC was observed in established mouse melanoma model. Curdlan treatment delayed tumor development and the suppressive effects of MDSC in tumor-bearing mice.(3) Figs have been traditionally used for its medicinal benefits as laxative, cardiovascular, respiratory, antispasmodic and anti-inflammatory remedies. The study uses polysaccharide of figs (FCPS) to stimulate DCs to study their impact on DC maturation and the ability to stimulate the immune responses and study its role in signaling pathways. Method:(1.1) the study was carried out by flow cytometry (FCM) to detect the expression of cell surface BMDC dectin-1receptor;(1.2) β-glucan was used to stimulate BMDC cells, and employed the use of FCM detection of cell surface expression of GITRL;(1.3) in vitro stimulation of BMDC with β-glucan and effector T cells (Teff), regulatory T cells (Treg) or co-cultured cells with effector T cells were done using [3H] TdR incorporation assay to detect β-glucan inhibition of Treg function and effects on T cell proliferation after stimulation of DCs.(1.4) GITRL and Th17proportions and number of related cytokines and transcription factor expression were also assayed by qRT-PCR and ELISA;(1.5) We established Melanoma mouse model by observing (3-glucan treatment after tumor development using flow cytometry and qRT-PCR detection in vivo alongside GITRL, Th17cells and changes in related cytokines.(2.1) MDSC cells were stimulated in vitro using Curdlan. FCM was used to detect MDSC cells and detect arginase activity was detected by modified ELISA;(2.2) establishment of the mouse melanoma model was done. The progress of tumor development was observed and measured, and FCM was used to detect MDSC cells infiltration of tumor-bearing mice.(3.1) the use of figs polysaccharide (FCPS) to stimulate BMDCs was done. FCPS activation of downstream signaling molecules was detected by Western-blot;(3.2) FCPS was used to stimulate DCs to detect the expression of inflammatory factors of BMDC and cell surface molecules CD40, CD80, CD86, MHCII;(3.3) In vitro FCPS stimulation of BMDC and effector T cells (Teff) was done using [3H] TdR incorporation assay to detect FCPS DC stimulation of effector T cell proliferation, thereby reflecting DC immunostimulatory capacity. Results:(1.1) using FCM detection, BMDCs expressed Dectin-1receptor;(1.2) FCM results showed, β-glucan treatment on BMDC can upregulate the expression of cell surface GITRL;(1.3) in vitro proliferation assay and immunosuppressive Treg function tests showed that β-glucans stimulate DC surface increase mGITRL through GITR/GITRL system to promote effector T cell proliferation and impair Treg suppression;(1.4) by FCM, qRT-PCR and ELISA we showed that GITRL can increase the number of Th17cells and the expression of IL-17and RORyt;(1.5) the results of tumor models show that β-glucan can delay the progress of tumor development, increased in vivo expression of DC surface GITRL and also enhancing the response of Th17cells.(2.1) FCM results showed that, Curdlan can significantly reduce the number of MDSC cells and expression of arginase;(2.2) in mice tumor model, we found that, Curdlan can delay the progress of melanoma development and significantly reduced MDSC cell infiltration in mice.(3.1) as detected by Western-blot, FCPS stimulated BMDC cells were capable of activating Syk activation to bind Dectin-1;(3.2) FCM and qRT-PCR results showed that, FCPS can promote BMDC cell activation and maturation, and promote DC cell IL-12, IFN-y, IL-6and IL-23mRNA expression levels; (3.3) In vitro co-culture experiments show that, FCPS can enhance the immune stimulatory capacity of DC cells and also promote the proliferation of effector T cells.Conclusion:(1) In vivo and in vitro experiments showed that, β-glucan can upregulate the expression of DC surface GITRL expression via Dectin-1thereby enhancing the immune response of Th17cells, and thus delay the development of tumors.(2) In vivo and in vitro experiments revealed that, Curdlan treated MDSCs can reduce the number of MDSCs and arginase levels, and thus delay the development of tumors.(3) In vitro experiments displayed that, FCPS can promote DC activation and maturation of cells through Dectin-1/Syk interactions and enhance the ability of DC to stimulate the immune cells thereby contributing to the proliferation of T cells.