PD-1 Expression On ILC2 Surface In Patients With Esophageal Squamous Cell Carcinoma And Its Clinical Significance

BackgroundEsophageal cancer is one of the malignant tumors with high incidence and mortality around the world.It ranks as seventh common mortality malignancy and it is one of the prominent causes of tumor-associated deaths worldwide.Esophageal cancer has a high incidence in China.The incidence of esophageal cancer in China is higher than that in the world.Although the diagnosis and treatment of esophageal cancer have made marked progress,the patients are often diagnosed at advanced stage because the patients at early stage usually have insidious onset and nonspecific symptoms.The delayed diagnosis is part of the reason for the low 5-year survival rate.Revealing the underlying mechanism of tumorigenesis and development of esophageal cancer may be fundamental to the clinical diagnosis and treatment.The development and progression of tumor are complex processes,during which tumor microenvironment plays an important role.The tumor microenvironment is comprised of tumor cells,immune cells,stromal cells and some cytokines.In the tumor microenvironment,the number and the functional status of immune cells play an important role in the tumor onset and progression.Innate lymphoid cells(ILCs)are newly discovered innate immune cells population,differentiated from hematopoietic common lymphoid progenitor(CLP),with antigen-non-specific receptors on its surface.As immune cells,ILCs regulate immunity by releasing cytokines or other mechanism.Based on cell phenotypes and secreted cytokines,ILCs are divided into three groups:ILC1,ILC2 and ILC3.In the presence of some kinds of cytokines,such as IL-33,ILC2 can secrete type 2 helper T cell cytokines,including IL-4,IL-5,IL-9 and IL-13.And in some relevant reports,ILC2 plays a prominent role in anti-parasitic infection,autoimmune disease and so on.Although it was reported that ILC2 are found in tumor tissue in recent years,the role of ILC2 on tumor progression is still inconclusive.As a significant immune checkpoint molecule,programmed cell death protein-1(PD-1)is mainly expressed on the surface of immune cells.With engagement of PD-L1 and PD-L2,PD-1 can inhibit the function of immune cells.Previous studies about PD-1 focused on adaptive immune cells.In recent years,growing research showed that PD-1 was important mediator in the occurrence and development of disease by regulating the function of innate immune cells.At present,some studies have confirmed that PD-1 expression of ILC3 was related to distant metastasis of tumor.However,there were few research studies involved PD-1-expression on ILC2in tumorigenesis and development of tumor.More and more studies suggest that ILCs play a prominent part in the initiation and development of tumor.However,the infiltration degree of ILC2 in tumor microenvironment of esophageal squamous cell carcinoma(ESCC),PD-1 expression on ILC2 and its clinical significance are still unknown.The subject is aimed to explore the infiltration degree of ILC2 in tumor tissue of ESCC,PD-1 expression on ILC2,PD-1 effect on ILC2 and the correlation between the proportion of PD-1~+ILC2and ESCC clinical parameter,in order to reveal the role of ILC2 in the initiation and development of tumor and provide theoretical basis for new clinical indicators.Method1.Peripheral blood(from 15 health donors and 44 patients with ESCC)and tumor tissue samples(from 44 patients with ESCC)were collected.Single cell suspension was acquired by using density gradient centrifugation and three-enzyme digestion respectively.Then flow cytometry was utilized to test the proportion of three subtypes of ILCs and the expression of PD-1.2.ILC2,PD-1~-ILC2 and PD-1~+ILC2 in peripheral blood and tumor tissue of patients with ESCC were sorted by using flow cytometry.The flow cytometry was used to detect the level of IL-5 and IL-13 expressed by ILC2.ELISA was performed to measure the level of IL-13 in ILC2 culture supernatant.3.ESCC cells(KYSE70)were cocultured with ILC2 or ILC2 supernatant. Luciferase Assay and flow cytometry were used to detect the apoptosis of KYSE70.KYSE70 cells were treated with ILC2 supernatant and recombinant human IL-13,Transwell assay was used to detect the migration of KYSE70.4.According to the expression level of PD-1 on ILC2,the patients were divided into low PD-1 expression group and high PD-1 expression group.Analysis the association between PD-1 expression and clinical parameters and prognosis of ESCC patients.Results1.The results of flow cytometry showed that the percentage of ILC2 and PD-1~+ ILC2 in tumor tissue of patients with ESCC had improved significantly compared with that in blood of patients and healthy donors.2.The results of flow cytometry and ELISA consistently showed that the expression level of IL-13 in ILC2 derived from tumor tissue was significantly higher than that derived from peripheral blood and the IL-13 level of PD-1~+ILC2 in tumor-tissue was higher than that of PD-1~-ILC2.3.The results of flow cytometry and Luciferase Assay verified that ILC2 and the culture supernatant of ILC2 had no effect on the apoptosis level on KYSE70.The results of transwell array proved that supernatant of ILC2 and rh IL-13 promoted the migration level of KYSE70.4.The results of clinical data showed that the expression of PD-1 on ILC2 was related to lymph node metastasis and TNM stage.The median survival time of low PD-1 expression group was longer than high PD-1 expression group.ConclusionCompared with peripheral blood of healthy donors,the proportion of ILC2 in peripheral blood and tumor tissue of ESCC patients was increased.Then the expression of PD-1 promoted secretion of IL-13,which further promoted the tumor invasion and metastasis.By contrast,the result of clinical data analysis confirmed that the expression of PD-1 on ILC2 was positive correlated with poor prognosis.