PD-L1 And PD-L2 Induce The High Expression Of IL-10 In Regulatory B Cells And The Related Mechanism In The Immune Escape Of Hypopharyngeal Carcinoma

Hypopharyngeal cell carcinoma is the most malignant tumor in head and neck,mostly consisted of poorly differentiated squamous cell carcinoma and early diagnosis is difficult.Hypopharyngeal cell carcinoma has the tendence to metastasize through lymphatic duct at early stage and is characterized by high aggressiveness,poor prognosis and easy relapse.Despite the improvement in combined therapy of sugery assisted by radiotherapy and chemotherapy,the five-year survial rate of hypopharyngeal cell carcinoma patients still remains around 50%.Because of the difficulty in early diagnosis,patients usually suffer large surgical range,which severely impair their quality of life.Based on this situation,it has become increasingly significant to deeply explore the mechanism of hypopharyngeal cell carcinoma and to search for important targeting factor favoring early diagnosis and more efficient treatment.Programmed death ligand 1(PD-L1)and 2(PD-L2)are important immune checkpoint ligands and both can promote tumor progression.They can bind with programmed cell death 1(PD-1)to inhibite the activation of effector T cells,leading to attenuated anti-tumor immunity,which facilitates the immune escape of tumor cells.Hypoxia inducible factor-1?(HIF-1?)is one of the upstream regulator factors of PD-L1 that is also able to promote tumour progression.Hypoxia inducible factor-2?(HIF-2?)and HIF-1? are members of the same family and likely share similiar functions.Regulatory B cell(Breg)is one of B cell subgroups and plays a role in immune escape.PD-L1 could induce Breg differentiation in some tumors.Breg inhibits the body's immune response and promotes tumor progression by secreting inhibitory cytokine IL-10.In this study,we detected the expression of PD-L1,PD-L2 and IL-10 in hypopharyngeal cell carcinoma and correlated squamous epithelum by immunohistochemistry and detected the co-expression of IL-10 and CD19 in hypopharyngeal cell carcinoma by immunofluorescence.We also discussed the mechanism of hypopharyngeal cell carcinoma progression by co-cultivation of Fa Du cells and peripheral blood lymphocytes.Objective:1.To investigate the expression of PD-L1,PD-L2 and IL-10 in hypopharyngeal cell carcinoma and correlated squamous epithelum and analyze the relationship between the their expression level and clinical status;2.To investigate the correlation of IL-10 expression with PD-L1 and PD-L2;3.To investigate the co-expression of IL-10 and CD19 in hypopharyngeal cell carcinoma;4.To investigate the effect of peripheral blood lymphocyte on expression of PD-L1,PD-L2,HIF-1? and HIF-2? in Fa Du cells on m RNA level,separately;5.To investigate the effect of Fa Du cells on IL-10 secretion by Breg cells.Methods:1.Paraffin sections were made from 33 hypopharyngeal carcinoma tissues and correlated squamous epithelum.Then the expression of PD-L1,PD-L2 and IL-10 were detected by immunohistochemistry and the co-expression of IL-10 and CD19 was detected by immunofluorescence;2.Fa Du cells and human peripheral blood lymphocytes were co-cultured and the m RNA expression levels of PD-L1,PD-L2,HIF-1? and HIF-2? in Fa Du cells were detected by Real-Time PCR;3.Fa Du cells and human peripheral blood lymphocytes were co-cultured and the expression level of IL-10 in peripheral blood cells was detected by ELISA.Results:1.PD-L1 and PD-L2 had different expression levels in hypopharyngeal cell carcinoma and were mainly expressed in cytoplasm.However,no expression were detected in squamous epithelum;2.IL-10 had different expression levels both in hypopharyngeal cell carcinoma and squamous epithelum and was mainly expressed in cytoplasm.Besides,IL-10 expression was significantly correlated with primary site and differentiation degree of tumor;3.IL-10 significantly correlated with PD-L2 in following groups: ?60-year-old group,N2 stage group,IV grade group and low differentiation group;4.CD19 and IL-10 were apparently co-expressed in hypopharyngeal cell carcinoma;5.Peripheral blood lymphocytes promoted the m RNA expression of PD-L1 and PD-L2 in FaDu cells,but not HIF-1? or HIF-2?;6.Fa Du cells promoted IL-10 expression in peripheral blood lymphocytes.Conclusion:1.PD-L1 and PD-L2 are highly expressed in hypopharyngeal cell carcinoma.They may become target factors for early diagnosis and more effective treatment of hypopharyngeal cell carcinoma;2.Peripheral blood lymphocytes participate in the immune escape pathway of hypopharyngeal cell carcinoma through PD-L1 and PD-L2;3.CD19+IL-10+Breg is the main type of regulatory B cell in hypopharyngeal cell carcinoma.It promotes hypopharyngeal cell carcinoma progression by highly expressing IL-10.