The Immune Micro-environment Of Hepatocellular Carcinoma

Objective: To learn the infiltration of immune cells and secretion of some cytokines in liver micro-environment in patients with primary hepatocellular carcinoma.Method:1.Collecting 76 cases of fresh tumor tissues (from the edge of cancer <1cm) and the corresponding non-tumor liver tissues(from the edge of cancer> 5cm) in primary hepatocellular carcinoma patients who received surgical resection, removing bloodstained and necrotic tissues, fully homogenating the samples by tissue samples apparatus, then centrifuging and preserving the supernatant, analyzing the percentage of CD4 + T cells, CD8 + T cells, Treg cells, B lymphocytes, NK, macrophages by flow cytometry. 2. At the same time, investigating the number and distribution of the above-mentioned immune cells by immunohistochemistry on specimens of 30 cases from these cases.3. Detecting the levels of Th1/Th2 type cytokines by CBA assay in the supernatants, the levels of TGF-β1 and VEGF by ELISA 4.analyzing data with patients clinical figures by SPSS.Results: 1. The percentages of CD3 + T cells, CD3 + CD4 + T cells, CD4 + CD25 + CD127-regulatory T cells and CD3 + CD45RO +, CD8 + CD45RO + memory T cells in tumor tissues were higher than the percentages in the corresponding non-tumor tissues in HCC patients (P <0.005; P <0.001; P <0.001; P <0.001; P <0.001), on the contrary, the percentage of CD3 + CD8 + T cells in tumor tissues was lower than the corresponding non- tumor tissues (P <0.001).The expression of CD69 which an early activation marker in CD3 + T lymphocytes and CD56 + NK cells in tumor tissues was lower compared with in the corresponding non- tumor tissues (P <0.005; P <0.001),but the expression of HLA-DR, a late activation marker in CD3 + T cell, in tumor tissues was higher than the corresponding non- tumor tissues (P <0.005). 2. The numbers of CD3 + T cells, CD4 + T cells, FoxP3+ regulatory T cells and CD45RO + memory T cells in tumor tissues were more than the corresponding non- tumor tissues (P <0.05);the number of CD8 + T cells in tumor tissue was fewer than the corresponding non- tumor tissues (P <0.05). The numbers of CD68 + macrophages and CD56 + NK cells in tumor tissues were fewer than in the corresponding non- tumor tissues (P <0.001; P <0.05) . 3. The levels of IL-10, TGF-β1 and VEGF in the supernatants in tumor tissues were higher than in the corresponding non- tumor tissues (P <0.005; P <0.001), even the level of VEGF in tumor tissues was positively correlated with tumor size. 4. The percentage of CD4 + CD25 + CD127-Treg cells was negatively correlated with the percentages of CD4 + T cells or CD8 + T cells (P <0.05), but positively correlated with the levels of IL-10, TGF-β1 (P <0.05). 5. In tumor tissues ,the percentages of CD3 + CD4 + T cells and CD4 + CD25 + CD127-Treg in less than 60-year-old age group were higher than in more than 60-year-old group (P <0.005; P <0.05),and the percentages of CD3 + CD8 + T cell in less than 60-year-old age group was lower than in more than 60-year-old group (P <0.05); the percentage of CD4 + CD25 + CD127-Treg cells in the HBsAg or HBeAg-positive group is higher than the corresponding HBsAg, HBeAg negative group (P <0.05 ), respectively. The percentages of immune cells were not correlated with tumor size, with or not violating of blood vessels, TNM stage, histological grade, with or not liver cirrhosis and levels of ALT/AST preoperatively .Conclusions: 1. Tumor can induce lymphocytes infiltration in tumor tissues. Even though HCC tissues had higher percentages of CD3 + T and CD4 + T cells compared with the non-tumor portion, but the proportion of CD8 + T cells which played an important role at the anti-tumor immune response was significantly decreased, while the proportion of Treg cells which exerted inhibitory effects on the immune response increased. At the same time the activations of T cells and NK cell were inhibited, resulting in the suppressive immune micro-environment in HCC.2. In HCC tissues, macrophages may be mainly M1-type and exert anti-tumor effects.3. Free cytokines in HCC tissues were inhibitory factors suppressing cell-mediated immune response, expressions of IL-10,TGF-β1 and VEGF inhibiting the immune increased in tumor micro-environment.