The Quantity,Function And Anti-tumor Effect Of Mucosal Associated Invariant T Cells In Patients With Bladder Cancer

Background:Bladder cancer is one of the most common malignant tumors in the urinary system.Recently,the incidence and mortality of bladder cancer have risen in China.The treatment of bladder cancer,especially of muscle invasive bladder cancer,is still not effective.After the comprehensive treatment of surgery,radiotherapy and chemotherapy,most patients still had local recurrence or distant metastasis,and eventually died of bladder cancer.Immunotherapy,as the fourth treatment for cancer after surgery,chemotherapy and radiotherapy,is becoming more and more mature.Some new type immunotherapy for bladder cancer has achieved good results in some small scale clinical trials.Cancer immunotherapy is the use of the human's immune system to treat cancer,which can be categorized as active and passive immunotherapy.The active one makes the immune system to attack tumor cells directly by targeting tumor-associated antigens.And the passive one enhances the existing anti-tumor responses by the use of monoclonal antibodies,lymphocytes or cytokines.Therefore,to fully understand the body's immune system,including immune cells,immune effector molecules and their interactions,especially to understand the immune system of cancer patients,is an important basis for improving the therapeutic efficacy of tumor immunotherapy.Mucosal associated invariant T(MAIT)cells are innate-like T cells expressing a semi-invariant T cell receptor(TCR),first found in the intestinal mucosa.MAIT cells occupy a large proportion in T cells,and widely distributed in human organs and tissues.After activation,MAIT cells can produce a variety of cytokines and cytotoxic products,thus causing immune effects.Previous studies showed that MAIT cells are closely related to bacterial infections,viral infections,and autoimmune diseases.It showed that MAIT cells have an antimicrobial effect,and can also cause disorder of mucosal immune in some patients with systemic lupus erythematosus and inflammatory bowel disease.A Few studies have found that MAIT cells have a close relationship with some cancers,such as colorectal cancer,gastric cancer and lung cancer.Unlike the increasing of the number of MAIT cells in colorectal cancer tissues,the IFN-y secreted by MAIT cells was reduced,and the MAIT cells in the peripheral blood did not change significantly or significantly reduced.MAIT cells can induce G2 phase arrest in colon cancer cell lines and have potential role in tumor immune surveillance.In conclusion,whether MAIT cells have antitumor effects is still not clear,and there are conflicting results in different studies,suggesting that MAIT cells may have a dual effect.Although MAIT cells are closely related to human tumors,there has been no study of MAIT cells in patients with bladder cancer.Here we want to study the quantity,function and anti-tumor effect of MAIT cells in patients with bladder cancer.Methods:1.A total of 75 newly diagnosed "bladder cancer" patients and 183 volunteers were enrolled in this study.All participants were excluded from infectious diseases,autoimmune diseases,other neoplastic diseases,and past medical history that could affect the outcome of the study.64 patients with pathologically confirmed bladder cancer were included in the final statistical analysis.Peripheral venous blood samples collected from the participants were isolated by density-gradient centrifugation using Ficoll solution.The isolated cells were stained with the fluorescent-labeled antibodies,and detected to confirm the quantity of MAIT by the flow cytometers.Or the isolated cells were stimulated by adding PMA/Ionomycin and GolgiPlug,then stained with antibodies and detected to confirm the function of MAIT.The fresh bladder cancer tissues from surgical resection were used to extract tumor infiltrating lymphocytes,which were stained with the fluorescent-labeled antibodies,and detected to confirmthe quantity of MAIT in bladder canceer tissues.2.The clinical features of tumors were collected from patients with bladder cancer,including the size and number of tumor found during operation,the histological grade and tumor invasion according to the WHO 2004 grading standards and the seventh edition of TNM staging method of UICC.The number and function of MAIT cells in peripheral blood of bladder cancer patients were analyzed according to the clinical features of tumors.3.pLVX-EF1a-Luc2/GFP-IRES-Puro Plasmids were constructed to prepare Luc2/GFP lentiviral vectors.The T24 cell line of expressing Luc2/GFP was obtained by infecting lentivirus.The expression of GFP and luciferase activity were detected by immunofluorescence and bioluminescence imaging system,which were used to confirm the stability of T24-Luc2/GFP cells and the cell killing assay platform.After MAIT cells were obtained by flow sorting and in vitro amplification,MAIT cells were co-cultured with T24-Luc2/GFP cells at different target effect ratios,and the PBMC control group was set up simultaneously.The killing activity of the cells in vitro was detected by bioluminescence imaging system.Supernatant after MAIT cells culture was added to T24-Luc2/GFP cells to observe the effect on cell growth.A xenograft bladder cancer model based on in vivo imaging technique was constructed by subcutaneous injection of T24-Luc2/GFP cells,and then MAIT cells were injected locally at the tumor site and the change of tumor size were "detected by bioluminescence imaging system.Results:1.The peripheral blood MAIT cells widely exist in healthy people from young to old,which accounts for 1.26%of the CD3+T cells(median;0.10%-7.87%),and 2.33x104/ml of the absolute number(median,0.15-14.55x104ml).With the increasing of age,the proportion of MAIT in CD3+T cells and their absolute numbers were significantly decreased.Correlation analysis showed that the proportion of MAIT cells in peripheral blood CD3+T cells in healthy subjects was negatively correlated with age(r=-0.345,p<0.001).In patients with bladder cancer,the proportion of MAIT in CD3+T cells in the peripheral blood was 1.05%(median,0.10%-5.70%),the absolute number was 0.92x104/ml(median,0.12-3.07x104ml),and there was no significant difference between the bladder cancer patients and the healthy controls matched for age and gender.Subgroup analysis showed that the MAIT cells were mainly CD 8+ subsets(median 72.35%,10.4%-90.0%),and a small number of CD4+ subgroups(median 5.74%,0.55%-16.0%),and there was no significant difference compared with healthy controls.MAIT cells were found in the bladder cancer tissues of 8 radical cystectomy patients,accounting for about 1.99%(median,1.01%-5.75%)of CD45+CD3+ cells.Study on the function of MAIT cells in peripheral blood of bladder cancer patients shown that the proportion of IFN-y producing MAIT cells were significantly higher than that in healthy controls(median:65.65%vs 29.75%,p<0.0001);the proportion of TNF-a producing MAIT cells were significantly higher than that in healthy controls(median:65.85%vs 28.52%,p<0.001);the proportion of IL-2 producing MAIT cells were significantly higher than that in healthy controls(median:13.10%vs 4.41%,p<0.0001);the proportion of IL-17 producing MAIT cells were not significantly different from that of healthy controls(median:0.86%vs 0.88%,p>0.05).2.Stratified analysis showed that the number of MAIT cells in peripheral blood of bladder cancer patients was not correlated with tumor size,number,histological grade and tumor invasion.The proportion of IFN-? producing MAIT cells were significantly correlated with tumor number(tumor multiple group vs single group:median72.65%vs 61.70%,p<0.05)and tumor invasion(invasive bladder tumor group vs non-invasive group:median 73.10%vs 62.50%,p<0.05).The proportion of TNF-??IL-2?IL-17 producing MAIT cells were not correlated with tumor size,number,histological grade and tumor invasion.3.The successfully construction of pLVX-EF1a-Luc2/GFP-IRES-Puro plasmid and Luc2/GFP lentiviral vector made the T24-Luc2/GFP cells prepared.The intensity of fluorescence signals in the T24-Luc2/GFP cells was positively correlated with the number of cells detected by chemiluminescence enzyme analyzer,and the signal was stable within 1-1.5h.That is to say,the stable T24-Luc2/GFP cells and the cell killing assay platform were successfully established.The co-culture of MAIT cells and T24-Luc2/GFP cells showed that with the increase of effector target ratio,the killing efficiency to target cell was enhanced.When the effector target ratio was 20:1 or 40:1,the killing efficiency of MAIT cells was significantly better than that of the same amount of PBMC(p<0.05,p<0.01).Supernatant after MAIT cells culture has a significant inhibitory effect on the growth of T24-Luc2/GFP cells.When the ratio of supernatant to fresh medium was 1:1,1:2 and 1:4,there were significant differences(p<0.01,p<0.05,p<0.05)compared with the control group.The results of in vivo experiments showed that the tumor size was significantly reduced by local injection of MAIT cells(p<0.05).Conclusions:1.Our study found that the number of MAIT cells in peripheral blood of healthy adults was negatively correlated with age.Compared with healthy people matched for age and gander,the number of MAIT cells in peripheral blood of bladder cancer patients did not change significantly.There are MAIT cells distributed in bladder cancer tissues.2.Our study found that the proportion of IFN-?,TNF-? and IL-2 producing MAIT cells in peripheral blood of bladder cancer patients increased significantly,while the proportion of IFN-y producing MAIT was positively related to tumor number and tumor invasion.3.Our study found that MAIT cells can kill human bladder cancer cell line T24 in vitro and in vivo.