TRAF6 Regulates The Immunosuppressive Function Of MDSCs In Tumor-bearing Mice

Objective:In this study,we tried to explore the regulation of TRAF6 on the immunosuppressive function of MDSCs and investigate the potential molecular mechanism.We aimed to propose novel ideas for tumor immunotherapy targeting MDSCs.Methods:(1)MDSCs derived from the spleen of wild-type(WT)mice and tumor-bearing(TB)mice were isolated and purified by immunomagnetic beads method,and MDSCs from tumor tissue of TB mice were isolated by flow cytometry(FCM).The purity of MDSCs was determined by FCM.Then we used qRT-PCR and Western-blot to detect the expression of TRAF6 in MDSCs derived from different sources.(2)The proliferative ability of CD4~+T cells was detected by CFSE staining.In order to compare the immunosuppressive capacity between MDSCs derived from spleen of TB mice and MDSCs from tumor tissue of TB mice,the inhibitory effect of MDSCs on proliferation of CD4~+T cells was detected.The mRNA expression levels of MDSCs-related effector molecules Arg1 and iNOS were detected by qRT-PCR.(3)To investigate the regulation of TRAF6 on the immunosuppressive function of MDSCs,specific siRNA was used to knock down the expression of TRAF6 in MDSCs from tumor tissues of TB mice,and then co-cultured with CD4~+T cells.The proliferation ability of CD4~+T cells was detected by CFSE staining to reflect the immunosuppressive function of MDSCs.Moreover,we detected the activity of Arg1 by colorimetric assay and the content of NO by Griess method.(4)Co-IP was used to detect whether TRAF6 bound to STAT3 in tumor tissue-derived MDSCs.We knocked down TRAF6 in tumor tissue-derived MDSCs with specific siRNA,and detected the levels of the K63-linked polyubiquitination and phosphorylation of STAT3 in MDSCs by Co-IP and Western-blot.(5)To investigate the effects of TRAF6 on the immunosuppressive function of MDSCs in vivo,we divided wild type C57BL/6 mice into control group and siTRAF6-MDSCs group.The mice in the control group were injected subcutaneously with 1×10~6 MDSCs transfected with siNC and1×10~6 Lewis,and the mice in the siTRAF6-MDSCs group were injected subcutaneously with1×10~6 MDSCs transfected with siTRAF6 and 1×10~6 Lewis.We constantly monitored the tumor growth.The mice were sacrificed on the 28nd day after inoculation of tumor cells.Moreover,we used FCM to identify the proportion of Th1 and CTL from draining lymph nodes,spleen and tumor tissue of TB mice.Results:(1)The purity of MDSCs isolated from spleen and tumor tissue of mice was over 95%.Compared with MDSCs from spleen of wild-type(WT)mice or MDSCs from spleen of tumor-bearing(TB)mice,the expression of TRAF6 in MDSCs from tumor tissue of TB mice was significantly increased(p<0.001).(2)MDSCs derived from spleen or tumor tissue of TB mice were co-cultured with CD4~+T cells of WT mice,respectively.Compared with the MDSCs derived from spleen of TB mice,the MDSCs from tumor tissue of TB mice had a stronger inhibitory effect on the proliferation of CD4~+T cells.Meanwhile,the expression levels of Arg1 and iNOS of tumor tissue-derived MDSCs were higher than that from MDSCs derived from spleen of TB mice.The results showed that the immunosuppressive function of MDSCs derived from tumor tissue was stronger than that of spleen-derived MDSCs of TB mice.(3)The tumor tissue-derived MDSCs transfected with siTRAF6 were co-cultured with CD4~+T cells,the proliferation of CD4~+T cells was higher than that of the transfected control group,which indicated that the inhibitory effect of MDSCs on proliferation of CD4~+T cells was significantly attenuated after knockdown of TRAF6 in MDSCs.After knockdown of TRAF6,the activity of Arg1 in MDSCs was decreased(p<0.05),while the content of NO in MDSCs did not change significantly.(4)TRAF6 bound to STAT3 in MDSCs from tumor tissue of TB mice.After knockdown of TRAF6 in tumor tissue-derived MDSCs,K63-linked polyubiquitination of STAT3 was significantly down-regulated.Silencing of TRAF6 remarkably decreased the levels of phosphorylated STAT3 in MDSCs from tumor tissue of TB mice.(5)Compared with control group,the tumor growth of mice in the siTRAF6-MDSCs group was evidently delayed,and the tumor volume and weight were significantly smaller(p<0.05).The proportion of Th1 cells in the draining lymph nodes of the control group was 3.63±0.23%,and the proportion of Th1 cells in the draining lymph nodes of the siTRAF6-MDSCs group was5.82±0.69%.Compared with control group,the proportion of Th1 cells in the draining lymph nodes of the siTRAF6-MDSCs group was up-regulated(p<0.05).The proportion of CTL in the tumor tissue of the control group was 2.47±0.35%,and the proportion of CTL in the tumor tissue of siTRAF6-MDSCs group was 3.93±0.34%.The proportion of CTL in the tumor tissue of the siTRAF6-MDSCs group was significantly increased as compared with the control group(p<0.05).Conclusions:The expression of TRAF6 in MDSCs derived from tumor tissue of tumor-bearing mice was significantly increased.TRAF6 promoted the immunosuppressive function of MDSCs by mediating K63-linked polyubiquitination of STAT3.