Investigation Of The Effect Of IL-15 On The Phenotype And Immune Response Of Tumor Infiltrating CD8+CD57+T Lymphocytes In NSCLC

Research background: The tumor microenvironment effectively inhibits the tumor infiltrating CD8+T lymphocytes through multiple mechanisms,and causes the immune escape.Numerous co-inhibitory immune checkpoint receptors expressed by T cells have been discovered in recent years,including PD-1,CTLA-4,TIM-3,LAG-3.etc,which are one of the most important mechanisms of tumor suppressing infiltrating effector T cells.Immune checkpoint PD-1/PD-L1 blocking therapy is considered to be the breakthrough in the field of treating advanced non-small cell lung cancer(NSCLC).However,due to the heterogeneity of the tumor microenvironment,including the components and status of the tumor-infiltrating immune cells,PD-1/PD-L1 inhibitors have significant effects only on a fraction of lung cancer patients.The density,distribution,and functional status of tumor-infiltrating CD8+T cells are the key factors that determine the efficacy of immunotherapy.Therefore,the identification and study of tumor-infiltrating CD8+cell subsets will help to find markers that predict the efficacy of immune checkpoint therapy,explore new immunotherapy targets,and elevate the efficacy of tumor immunotherapy further.The most and in-depth research on tumor-infiltrating CD8+T cells is the exhausted CD8+T cells(Tex)that express high levels of PD-1.Anti-PD-1/PD-L1 antibodies block PD-1 signaling pathways that inhibit the T cell function,thereby restoring the immune response of Tex cells and exerting their tumor-killing effects.However,not all CD8+T lymphocytes positive for PD-1 in tumor tissues are Tex.The primitive naive T lymphocytes will further differentiate into effector T cells during the process of being stimulated by the antigen.Among them,T lymphocytes expressing CD57 molecules have higher cytotoxic capacity.Repeated antigen stimulation during chronic infection will promote the formation and accumulation of CD8+CD57+T cells which play a crucial role in the fight against chronic viral infections.However,the role of this cell subset in anti-tumor immunity remains unclear.Research purposes: Systematically study the cellular phenotype and immune response of CD8+CD57+T cells in peripheral blood,tumor tissues,and lymph nodes of patients with NSCLC.Reveal the key molecules that promote the effector function of tumor infiltrating CD8+CD57+T cells,explore the new immunotherapy target,and improve the efficacy of immunotherapy in lung cancer.Experimental methods: In this study,tumor tissues,peripheral blood and lymph nodes of patients with NSCLC were collected in the Department of Thoracic Surgery of Wuhan Tongji Hospital.To avoid the influence of enzyme digestion on the phenotype of lymphocytes,single-cell suspensions were obtained by manually mechanical methods and density gradient centrifugation was used to extract mononuclear cells(PBMC),tumor-infiltrating lymphocytes(TIL)and lymphocytes from peripheral blood,tumor tissues,and lymph nodes respectively.CD8+CD57+or CD8+CD57-T lymphocytes were purified by using multisort magnetic beads.Use PMA plus Ionomycin or anti-CD3 plus anti-CD28 method to cultivate and activate lymphocytes mixture.By using the method of cell line-based redirected T cytotoxicity assay,the cytolytic ability of CD8+CD57+and CD8+CD57-T lymphocytes were measured.The phenotype,effector function,proliferation ability and related transcription factors expression in CD8+CD57+T cells were analyzed and evaluated by Flow Cytometry.RT-q PCR and Western blot technique were used to investigate that interferon-? regulates the expression of IL-15 and its mechanism.Experimental results: The proportion of CD8+CD57+T lymphocytes in total CD8+T cells in tumor microenvironment is lower than that in peripheral blood(15.10% ± 7.74% versus19.04% ± 10.53%);Compared with peripheral blood and tumor tissue,the proportion of CD8+CD57+T cells is the lowest in lymph nodes,accounting for about 6?8%.CD8+T cells activated by antigen can be differentiated into various types of memory cells and effector cells.According to whether T cells express CD27,CD28,KLRG-1,etc,the differentiation stage of T cells can be evaluated preliminarily.In the process of cell differentiation and functional maturation,the expression of co-stimulatory CD27 and CD28 molecules is gradually lost,and the cells that obtain KLRG-1 expression are terminally differentiated effector cells and have mature immune response functions.In peripheral blood,CD8+CD57+T lymphocytes barely express CD27 and CD28 and express high levels of KLRG-1.However,some CD8+CD57+T lymphocytes in lymph nodes and tumor tissues express both CD27 and CD28 molecules,especially in lymph nodes;and CD8+CD57+T lymphocytes in tumor tissues and lymph nodes do not express KLRG-1.Using the CD45 RA and CCR7 molecule to define the differentiation stage of this cell subset,we found that in tumor tissues and lymph nodes,more than 90% of CD8+CD57+T lymphocytes are effector memory T lymphocytes(CD45RA-CCR7-,TEM);In the peripheral blood of NSCLC patients,about 50% of CD8+CD57+T lymphocytes are terminally differentiated effector cells expressing CD45RA(CD45RA+CCR7-,TEMRA).Tumor-infiltrating CD8+CD57+T cells are different from exhausted T cells in cell phenotype and do not express LAG-3,TIM-3,CTLA-4 and other inhibitory receptors.Cytotoxicity analysis found that CD8+CD57+T lymphocytes in isolated lymph nodes and tumor tissues do not have cell killing function,while CD8+CD57+T lymphocytes in peripheral blood can induce 20?30% early apoptosis in P815 cells.We further investigate the expression of effector molecules and related transcription factors in CD8+CD57+T cells,we found that the expression of perforin in CD8+CD57+T lymphocytes in tumor tissues and lymph nodes is impaired,and the expression of the transcription factor T-bet,which is closely related to T cell differentiation and function is also at low level.As an important cytokine,IL-15 is related to the differentiation and function of CD8+T lymphocytes.IL-15 plays an important role in the maintenance of tumor-infiltrating CD8+T cells in the tumor environment.Our in vitro experiments found that interferon-? can up-regulate the expression of IL-15 in tumor cells through the JAK/STAT1/IRF-1 pathway.The use of exogenous IL-15 can significantly increase the expression of Perforin and T-bet in tumor-infiltrating CD8+T lymphocytes,and improve the killing ability of this group of cells.Conclusions: The above results suggested that there are incompletely differentiated effector CD8+CD57+T lymphocytes in tumor tissues.The tumor microenvironment may lead to the impaired differentiation and dysfunction of CD8+CD57+T lymphocytes.IL-15 can promote tumor-infiltrating CD8+CD57+T lymphocyte differentiation and immune response,and interferon-? can up-regulate the expression of IL-15 in tumor cells through the JAK/STAT1/IRF-1 pathway.On the one hand,our research work reveals a new mechanism of tumor immune escape,that is,the tumor microenvironment hinders the differentiation and functional maturation of activated T cells.Moreover,IL-15 can promote the differentiation and functional maturity of CD8+CD57+T cells.Therefore,it provides a certain theoretical basis and a new strategy for the combination of immune checkpoint inhibitors and IL-15 to improve the effectiveness of immunotherapy.