The Study Of B7-H1/PD-1 Inhibitory Pathway And Relationship To Regulatory T Cells In Human Colon Carcinoma

Objective:To investigate the level of B7-H1&PD-1 expression in human colon carcinoma, and its correlationship with Regulatory T Cells (T_Reg) high infiltrated in tumor microenvironment?Method:Western Blot was used to investigate the expression of B7-H1 in colon carcinoma and its adjacent normal tissue, in which the expression of B7-H1 molecules on the surface of macrophages was measured by using flow cytometry as well. And also the cellular localization of PD-1⁺ T cells was detected by fluorescence immunohistochemistry. Using the flow cytometry Regulatory T cells would be found infiltrating in colon carcinoma and its adjacent normal tissue. The expression of IL-10 molecules expressed on CD4⁺CD25⁺ T cells and on CD4⁺CD25^- T cells was measured in the colon carcinoma microenvironment by using Western Blot. ELISA was also used to investigate the expression of IL-10 in the culture environment of CD4⁺CD25⁺ T cells or CD4⁺CD25^- T cells from the blood of healthy donors, stimulated with anti-human CD3 and anti-human CD28. Co-cultured CD14⁺ cells from the blood of healthy donors and T_Reg from the tissue of colon carcinoma with antibody against human IL-10 or not, stimulated with anti-human CD3 and anti-human CD28 for 5 days, CD14⁺ cells were harvested and B7-H1 expression was determined by FACS. Co-cultured Na(i|¨)ve T cells marked with CFSE and CD14⁺ cells from the tissue of colon cancer or the blood of healthy donors which were co-cultured with T_Reg for 5 days and high expressed of B7-H1, with antibody against human B7-H1 or antibody against human PD-1 or not, stimulated with anti-human CD3 and anti-human CD28 for 2 days, percentages of CFSE^dim were determined by FACS.Results:1. The data of Western Blot indicated that B7-H1 molecule can be detected in all of patients with colon carcinoma and the expression level is significantly higher in colon carcinoma than in its adjacent normal tissue.2. B7-H1 was found express on the surface of macrophages from both colon cancer and adjacent normal tissue by flow cytometry, and the expression level is significantly higher in carcinoma compared to its adjacent normal tissue with percentages of 38±4% and 5±2% or MFI of 1900±20 and 250±100 respectively.3. FACS confirmed that PD-1 was mainly expressed on CD8⁺ T cells, and the expression were significantly higher in carcinoma than its adjacent normal tissue with the percentages of 51±6% and 6±2%.4. The percentage of T cells'CFSE^dim determined by the flow cytometry shown us that the proliferation of T cells co-cultured with macrophages from the tissue of colon cancer could be inhibited, with the percentage (32±8%) smaller than the couple of na(i|¨)ve T cultured alone (63±7%) or co-cultured with macrophages from the tissue of the adjacent normal tissue (55±8%). Blocking B7-H1/PD-1 pathway by using neutralizing antibodies, the proliferation of T cell could be recovered, with the percentage using the antibody of anti-human B7-H1 51±10% or PD-1 54±11% respectively.5. Foxp3⁺ T cells were determined both in colon cancer and adjacent normal tissue, and the percentage of in CD3⁺CD4⁺ cells from colon cancer (18±5%) was higher from adjacent normal tissue (5±3%) by flow cytometry. 6. Western Blot investigated that IL-10 was higher expressed in CD4⁺CD25⁺ T cells than CD4⁺CD25^- T cells from tissue of colon Carcinoma. ELISA was also confirmed that IL-10 was higher expressed in CD4⁺CD25⁺ T cells (180±27pg/ml) which was cultured and stimulated with anti-human CD3 and CD28 than in CD4⁺CD25^- T cells (52±9pg/ml).7. The data of FACS indicated that B7-H1 expressed on monocytes in the group which the monocytes co-cultured with T_Reg from the tissue of colon carcinoma (28±3%, MFI: 150±20) was more than the control group without T_Reg from the tissue of colon carcinoma (5±1%, MFI: 1200±80) and the group with anti-human IL-10 (6±2%, MFI: 350±50).8. The percentage of T cells'CFSE^dim determined by the flow cytometry shown us that the proliferation of T cells co-cultured with monocytes primed with induction of B7-H1 high expressed could be inhibited, similar as na(i|¨)ve T cells co-cultured with macrophages from the tissue of colon cancer, with the percentage (30±5%) smaller than the couple of na(i|¨)ve T cultured alone (60±7%) or co-cultured with monocytes from t without B7-H1induction (64±11%). Importantly, blocking monocytes B7-H1 interaction with PD-1+CD8+ cells PD-1 by using neutralizing antibodies, the i proliferation of T cell could be recovered too, with the percentage using the antibody of anti-human B7-H1 52±8% or PD-1 55±5% respectively.Conclusion:1. In colon cancer microenvironment B7-H1 was high expressed on macrophages and PD-1 was high expressed on CD8+ T cells.2. Regulatory T Cells (T_Reg) were abnormal increased in colon cancer microenvironment, and they could enrich the expression of B7-H1 on monocytes by high expression of IL-10.3. The proliferation of T cells would be inhibited through the axis of B7-H1/PD-1 pathway, which contributed to tumor immune escape.4. Blocking the axis of B7-H1/PD-1 pathway by using neutralizing antibodies, the proliferation of T cell could be recovered, which may become one way of adjuvant immune treatment in colon cancer in the future.