Tumor-derived TGF-β Restricts CD8~+ T Cell Migration From Lymph Nodes To Tumors

Background and purposeThe formation of tumors is actually the result of the body’s immune cells failing to eliminate the tumor cells.The immune system plays the role of recognizing and killing tumor cells,encircling and eliminating tumor cells.Among them,CD8+ T cells are the main effector cells in the body’s anti-tumor immune response and play an extremely important role in killing tumors.However,tumor cells use their own highly mutated characteristics to evade tumor immune surveillance,and rely on immunosuppressive cells and inhibitory cytokines to build a powerful immunosuppressive microenvironment to resist the body’s immune response.In the process of tumor occurrence and development,the dynamic process of tumor cell resistance and suppression of the immune system is complex.This change is not limited to the tumor microenvironment but also involves the entire body,including the lymph nodes that activate and then deliver CD8+ T cells to the tumor.The activity and quantity of tumor-infiltrating lymphocytes(TILs)determine the growth rate and outcome of Tumor.Studies have shown that T cells can not inhibit tumor progression even in,"hot tumors" with abundant immune cell infiltration and good activity,suggesting that changes in the external immune state of tumor microenvironment also greatly affect the anti-tumor activity of T cells.Studies have shown that the secreted factors in the tumor microenvironment like transforming growth factor-β(TGF-β),can significantly reduce the anti-tumor immune response.TGF-β has strong immunosuppressive function and is secreted by tumor cells,stromal cells and immunosuppressive cells.Blockade of TGF-β can increase the number of tumor-infiltrated CD8+T cell.However,the factors affecting CD8+ T infiltration remain to be explored.In this study,we explored the changes of lymph nodes and tumor-infiltrating CD8+ T cells in early and late tumor models,and found that tumor-infiltrating CD8+T cells had strong killing functions in the early stage of tumor formation,and were exhausted and function decline in the late stage,while CD8+ T cells were always in a low infiltrating state in the early or late stage tumor.However,the movement ability of CD8+T cells in the drainage lymphatic node to the tumor was always inhibited in the early or late stage of tumor formation.Based on this finding,this study explores the reasons and mechanisms of tumor inhibition of CD8+T cell infiltration.Methods:1.Changes in the activation,proliferation and killing function of tumor-infiltrating T cells in early and late stages of tumor formation were detected by flow cytometry.2.The number of tumor-infiltrating T cells was detected by flow cytometry.3.Flow cytometry was used to detect the function and quantity changes of spleen T cells in the early stage of tumor formation.4.Transwell assay was used to verify the migration function of spleen T cells in the early stage of tumor formation.5.Flow cytometry was used to detect the change of T cell function and number in drainage lymph nodes and non-tumor drainage lymph nodes in the early stage of tumor formation.6.Transwell assay was used to detect the migration function of T cells in draining lymph nodes and non-draining lymph nodes in the early stage of tumor formation.7.Flow cytometry was used to detect the expression of CCR7 in tumor draining lymph nodes of mice.8.Transwell experiment was used to verify the changes of T cell migration function after treatment with TGF-β recombinant protein and inhibitor.9.Flow cytometry was used to detect the expression changes of CCR7 in T cells treated with TGF-β recombinant protein and inhibitor.Results:1.In the early stage of tumor formation,tumor infiltrating CD8+T cells showed strong activation,proliferation and killing functions;2.The numbers of infiltrating CD8+ T cells were low in both early and advanced tumors;3.Tumors had no significant effect on the function and number of CD8+ T cells in spleen;4.At the early stage of tumor formation,CD8+T cells in draining lymph nodes had strong killing,activating and proliferating functions;5.In the early stage of tumor formation,the number of CD8+T cells in the draining lymph nodes was increased,but their migration function was inhibited;6.The expression of CCR7 in CD8+ T cells was increased in tumor draining lymph nodes;7.TGF-β inhibited the migration of CD8+ T cells by promoting CCR7 expression.Conclusions:1.In the early stage of tumor formation,tumor infiltration of CD8+T cells has strong killing function,but weak infiltration;2.The inhibition of CD8+ T cell egress from tumor draining lymph nodes contributes to the limited tumor infiltration of T cells;3.Tumor-derived TGF-β inhibits the egress of T cells by promoting the expression of CCR7.