| Part 1 Distribution and clinical significance of LAP+CD4+T lymphocyte in immunol micro-environment of colorectal cancer ABSTRACTObjiective:To detect the distribution of LAP+CD4+T cells in tumor micro-environment of patients with colorectal cancer, the correlation with CD4+CD25+Treg and clinicopathologic factors were analyzed. Explore the role of LAP+CD4+T cells in colorectal cancer occurrence and development.Methods:From January 2014 to March 2014,50 patients with different stages of CRC, who received surgical treatment in first affiliated hospital of guangxi medical university, department of colorectal surgery, were enrolled in this study. Peripheral blood, colorectal tumor, and nontumor tissues were obtained from 50 patients undergoing primary tumor resection for colorectal adenocarcinoma. The nontumor tissue was obtained near the resection margin of the specimen, which was proved to be free of tumors as diagnosed in the routine pathologic examination. During the same period we collectted the peripheral blood of 25 healthy donors (HD) as control group. Tumor tissue and nontumor tissues were digested by enzymatic means. Peripheral blood mononuclear cells (PBMC) from these patients were isolated by the Ficoll/Paque PLUS density gradient centrifugation method, tumor-infiltrating lymphocytes were isolated over a discontinuous Ficoll gradient method. The percentages of LAP+CD4+T cells and CD4+CD25+Treg cells were measured by flow cytometry. The percentages of LAP+CD4+T cells in peripheral blood of CRC and HD were compared. The percentages of LAP+CD4+T cells in tumor and nontumor tissues were compared; Analyze its correlation with CD4+CD25+Treg cells and clinicopathological factors.Results:The percentages of LAP+CD4+T cells in peripheral blood of patients with CRC were significantly higher than HD(CRC vs. HD:9.44%±3.18% vs. 1.49%±1.00%, P=0.000), and in tumor tissue when compared with nontumor tissue (tumo vs. nontumor tissue:11.76%±3.74% vs.3.87%±1.64%,P=0.000); The level of LAP+CD4+T cells was highest in the tumor tissue; Correlative analysis showed that there was a positive correlation between LAP+CD4+T cells and CD4+CD25+Treg, r=0.327, P=0.02; The percentages of LAP+CD4+T cells were correlative with TNM stage, metastasis, level of CEA, P<0.05.Conclusions:The percentages of LAP+CD4+T cells were increased in peripheral blood and tumor tissues in CRC. Correlative analysis showed that there was a positive correlation between LAP+CD4+T cells and CD4+CD25+Treg. The percentages of LAP+CD4+T cells in tumor tissues were correlative with TNM stage, metastasis, level of CEA, P<0.05; These observations demonstrate that LAP+CD4+T cells could accumulate in the tumor microenvironment and play an important role in tumor progression in patients with CRC.Part 2 The phenotypes and cytokine profiles of LAP+CD4+T cells in colorectal tumor tissues ABSTRACTObjiective:The phenotypes and cytokine profiles of LAP+CD4+T cells in colorectal tumor tissues were analyzed. This study was designed to investigate the molecular mechanisms of LAP+CD4+T cells in colorectal tumor microenvironment.Methods:From April 2014 to June 2014,33 patients with different stages of CRC, who received surgical treatment in first affiliated hospital of guangxi medical university, department of colorectal surgery, were enrolled in this study, colorectal tumor tissues were obtained and digested by enzymatic means. tumor-infiltrating lymphocytes were isolated over a discontinuous ficoll gradient method. Expression of CCR7, CD45RA, Foxp3, CTLA-4, CCR4 and CCR5 were analyzed by flow cytometry. LAP+CD4+T cells and LAP-CD4+T cells were isolated by MACS (magnetic cell sorting), The purity of LAP+CD4+T cells were analyzed by flow cytometry (FCM). The activity of LAP+CD4+T cells and LAP-CD4+T cells were measured by mtrypan blue staining. RT-qPCR technique was applied to detected the expression of cytokine IL-2, IL-4, IL-10, IL-17, IFN-γ and TGF-β. Luminex liquichip technology was applied to detect concentration of cytokine IL-2, IL-4, IL-10, IL-17 and IFN-γ. The concentration of TGF-β was measured by ELISA.Results:The result of FCM show that LAP+CD4+T cells mainly displayed the central memory (CM) phenotype in the peripheral blood in both HDs and patients with CRC, the nontumor tissue also mainly displayed the central memory (CM) phenotype. However, a predominant effector memory (EM) phenotype was found in tumor tissue compared with nontumor tissue. Compared with LAP-CD4+T cells, these LAP+CD4+T cells lack Foxp3 but expressed high levels of CTLA-4, CCR4 and CCR5 (P<0.05); After being sorted by MACS, the enrichment rate of CD4+T cells was 93.48±2.20%, LAP+CD4+T cells was 95.02±2.87%, LAP-CD4+T cells was 94.75±2.76% respectively; Staining by Trypan Blue, the motility rate of LAP+CD4+T cells was 90.16±4.26%, LAP-CD4+T cells was 92.99±2.98%. The RT-qPCR result showed that LAP+CD4+T cells expressed high levels of IL-10 and TGF-β mRNA compared with LAP-CD4+T cells. Result of Liquichip and ELISA showed that LAP+CD4+T cells produced significantly higher levels of IL-10 and TGF-β(P <0.05)Conclusions:These LAP+CD4+T cells lack of Foxp3, but high expression of CTLA-4, CCR4 and CCR5, it was different from traditional CD4+CD25+Treg, has a unique phenotypic characteristics. High expression of immunosuppressive cytokines IL-10 and TGF-β may be involved in tumor immune escape.Part 3 In vitro study on immune regulation mechanism of LAP+CD4+T cells Objective:To investigate the immune regulation mechanism of LAP+CD4+T cells in vitro and explore its immunosuppressive pathways and mechanism on effector T cells.Methods:From July 2014 to August 2014,12 patients with different stages of CRC, who received surgical treatment in first affiliated hospital of guangxi medical university, department of colorectal surgery, were enrolled in this study, colorectal tumor tissues were obtained and digested by enzymatic means. tumor-infiltrating lymphocytes were isolated over a discontinuous ficoll gradient method. LAP+CD4+T cells and LAP-CD4+T cells were isolated by MACS (magnetic cell sorting), These cells were activated with anti-CD3 mAbs, anti-CD28 mAbs and human IL-2 for 72 hours in single culture and coculture at a 1:1 ratio. CKK-8 was used to detect the proliferation of these cells, transwell experiment were conducted to verify whether LAP+CD4+T cells could inhibit the proliferation of LAP-CD4+T cells by non-contact. The cytokines antagonistic experiment were conducted to verify whether anti-IL-10 mAbs and anti-TGF-β mAbs could reverse the suppressive properties of LAP+CD4+T cells.Results:After activated with anti-CD3, anti-CD28 mAbs, human IL-2 for 72 hours in vitro, the OD value of LAP-CD4+T cells were significantly higher than LAP+CD4+Tcells(0.85±0.16 vs.0.32±0.11,P<0.001); The OD value of single culture group was significantly higher than coculture group(0.87±0.21 vs. 0.48±0.18, P< 0.05). The results of transwell experiment shows that LAP+CD4+T cells could inhibit the proliferation of LAP-CD4+T cells by non-contact. The antagonistic experiment shows that anti-IL-10 mAbs and anti-TGF-β mAbs could reverse the suppressive properties of LAP+CD4+T cells partly.Conclusions:LAP+CD4+T cells have the characteristics of immune anergy and immune inhibition. The suppressive properties of LAP+CD4+T cells were mediated by IL-10 and TGF-β; anti-IL-lOmAbs and anti-TGF-β mAbs could reverse its suppressive properties partly. Whereas the suppressive function of CD4+LAP+T cells was both cell contact and soluble factor dependent. |
PDF Full Text Request