Research Of Costimulatory Molecule B7-H1 On The Immunoescape And Immunotherapy Of Bladder Cancer

Interactions between the immune system and malignant cells play an important role intumorigenesis.Failure of the immune system to detect and reject transformed cells maylead to tumor development and tumors can use multiple mechanisms to escape fromimmune-mediated rejection.The study of tumor immuoescape mechanisms is always a hotspot and nodus on the way of tumor treatment.T-cell mediated cytoimmunity is a main form of host antitumor immunity.Recognitionand presentation of tumor antigen and activation of tumor specific T-cell is necessary forthe start of tumor immune response.Tumor cells can promote the anergy or tolerance andeven apoptosis of T cells in order to escape host immune destruction by overexpressionor/and absence some immune molecules.B7-H1 is a recently discovered T-cell suppressivecostimulatory molecule that has been implicated as an important molecule in tumorimmunoescape.It can inhibit immune responses by inducing T-cell apoptosis,impairingcytokine production,and diminishing the cytotoxicity of activated T cells.A lot of humancancers,including lung,ovarian,colon,breast,kidney,lymphoma,and melanoma,havenow been reported to aberrantly express B7-H1 and the expression of B7-H1 was stronglyassociated with the genesis,development and aggressive biological behavior of tumors.Bladder cancer is the most common malignant tumor of urology in China.The highrecurrence rate and aggressive biological behavior after operation indicate that there are still some mechanisms which can promote bladder cancer cells escaping the lethal effect ofimmune cells.B7-H1 has been a hot spot in the field of tumor research for its importantrole in tumor immunoescape.However,it is not clear that whether B7-H1 is aberrantlyexpressed in human bladder cancer,whether and how B7-H1 plays an important role inimmunoescape of bladder cancer and whether the manipulation of B7-H1 could become abeneficial target for immunotherapy in human bladder cancer.Therefore,this research investigated the expression of B7-H1 in bladder cancer tissuesand the effects of B7-H1 overexpression on immunoescape of bladder cancer in vitro.Moreover,we further investigated the effects of B7-H1 blockade on the antitumorimmunity mediated by tumor antigen specific CTL and two immune effector cells (CD3AKcells and CD3/CD28-costimulated T cells) in order to explore a new strategy ofimmunotherapy in bladder cancer.PartⅠExpression of B7-H1 in bladder cancer tissue and itsclinical significanceObjective:To investigate B7-H1 mRNA and protein expression in bladder cancer tissueand its clinical significance.Methods:RT-PCR method was used to detect B7-H1 mRNA expression in 20 cases ofbladder cancer and immunohistochemical method was used to detect B7-H1protein and FasL protein expression in 50 cases of bladder cancer.Therelationship among B7-H1 expression,biological characters of bladder cancerand FasL expression was also analyzed.Meanwhile,the relationship betweenB7-H1 expression and survival time of bladder cancer patients was alsodetected using survival analysis.Results:There was no B7-H1 expression in nomal bladder tissue.However,B7-H1 expression increased significantly in bladder cancer.B7-H 1 mRNA and proteinexpression were strongly associated with pathological grade and clinical stageof bladder cancer.Meanwhile B7-H1 protein expression was associated withrecurrence of bladder cancer and had a positive correlation with FasL proteinexpression.The survival rate was significantly worse in patients with B7-H1protein positive expression than in those with B7-H1 protein negativeexpression and multivariable analysis revealed that B7-H1 protein expressioncould be regarded as an independent factor in evaluating the prognosis ofbladder cancer.Conclusion:B7-H1 expression is strongly associated with neoplastic progression andpoor prognosis of bladder cancer.B7-H1 may become one of parameters forunderstanding the biological behavior of bladder cancer.The basis for theseassociations may relate to the recognized ability of B7-H1 to involve in tumorimmune escape by upregulation of FasL and then inducing T-cell apoptosis.PartⅡExpression of B7-H1 on BIU-87 cell line and the inductionby LPS in vitroObjective:To investigate B7-H1 mRNA and protein expression on BIU-87 cell line and theinduction by LPS in vitro.To simulate bladder cancer microenvironment withLPS-induced high expression of B7-H1 on BIU-87 cell line and makepreparation for the next research.Methods:B7-H1 mRNA and protein on BIU-87 cells were detected by RT-PCR andWestern blot methods respectively.Then various concentrations of LPS wereadded into the culture system to induce B7-H1 expression on BIU-87 cells. After that B7-H 1 mRNA and protein expression were also detected by RT-PCRand Western blot method respectively.Results:B7-H1 mRNA and protein were constitutively expressed on BIU-87 cells at alow level.But after induced by LPS of various concentrations,B7-H1 mRNAand protein were further up-regulated in a concentration-dependent manner,with the most pronounced effect observed at 1μg/ml.Conclusion:B7-H1 was constitutively expressed on BIU-87 cells at a low level and wasfurther up-regulated after induced by LPS.We could get B7-H1 high expressedBIU-87 cells by LPS induction and then simulate bladder cancermicroenvironment in vitro.PartⅢEffect of B7-H1 on immunoescape of bladder cancer invitro and tumor immunotherapy by B7-H1 blockadeObjective:To investigate the effect of B7-H1 on immunoescape of bladder cancer and theeffect of B7-H1 blockade on the antitumor immunity mediated by tumorantigen specific CTL in vitro.Methods:T cells were isolated from peripheral blood and induced to tumor antigenspecific CTL by stimulating with mitomycin inactivated BIU-87 ceils.Andthen tumor antigen specific CTL were co-cultured with BIU-87 cells whichhighly expressed B7-H1 by LPS induction.The cytotoxicity of tumor antigenspecific CTL against BIU-87 cells was measured by MTT method andapoptotic rate of tumor antigen specific CTL was measured by flow cytometrywith PI labeling technique.After that B7-H1 mAbs was used to block B7-H1pathway.The cytotoxicity and apoptotic rate of tumor antigen specific CTL were measured by MTT method and flow cytometry respectively.Results:After co-cultured with BIU-87 cells which highly expressed B7-H1,thecytotoxicity of tumor antigen specific CTL against BIU-87 cells was depressedobviously.But its apoptotic rate increased greatly and its survival time in vitrowas significantly shortened.After B7-H1 blockade,the cytotoxicity of tumorantigen specific CTL returned to a high level and its apoptotic rate decreasedobviously in a concentration-dependent manner.Conclusion:BIU-87 cells can escape the killing effect of tumor antigen specific CTL byinducing CTL apoptosis through B7-H1 pathway.B7-H1 may play animportant role in immunoescape of bladder cancer and the manipulation ofB7-H 1 may become a beneficial target for immunotherapy in bladder cancer.PartⅣEffects of B7-H1 blockade on the cytotoxicity of CD3AK cellsand CD3/CD28-costimulated T cells againstbladder cancer cells in vitroObjective:To investigate the effects of B7-H1 blockade on the cytotoxicity of CD3AKcells and CD3/CD28-costimulated T cells against bladder cancer cells in vitro.Methods:Two immune effector cells,CD3AK cells and CD3/CD28-costimulated T cellswere produced by normal human peripheral blood lymphocytes stimulated withCD3 mAbs and CD3 plus CD28 mAbs respectively.Then B7-H1 mAbs wasadded to block B7-H1 pathway.The proliferation of these two immune effectorcells were measured by ~3H-thymidine incorporation assay and IFN-γ,TNF-αand IL-10 secretion were measured by ELISA method.Meanwhile,thecytotoxicity of these two immune effector cells against BIU-87 cells was measured by MTT method.Results:Blockade of B7-H1 greatly promoted the proliferation of CD3AK cells andCD3/CD28-costimulated T cells and extended their survival time in vitro.Italso significantly enhanced their IFN-γand TNF-αsecretion but suppressedIL-10 secretion.Meanwhile,the cytotoxicity of CD3AK cells andCD3/CD28-costimulated T cells against BIU-87 cells was significantlyenhanced.Conclusion:Blockade of B7-H1 can promote the proliferation and activation of CD3AKcells and CD3/CD28-costimulated T cells.It can also improve the antitumorimmunity mediated by them against bladder cancer.The manipulation ofB7-H1 may become a new strategy to improve the therapeutic effect ofadoptive cellular immunotherapy against bladder cancer.