The Significance Of TNFR2 Signaling In The Progression Of Malignant Pleural Effusion

Background:Malignant pleural effusion(MPE)is a common,debilitating complication of the spread of advanced lung cancer to pleura.Regulatory T cells(Tregs)and tumor cells are the major cellular mechanisms that mediate the immunosuppression in MPE by dampening the anti-tumor response,and play predominant roles in the progression of MPE.Previous studies have shown that tumor necrosis factor receptor type II(TNFR2)are highly expressed on tumor-associated Tregs and ovarian cancer,mediating the effect of tumor necrosis factor(TNF),which contibuted to the immunosuppression in the tumor tissues.However,the significance of TNFR2 signalings in the progression of MPE remains unknown.Methods:The distribution of TNFR2~+cells in Tregs and effector T cells(Teffs)in PE,including MPE and tuberculosis pleural effusion(TPE),as well as peripheral blood(PB)were determined.The associations between frequencies of TNFR2~+Tregs in MPE and the clinical characteristics of related patients were investigated as well.The immunosuppressive phenotype of TNFR2~+Tregs in MPE was analyzed.Meanwhile,the effects of TNFR2 agonist and the TNF-TNFR2 interaction on the immunosuppressive function of Tregs was explored.Besides,the source of TNF in MPE was identified.The expression of chemokine receptors on TNFR2~+Tregs were analyzed and the concentration of the corresponding chemokines in MPE were detected by ELISA.The mechanisms of TNFR2~+Tregs attracted to MPE were investigated in vitro.The expression of TNFR2 on primary tumor cells isolated from MPE and lung adenocarcinoma cell lines were detected by flow cytometry.The effect of TNFR2 on the proliferation and apoptosis of tumor cells was also tested.The significance of TNFR2 expressed by tumor cells on the immune microenvironment of MPE were investigated.Finally,the safety and efficacy of TNFR2targeted therapy were evaluated in MPE model mice.Results:(1)The level of TNFR2 expression on Tregs in MPE was markedly higher compared with the levels expressed in MPE Teffs,PB Tregs,or TPE Tregs.(2)The frequencies of TNFR2~+Tregs were positively correlated with the number of tumor cells in MPE,as well as the volume of MPE.High frequencies of TNFR2~+Tregs in MPE indicated short survival time and poor performance status for MPE patients.(3)Compared to TNFR2~-Tregs,TNFR2~+Tregs expressed higher levels of immunosuppressive molecules,including T cell immunoreceptor with Ig and ITIM domains(TIGIT),zinc finger protein Helios,cytotoxic T lymphocyte-associated protein 4(CTLA-4)and programmed cell death-ligand 1(PD-L1).(4)Interacting with TNFR2,TNFR2 agonist and tumor necrosis factor(TNF)enhanced the suppressive capacity of Tregs by up-regulating TIGIT,Helios,CTLA-4 and PD-L1 expression.(5)T helper 1(Th1)and T helper 17(Th17)cells are the major source of TNF in MPE,suggesting that MPE Teffs may paradoxically promote tumor growth by boosting MPE Treg activity via the TNF-TNFR2 pathway.(6)Compared with TNFR2~-Tregs,CXC-type chemokine receptor 6(CXCR6),CC type chemokine receptor 4(CCR4)and CC type chemokine receptor 6(CCR6)was upregulated in TNFR2~+Tregs.Moreover,there was a positive correlation between the expression of CXCR6 and TNFR2.(7)The concentrations of CXC-type chemokine ligand 16(CXCL-16),CC type chemokine ligand 20(CCL-20)and CC type chemokine ligand 22(CCL-22)in MPE were much higer compared with paired PB,while the concentration of CXCL-16 was the highest among them.(8)After blocking the activity of CXCL-16 in MPE,the chemotactic effect of MPE on TNFR2~+Tregs was significantly reduced.(9)TNFR2 was highly expressed in lung adenocarcinoma cell lines as well as the tumor cells isolated from patients with MPE caused by lung adenocarcinoma.The apoptosis of tumor cells induced by IFN-?was significantly decreased after TNFR2 agonist treatment.(10)TNF-TNFR2 interaction promotes the secretion of IL-33,CCL-5,CXCL-1 and CXCL-10 in tumor cells.(11)Overexpression of TNFR2 in tumor cells increased the infiltration of Tregs in MPE.(12)In MPE mice model,the volume of MPE were markdly reduced and the survival time were significantly prolonged after TNFR2 blockade in vivo.(13)In addition,the proportions of interferon gamma(IFN-?)-producing cytotoxic T lymphocytes(CTLs)present in MPEwere increased significantly after TNFR2 blockade,while the number of CD8~+CTL present in periphery were not affected.Conclusions:Our data expanded the immunosuppressive mechanisms induced by Tregs and tumor cells in MPE,and provide a novel insight for the diagnosis,disease evaluation and treatment of MPE.