Antitumor Effects And Mechanisms Of MUC1 Peptide-Loaded TRAF6-Overexpressing BMDCs

Dendritic cells(DCs)are professional antigen-presenting cells(APCs)that play a key role in antitumor immune responses.Considering that many factors in the tumor microenvironment(TME)inhibit developing DCs precursor cells and prevent their differentiation into mature DCs,they cannot elicit antitumor immune responses.Therefore,cancer is usually treated clinically by isolating or generating and expanding autologous DCs in vitro,then manipulating them and reinfusing them into the patient.The maturity of DCs is the main factor that determines the success or failure of DCs vaccines.But the insufficient maturity of DCs is one of the main problems faced by DCs vaccines.Although there are many ways to stimulate the activation and maturation of DCs,it is still insufficient to stimulate the full maturation of DCs.Therefore,more effective methods are highly needed to promote the maturation of DCs and overcome the immunosuppressive microenvironment.Tumor necrosis factor(TNF)receptor-associated factor 6(TRAF6),an E3ubiquitin ligase,is a signal transducer shared by the interleukin 1 receptor(IL-1R)/Toll-like receptor(TLR)family and the TNFR superfamily.TRAF6-mediated signaling has been shown to be critical for the development,homeostasis,and activation of DCs.TRAF6-deficient DCs show marked defects in surface molecule upregulation and severe Th1 cytokine deficiency.Therefore,we intend to use TRAF6 as an entry point to overexpress TRAF6 in BMDCs to improve the maturity and therapeutic effect of DCs,and to explore the feasibility and antitumor mechanism of TRAF6 as a DCs vaccine.The effect of TRAF6 on the maturation of BMDCsTo study the relationship between TRAF6 and BMDCs maturation,we first stimulated BMDCs with TLR2 agonist Pam3Csk4,TLR4 agonist LPS and TLR9agonist Cp G 1826 alone or in combination.Further,we knocked out TRAF6 in BMDCs by CRISPR/CAS9 technology to observe the maturation of BMDCs.1.TLR agonist-induced elevation of TRAF6 correlates with maturation of BMDCsThe results showed that the expression level of TRAF6,the expression of surface CD40,CD80,CD86,MHC I and MHC II molecules,and the secretion level of IL-12in BMDCs stimulated by TLR agonist were significantly increased in each group.The above results suggest that the expression of TRAF6 is related to the maturity of BMDCs,and the maturity of BMDCs induced by LPS is higher,which can be used as a positive control for subsequent experiments.2.TRAF6 knockout reduces the maturity of BMDCsWe first knocked out TRAF6 in BMDCs by using CRISPR/CAS9 technology.The surface molecule expression and IL-12 secretion level of BMDCs was detected.The results showed that compared with NC group,TRAF6 knockout BMDCs significantly downregulated the expressions of CD40,CD80,CD86 and MHC class I molecules on the surface of BMDCs.Compared with LPS group,LPS-stimulated TRAF6 knockout BMDCs significantly downregulated the expression of CD40,CD80,CD86 and MHC class I molecules and the secretion level of IL-12.After co-culturing BMDCs with CD4~+T cells,compared with the LPS group,LPS-stimulated TRAF6 knockout BMDCs significantly reduced the proliferation level of CD4~+T cells and the secretion level of IFN-γ.There was no significant difference observed on the secretion level of IL-4among the groups.The above results suggest that LPS can induce the maturation of BMDCs.Knockdown of TRAF6 in BMDCs can inhibit the LPS-induced maturation of BMDCs.LPS-stimulated BMDCs can promote CD4~+T cell activation to Th1 type,but this activation is inhibited in TRAF6-knockout BMDCs.Construction and maturation of TRAF6-overexpressing BMDCsTo use TRAF6-overexpressing BMDCs as DCs vaccine for antitumor therapy,we first constructed TRAF6-overexpressing BMDCs to explore the changes in their maturation in vitro.1.Construction of TRAF6-overexpressing BMDCsA lentiviral vector overexpressing TRAF6 was constructed,packaged into a lentiviral transfection solution,and transfected into BMDCs.The constructed TRAF6-overexpressing BMDCs were analyzed by fluorescence microscopy,q RT-PCR and Western Blotting.The results showed that the transfection efficiency of TRAF6-overexpressing lentivirus was over 90%.It could significantly overexpress TRAF6 and activate the downstream JNK protein expression levels and phosphorylation levels of JNK,ERK and IκB.The above results suggest that we have successfully constructed TRAF6-overexpressing BMDCs.2.TRAF6 overexpression increases the maturity of BMDCsWe first detected the expression of molecules on the surface of BMDCs and the secretion level of IL-12.It was found that compared with the NC group,the TRAF6-overexpressing BMDCs significantly increased the expressions of CD40,CD80,CD86and MHC I molecules and the expression of IL-12.Compared with the LPS group,the expressions of CD40,CD80,CD86 and MHC I molecules and the secretion level of IL-12 were also significantly increased in TRAF6-overexpressed BMDCs stimulated by LPS group.After co-culturing BMDCs with CD4~+T cells,compared with the NC group,the cell proliferation level and IFN-γsecretion level of the remaining groups were significantly increased.Compared with the LPS group,the LPS-stimulated TRAF6-overexpressing BMDCs also significantly increased cell proliferation and IFN-γsecretion levels.There was no significant difference observed on the secretion level of IL-4 among the groups.The above results suggest that overexpression of TRAF6 in BMDCs can further promote the maturation of LPS-induced BMDCs.It can further promote the activation of CD4~+T cells to Th1-type cells induced by LPS-stimulated BMDCs.The antitumor effect and mechanism of TRAF6-overexpressing BMDCs loaded with MUC1 polypeptide in vivoTo further study the antitumor effect of TRAF6-overexpressed BMDCs as a vaccine in vivo,we used MUC1 polypeptide as the loading antigen of BMDCs to prepare a TRAF6-overexpressing BMDCs vaccine loaded with MUC1 peptide.B16melanoma cells expressing human MUC1(B16-MUC1)was used as a tumor-bearing mouse model to study its antitumor effect and mechanism.1.Preparation of MUC1 peptide-loaded TRAF6-overexpressing BMDCs vaccineBMDCs were transfected with TRAF6-overexpressing lentiviral transfection solution for 36 h,and MUC1 peptide was added to BMDCs for 12 h,so that BMDCs were loaded with MUC1 peptide.2.MUC1 peptide-loaded TRAF6-overexpressing BMDCs can inhibit the growth of B16-MUC1 melanoma in miceWe firstly injected B16-MUC1 cells into mice subcutaneously to establish a tumor-bearing mouse model.On the 7th and 14th days after the tumor-bearing,immunotherapy of BMDCs was performed twice.We measured and calculated the tumor volume.We found that the tumor size of TRAF6~+DC group and TRAF6~+DC+MUC1 group was significantly smaller than that of Control DC group and Control DC+MUC1 Group.In addition,the tumor size of TRAF6~+DC+MUC1 group was also significantly higher than that of Control DC+MUC1 group.Among them,tumor inhibition rate of TRAF6~+DC+MUC1 group was the highest(83.79%).The above results suggest that MUC1 peptide-loaded TRAF6-overexpressing BMDCs can effectively inhibit tumor growth.3.In tumor-bearing mice,injected BMDCs can be distributed to the spleen,lymph nodes and tumorsTo analyze the distribution of injected BMDCs in tumor-bearing mice,the proportion of GFP~+cells and CD11c~+MHC II~+DCs in the spleen,lymph nodes and tumor microenvironment of mice was detected by flow cytometry.The proportion of GFP~+cells and CD11c~+MHC II~+DCs in the spleen,lymph nodes and tumor microenvironment of mice in each group was significantly increased.The above results suggest that injected BMDCs can migrate into the spleen,lymph nodes and tumor microenvironment.4.The effect of MUC1 peptide-loaded TRAF6-overexpressing BMDCs on T cell responses in tumor-bearing mice.(1)The proportion of Th1 cells and Tc1 cells in the spleen and lymph nodes of mice was detected by flow cytometry.The results showed that the proportion of Th1cells and Tc1 cells in the TRAF6~+DC group was significantly higher than that in the Control DC group,and the Th1 cells and Tc1 cells in the TRAF6~+DC+MUC1 group.The proportion of cells was significantly higher than that in the Control DC+MUC1group.(2)We coated with MUC1 peptide and detected MUC1-specific Ig G in the serum of tumor-bearing mice by ELISA.The results showed that compared with the Control DC group,the antibody levels of Ig G,Ig G1 and Ig G2c and the ratio of Ig G2c/Ig G1 in the TRAF6~+DC group were significantly increased.Similarly,compared with the Control DC+MUC1 group,the antibody levels of Ig G,Ig G1 and Ig G2c and the Ig G2c/Ig G1 ratio were also significantly increased in the TRAF6~+DC+MUC1 group.It is indicated that MUC1 peptide-loaded TRAF6-overexpressing BMDCs can induce MUC1-specific humoral and Th1-type immune responses.(3)After culturing splenocytes with MUC1 peptide for 5 days,the proliferation level of MUC1-specific splenocytes was detected by WST-1.The results showed that the proliferation level of spleen cells in TRAF6~+DC group was significantly higher than that in Control DC group,and the proliferation level of splenocytes in TRAF6~+DC+MUC1 group was significantly higher than that in Control DC+MUC1 group.The MUC1-specific secretion levels of IFN-γand IL-4 were detected by ELISA.The results showed that compared with the Control DC group,the TRAF6~+DC group significantly increased the secretion level of IFN-γand significantly decreased the secretion level of IL-4.Similarly,compared with the Control DC+MUC1 group,the TRAF6~+DC+MUC1 group also significantly increased the secretion level of IFN-γand significantly decreased the secretion level of IL-4.(4)We used spleen cells after culturing with MUC1 polypeptide for 5 days as effector cells and B16-MUC1 cells as target cells.The MUC1-specific CTL killing level was detected by RTCA.The results showed that the killing level of CTL in the TRAF6~+DC group was significantly higher than that in the Control DC group.The killing level of CTL in the TRAF6~+DC+MUC1 group was also significantly higher than that in the Control DC+MUC1 group.(5)The proportion of Tregs in mouse spleen,lymph nodes,and tumor microenvironment was examined by flow cytometry.It was found that the proportion of Tregs in the spleen and lymph nodes did not differ much between the groups.In the tumor microenvironment,the proportion of Tregs in the TRAF6~+DC group was significantly lower than that in the Control DC group,and the proportion of Tregs in the TRAF6~+DC+MUC1 group was significantly lower than that in the Control DC+MUC1 group.The above results suggest that TRAF6-overexpressing BMDCs loaded with MUC1 peptide can not only induce MUC1-specific humoral immune responses in mice,but also induce a strong Th1-type immune response and CTL killing activity,which can significantly reduce tumor-bearing mice.In particular,the proportion of Tregs in the tumor microenvironment.5.The effect of MUC1 peptide-loaded TRAF6-overexpressing BMDCs on the survival of tumor-bearing miceTo further evaluate the effect of MUC1 peptide-loaded TRAF6-overexpressing BMDCs on the survival of tumor-bearing mice,a total of 60 days were observed after melanoma transplantation,and a graph was drawn according to the survival of the mice.Mice in the TRAF6~+DC+MUC1 group had the highest survival rate of 40%.Compared with the Control DC group and the Control DC+MUC1 group,there was a statistical difference.This was followed by the TRAF6~+DC group with a survival rate of 20%.All the mice in the other groups died before the end of the 60-day observation.The above results suggest that MUC1 peptide-loaded TRAF6-overexpressing BMDCs can effectively prolong the survival period of mice.Conclusions:The results of this study indicate that TRAF6 plays a key role in the maturation of BMDCs,and overexpression of TRAF6 can further improve the maturation of DCs based on the existing methods of stimulating the maturation of DCs.The antitumor effect can inhibit tumor growth in tumor-bearing mice by increasing the immune response of T cells,thereby prolonging the survival period of mice.In this study,TRAF6-overexpressed BMDCs were used for the first time in tumor-bearing mice to conduct antitumor research,suggesting the feasibility of TRAF6-overexpressing BMDCs in the treatment of tumors,and indicating the application prospects of TRAF6-modified BMDCs as vaccines.This study set up a theoretical and experimental foundations for the feasibility of future clinical application.