Regulatory Mechanism Of OX40 Signal Induce Th9 Cell Differentiation And Tumor Immunization In Brest Cancer

Breast cancer is not only the most common female malignant tumor,but also the leading cause of cancer death among women.Although traditional surgical treatment,radiotherapy,chemotherapy,and endocrine therapy have been widely used in clinical,but there are still recurrences and metastases in breast cancer.Recently,with the deepening understanding of tumor immune escape mechanisms in tumor occurrence and development,and with the profound discovery of the effector immune cells,cytokines and surface molecules in immune surveillance and immune killing system in internal environment,the discussion of breast cancer tumor immunotherapy has become a frontier topic in this field.In our previous study,we found that the percentage of Th9 cells and the expression of OX40 molecules on the surface of CD4+T cells in peripheral blood of invasive breast cancer patients were lower than those of benign fibroadenoma patients and healthy volunteers.Compared to other CD4+T cell subsets,Th9 cells have specific tumor suppressive effects;the OX40 molecule and its signal which belong to the Tumor Necrosis Factor Receptor(TNFR)superfamily could promote the proliferation and differentiation of activated T cells and is an important costimulatory signal for long-term survival of memory T cells.In this contribution,we focus on the positive molecule OX40 which promotes the immune response and Th9 cells differentiation,to explore OX40 molecule and its signal to induce Th9 cell differentiation and the mechanism in anti-breast cancer process by immunology,cell biology,animal models histopathology methods.Part Ⅰ,To study the differentiation of Th9 cells,the secretion of IL-9,the expression of PU.1 and the killing ability on breast cancer cell line 4T1 induced by OX40 signal by flow cytometry,Real-time PCR,T cell and tumor cells co-incubation experiments.The results show that:(1)42h,25ng/ml rhOX40L stimulated OX40 signal could induce high levels of Th9 cells,and the differences were statistically significant(P<0.0001;P<0.0001);(2)Under the optimal induction conditions,the levels of IL-9 and PU.1 protein expression were significantly increased(P<0.0001;P<0.0001);effectively increase the mRNA levels of IL-9 and PU.1(P<0.0001;P=0.019);(3)CD4+T cell killing ability experiment showed that OX40 signal could significantly increase the expression of perforin,and the difference was statistically significant(P<0.0001);Direct and indirect co-culture experiments of Th9 cells and 4T1 breast cancer showed that the activation of OX40 signal can significantly inhibit tumor proliferation(P=0.0454;P=0.0274)and induce early apoptosis of tumor cells(P=0.0001).Part Ⅱ,To study and analyze the expression of related signaling molecules and transcription factors after OX40 signal excitation and blockade,and discuss the possible regulatory mechanisms and signaling pathways of OX40 signaling on Th9 cells differentiation and the effects on immune response by Real time PCR technology and TRAF6 gene knockout.The results show that:(1)Real-time PCR results showed that stimulation of OX40 signal could up-regulate the expression of TRAF6 mRNA in 12h(P=0.0427),24h up-regulates PU.1 mRNA(P=0.0019)and BATF mRNA(P=0.023);(2)TRAF6 gene knockout could lead to the inhibition of Th9 cell differentiation and the decrease of the transcription factor PU.1 protein level(P=0.0011,P=0.0163);(3)The excitation of OX40 signal could significantly promote the increase of TEM ratio(P=0.0012),but the promotion of TCM was not obvious(P=0.1488),and both can promote the secretion of higher levels of IL-9 in TEM and TCM(P<0.0001,P<0.0001);(4)The precentage of TEM could be affect by TRAF6 gene knockout(P=0.0082).Part Ⅲ,We used the 4T1 in situ breast cancer animal models to explore the effect of OX40 signal-stimulated CD4+T cells on adoptive immunotherapy;Whole Genome Sequencing was used to analyze the changes of genes related to Th9 cell differentiation and function in the OX40 signal-stimulated group.The results show that:(1)The tumor growth of tumor-bearing mice in OX40 excitation treatment group were inhibited(P=0.0370;P=0.0382);(2)The secretion of IL-9,IL-21,IFN-y and IL-4 in the OX40-stimulated treatment group were up-regulated(P=0.0343,P=0.0164,P=0.0048,P=0.0006),while the secretion of IL-17 were down-regulated(P=0.0427);(3)The tumor-bearing mice in the OX40-stimulated treatment group had higher levels of CD4+T cells than ThO treatment group and Th9 treatment group(P=0.0218),and their surface negative costimulatory molecule PD-1 expression and Tregs transcription factor Foxp3 were all down-regulated(P=0.0057,P=0.0052);(4)The percentage of NK cells and TEM were all increased(P=0.0179,P=0.0227);while the percentage of B cells were down-regulated(P=0.0048);(5)Transcriptome sequencing results show that OX40 signal could up-regulate Th9-related cytokines IL-9,IL-21,etc,as well as perforin and other functional molecules.In summary,the stimulation of OX40 signal can induce Th9 cell differentiation,by promoting the secretion of cytokine IL-9 and up-regulating the expression of transcription factor PU.1 at the protein and gene levels;at the same time,the excitation of OX40 signal can also enhance the secretion of perforin in Th9 cells,directly and indirectly inhibit the proliferation of breast cancer cell line 4T1 and induce its early apoptosis;in addition,OX40 signal can significantly promote the increase of TEM ratio and can also significantly promote the high level of IL-9 expression on TEM and TCM;TRAF6 gene knockout experiments confirmed that the blocking of OX40 signaling could reduce IL-9 secretion and down-regulate the expression of the transcription factor PU.1;TRAF6 gene knockout experiments also confirmed the existence of the OX40-TRAF6-PU.1 pathway during Th9 cell differentiation and the TEM formation was affected;Whole-genome sequencing results confirmed that the activation of OX40 signal can up-regulate Th9 cell-related cytokines and function-related molecules.In addition,in vivo experiments confirmed that non-tumor specific adoptive immunotherapy stimulated by OX40 signal could inhibit tumor growth and secrete higher levels of IL-9,IL-21 and other Th9 cell-related cytokines and down-regulate the expression of PD-1 and Tregs transcription factor Foxp3.The above-mentioned in vitro and in vivo experiments provide new ideas for the molecular biology transformation of Th9 cells and OX40 signal-mediated adoptive immunotherapy of Th9 cells in breast cancer treatment.