Study On The Expression And Significance Of CTLA-4,PD-1 And TIM-3 On T Cell In NSCLC Patients

As tumor immunology and molecular biology continue to advance in recent decades,It is recognized that dynamic changes of the immune state of the body and the immune micro-environment in the tumor run through the process of tumor genesis and development.As early as 1970 s,Some scholars put forward the theory of immune surveillance which elaborated the cellular immune system recognize and kill abnormal cells to maintain normal cell proliferation and transfer of genetic material under normal circumstances;When the body's immune function inhibition or abnormal cell evolution,the immune system is unable to identify abnormal cells,and abnormal cell proliferation and leading tumor.The theory of tumor immune editing emerging in the early twenty-first Century is the background of the research on tumor immunity in the past ten years,there are three immune states in the process of tumor genesis and development are described: Clear-balance-escape;It is considered that the immune system is a dynamic process of changing and being changed.The dynamic changes of T lymphocyte phenotype and function play an important role in process.In the stage of immune escape,tumor cells can escape the recognition and killing effect of T cells,which lies in the large mount of immunosuppressive molecules and inhibitory T cells in the body and tumor tissue.Immunotherapy opens a new milestone in the history of cancer therapy,its target of treatment and the cause of the failure are tumor immunosuppression,especially in the microenvironment of tumor.large amounts of regulatory T cells(T-regs)and myeloid derived suppressor cells(MDSCs)were accumulated in the tumormicroenvironment;exhausted T cell induced by the high expression of many inhibitory molecules and the abnormal secretion of cytokines building up an inhibitory immune environment,by which tumor cells escape the immune system.T-reg cells is an important immune suppressor cells,can inhibit cytomoxic T lymphocyte(CTL)cells,natural killer(NK)cells and dendritic cells(DC)and other immune effector cell functions is the key factor for tumor immune escape.T-reg cells were induced by tumor cells,It has been proved that the number of T-reg cells increased in many kinds of tumors,and then inhibit the anti-tumor response,and the level of T-reg cells was negatively correlated with tumor progression and prognosis.The results showed that the proportion of T-reg cells and peripheral blood T-reg cells in non small cell lung cancer(NSCLC)patients was significantly higher than that in normal subjects,and the proportion of T-reg cells was correlated with tumor stage and prognosis.However,in NSCLC patients,the relationship between the expression of T-reg cell surface inhibitory molecules and tumor progression has not been reported.In addition to inhibitory T cells such as regulatory T cells and myeloid derived suppressor cells,there are a large amount of T cells without immune function in body and in tumor,recognized as the exhausted T cells,which is characterized by high expression of inhibitory molecules or inhibitory receptor on the surface.Like cytotoxic T-lymphocyte associated antigen-4(CTLA-4),programmed death-1(PD-1),T cell immunoglobulin and mucin domain-3(TIM-3),as checkpoint Regulating the function,proliferation and apoptosis of immune cells.Under normal circumstances,the immune checkpoint is involved in maintaining self tolerance and protecting normal tissues from the immune system.In patients with cancer,because of the insufficient exposure to the effective tumor antigen for a long time,lymphocytes canexpress some inhibitory molecules,thus leading to the dysfunction of tumor specific T cells,which is characterized by T cell proliferation disorder or at the same time reduce the secretion of cytokines such as IL-2,IL-10,IFN-?.These immune checkpoint proteins are potential targets for cancer immunotherapy.A large number of clinical data show that antibodies against co inhibitory signal can enhance the response of tumor specific T cells.The inhibitory effects of immunosuppressive molecules are not independent,The function and expression of inhibitory molecules on the surface of the same lymphocytes or different types of lymphocytes induce and regulate each other;the downstream channels are interlaced and influence each other,together constitute a lot of intersection of inhibitory molecular networks.Therefore,in NSCLC patients,CTLA-4,PD-1 and TIM-3 in different types of T cell surface expression level? What is the relationship between these three negative costimulatory molecules? What is the relationship between the clinical and pathological features of the patients? How does the expression level change in perioperative period? To explore these issues,we designed the study,including three parts:Part one: The the levels of CTLA-4,PD-1,TIM-3 expressed on CD3+T cells,CD4+T cells,CD8+T cells and T-reg cells in peripheral blood of 64 NSCLC patients and 18 healthy volunteers were detected by flow cytometry(FCM).analyze the relationship between immune indexes and the clinical pathological features and the correlation between the immune indexes.Results showed that: the ratio of CD4+T cells and CD4+/CD8+ in peripheral blood was significantly lower than that in healthy volunteers.The ratio of T-reg/CD4+ and T-reg cells significantly increased in patients as compared with healthy controls.The proportion of PD-1+ cells and TIM-3+ cells in CD3+T cells and CD4+T cells was higher than that in healthy group;the ratio of CTLA-4+ and PD-1+ cells in CD8+ T cells was higher than that in healthy group;the ratio of CTLA-4+ and TIM-3+ cells in T-reg cells was higher than that in healthy.T-reg/CD4+ associated with TNM staging in patients with NSCLC;the proportion of PD-1 and TIM-3+ cells in CD4+T cells was correlated with lymph node metastasis in patients with NSCLC.The rate of T-reg in CD4 and the expression rate of PD-1 in CD4+T cells,the expression rate of TIM-3 in T-reg cells and the expression rate of CTLA-4 in T-reg cells showed positive correlation;the rate of T-reg in CD3 and the expression rate of CTLA-4 in T-reg cells showed positive correlation;the expression of CTLA-4+ in T-reg cells was positively correlated with the expression of TIM-3+ in T-reg cells The expression rate of PD-1 in CD8+T cells was positively correlated with the expression rate of TIM-3 in CD8+T cells.Part two: The the levels of PD-1,TIM-3 expressed on tumor infiltrating T lymphocytes in tumor tissue was detected by multiplex quantitative Immunofluorescent(QIF).To analyze the co-expression phenomenon of PD-1 and TIM-3 in tumor infiltrating T lymphocytes and the relationship between these indexes and clinical pathological features.Results showed that: the levels of PD-1,TIM-3 expressed on tumor infiltrating T lymphocytes of NSCLC tumor tissue is high,and TIM-3 and PD-1 co-expression phenomenon is ubiquitous.The ratio of PD-1+/CD3+T and TIM-3+/CD3+T cells was significantly increased in tumor tissue as compared with in Peripheral blood.The ratio of TIM-3+/CD3+T cells in tumor tissue was relevant with in Peripheral blood.PD-1+TIM-3+TIL represents the main component of tumor infiltrating T lymphocytes.PD-1+/CD3+T cells ratio in tumor tissue was associated with lymph node metastasis,TIM-3+/CD3+T cells ratio in tumor tissue was correlated with tumor diameter,The ratio of PD-1+TIM-3-T/CD3+T cells was correlated with lymph node metastasis,PD-1+TIM-3+TIL/CD3+T was associated with lymph node metastasis and tumor stage.Part three: Flow cytometry was used to detected expression levels of CTLA-4,PD-1 and TIM-3 on CD4+T cells,CD8+T cells,T-reg cells surface in the peripheral blood in 41 patients with NSCLC at 1 day before surgery,1 day,3 days,7 days,28 days after surgery.To analyze the changes of the expression of co-inhibitory molecules at different time points,and to explore the relationship between immune state and surgical treatment.Results showed that: compared with 1 day preoperative,the levels of CD3+T cells percentage,CD4+T cells percentage and CD4+/CD8+ratio in peripheral blood were significantly decreased in 1 day,3 days,7 days postoperative.The level of CD3+ percentage,CD4+ percentage and CD4+/CD8+ratio on 28 days postoperative was significantly higher than those on 1 day preoperative.The percentage of CD8+ is lower on 28 days after operation than that before operation.The proportion of T-reg/CD3+T in peripheral blood at different time points after operation was lower than that before operation.The ratio of T-reg/CD4+T cell on 28 days after surgery was significantly lower than that before surgery;the ratio of T-reg/CD4+T cell on 1 day after operation was higher than before operation.Compared with the 1 day before operation,the ratio of CTLA-4 on CD3+T cells,CD4+T cells,CD8+T cells,T-reg cells on 7 days,28 days postoperative were significantly decreased.At each time point after operation,the ratio of PD-1 on CD3+T cells,CD4+T cells,CD8+T cells,T-reg cells was significantly lower than that before operation.Compared with 1 day preoperative,the ratio of TIM-3 on CD3+T cells,CD4+T cells,CD8+T cells,T-reg cells on 7 days,28 days postoperative were significantly decreased.Compared with healthy volunteers,the levels of CTLA-4 and(or)PD-1 and(or)TIM-3 expressed on lymphocytes surface were increased in peripheral blood of patients with NSCLC;elevated levels of co-inhibitory molecules and inhibitory cells were associated with TNM stage or lymph node metastasis in patients with NSCLC;suggesting that NSCLC patients are in the state of immuno-suppression,and the immuno-suppressive factors in peripheral blood may promote tumor metastasis.The correlation between TIM-3 and CTLA-4 on the surface of T-reg suggested that TIM-3 may be a surface marker of T-reg cells;CD4+PD-1+T cells may be involved in the regulation of T-reg cell proliferation and transformation.The co expression of TIM-3 and PD1 in tumor tissues revealed that most of the tumor infiltrating T lymphocytes were highly exhausted T cells,which Involved in tumor cell immune escape,promote disease progression.The application of multicolor labeling immunofluorescence technique and multi spectral tissue imaging analysis system may be helpful to exploring tumor immune suppression network,and providing facilitate for exploring new targets and the optimal application of immune checkpoint inhibitor.Surgery may inhibit the immune function of a short time,but also can effectively reduce the expression of co-inhibitory molecules,and reduce the tumor derived immunosuppression,Before operation and immediately following operation may be appropriate for the application of the immune checkpoint inhibitors.