Expression Of Immune Checkpoint Molecules And Characteristics Of TILs In The Patients Of Henan With Esophageal Squamous Cell Carcinoma

Esophageal Carcinoma is one of the most common gastrointestinal tumors in the world.It ranks eighth among all tumors,especially in China,and the incidence in the north of henan province especially in linzhou,is the highest in the world.Esophageal squamous cell carcinoma or ESCC is the most common histological type of esophageal carcinoma in China,accounting for more than 90%of all diagnosed esophageal carcinoma patients.Many patients are found to be in advanced stage and lose the best chance of treatment because of the lack of effective early diagnosis methods.ESCC has a high mortality rate although surgical treatments have improved in recent years.It is ranking the sixth among all tumors,with a five-year survival rate of about 15 to 34 percent.It seriously affects quality of life of patients and physical health.Its pathogenesis is related to the patient’s lifestyle,diet,long-term chronic stimulation and genes,but it is still unclear concerning the exact molecular mechanisms.Therefore,it is an important task to study the pathogenesis of esophageal carcinoma and its appropriate treatment.Tumor microenvironment fosters tumor growth formed by the interaction between tumor occurrence,progression and metastasis and the surrounding environment.It is characterized by low oxygen and high permeability,which has a great impact on human immune system.T cells can also be activated in the early stages of a tumor.However,with the continuous progression of the tumor and the continuous exposure of antigen,the function of T cells and the differentiation program of memory T cells could enter into exhaustion phenotype.The ability of T cells to produce cytokines can be significantly reduced,and effector function as well as proliferation and self-renewal ability can be servely impaired.When dysfunction of T cell or apoptosis occurs,it therefore prevents differentiation into anti-tunor memory T cells.After T cell exhaustion occurs,a number of immune checkpoints and the mainly the inhibitory molecules such as programmed cell death protein 1(PD-1),cytotoxic T lymphocyte-associated antigen 4(CTLA-4),T cell immunoglobulin mucin 3(TIM-3),Lymphocyte activation gene-3(LAG-3),B and T lymphocyte attenuator(BTLA),A2A adenosine receptor(A2AR),etc.The high expression of these immune checkpoints will interact with their respective ligands to secrete a large number of cytokines,leading to tumor progression.Regarding the development of immunotherapy research,T-cell immunotherapy is the most important and promising method to treat tumor in the present stage.Thereapies targeting immune checkpoint inhibitors,represented by PD-1 and CTLA-4 inhibitors,offer hope to many cancer patients,but many patients were found to be ineffective.Therefore,understanding the causes of treatment failure is key to current research.In this study,we collected tissue samples from patients with ESCC and selected6 immune checkpoint molecules.Immunohistochemistry was used to determine the expression of various immune checkpoint molecules in esophageal squamous cell carcinoma tissues.We analyzed the correlations between these molecules and various clinic pathological characteristics,as well as how expression of these moelcules could impact survival rates of patients.Western blot and q RT-PCR were used for quantitative detection.The possible mechanism was further explored to understand the effect of immunosuppressive molecules on esophageal squamous cell carcinoma,by means of revealing at genomic level how the T cells could respond to micro-environment inside the tumor.We isolated T lymphocytes or T cells from blood and cancer tissue and and performed transcriptomic sequencing.The gene expression status of the two type of T cells were analyzed for differential expression of tumor-related cytokines,chemokines and T cell activation pathways at the transcriptomic level.Furthermore,NFATC2 was detected in invasive lymphocytes of patients with esophageal squamous cell carcinoma,study the correlation between NFATC2 and immune checkpoint molecules.Nfatc2 was knocked out in C57BL/6mice.Objective to compare the expression of PD-1,TIM-3 and BTLA between Nfatc2 deficient mice and wild-type mice,providing basic and supporting resources for follow-up work,and further revealing the role of T cells in tumor regulation.Part I Expression levels and clinical significance of immunocheckpoint molecules in esophageal squamous cell carcinomaMethodsApproved by the ethics committee of the first affiliated hospital of Xinxiang Medical University,the paraffin specimens of 161 patients with esophageal squamous cell carcinoma who received radical resection of esophageal cancer in the hospital from February 2014 to December 2015 were retrospectively studied.None of the patients received preoperative treatment.The expression of PD-1,CTLA-4,TIM-3,LAG-3,BTLA and A2AR in all samples was determined by immunohistochemistry.χ~2 test was used to compare the correlations between immune checkpoint molecules and clinicopathological features.Kaplan-Meier method was used to obtain the survival curves,and Log-rank method was used to test and compare the differences of survival curves among different groups.Cox proportional regression risk model was used to analyze univariate and multivariate survival factors.The independent risk factors PD-1,TIM-3 and BTLA were detected by Western blot and q RT-PCR.Results1.The positive expression of PD-1 is closely related to the age of the patient and the presence of metastasis in the surrounding lymph nodes;the positive expression of CTLA-4 is closely related to the alcohol consumption of the patient and the presence of metastasis in the surrounding lymph nodes;and the positive expression of TIM-3 is closely related to the depth of tumor infiltration and the presence of metastasis in the surrounding lymph nodes;LAG-3 positive expression and tumor pathological type,differentiation degree and lymph node metastasis is closely related to positive BTLA expression;alcohol consumption,tumor differentiation,tumor infiltration depth and surrounding lymph node metastasis is closely related to A2AR positive expression and the patients age,tumor differentiation degree and lymph node metastasis(P<0.05).2.Univariate survival analysis showed that the degree of differentiation,depth of infiltration,lymph node metastasis,PD-1,CTLA-4,TIM-3,BTLA and A2AR were risk factors affecting the total survival of patients with ESCC(P<0.05).The results of multivariate survival analysis showed:tumor differentiation(HR=0.294,95.0%CI:1.055-1.707,P=0.017),lymph node metastasis(HR=0.897,95.0%CI:1.654-3.637,P=0.001),PD-1(HR=0.543,95.0%CI:1.075-2.757,P=0.024),TIM-3(HR=0.788,95.0%CI:1.203-4.018,P=0.001)and BTLA(HR=0.563,95.0%CI:1.075-2.868,P=0.025)can be used as independent prognostic factors for overall survival of patients with ESCC.3.Western blot and q RT-PCR quantitative detection showed,the expressions of PD-1,TIM-3 and BTLA in ESCC were significantly higher than those in adjacent tissues.4.The co-expression of PD-1 and TIM-3,PD-1 and BTLA,TIM-3 and BTLA significantly reduced the survival time of patients with ESCC(P<0.05).ConclusionThe degree of tumor differentiation,depth of infiltration,lymph node metastasis,PD-1,CTLA-4,TIM-3,BTLA,and A2AR in patients with ESCC were risk factors affecting total survival,while the degree of tumor differentiation,lymph node metastasis,and PD-1,TIM-3,BTLA were independent prognostic factors affecting total survival of patients with esophageal squamous cell carcinoma.The co-expression of PD-1 and TIM-3,PD-1 and BTLA,TIM-3 and BTLA significantly reduced the survival time of patients with ESCC,providing a theoretical basis for the combinational use of drugs targeting dual immunocheckpoint molecules to achieve more effective treatments.PartⅡGene expression characteristics of peripheral blood and tumor infiltrating T lymphocytes in patients with esophageal squamous cell carcinomaMethodsApproved by the ethics committee of the first affiliated hospital of Xinxiang Medical University,7 surgical patients were selected and peripheral venous blood was extracted 2 hours before surgery.Lymphocyte layers in peripheral blood were collected by density gradient centrifugation method,and tumor infiltrating lymphocytes in fresh ESCC tissues were isolated and extracted.The recorded data was analyzed using version 10.0 of FLOWJO.CD3-positive cells in peripheral blood and tumor were separated by magnetic beads,and RNA was extracted from 14samples of trace cells and sequenced,so as to compare the transcriptome levels of peripheral blood and tumor infiltrating lymphocytes.The selected differentially expressed genes were enriched in specific signaling pathways to analyze the involvement of specific pathways in the mutual regulation of tumor microenvironment and T lymphocytes.The high expression gene NFATc2 was detected by immunohistochemistry,the relationship between NFATC2 and PD-1,TIM-3,BTLA was analyzed.The expression of NFATC2 phosphorylation in tumor infiltrating lymphocytes and peripheral blood lymphocytes was detected by flow cytometry,Nfatc2 was knocked out in C57BL/6 mice,T cells were stimulated to induce the expression of inhibitory molecules.The expressions of PD-1,TIM-3 and BTLA were compared between Nfatc2 deficient mice and wild-type mice.Ruslts1.There were a large number of differentially expressed genes found between blood T cells and tumor T cells from patients.The number of differentially expressed genes in each group ranged from 8048 to 12819,and the average number of differentially expressed genes was 11,477.2.The correlation of the same tissue is good,but the correlation between tumor and peripheral blood is relatively weak,with obvious difference.3.The expression of NFATC2 and PD-1,TIM-3 and BTLA were positively correlated in ESCC(P<0.05).4.The phosphorylation levels of NFATc2 in CD4 and CD8 T cells in tumor tissue were significantly higher than those in peripheral blood(P<0.05).5.The expression of PD-1,TIM-3 and BTLA decreased significantly in Nfatc2deficient mice(P<0.001).ConclusionThis study based on the same esophageal squamous carcinoma patients,by comparing T cell transcriptomic features between peripheral blood T cells and tumor infiltrating T cells,found that T cell gene expression in tumor tissue and peripheral blood T cells had enormous difference.More importantly such genome-wide RNA sequencing results proved that the tumor microenvironment has enormous regulation on function of T cells.The expression of NFATC2 was positively correlated with PD-1,TIM-3 and BTLA.Nfatc2 knockout significantly reduced the expression of PD-1,TIM-3 and BTLA.These results suggest that NFATC2 is an important transcription factor regulating T cell inhibitory receptor.