Dominant Negative TGF-β Receptor Type Ⅱ In T Lymphocytes Promotes Anti-tumor Immunity By Modulating T Cell Subsets And Enhancing CTL Responses

Cancer therapies that aim to directly kill cancer cells include traditional therapies,such as radiotherapy and chemotherapy,and new therapeutic strategies,including genetic therapy and cancer immunotherapy.Cancer immunotherapy,which mainly includes antibody therapy,cellular immunotherapy and cytokine therapy,enhance anti-tumor immunity so that cancerous cells can be eliminated in a timely and effective manner.In practice,tumor immunotherapy has shown incomparable advantages of traditional treatment methods,with less damage to healthy organs of tumor patients,less adverse reactions during treatment,and more effective removal of residual tumor cells.In the tumor microenvironment,cells that play an anti-tumor immune function mainly include T cells,B cells,and macrophages.Immune cells and related stromal components recruited and activated by tumor cells begin to aggregate in the early stages of tumor growth,forming a microenvironment that inhibits tumor growth.However,with continuous tumor antigen stimulation and immune activation,the anti-tumor immune effector molecules and cells in the tumor immune microenvironment are depleted,forming an immunosuppressive tumor immune microenvironment,which instead promotes tumor progression.This immunosuppressive tumor immune microenvironment is present in many different tumor types,including lung cancer,colorectal cancer,melanoma,fibrosarcoma,and breast cancer.Therefore,restoring the anti-tumor ability of the tumor immune microenvironment and strengthening anti-tumor immunity are the keys to tumor immunotherapy.Immunotherapy drugs that stimulate tumor-reactive cytotoxic T lymphocytes（CTLs）are effective in some patients.However,acquired resistance to these drugs frequently develops.Therefore,novel strategies to induce anti-tumor immunity are required to improve patient outcome.Transforming growth factor-β（TGF-β）is a multifunctional regulatory cytokine with multiple immunosuppressive properties.TGF-βinhibits T cell proliferation and differentiation into helper T cells and CTLs and inhibits the stimulation of T cells by antigen-presenting cells.CD8⁺T cells activated in the presence of TGF-βdo not acquire CTL function,which may be because TGF-βinhibits the expression of multiple effector molecules of CTLs,including granzyme A（Gzm A）,granzyme B（Gzm B）,perforin and IFN-γ.Together these findings demonstrate an important role for TGF-βin tumor evasion,indicating that TGF-βmay be a possible target for cancer immunotherapy.TGF-βneutralizing antibody treatments and drugs for TGF-βblockade have achieved some results,but a considerable proportion of cancers still cannot be cured by neutralizing antibodies or small molecule drugs.Immunotherapy drugs for TGF-βblockade,such as neutralizing antibodies,are usually not cell-selective and block the TGF-βpathway in various cell types.Although TGF-βblockade affects all TGF-β-responsive cells and changes the tumor microenvironment.The lack of TGF-βsignaling results in limited changes in key cells such as T cells in the tumor microenvironment and brings greater side effects.Considering that the immunosuppressive and tumor evasion effects of TGF-βare mainly achieved by its inhibition of the proliferation of T cells and differentiation of T cells into Th1 and CTLs,the suppression of TGF-βsignaling specifically in T cells may be an effective strategy to induce anti-tumor immunity and inhibit tumor evasion.In addition,the role of TGF-βin the development,maintenance,and induction of regulatory T cells（Tregs）has at attracted much attention.It has been confirmed that TGF-βcan promote the conversion of CD4⁺T cells to Tregs in vitro,but the role of TGF-βon Tregs under physiological conditions is still controversial.Tregs can actively suppress immune responses and maintain immune tolerance,playing a role in tumor escape.Therefore,it is also worth paying attention to whether the suppression of TGF-βsignaling specifically in T cells reduces the proportion of Tregs and then enhance anti-tumor immunity.T-bet,which is encoded by the Tbx21 gene,is a member of the T-box family of transcription factors.Previous studies have shown that TGF-βinhibits the expression of T-bet and in this way inhibits the differentiation of CD4⁺T cells.In addition,T-bet transcriptionally regulates genes encoding effector molecules of CTLs,Gzm A,Gzm B,perforin and IFN-γ,secreted by T cells.Therefore,strategies that prevent TGF-β-mediated T-bet inhibition may result in increased synthesis of Gzm B,IFN-γand other effector molecules and induced anti-tumor immunity of T cells.In this study,we used transgenic mice genetically engineered to overexpress a dominant-negative form of TGF-βreceptor type Ⅱ（dnTGF-βRⅡ）under control of the CD4 promoter,which renders both T cell subsets,but not other cells,insensitive to TGF-βsignaling.In many different tumor types,such as lung cancer,colorectal cancer,melanoma,fibrosarcoma,and breast cancer,an immunosuppressive tumor immune microenvironment exists.Therefore,this paper attempts to use the lung cancer cell line LLC and fibrosarcoma cell line MCA205 to establish lung cancer and fibrosarcoma tumor-bearing models in WT and dnTGF-βRⅡ mice,and explore whether targeting T cells to specifically block TGF-βsignaling can enhance anti-tumor effects,inhibit tumor growth,and explore its mechanism.We confirmed that dominant negative TGF-βreceptor type Ⅱ in T lymphocytes promotes the proliferation of T cells,and significantly increases the levels of CD4⁺,CD8⁺T cells and effector T cells,and markedly enhances the response of CTL,which may be related to the up-regulation of T-bet expression.We also found that dominant negative TGF-βreceptor type Ⅱ in T lymphocytes decreased the level of Tregs in tumor-bearing mice,which may be one of the reasons for enhancing anti-tumor immunity.Our findings demonstrated that T cell–specific blockade of TGF-βsignaling has a strong therapeutic potential to shift the balance of the immune response in favor of anti-tumor immunity.ObjectiveInvestigating whether targeting T cells to specifically block TGF-βcan enhance anti-tumor immunity,inhibit tumor growth,and explore its mechanism of enhancing anti-tumor immunity will provide new evidence and experimental basis for targeted anti-tumor immunotherapy.MethodsEstablishment of a mouse subcutaneous tumor-bearing model and assessment of tumor burden:a number of wild type（WT）mice and dnTGF-βRⅡ transgenic mice were randomly selected and divided into WT group and dnTGF-βRⅡ group,and 1×10~6mouse lung cancer cell lines LLC in logarithmic growth phase were selected.The mice were inoculated subcutaneously on the backs of the two groups of mice.When the tumors on the backs of the mice grew to be visible to the naked eye,the body weight and tumor volume changes of the mice were recorded every other day,and the mice were sacrificed when the volume of the subcutaneous tumors on the backs of the mice reached a maximum of 2000 mm~3.The same number of WT mice and dnTGF-βRⅡ transgenic mice were also taken,and 1×10~5 mouse fibrosarcoma cell line MCA205 in logarithmic growth phase was subcutaneously inoculated on the back in the same way,and the body weight and tumor volume changes of the mice were recorded every other day.After the mice were sacrificed,their dorsal skin was cut,and tumor tissue was dissected,weighed and recorded.HE staining was used to detect the pathological characteristics of mouse tumor tissues,immunohistochemistry was used to detect the expression of Caspase-3,TUNEL staining and laser confocal microscopy were used to observe the apoptosis of mouse tumor tissues,and Ki67 staining and semi-quantitatively analyze was used to detect the level of proliferation of cells in a tissue.Detection and evaluation of anti-tumor immunity in mice:the method of collagenase digestion and density gradient centrifugation was used for mouse tumor tissue,and the method of grinding and density gradient centrifugation was used for mouse spleen tissue.Mononuclear cells were isolated from mouse tumor tissue and spleen tissue respectively.The proportion of each cell subset was detected by cytometry.CD3,CD4,and CD8staining were used to detect the proportions of CD4⁺T cells and CD8⁺T cells in mice;CD44 and CD62L staining were used to detect the proportions of effector CD4⁺T cells and CD8⁺T cells;Ki67 staining was to detect the proliferation of CD4⁺T cells and CD8⁺T cells.CD4,CD25,Foxp3 staining was used to detect the proportion of Tregs in mice.Gzm A,Gzm B and Prf staining were used to detect the levels of CTL multi-effectors secreted by mouse CD4⁺T cells and CD8⁺T cells.The serum and fresh tumor tissue of the mice were collected,and the levels of IFN-γin the serum and tumor tissue of the mice were detected by ELISA.The primary CD3⁺T cells in the spleen of WT mice and dnTGF-βRⅡ transgenic mice were obtained by flow sorting,RNA was extracted,and the transcription levels level of Gzm A,Gzm B,Prf,and IFN-γwere detected by quantitative polymerase chain reaction（QPCR）.The same method was used to extract RNA from mouse tumor tissue,and QPCR was used to detect the transcription levels of Gzm A,Gzm B,Prf,and IFN-γin mouse tumor tissue.The effect and mechanism of dnTGF-βRⅡ on the antitumor effect of T cells in vitro:the primary CD3⁺T cells in the spleen of WT mice and dnTGF-βRⅡ transgenic mice were obtained by flow sorting,stimulated with Con A for 6 hours（a blank control group was also set）,and mixed with 1×10~5（12-well plate）or 5×10~5（6-well plate）of MCA205 cells in logarithmic growth phase were co-cultured in direct contact for 48 hours.After co-culture,the proliferation level of MCA205 was detected by high-content imaging system,Annexin V-PI staining was used,and the apoptosis level of MCA205 cells was detected by flow cytometry.The primary CD3⁺T cells in the spleen of WT mice and dnTGF-βRⅡ transgenic mice were obtained by flow sorting,RNA was extracted,and the transcription level of T-bet was detected by QPCR.The same method was used to extract RNA from mouse tumor tissue,and QPCR was used to detect the transcription level of T-bet in mouse tumor tissue.Results1.Establishment of a mouse subcutaneous tumor-bearing model and assessment of tumor burden1.1 Mouse body weight,tumor tissue volume and weightIn the lung cancer model,there was no significant difference in body weight between the WT group and the dnTGF-βRⅡ group,and compared with the WT mice,the dnTGF-βRⅡ group had smaller tumor volume and slow tumor growth rate.Furthermore,compared with WT mice,mice in the dnTGF-βRⅡ group had smaller tumor weights and lower tumor burden.Similar results were observed in a fibrosarcoma model.1.2 Histopathology of mouse tumorIn the lung cancer model,HE staining showed that compared with the WT group,the tumor tissue of the mice in the dnTGF-βRⅡ group exhibited cell shrinkage,sparse arrangement and fragmentation,and extensive necrosis was seen within the tumor.Similar results were observed in a fibrosarcoma model.1.3 Apoptosis and proliferation in mouse tumor tissueIn the lung cancer model,TUNEL staining and Caspase-3 immunohistochemistry showed that compared with the WT group,there was more apoptosis in the tumor tissue of the mice in the dnTGF-βRⅡ group.In addition,Ki67 staining showed that the level of cell proliferation was higher in tumor tissues of WT mice compared with dnTGF-βRⅡ group.Similar results were observed in a fibrosarcoma model.2.Detection and evaluation of anti-tumor immunity in mice2.1 Effects of dnTGF-βRⅡ on the proliferation of mouse CD4⁺T cells and CD8⁺T cellsIn the lung cancer model,the results of flow cytometry showed that compared with the WT group,the proportion of CD4⁺T cells and CD8⁺T cells in the tumor tissue and spleen in the dnTGF-βRⅡ group was higher.The proportion of Ki67⁺CD4⁺and Ki67⁺CD8⁺T cells in tumor tissue and spleen in dnTGF-βRⅡ group was higher,indicating that the proliferation of CD4⁺T cells and CD8⁺T cells in dnTGF-βRⅡ group was more.In the fibrosarcoma model,similar results were observed.2.2 Effects of dnTGF-βRⅡ on effector CD4⁺T cells and CD8⁺T cells in miceIn the lung cancer model,flow analysis results showed that compared with the WT group,the ratio of effector CD4⁺T cells（CD44⁺CD62L^-CD4⁺）and effector CD8⁺T cells（CD44⁺CD62L^-CD8⁺）was higher;CD4⁺T cells and CD8⁺differentiation into effector T cells is increased.In the fibrosarcoma model,similar results were observed.2.3 Effects of dnTGF-βRⅡ on the function of mouse Tregs cells and CTLIn the lung cancer model,the results of flow analysis showed that compared with the WT group,the proportion of Tregs in the tumor tissue and spleen in the dnTGF-βRⅡ group was lower,and the levels of CD4⁺T cells and CD8⁺T cells secreted CTL pleiotropic factors were increase.QPCR results showed that compared with the WT group,the m RNA levels of synthetic CTL pleiotropic factors in the tumor tissue and T cells in the dnTGF-βRⅡ group were higher.In addition,the ELISA results showed that compared with the WT group,the levels of IFN-γin the serum and tumor tissue in the dnTGF-βRⅡ group were higher.In the fibrosarcoma model,similar results were observed.3.The effect and mechanism of dnTGF-βRⅡ on the antitumor effect of T cells in vitro3.1 The effects of dnTGF-βRⅡ on T cells inhibiting tumor cell proliferation in vitroT cells were co-cultured with MCA205 cells in vitro,and high-content imaging results showed that regardless of whether T cells were pretreated with Con A,the total number of MCA205 cells in the dnTGF-βRⅡ group was less than that in the WT group,and the proliferation was more inhibited significantly.3.2 The effects of dnTGF-βRⅡ on tumor cells apoptosis by T cells in vitroT cells were co-cultured with MCA205 cells in vitro,and Annexin V-PI staining was analyzed by flow cytometry.The results showed that after T cells were pretreated with Con A for 6 hours,compared with the WT group,the total apoptotic ratio of MCA205cells in the dnTGF-βRⅡ group was higher.3.3 The effect of dnTGF-βRⅡ on T-betQPCR results showed that compared with the WT group,the m RNA levels of T-bet in the tumor tissue and spleen-derived T cells of the mice in the dnTGF-βRⅡ group were increased,and the level of T-bet in T cells increased was more significantly.Conclusions1.dnTGF-βRⅡ reduced tumor burden in mice,promoted tumor tissue necrosis,decreased the proportion of KI67⁺cells in tumor tissue,and increased the proportion of TUNEL⁺cells and the expression of Caspase-3;2.dnTGF-βRⅡ promoted the killing of tumor cells by T cells in vitro,increases the proportion of CD4⁺,CD8⁺,KI67⁺CD4⁺,KI67⁺CD8⁺and CD44⁺CD62L^-CD4⁺,CD44⁺CD62L^-CD8⁺T cells in tumor tissue and spleen of mice,and increases the expression of effector factors of CTL,reduces the proportion of Tregs in mouse spleen and tumor tissue,and enhances anti-tumor immunity;3.The increased secretion of CTL multi-effector molecules caused by dnTGF-βRⅡ may be related to the promotion of T-bet expression.