The Effects Of TNF-α On Tc9 Cell Differentiation And Antitumor Activity

Malignant tumors are one of the main diseases threatening human life and health at present.The treatment methods of malignant tumors mainly include surgical treatment,chemotherapy,and radiotherapy.In recent years,as one of the techniques of tumor biotherapy,tumor immunotherapy has become a research hotspot of researchers due to its advantages of small side effects.Tumor immunotherapy is a treatment method that targets the body’s autoimmune system.It can achieve the purpose of tumor treatment by improving the anti-tumor ability of the human immune system.Adoptive cell immunotherapy is one of the methods of tumor immunotherapy,which is to inject sensitized lymphocytes into patients with low cellular immune function(such as cancer patients)to improve their anti-tumor immunity.Cytotoxic T lymphocytes(CTLs)are traditional tumor killing effector T cells,which can directly kill tumor cells and are the main effector T cells currently used in adoptive immunotherapy of tumors.Cytotoxic T cells 9(Tc9 cells)are a new kind of cytotoxic T cells subset which were discovered in recent years.Compared with the Tc1cells used in clinical treatment,Tc9 cells can survive longer in vivo.In addition,studies have shown that although the direct killing effect of Tc9 cells on tumors in vitro is weak,Tc9 cells can differentiate into Tc1 cells in vivo,which benefits to increase their anti-tumor capabilities.Therefore,Tc9 cells are expected to be more effective effector T cells in adoptive cell therapy.Tumor necrosis factor-α(TNF-α),a member of the TNF family,is a cytokine with a variety of biological functions,which can not only induce the production of inflammatory cytokines,but also participate in cell apoptosis and other cell responses.Previous studies have shown that TNF-αaffects the production and proliferation of helper T cells(Th cells).Since Th and Tc cells of the same subtype have similar polarization environments,and Tc9 cells have great potential in adoptive cell therapy,in this study,we will explore the effect of TNF-αon Tc9 cells in order to induce and amplify tumor-specific Tc9 cells more effectively and improve the expression level of its effector molecules(such as IL-9),to enhance its anti-tumor abilities.The study is mainly divided into the following three parts:Part 1 Induction of Tc9 cells by TNF-αin vitroObjective:To investigate the effects of TNF-αon Tc9 cells differentiation,survival,and proliferation in vitro.Methods:(1)Na?ve CD8~+T cells were cultured under the Tc0、Tc9 conditions with or without the addition of TNF-αfor 2 days,and the mRNA and protein levels of IL-9 were analyzed by qPCR and flow cytometry;(2)Place the sorted CD8+T cells under the same polarization environment as(1).After 2 days in vitro culture,the expression of T cell-related cytokines,transcription factors and differentiation factors were detected by qPCR.(3)Place the sorted CD8+T cells under the same polarization environment as(1).After 2 days in vitro culture,the flow cytometry was used to detected the percentage of Annexin V~+CD8~+T cells and Ki67~+CD8~+T cells.Results:(1)TNF-αtreatment increased the expression of IL-9 mRNA and protein in Tc9 cells;(2)TNF-αtreatment did not affect the expression of other Tc-related transcription factors and cytokines;both the mRNA and protein expression levels of CD44 were significantly increased in TNFα-induced Tc9 cells compared to regular Tc9 cells;(3)Annexin V staining detected less cell apoptosis of TNFα-induced Tc9 cells than regular Tc9 cells,and the frequency of Ki67~+CD8~+T cells increased in TNFα-Tc9 cells as compared to regular Tc9 cells.Conclusion:TNF-αpromotes Tc9 cell differentiation and increases Tc9 cell survival and proliferation in vitro.Part 2 Effects of TNF-αon Tc9 cell antitumor efficacy in vivoObjective:To examined the antitumor efficacy of TNFα-treatment Tc9 cells.Methods:(1)We generated Tc9 and TNFα-induced Tc9 cells from OT-I mice in vitro and cells were used to treat B16-OVA-bearing C57BL/6 mice.Tumor growth was compared between the different groups;(2)Tc9 cells and TNFα-induced Tc9 cells from OT-I mice were transfused to B16-OVA lung metastasis C57BL/6 mice.CD8~+T cells from the lung tumor tissues were collected and analyzed;(3)To explore the cytotoxicity of TNFα-Tc9 cells in vivo,OT-I Tc9 cells and TNFα-induced Tc9 cells were transfused to C57BL/6 mice with target/non-target cells.Six hours after cell injection,mice were sacrificed and splenocytes were collected and analyzed by flow cytometry.Results:(1)TNFα-treatment Tc9 cell exhibited greater inhibition on melanoma tumor growth than regular Tc9 cells;(2)Cells from mice receiving TNFα-Tc9 cells expressed higher levels of Il9 than mice transfused with regular Tc9 cells or PBS controls;(3)TNF-αtreatment significantly increased Tc9 cell CTL activity in vivo.Conclusion:TNF-αtreatment improves the antitumor efficacy of Tc9 cells.Part 3 Mechanism of TNF-αpromoting Tc9 cell differentiationObjective:To investigate the mechanism of TNF-αpromoting Tc9 cell differentiation.Methods:(1)Na?ve CD8~+T cells were cultured under Tc9 polarizing conditions in the presence of TNFR1(αR1)or TNFR2(αR2)blocking antibodies or an isotype control IgG(IgG)with or without the addition of TNF-α.After 2 days,the mRNA and protein levels of IL-9 were analyzed by qPCR and flow cytometry;(2)Place the sorted CD8+T cells under the same polarization environment as(1)for 2 days.The percentage of Annexin V-stained T cells and CFSE-stained T cells were analyzed by flow cytometry;(3)Na?ve CD8~+T cells were cultured under Tc9 polarizing conditions with or without the addition of TNF-αfor 3 hours.Western blots examined the protein levels of the phosphorylated STAT5(p-STAT5)and IKKα/β(p-IKKα/β)in T cells;na?ve CD8~+T cells were cultured under Tc9 polarizing conditions in the presence or absence of TNF-αwith or without(DMSO)the addition of STAT5 inhibitor(STAT5i)or NF-κB inhibitor Bortezomib(Bor)for 2 days.IL-9 expression in T cells was analyzed by qPCR and flow cytometry.Results:(1)The addition ofαR2 but notαR1 largely abrogated the up-regulation of Tc9 cell differentiation and IL-9 expression in TNFα-Tc9 cells;(2)The addition ofαR2 but notαR1 inhibited TNFα-induced Tc9 survival and proliferation;(3)Western blots detected increased protein levels of p-STAT5 and p-IKKα/βin TNFα-treated T cells compared to untreated controls;the lower expression of IL-9 mRNA and protein were detected in TNFα-Tc9 cells with the addition of STAT5i or Bortezomib compared to the regular Tc9 cells and DMSO controls.Conclusion:TNF-αstimulates Tc9 cell differentiation through TNFR2-mediated signaling.STAT5 and NF-κB signaling pathways are involved in the process of TNF-αpromoting Tc9 cell differentiation.