Regulatory T Cells Affect The Anti-Tumor Activity Of Oligoclonal Tumor-Infiltrating Lymphocytes From Human Hepatocellular Carcinoma

[Background and Objective]Hepatocellular carcinoma is a common malignant tumor in China. Only surgical treatment is not ideal and suitable for the middle and terminal cancer patients.Therefore, academician Wu Mengchao proposed that the comprehensive treatments were preferred for advanced liver cancer patients. Adoptive cellular immunotherapy (ACI) is one of the comprehensive treatments of liver cancer, and the tumor infiltrating lymphocyte (TIL) cells are the main effectors in ACI. Because of its specificity and efficiency in anti-tumor therapy, clinical reasearchers has paid more attentioned to ACI treatment.How to access a large number of TIL cells which have high anti-tumor activities is critical for ACI technology. Although the preparation methods of TIL can obtain a large number of cells by now, they still have some problems, such as unable to selectively amplify the TIL groups with strong anti-tumor activity. Therefore, the study attempts to establish an oligoclonal TIL culture method for the selective amplification of a strong anti-tumor activity of TIL cell groups, as well as to to test and verify the relationship between anti-tumor activity of TIL and regulatory T cells (T regulation cell, Treg).Therefore, provide a theoretical basis and experimental basis for the oligoclonal cultivation method of TIL.[Content and Methods]1,Established method of TIL's isolation, culture and assessment: Hepatocellular carcinoma tissue blocks (diameter of about 2 mm) were took from different parts of a fresh tumor tissue. The sampling region covered the central area and boundary areas between tumor and normal tissue (2 mm inside and outside the boundary line). The tissue blocks were placed in a 24-well plate then a specific medium were added into every wells. The plate were stored at 5% CO2 and 37℃condition for culture. In order to identify oligoclone TIL's anti-tumor activity, the enzyme-linked immunosorbent assay were used to detect the secretion of IFN-γ.2,The distribution characteristics of Treg in tumor tissue:The distribution and density of Treg in hepatocellular carcinoma were detected by using Immunohistochemistry method (IHC). Meanwhile the experimental data were analysis by statistical process. 3,Relationship between the proportion of Treg in TIL and anti-tumor activity of TIL: The enzyme-linked immunosorbent assay was used to detect the secretion of IFN-γand flow cytometry were used to detect the proportion of Treg in oligoclonal TIL culture.The experimental data were analysis by statistical process.4,Relationship between the proportion of Treg in TIL and the culture time of TIL: The oligoclonal TIL cells were cultured and observed in a sequential order.Cells were collected respectively when cultivated at the second week, third week, fourth week and the fifth week. The proportions of Treg in TIL were detected by flow cytometry and all the experimental data were analysis by statistical process.[Results]1,Isolation and culture of TILDuring the oligoclonal culture of TIL, TIL gradually moved out of the tumor tissue then a dense cluster of lymphocytes were formed surround the tumor tissue blocks. With the extension of the culture time, the numbers of lymphocytes were increased in culture plate. Approximately 2 weeks later, lymphocytes were converged together in every well of 24-well plates.2,The distribution characteristics of Treg in tumor tissueBy immunohistochemical stain method, FoxP3+ cells were distributed unevenly in the whole tumor tissue slices. In a single high power field, the number of Treg in the solid parts of tumor was 34.32±6.24; however, the number of Treg was 4.32±2.66 in non-tumor tissues which close to tumor. The results indicate that the numbers of Treg were different between the central area and peripheral area(P=0.009<0.05).3,Relationship between the proportion of Treg in TIL and anti-tumor activity of TILIFN-γwas the main cytokines which showed the tumor-killing effect and released by TIL. The percentage of IFN-γwas positively correlated with TIL anti-tumor activity. Therefore, detected the emissions of IFN-γin oligoclonal culture of TIL can analyzed the strength of its anti-tumor activity. This partial results showed that the proportion of Treg in TIL and the emissions of IFN-γhad a negative correlation (P<0.001), correlation coefficient was -0.650. 4,Relationship between the proportion of Treg in TIL and the culture time of TILBy flow cytometry technology, the proportion of Treg in TIL cells which collected in a sequential order was analysis. Results showed that compared with the second week's data, proportion of Treg in TIL decreased significantly in the third week (P<0.001). On the week 4, Treg proportion of TIL decreased significantly (P=0.001) compared with the third week's data. Compared with Fourth and fifth week's data of the Treg's percentage in TIL, the differences are not statistically significant (P= 0.148).[Conclusions]1,Oligoclonal TIL culture method can selectively amplify the TILs with high anti-tumor activity.2,The density of Treg is much higher in the internal area of tumor tissues than in the peripheral area. A large number of Treg distributed in tumor interstitial of hepatocellular carcinoma. While in the peripheral area, Treg were infiltrating sparsely and dispersively.3,The percentage of Treg in TIL was inversely proportional to anti-tumor activity of TIL.4,During the process of TIL cultivation in vitro, the proportion of Treg was declined with the extension of culture time.