Research Of Detecting Regulatory T Cell In The Peripheral Blood Of Patients And Adoptive Transfer Retro Nectin Activated NK Cells In Vitro In Pancreatic Cancer

[English Abstract]Research o f Detecting Regulatory T Cell in the Peripheral Blood of Patients and adoptive transfer RetroNectin activated NK cells in vitro in Pancreatic cancer M.D. Student:zhou jian guo Supervisor:zhao pingAbdominal Surgery Department, Cancer Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China[Background and Purpose]Immunotherapy is an important mode of treatment for cancer. Regulatory T cells (Treg) is an immunosuppressive cell, which plays an important role in tumor immunity. How to define immunosuppressed state of the tumor-bearing individuals associated with clinicopathologic features and therapeutic efficacy, and How to effectively acquire proliferation and broad-spectrum effector cells in vitro have been the main problems in cancer immunotherapy. In this study, the impact and significance of immunosuppressed state of pancreatic cancer is discussed by detecting the expression level of Treg in peripheral blood of pancreatic cancer and inhibitory effect of the highly efficient natural killer cells cultured by RetroNectin method in vitro.[Materials and Methods]62 peripheral blood samples of pancreatic cancer patients and 30 samples of healthy volunteers were collected from October 2006 to March 2009 in our hospital. Ratio difference of CD4+CD25high regulatory T cells was determined by flow cytometry. The relationship between the prevalence of Treg cells and pancreatic cancer staging and prognosis were Analysed. CD56+T cells of peripheral blood were acquired by immunomagnetic bead separation, and then NK cell were cultured by RetroNectin method. killing assay in vitro with JF-305 human pancreatic cancer cells was detected by MTS method. The impact of RetroNectin method for the killing of effector cells was Analysed by detecting phenotype, proliferation and anti-determined kill rate.[Results]The prevalence of CD4+CD25high regulatory T cells in CD4+T cells in the peripheral blood of patients with pancreatic carcinoma was(15.16±13.97)%, which was significantly higher than the level of healthy controls (3.38±1.70)%(P＜0.001).The proportions of Treg cells in the patients with pancreatic carcinoma in stageⅠ-Ⅱ(14.53±6.95)%(n=13) stageⅢ(17.75±18.66)%(n=31), stageⅣ(18.48±21.27)% (n=18), were all significantly higher when compared with healthy controls (P<0.01). Univariate analysis showed that there was no relationship between the prevalence of Treg cells with age, sex, CA199, bilirubin level and TNM staging. The proportion of CD4+CD25high regulatory T cells at the the 7th day after operation was signigicanfly lower than that level before operation with radical resection or palliation treatment. Univariate analysis didn't showed that it's the relationship with prognosis. Multivariate analysis indicated that only TNM stage was independent prognostic factor that influenced survival.Results in vitro showed that Proliferative activity of effector cells increased 371-folds, which cultured by Anti-CD3 antibody and RetroNectin method. while the detection changed significantly in phenotype. CD56+T/CD56+CD16+T cell ratio was significantly higher than control group and Anti-CD3 antibody+RetroNectin group. Killing assay was observed with effector:target ratios from 10 to 40.The results of showed that RetroNectin group's kill rate were significantly different compared with other groups on every different effector target ratio.[Conclusion]Prevalence of Treg cells in peripheral blood of patients with pancreatic cancer significantly increased when compared with normal donors. Immunosuppression in patients with pancreatic cancer may be closely related to the increase in Treg cells. In compare with the proportion before resection and at first week after operation,the proportion of CD4+CD25high regulatory T cells descends by radical resection or palliation treatment. The proportion of CD4+CD25high regulatory T in peripheral blood was not related to prognosis.Further experiments showed that the immune killing effect of the NK cells cultured by RetroNectin method.be enhanced significantly. When added Anti-CD3 antibody, Proliferative activity of effector cells increased. RetroNectin method can be more efficient access to effector cells. This study shows good prospect of Culturing effector cells by combining removal of Treg cells and efficiently RetroNectin method for immunotherapy is a feasible idea. It may provide theoretical and experimental basis for building a new adoptive immunotherapy model for pancreatic cancer in future.