The Study Of B7-H3 On The Function Of γδT Cells In Colon Cancer And Its Mechanism

The 2021 Global Cancer Statistics Report shows that colon cancer has become the third most common cancer in the world and ranks second in tumor-related deaths.In China,the incidence and mortality of colon cancer have exhibited sustained growth over recent decades.Although improvements in screening programs and treatment patterns have been made,the five-year survival rate of colon cancer patients with distant metastases is only 10%.Therefore,there is an urgent need for new treatment options for the treatment of colon cancer.Tumor immunotherapy,including active immunotherapy,passive immunotherapy and immune checkpoint blocking,has become a new cancer treatment research direction and received significant attention.γδ T cells constitute approximately 5%of all circulating T cells and play a key role in innate and adaptive immune surveillance.Vγ9Vδ2T cells are the main subgroup(50-90%)of human peripheral blood γδT cells.They have a high anti-tumor ability and kill various tumor cells in vitro,but shown limited success in clinical trials of solid tumors.Therefore,it is necessary to study how to improve the killing effect of γδT cells on solid tumors.As an important member of the B7 superfamily,B7-H3 has been shown to regulate the biological functions of immune cells,including macrophages,NK cells,CD4+T cells and CD8+T cells,and play a dual role in regulating innate and adaptive immune responses.However,there is no report on the influence and potential mechanism of B7-H3 on the biological function of γδT cells in colon cancer.Therefore,we take colon cancer as the research object to explore whether and how B7-H3 regulates the properties of γδT cells and its anti-tumor against on colon cancer,in order to provide a new treatment idea and plan for future colon cancer immunotherapy.Part Ⅰ B7-H3 and γδT cells affect the development of colon cancerObject:To analyze the expression of γδT cells in the microenvironment of colon cancer,to screen the most suitable immune checkpoint molecules on γδT cells,and to study the correlation between γδT cells and B7-H3 in colon cancer.Methods:Taking colon cancer patients as the research object,the proportion of γδT cells in the peripheral blood and tumor tissues were detected;flow cytometry was used to detect the proportion of various immune checkpoint molecular subtypes(B7-H3+γδT cells,PD-1+ γδT cells,PD-L1+γδT cells,Trem-1+γδT cells,Tim-3+ γδT cells)in circulating γδT cells in colon cancer patients,and screen out the molecular subtype with the highest cell proportion;re-test the proportion of molecular subtype in γδT cells in the peripheral blood and tumor tissues of colon cancer patients;collect clinical data to analyze the clinical significance of γδT cells and the selected molecular subtype in colon cancer.Results:(1)Compared with healthy volunteers,the proportion of γδT cells in the peripheral blood of colon cancer patients was significantly reduced;compared with normal tissues adjacent to the cancer,the proportion of γδT cells infiltration in the tumor tissues of patients was also significantly decreased;(2)The proportion of B7-H3+γδT cells in circulating γδ T cells in colon cancer patients was the highest;(3)Compared with healthy volunteers or normal tissues adjacent to cancer,the proportion of B7-H3+γδT cells in circulating γδ T cells and tumor-infiltrating γδ T cells was significantly increased in colon cancer patients;(4)γδT cells and B7-H3+γδT cells are closely related to the distant metastasis and TNM staging of colon cancer.Conclusion:This part of the study found that the proportion of γδT cells was significantly decreased in colon cancer,but the proportion of B7-H3+ γδT cells was increased,indicated that B7-H3 may be involved in the regulation of γδT cells in colon cancer.Part Ⅱ The effect of B7-H3 on the activity and function of γδT cells in colon cancerObject:To analyze the effect of B7-H3 on the biological function of γδT cells in colon cancer.Methods:Using the B7-H3 antibody MIH35 to block the function of B7-H3 on the cell membrane of Vδ2T cells or transfecting Vδ2T cells with B7-H3 small interfering RNA(siRNA)to reduce the expression of B7-H3 on their surface and using the B7-H3 activator 4H7 to stimulate Vδ2T cells,flow cytometry was used to analyze the influence of B7-H3 on the proliferation,apoptosis,activation and differentiation of Vδ2T cells;to detect the expression of γδT cell functional subtypes in peripheral blood and tumor tissues of colon cancer,and to analyze the correlation between IFN-γ+ γδT cells and B7-H3+ γδT cells;use CCK-8 method and cell clustering function to detect the effect of B7-H3 on Vδ2T killing tumor cells(HCT116,RKO and SW480 cells).Results:(1)Reducing the expression of B7-H3 or blocking the function of B7-H3 significantly promoted the proliferation,the expression of activation markers(CD25 and CD69)and the differentiation of IFN-γ+γδT cells of Vδ2T cells,and inhibited the apoptosis of Vδ2T cells;while stimulating the expression of B7-H3,the opposite results were observed;(2)Compared with the peripheral blood in healthy volunteers or normal tissues adjacent to the cancer in colon cancer patients,the proportion of IFN-γ+γδT cells in circulating γδT cells and tumor infiltrating γδT cells of colon cancer patients was significantly reduced,there is an negatively correlation between the expression of B7-H3 and IFN-γ in γδT cells;(3)B7H3 inhibited the killing function of γδT cells against on colon cancer cell lines HCT116,RKO or SW480.Conclusion:B7-H3 inhibited the proliferation,activation,the differentiation of IFN-γ+γδT cells and anti-tumor function of γδT cells in colon cancer,and promoted the apoptosis of γδT cells.Part Ⅲ The mechanism of B7-H3 regulating the anti-tumor function of γδT cells in colon cancerObject:To explore and verify the relevant mechanisms of B7-H3 regulating the antitumor function of γδT cells,and to analyze its actual therapeutic effect in vivo.Methods:Taking tumor cells HCT116 and RKO as the research objects,IFN-γantibody was added to Vδ2T cells transfected with B7-H3 siRNA,and the cytotoxicity of Vδ2T cells was observed by CCK-8 method and cell clustering ability.Human recombinant IFN-y factor was added to Vδ2T cells pre-stimulated with 4H7,and the cytotoxicity of Vδ2T cells was observed again by CCK-8 method and cell clustering ability.RT-qPCR method was used to screen the key genes that can regulate the production of IFN-γ in Vδ2T cells;Vδ2T cells co-transfected with the small interfering RNA about transcription factor and B7H3 siRNA were used to observe the expression of B7-H3,IFN-y and the key gene at the protein level by flow cytometry and Western blot.ELISA was used to evaluate the influence of B7-H3 on the secretion of perforin and granzyme B in Vδ2T cells;adding perforin inhibitor CMA and granzyme B inhibitor BCL-2 to Vδ2T cells transfected with B7-H3 siRNA,or adding human recombinant granzyme B factor to Vδ2T cells pre-stimulated by 4H7,observed the effect of anti-tumor function of Vδ2T cells.While SCID mice were used to construct humanized colon cancer tumor-bearing models,and sequentially injected B7H3 blocking antibody MIH35 and Vδ2T cells,we recorded the tumor growth curve and tumor weight,and analyzed the expression of IFN-γ and granzyme B in Vδ2T cells in peripheral blood and tumor tissues by flow cytometry.Results:(1)IFN-y antibody reversed the increased cytotoxicity of Vδ2T cells induced by B7-H3 siRNA against on colon cancer cells,while recombinant human IFN-γ factor offset the inhibition of 4H7 stimulation on the cytotoxicity of Vδ2T cells against on colon cancer cells.(2)B7-H3 inhibited the production of IFN-y in Vδ2T cells by regulating the expression of the transcription factor T-bet.(3)B7-H3 inhibited the secretion of perforin and granzyme B in Vδ2T cells.(4)CMA or BCL-2 treatment reversed the increase in the killing function of Vδ2T cells induced by B7-H3 siRNA,while the addition of recombinant human granzyme B significantly enhanced the decreased anti-tumor ability of Vδ2T cells after 4H7 stimulation.(5)Animal experiments showed that MIH35 and γδT cells combined treatment has the best therapeutic effect,and found B7-H3 can inhibit the secretion of IFN-y and granzyme B from γδT cell in mice.Conclusion:B7-H3 inhibited the secretion of IFN-y from Vδ2T cells by downregulating the expression of T-bet,thereby inhibited the cytotoxicity of Vδ2T cells.It can also inhibit the anti-tumor function of Vδ2T cells by inhibiting the expression of perforin and granzyme B.In summary,we found that the proportion of γδT cells in the peripheral blood and tissues in colon cancer patients was significantly decreased,while the proportion of B7-H3+γδT cells in γδT cells was significantly increased,and both of them are closely related to the distant metastasis of colon cancer.B7-H3 inhibited the proliferation,activation,the differentiation of IFN-γ+γδT subtypes and cytotoxicity of γδT cells,and promoted the apoptosis of γδT cells.The combined treatment of B7-H3 inhibitor MIH35 and γδT cells significantly slowed the progression of colon cancer in SCID mice.B7-H3 inhibited the antitumor ability of γδ T cells on colon cancer by regulating the secretion of IFN-γ and perforin/granzyme B.This indicated that B7-H3+γδT cells can be used as a potential marker of colon cancer prognosis and immune efficacy,and Vδ2T cells combined with inhibiting or blocking B7-H3 represents a potential immunotherapeutic approach for colon cancer.