Study Of The Anti-tumor Activity And Its Mechanisms Of Oligoclonal Tumor-infiltrating Lymphocytes In H22Tumor-bearing Mice

[OBJECTIVE]Tumor-infiltrating lymphocytes (TIL), the major effector cells with specific and efficient anti-tumor effect of adoptive cellular immunotherapy (ACI), are the heterogeneity lymphocyte subsets exist in tumor interstitium. However, the anti-tumor activity of the freshly extracted and expanded TIL via the conventional method is low, and the clinical efficacy of TIL is not satisfactory. Studies have shown that TIL extracted by conventional way contain a certain number of regulatory T cells (Treg), which can inhibit the anti-tumor activity of TIL. But TGF-beta, IL-10can induce the proliferation of Treg and lead to the TIL-mediated immune bias, which is the important reason of getting the anti-tumor treatment of TIL into trouble. Therefore, how to get a large number of TIL cells with strong anti-tumor activity will be the key to adoptive immunotherapy. This project aims to establish an oligoclonal TIL culture method which can selectively expand TIL cells contain less Treg and with strong anti-tumor activity. Finding out the relationship between the anti-tumor activity of TIL and various kinds of cytokines by experiment in vitro and studying on the anti-hepatoma effect of TIL in mice, which will provide the theoretical and experimental basis for culturing and expanding oligoclonal hepatocellular carcinoma infiltrating lymphocytes to adoptive immunotherapy.[Contents and methods]1. Set up the animal model of hepatocarcinoma H22tumor-bearing miceCollect murine logarithmic growth phase hepatoma cell line H22cells to adjust the cell concentration. They were injected into male BALB/C mice forelimb axillaries skin. The inoculation density was106to107,0.2mL/mouse. Select the average tumor diameter about1.0cm to experiment of TIL extraction and adoptive therapy trial in vivo.2. The separation, cultivation and identification of liver-infiltrating lymphocytesOligoclonal culture method:take the different parts of freshly isolated mice tumor with a volume of2mm3, range from the center to the border region of the tumor (around tumor boundary line about2mm). These tissues cultured in24-well plates with the particular medium and maintained at37℃in an incubator with a5%CO2atmosphere. Conventional culture method:take the different parts of freshly isolated mice tumor with a volume of2mm3, range from the center to the border region of the tumor (around tumor boundary line about2mm). These tissues were digested by various digestive enzymes and passed through a200-gauge stainless steel mesh. Lymphocytes were separated by lymphocyte separation medium via density gradient centrifugation and viable cells were counted with0.2%trypan blue staining, after culture and amplification.3. Adoptive cellular immunotherapySet up tumor-bearing model via murine H22hepatocarcinoma, and select18ectopic xenograft mice with tumor diameter about1.0cm, which were randomly divided into control group, the conventional TIL treatment group and oligoclonal TIL treatment group. These mice were respectively treated with PBS, routinely cultured TIL and oligoclonal cultured TIL by tail intravenous injection, and then observed the growth of mice and the tumor volume. They were sacrificed after30days, and we detected the expression of TGF-beta, IL-10、IFN-y in peripheral blood and FoxP3and IL-17in tumor, and measured tumor weight and the histopathological changes of tumor tissue. All data were statistically analysed.4. the main detection method(1) Detection the purity of TILCD8-positive cells were determined by FCM to identify the purity of TIL separated by different methods.(2) TIL in vitro cytotoxicity assaysTIL were as effector cells and H22were as target cells, in vitro at10:1effector:target ratio, and then we detected the anti-tumor activity of TIL via the different separation methods by tetramethyl-tetrazolium (MTT) test.(3) Determination the expression levels of cytokines in peripheral bloodDetect the expression levels of IFN-y, TGF-beta, IL-10in peripheral blood of mice in the different treatment groups by Enzyme-linked immunosorbent assay (ELISA) method. The experimental data were statistically analyzed.(4) Detection the infiltration of lymphocytes and expression levels of related cytokines in tumor tissueDetect the diversity of lymphocytic infiltration of tumor tissue in mice with different treatment by H&E staining, and detect the FoxP3, IL-17expression levels of tumor tissue in mice with different treatment by immunohistochemical stain. All experimental data were statistically analyzed.[Results]1. Set up the animal model of hepatocarcinoma H22tumor-bearing miceWe observed the growth of tumor in BABL/C mice after3to4days by subcutaneous injection of H22hepatoma cells. We measured tumor diameter about1.0cm with a vernier caliper aftert2weeks.2. The isolation culture and identification of oligoclonal carcinoma infiltrating lymphocytesWe could see the TIL had isolated from the tumor mass with dense lymphocyte population around them in24hours by oligoclonal TIL culture method. The number of TIL cells were growing with time, which were up to1.2×106/mL after2weeks. CD8-positive cells were determined by FCM. The results showed that the CD8-positive cells were (71.25+9.09)%through oligoclonal culture method, which were much higher than (41.89+5.57)%by conventional culture method, and the difference was statistically significant (P <0.01).3. The cytotoxic effect of TIL cell on H22in vitroWe detected the cytotoxic activity of TIL by different culture ways on murine H22hepatoma cells by MTT. The results showed that after cultured for2weeks the oligoclonal and conventional cultured TIL showed the cytotoxic activity of H22cells, the killing rate is (72.56±6.69)%,(46.24±4.03)%, respectively, the difference was statistically significant (P <0.01).4. The inhibitory effect of TIL cells to murine hepatoma xenograftsThe growth of tumor in tumor-bearing mice was suppressed after getting TIL treatment. The tumor volume of the the conventional TIL treatment group and oligoclonal TIL treatment group were(1.172±0.085)cm3,(0.891±0.061)cm3,the difference was statistically significant (P<0.05); tumor weight (1.31±0.22)g,(0.91±0.09)g, the difference was statistically significant (P<0.05). The inhibition rates were (46.13±9.21)%,(62.52±3.76)%, the difference was statistically significant (P<0.05). Compared to the control group (1.714±0.040)cm3,(2.43±0.17)g, the difference was statistically significant (P<0.01).5. Determination the expression levels of cytokinesDetection the expression levels of cytokines in the peripheral blood serum by ELIS A, the expression levels of TGF-beta in the conventional TIL treatment group and oligoclonal TIL treatment group were (241.44±56.01)pg/mL,(65.73±44.79)pg/mL, the difference was statistically significant (P<0.05); the IL-10were (166.52±59.20)pg/mL,(36.66±18.63) pg/mL, the difference was statistically significant (P<0.01); which were lower than the control group (388.21±18.67)pg/mL,(290.74±65.91)pg/mL, the difference was statistically significant (p<0.05); but the expression of IFN-y (538.57±103.43)pg/mL,(876.32±114.52) pg/mL was higher than the control (106.66±2.93)pg/mL, the difference was statistically significant (P<0.05).6. Detection the infiltration of lymphocytes and expression levels of related cytokines in tumor tissueDetect the diversity of lymphocytic infiltration of tumor tissue in mice with different treatment by H&E staining. The result showed that the infiltration of lymphocytes positive rate in tumor of TIL group were higher than the control group after30-day-treatment with TIL(P<0.01),but in the conventional TIL treatment group and oligoclonal TIL treatment group were not different(P=0.330)Detect the FoxP3, IL-17expression levels of tumor tissue in mice with different treatment by immunohistochemical stain. The result showed the positive rate about FoxP3and IL-17in tumor of the control group were the highest than TIL treatment group after30-day-treatment with TIL (P<0.01), and the FoxP3positive rate in tumor of the conventional group was higher than oligoclonal TIL treatment group(P<0.05), the IL-17positive rate in tumor of the conventional TIL treatment group and oligoclonal TIL treatment group were not different(P=0.682)[Conclusion] Compared with conventional TIL culture method, we could obtain much higher concentration of TIL by oligoclonal separation method. Not only the cytotoxic activity of TIL was high on H22in vitro, but also the inhibitory effect was strong on H22hepatoma xenografts growth, which could increase the expression of IFN-y and reduce the secretion of TGF-beta, IL-10, promoting Thl cell immune response and inhibition the proliferation of Treg cells, and then inhibited of tumor growth in vivo.