Background:TOSO,also named Fas inhibitory molecule 3(FAIM3),has recently been identified as an immunoglobulin M(IgM)Fc receptor(FcμR).Previous studies have shown that TOSO is specifically over-expressed in chronic ly...Background:TOSO,also named Fas inhibitory molecule 3(FAIM3),has recently been identified as an immunoglobulin M(IgM)Fc receptor(FcμR).Previous studies have shown that TOSO is specifically over-expressed in chronic lymphocytic leukemia(CLL).However,the functions of TOSO in CLL remain unknown.The B-cell receptor(BCR)signaling pathway has been reported to be constitutively activated in CLL.Here,we aimed to investigate the functions of TOSO in the BCR signaling pathway and the pathogenesis of CLL.Methods:We over-expressed TOSO in B-cell lymphoma cell lines(Granta-519 and Z138)by lentiviral transduction and knocked down TOSO by siRNA in primary CLL cells.The over-expression and knockdown of TOSO were confirmed at the RNA level by polymerase chain reaction and protein level by Western blotting.Co-immunoprecipitation with TOSO antibody followed by liquid chromatography coupled with tandem mass spectrometry(IP/LCMS)was used to identify TOSO interacting proteins.Western blotting was performed to detect the activation status of BCR signaling pathways as well as B-cell lymphoma 2(BCL-2).Flow cytometry was used to examine the apoptosis of TOSO-over-expressing B lymphoma cell lines and TOSO-down-regulated CLL cells via the staining of Annexin V and 7-AAD.One-way analyses of variance were used for intergroup comparisons,while independent samples t tests were used for two-sample comparisons.Results:From IP/LCMS,we identified spleen tyrosine kinase(SYK)as a crucial candidate of TOSO-interacting protein and confirmed it by co-immunoprecipitation.After stimulation with anti-IgM,TOSO over-expression increased the phosphorylation of SYK,and subsequently activated the BCR signaling pathway,which could be reversed by a SYK inhibitor.TOSO knockdown in primary CLL cells resulted in reduced SYK phosphorylation as well as attenuated BCR signaling pathway.The apoptosis rates of the Granta-519 and Z138 cells expressing TOSO were(8.46±2.90)%and(4.20±1.21)%,respectively,significantly lower than the rates of the control groups,which were(25.20±4.60)%and(19.72±1.10)%,respectively(P<0.05 for both).The apoptosis rate was reduced after knocking down TOSO in the primary CLL cells.In addition,we also found that TOSO down-regulation in primary cells from CLL patients led to decreased expression of BCL-2 as well as lower apoptosis,and vice versa in the cell line.Conclusions:TOSO might be involved in the pathogenesis of CLL by interacting with SYK,enhancing the BCR signaling pathway,and inducing apoptosis resistance.展开更多
T cell activation-induced cell death(AICD),that involves the induction of Fas-mediated apoptosis,is very important for the maintenance of immune homeostasis.TOSO was firstly described as an inhibitor of Fasmediated ap...T cell activation-induced cell death(AICD),that involves the induction of Fas-mediated apoptosis,is very important for the maintenance of immune homeostasis.TOSO was firstly described as an inhibitor of Fasmediated apoptosis and overexpressed in chronic lymphocytic leukemia.Recently,TOSO was identified as IgM FcR.In this study,we produced anti-TOSO monoclonal antibody(mAb)that could block the binding of IgM to TOSO and found that T cell apoptosis is negatively correlated with TOSO expression during T cell activation.Treatment of activated T cells with anti-TOSO blocking mAb promoted T cell AICD in in vitro AICD model,and treatment of xenogeneic-GVHD mice with the antibody also increased the sensitivity of activated T cells to Fasinduced apoptosis,which was accompanied by reduction of c-FLIPL expression and up-regulation of AP-1 complex.In summary,our data indicate the anti-apoptotic effect of TOSO in T cell AICD and open up new therapeutic prospects for the treatment of hematologic malignancies and immune disorders.展开更多
基金This work was supported by grants from the National Nature Science Foundation of China(Nos.81200395 and 81970187)Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences grants(No.2017-I2M-3-018)Fundamental Application and Advanced and Technology Research Program of Tianjin(No.15JCYBJC25100)。
文摘Background:TOSO,also named Fas inhibitory molecule 3(FAIM3),has recently been identified as an immunoglobulin M(IgM)Fc receptor(FcμR).Previous studies have shown that TOSO is specifically over-expressed in chronic lymphocytic leukemia(CLL).However,the functions of TOSO in CLL remain unknown.The B-cell receptor(BCR)signaling pathway has been reported to be constitutively activated in CLL.Here,we aimed to investigate the functions of TOSO in the BCR signaling pathway and the pathogenesis of CLL.Methods:We over-expressed TOSO in B-cell lymphoma cell lines(Granta-519 and Z138)by lentiviral transduction and knocked down TOSO by siRNA in primary CLL cells.The over-expression and knockdown of TOSO were confirmed at the RNA level by polymerase chain reaction and protein level by Western blotting.Co-immunoprecipitation with TOSO antibody followed by liquid chromatography coupled with tandem mass spectrometry(IP/LCMS)was used to identify TOSO interacting proteins.Western blotting was performed to detect the activation status of BCR signaling pathways as well as B-cell lymphoma 2(BCL-2).Flow cytometry was used to examine the apoptosis of TOSO-over-expressing B lymphoma cell lines and TOSO-down-regulated CLL cells via the staining of Annexin V and 7-AAD.One-way analyses of variance were used for intergroup comparisons,while independent samples t tests were used for two-sample comparisons.Results:From IP/LCMS,we identified spleen tyrosine kinase(SYK)as a crucial candidate of TOSO-interacting protein and confirmed it by co-immunoprecipitation.After stimulation with anti-IgM,TOSO over-expression increased the phosphorylation of SYK,and subsequently activated the BCR signaling pathway,which could be reversed by a SYK inhibitor.TOSO knockdown in primary CLL cells resulted in reduced SYK phosphorylation as well as attenuated BCR signaling pathway.The apoptosis rates of the Granta-519 and Z138 cells expressing TOSO were(8.46±2.90)%and(4.20±1.21)%,respectively,significantly lower than the rates of the control groups,which were(25.20±4.60)%and(19.72±1.10)%,respectively(P<0.05 for both).The apoptosis rate was reduced after knocking down TOSO in the primary CLL cells.In addition,we also found that TOSO down-regulation in primary cells from CLL patients led to decreased expression of BCL-2 as well as lower apoptosis,and vice versa in the cell line.Conclusions:TOSO might be involved in the pathogenesis of CLL by interacting with SYK,enhancing the BCR signaling pathway,and inducing apoptosis resistance.
基金supported by the National Key Basic Research Program of China(2012CB917101)the National Natural Science Foundation of China(30771968 and 81000920)
文摘T cell activation-induced cell death(AICD),that involves the induction of Fas-mediated apoptosis,is very important for the maintenance of immune homeostasis.TOSO was firstly described as an inhibitor of Fasmediated apoptosis and overexpressed in chronic lymphocytic leukemia.Recently,TOSO was identified as IgM FcR.In this study,we produced anti-TOSO monoclonal antibody(mAb)that could block the binding of IgM to TOSO and found that T cell apoptosis is negatively correlated with TOSO expression during T cell activation.Treatment of activated T cells with anti-TOSO blocking mAb promoted T cell AICD in in vitro AICD model,and treatment of xenogeneic-GVHD mice with the antibody also increased the sensitivity of activated T cells to Fasinduced apoptosis,which was accompanied by reduction of c-FLIPL expression and up-regulation of AP-1 complex.In summary,our data indicate the anti-apoptotic effect of TOSO in T cell AICD and open up new therapeutic prospects for the treatment of hematologic malignancies and immune disorders.
