| Malignant tumors are one of the major diseases that threaten human life and health in modern times.Strengthening prevention and treatment of malignant tumors has become a global health strategy problem in today's world.The traditional malignant tumor treatment methods include surgery,radiotherapy,and chemotherapy.In recent years,the immunotherapy of tumors has become the main direction for more and more experts and scholars to study.It is different from traditional treatment methods and has a good application prospect.Dendritic cells(DCs)are the main antigen-presenting cells(APCs)of the human body and play a very important role in inducing the body's anti-tumor immunity.DCs exert anti-tumor effects by inducing effector T cells in vivo.In vivo anti-tumor immunity includes cellular immunity and humoral immunity,and cellular immunity plays a crucial role in mediating the anti-tumor effects of DC vaccines.In vivo anti-tumor effect T cells mainly include CD4+ helper T cells and CD8+ cytotoxic T cells.Similar to Th subsets,naive CD8+ T cells can also differentiate into Tcl,Tc2,Tcl7,and regulatory CD8+ T cells under different polarization conditions,as well as a newly discovered subpopulation of Tc9 cells.Studies have shown that IL-9-secreting Tc9 cells have strong and long-lasting tumor therapeutic activity in vivo,and Tc9 anti-tumor activity is significantly better than Tc1.IL-33 is a multifunctional cytokine.Previous studies have confirmed that IL-33 promotes CD8+ T,ILC2,Th2,and Th1 cell responses to be beneficial for anti-tumor immunity,and IL-33 can also promote the production of Treg,which is not good for anti-tumor immunity.Therefore,in this study,we will further investigate the role of IL-33 in inducing Tc9 and anti-tumor immunity induced by BMDC.Research ismainly divided into the following three parts:Part 1 Induction of Tc9 by IL-33 combined with BMDCs in vitroObjective: To investigate the effect of IL-33 combined with BMDCs on the differentiation,survival and proliferation of Tc9 cells in vitro,and to clarify the induction of IL-33 combined with BMDCs on Tc9 cellsMethods:(1)Under conditions of polarization with Tc0 and Tc9,with or without addition of IL-33,naive CD8+ T cells were co-cultured with BMDCs.By measuring the expression levels of IL9 m RNA,Tc9-related transcription factors Spi1 and Irf4,the effect of IL-33 addition on Tc9 cells was examined.The level of IFNr m RNA secreted by Tc1 cells and the expression level of the related transcription factor Tbx21 were also examined.(2)Under the condition of polarization with Tc0 and Tc9,with or without adding IL-33,the primary CD8+ T cells were co-cultured with BMDCs.Explore the phenotype of IL-33 treated Tc9 cells.Results:(1)The addition of IL-33 can promote the production of IL-9 secreting Tc9 cells and increase the m RNA level of Il9 in Tc9 cells.In addition,addition of IL-33 increased Tc9 cells expression levels of their associated transcription factors Spi1 and Irf4,but had no effect on the Tc1-related transcription factor Tbx21.IL-33-treated Tc9 cells express high levels of Gzmb.Interestingly,the addition of IL-33 promotes the expression of IL-33 receptor ST2 in Tc9 cells.(2)Compared with Tc0 cells,Tc9 cells expressed less Pdcd1,and after adding IL-33,the expression of PD-1 in Tc0 and Tc9 cells was decreased at m RNA level and protein level.In addition,the addition of IL-33 can increase the m RNA levels of IL-2 in Tc0 and Tc9 cells.Conclusion: IL-33 promotes differentiation,survival and proliferation of Tc9 cells in vitro.Part 2 Effect of IL-33 Combined with BMDCs on Inducing Tc9/1 Cells in VivoObjective: To investigate the effect of IL-33 combined with BMDCs on Tc9/1cells in vivo.Methods: We used OVA peptide-loaded BMDCs with or without IL-33 immunized OT-I mice.The experiment was divided into 3 groups: 1)PBS,2)BMDCs,3)BMDCs+IL-33.The expression of Tc9 and Tc1 cells in CD8+ T cells of spleen and lymph nodes of OT-I mice was detected by FCM,ELISA and q PCR methods.Related cytokines and transcription factors are also detected.In addition,we also examined the cytotoxicity of tumor-specific CD8+ T cells induced with BMDCs.Results: Compared to the PBS,mice immunized with BMDCs alone had a greater number of CD8+ T cells(Tc9)expressing IL-9,whereas mice immunized with BMDCs combined with IL-33 had a greater number of Tc9 cells.ELISA and/or q PCR further confirmed that BMDCs plus IL-33 can increase IL-9 expression in mice.In addition,BMDCs plus IL-33-immunized mouse-derived T cells expressed significantly higher levels of Spi1 and Irf4 than BMDCs or PBS controls.We also detected T cell-expressing anti-tumor effectors IFN-? and Gzm B in mice.We found that BMDCs plus IL-33 immunized mice had more CD8+ T cells expressing IFN-?and Gzm B,with higher levels of protein or m RNA levels of IFN-gamma and Gzmb.In addition,CD8+ T cells from BMDCs plus IL-33 immunized mice also expressed Tbx21 at a higher level than BMDCs or PBS controls.It is worth noting that the addition of IL-33 can increase the cytotoxicity of tumor-specific CD8+ T cells induced with BMDCs.Conclusion: IL-33 promotes BMDCs to induce antitumor Tc9 and Tc1 cells in vivo.Part 3 Effects of IL-33 on the Proliferation Ability of Tc Cells and Its antitumor efficacy in vivoObjective: To investigate the effect of IL-33 on the proliferation of Tc9 cells and the anti-tumor effect of IL-33 in vivo.Methods: We used OVA peptide-loaded BMDCs with or without IL-33 immunized OT-I mice.The experiment was divided into 3 groups: 1)PBS,2)BMDCs,3)BMDCs+IL-33.The expression of IL-33 receptor ST2 by CD8+ T cells in the spleen and lymph nodes of OT-I mice was detected by FCM and q PCRmethods..The proliferation of CD8+ T cells in the spleen and lymph nodes of OT-I mice was detected by FCM.The Tc cell phenotype of CD8+ T cells in the spleen and lymph nodes of OT-I mice was detected by FCM and q PCR.Then,we used OT-I mouse B16-OVA melanoma as a model to perform DCs vaccine immunotherapy to study the antitumor immunity of IL-33 combined with BMDCs in vivo.Results: We found that BMDCs plus IL-33-immune mouse splenocytes had more ST2-expressing CD8+(ST2+CD8+)T cells and higher St2 m RNA levels compared to BMDCs or PBS controls.The mice immunized with BMDCs and the PBS control group had a considerable number of CD8+ T cells,while the BMDCs plus IL-33 immunized mice had significantly higher CD8+ T cells than the BMDCs or PBS groups.We also analyzed the expression of Ki67 in splenic T cells.Similarly,IL-33 could increase the expression of Ki67 in BMDCs induced Tc cells.Immunization of BMDCs promoted CD8+ T cells to express PD-1 and LAG3 compared to PBS control.However,immunization with BMDCs plus IL-33 strongly inhibited CD8+ T cells expressing PD-1,LAG3 and 2B4.In addition,IL-2 levels in CD8+ T cells of mice immunized with BMDCs plus IL-33 were higher than those of BMDCs alone or PBS control mice.Finally,DCs vaccine immunotherapy experiments showed that BMDCs plus IL-33 had a stronger inhibitory effect on melanoma growth.Conclusion: IL-33 promotes the proliferation of Tc cells in vivo,inhibits the depletion of CD8+ T cells activated by BMDCs,and promotes the anti-tumor effects induced by BMDCs. |
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