| Gastric cancer is one of the most common cancer in China, which seriously affects the patients’ quality of life. The 5-year relative survival rate of gastric cancer has not improved during the past 35 years, and remains stubbornly at 20-30%. Unfortunately, because gastric cancer causes few symptoms in its early development and lack of both early detection methods and effective medical treatment for advanced stages of the disease. Moreover, even after having a gastric tumor surgically removed, survival of patients with advanced gastric cancer remains low. Thus, there is an urgent need to develop new therapies to control gastric cancer. Immunotherapy is a hot spot in the present study, which will bring new hopes for the treatment of tumors.Recently, some co-stimulatory molecules were found in a variety of abnormal expression in tumor. CD40/CD40 L interaction regulates many features of cellular and humoral immunity such as T cell-mediated activation of DCs, T cell priming, proliferation of B cells, immunoglobulin synthesis, isotype switching, and germinal center formation. It is well known that CD40 was expressed on the lymphatic system of malignant cells, including almost all the B cell malignancies. It is also expressed on solid tumors such as bladder, renal, pancreatic, nasopharynx and breast carcinomas, as well as in prostate, colon, and lung cancers. Many clinical studies showed that the expression of CD40 is closely associated with tumor progression and metastasis. Compelling evidence indicates that costimulatory molecules not only provide crucial positive signals to stimulate and support T-cell activation, but can also offer negative signals that control and suppress T-cell responses, which Participate in the tumor immune escape. Among these suppressor cells, T regulatory cells（Treg） and myeloid-derived suppressor cells（MDSC） have been shown to increase significantly in hosts with advanced malignancies. Tregs play an essential role in the maintenance of self-tolerance and may impede antitumor immune responses. MDSCs contribute to tumor- associated immune dysfunctions through a myriad of mechanisms, including production of nitric oxide, arginine depletion, and production of reactive oxygen species. CD40 is essential not only for MDSC-mediated immune suppression but also for tumor-specific Treg expansion. Our study discusses the effect of CD40 on regulating the biological functions of MDSC and relevant mechanisms, which will provide a new strategy to ablate tumoral immune suppression and thereby heighten responses to immunotherapy.PartⅠCD40 expression on MDSC in a mouse model of gastric cancer and Its relationship with tumor progressionObjective: To detect the expression levels of CD40 on MDSC that is derived from spleen and tumor. Then, we analyzed the correlation of CD40 expression with the gastric cancer growth. Methods: We will use C57BL/6j mice challenged i.d. with mouse gastric cancer cell line MFC. Every 5 days, the transplanted tumor and spleen will be isolated from these mice. The frequency of MDSC and CD40 will be determined with flow cytometry. The correlation was analyzed with statistic software. Results: Compared with the normal mice MDSC derived from spleen, the expression of CD40 on spleen MDSC derived from tumor-burdened mice CD40 was higher（P=0.0024）. The proportion of MDSC gradually increased during the tumor growth. However, the expression levels of CD40 on MDSC were gradually decreased. MDSC absolute value increases and thereby the expression of the surface molecule CD40 is relatively increased. We also found that the tumor growth of WT mice around 5- 6 days after MFC injection, and CD40-/- mice were about 7 days. The tumor of CD40-/- mice had a lower growth speed and smaller size than WT mice. Taken together, our data indicated that CD40 was an important marker of tumor-associated MDSC, and correlation with tumor progression.Part Ⅱ The effect of CD40 signal on regulating MDSC biological functionObjective: To investigate the role of CD40 in MDSC apoptosis, inhibition of T cell proliferation and induction of Treg. Methods: T cells and MDSCs were isolated by magnetic bead from mice. After MDSCs were stimulated with IL-4、IL-6、IL-10、TNF-α、GM-CSF、IFN-γ、LPS、PEG2 or MFC for 24 h、48h、72h,expression of CD40（flow cytometry） will be measured. MDSCs were inoculated in the presence or absence of anti-CD40 for 24 h. The apoptosis rate of MDSCs were determined with flow cytometry. Graded numbers of MDSC and CFSE-labled T cells will be stimulated with CD3/CD28 m Abs for three days. Proliferation（CFSE dilution）, and expression of IL-4,IL-17,IFN-γ and Foxp3（flow cytometry） will be measured. Results: Here we show that, the expression of CD40 on MDSC was upregulated after LPS stimulated, especially after 48 h stimulated. CD40 can significantly inhibit apoptosis of MDSC in gatric cancer microenvironment. CD40 promote MDSC-mediated inhibition of T cell proliferation. CD40 involved in the MDSC-mediated induction of Treg-differentiation and promote Foxp3 expression. Taken together, we demonstrated that CD40 can promote the immune suppression of MDSC.PartⅢ Differential Expression Gene of CD40-/-and CD40low Gastric Tumor Bearing Mouse Spleen Cell Screened by Genechip TechniqueObjective: To screen differential expression gene of CD40-/-and CD40 iow gastric tumor bearing mouse spleen cell by Genechip technique. Analysis the possible molecular mechanisms of CD40-regulated MDSC. Methods: After Total RNA was extracted using Trizol. Genechip testing was commissioned by Capital Bio Corporation in Beijing. Results: The results show that there are differences between the two groups of gene expression profiles. 2890 genes were differentially expressed in gene profile. 519 genes were up-regulated and 1419 were down-regulated. The expression trend of six genes for further verification was consistent with array（3 up-regulated and 3 down-regulated）. Conclusions: CD40 is an important marker of tumor-associated MDSC and can regulate the MDSC-mediated immune suppression, which may be closely related with the tumor growth and development. |
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