| Research Objectives: Renal cell carcinoma(RCC)is one of the most common tumor in urinary system.It ranked seventh in the incidence of malignant tumor in male,and was rising at the rate of 2-4% every year.Surgery is the main treatment for patients with RCC,and the good curative effect had been obtained in early phase of RCC.But for a large number of patients with RCC,recurrence and metastasis are still the main factors influencing the patients' survival rate and mortality after resection of the primary lesions.RCCs presented high resistance to radiation and chemotherapy.Once metastases occur,the 5-year survival rate is only 5-10%.Therefore,it is of great significance to further explore new methods of anti-RCC and enhance the anti-cancer ability of human body.T cell immune globulin mucin molecular 3(Tim-3),is a member of the immunoglobulin super family that presents high-level expression in fully-differentiated Th1 lymphocytes,and it is a specific cell markers of Th1 CD4+ cells.Tim-3 can inhibit the production of IL-2 and gamma-IFN,then accelerate the proliferation of tumor cells.Studies have shown that Tim-3 heavily express in the CD4+ and CD8+ T cells of partial patients and animals with cancer(gastric cancer,colorectal cancer,etc.),which have the effect of negatively regulating T cell immune response,and lead to the death of T lymphocytes and weakness of its function,eventually the body's anti-tumor immunity were weakened.But it has not been reported what role Tim-3 plays in patients with RCC,which immune cells Tim-3 can regulate and what the specific mechanism is.Polymorphism was defined as two or more discrete variant genotypes or alleles simultaneously and frequently exist in a biological group,with single nucleotide polymorphism being one of the most common type.Gene polymorphism in clinical practice mainly reflects in clarifying the body`s susceptibility and tolerance to disease and poison,the diversity of clinical manifestations of diseases,as well as the reactivity to drug therapy.Now the polymorphisms about Tim-3 have been reported,but the correlation between Tim-3 and the susceptibility of RCC has not been reported yet.Based on the above-mentioned elucidation,this study aims to investigate the relationship between the polymorphism of Tim-3 and RCC susceptibility,to analyze the distribution of Tim-3 and explore the relationship between the Tim-3 and Th1,CD4 +,CD8 + Treg function,regulatory B cells and other immune cells in RCC patients with different stages,then to explore the function and mechanism of how Tim-3 is involved in cellular immunity,and to further clarify whether the function of Tim-3 is different in in RCC patients with different stages.Through all these designed jobs,this study try to lay theoretical basis for exploring the new treatment on kidney cancer.Method: Part 1:We collected the blood samples from 322 patients with kidney cancer and 402 healthy persons,sequenced the three polymorphisms of genes(-1516-G/T-574-G/T,and + 4259 T/G)of Tim-3 for genotyping by polymerase chain reaction-restriction fragment length polymorphism analysis technique,then we evaluated whether there was correlation between the polymorphisms of Tim-3 and the susceptibility of kidney cancer in China.Part 2: We collected the tumor tissue and peripheral blood samples from 30 patients with kidney cancer,10 healthy volunteers as control group.Then using flow cytometry,real time quantitative PCR techniques,we did the research on the expression and function of Tim-3 on TIL CD4+ T cells and TIL CD8+,which is used to clarify the role of Tim-3 in tumor infiltrating lymphocytes in the kidney cancer.Part 3:We respectively collected tumor tissue and peripheral blood samples from 8(I + II stage)patients and 7(III + IV stage)patients with kidney cancer,and documented tumor size and presence of distant metastasis and so on.We used cell separation,ELISAs and T cell proliferation and inducing inflammatory cytokines detection methods,to explore distribution of all kinds of IL-10+ cells in peripheral blood and tumor tissue,the phenotypic of IL-10 expression B cells in tumor infiltrating lymphocytes,secretion level of antibodies in tumor infiltrating type B cell,immunosuppression function of IL-10 expression B cell in tumor tissue,and its relevance to kidney cancer clinical progress,Tim – 3,respectively.Results: Part 1(1)The prevalence of TIM-3-1516G/T SNP did not show any significant difference between the RCC cases and the controls,but The-574 GT and + 4259 TG genotypes had significantly higher patients numbers than controls.Similarly,the-574 T allele and + 4259 G allele also increased in RCC patients;(2)The TIM-3-574G/T and + 4259T/G SNPs were in strong LD with each other,whereas the-1516G/T and-574G/T,or-1516G/T and + 4259T/G were not in LD.Among them,a significantly higher frequency of TTG haplotype was observed in the RCC case group than in the control group;(3)The prevalence of + 4259 G allele was significantly increased in the patients with metastasis than the cases without metastasis,while these polymorphisms did not show any significant differences in RCC cases with diabetes or tumor histology status.Part 2(1)The expression of Tim-3 was significantly up-regulated on TIL CD4+ T cells and TIL CD8+ T cells in RCC patients,and Tim-3 on TILs is correlated with higher stages of RC;(2)The expression of GATA3 and IFN-? was significantly lower in the TIL Tim-3(+)fraction,but the expression of T-bet was significantly lower higher.The research showed that phosphorylation of STAT5 in TIL Tim-3+ CD8+ T cells decreased by 60.5 % compared to the Tim-3(-)fraction,whereas phosphorylation of p38 in TIL Tim-3(+)fraction decreased by 30.6 % than in the Tim-3(-)fraction.These results suggest impaired Stat5 and p38 signaling pathways in Tim-3(+)TIL T cells of RCC.(3)Data revealed that percentage of CFSE(dim)cells was significantly increased in s Tim-3-treated TIL CD4+ T and CD8+ T cells compared to controls,indicating that blocking Tim-3 pathway may enhance TIL proliferations.In addition,presence of s Tim-3 clearly up-regulated expression of IFN-? in both TIL CD4+ T and CD8+ T cells,suggesting an elevated function of these cells.Part 3(1)Flow cytometry analysis demonstrated that IL-10-producing cells were found in both T cell(CD3+)and B cell(CD19+)compartments of circulating lymphocytes and tumor infiltrating lymphocytes.The relative distribu-tion of IL-10+ cells in the T cell,B cell,or other(non-T non-B)cell compartments varied by patient,but the relative contribution of IL-10 by B cells was higher in the tumor-infiltrating fraction compared to the circulating fraction.Aslo,T cells and B cells,and found that tumor infiltrating lymphocytes had significantly up-regulated IL-10+ cells compared to their peripheral blood counterparts.No significant corre-lation between the frequencies of peripheral blood IL-10+ cells and tumor infiltrating IL-10+ cells were found,either in total lymphocytes or in T cells and B cells;(2)Compared to that in the peripheral blood,the secretion of IL-10 was increased significantly in tumor infiltrating lymphocytes and the relative contribution of IL-10 from B cells,as a ratio of IL-10 by B cells to IL-10 by T cells,is significantly elevated in tumor;(3)IL-10-expressing B cells had lower CD19 and CD20 expression and higher CD27 expression than non-IL-10-expressing B cells,and were Ig Dnegative and Ig M-negative.Secreted Ig G and Ig A were detected in the supernatant of tumor-infiltrating B cells without stimulation,and were present in higher concentration than that in the supernatant of autologous blood B cells.The relative concentration of Ig A as a fraction of total adaptive antibodies(Ig G+Ig A),in particular,was significantly increased in tumor compared to blood;(4)The frequency of IL-10+ cells in tumor-infiltrating B cells is inversely correlated with the frequency of IFN-?-and TNF-?-producing tumor-infiltrating T cells,and Tim-3 is up-regulated in patients with high frequencies of IL-10+ tumor-infiltrating B cells.Compared to those cultured with blood B cell supernatant,T cells cultured with tumor infiltrating B cell supernatant had significantly reduced IFN-?production and T cell proliferation;(5)The frequency of IL-10-producing B cells was positively correlated with resected tumor size,stage III + stage IV tumors contained significantly higher frequencies of IL-10-producing B cells than stage I+ stage II tumors,and this difference was not due to sex differences between the stage I and stage II group and the stage III +stage IV group.Conclusion This study proved for the first time that Tim-3 SNPs and haplotype increased the risk of RCC in Chinese,and + 4259T/G SNPs were closely associated with metastates of RCC.In tissues of kidney cancer,expression level of Tim-3 affected T cell dysfunction and has obvious inhibitory effect on cytokines secretion of immune cell and killing tumor cells.B cell secreting IL-10 is a new subtype in plasma cells and plays a role of immunosuppression on T cell.Tim-3 may be one of functioning targets of B cell,namely B cells inhibited the function of T cells by upregulated the expression of Tim-3 in T cells,which eventually caused immune escape of tumors.These results may provide a theoretical basis to use Tim-3 as a promising way in the therapy of RCC,and may suggest that adumbrative antibodies against Tim-3 would become a new immunotherapy method for treating renal cell carcinoma in the future,and provide a new path for the treatment of renal cancer. |
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