The Study Of B7-H1/PD-1Pathway In Diffuse Large B Cell Lymphoma

Part Ⅰ The expression of B7-H1in human diffuse large B cell lymphoma(DLBCL)Objective:To analyze the expression of B7-H1in human DLBCL.Methods:54human DLBCL tumor tissue (DLBCL tissue of IPI<2points have31cases and DLBCL tissue of IPI>2points have23cases) and14peritumoral normal lymphatic tissue were got from patients with DLBCL after operation. And it is analyzed through the following methods:1. Using Western Blot to analyze the expression of B7-H1in human DLBCL tissue and peritumoral normal lymphatic tissue.2. Make a comparison of the expression of B7-H1on the surface of HLA-DR+CD14+macrophages cell and the percentage of CD4+/C D8+T cell, HLA-DR+CD11c+Lineage-Myeloid DC (mDC), HLA-DR+CD123+Lineage- Plasmacytoid DC (pDC), CD4+CD25+Foxp3+Regulatory T cells (CD4+CD25+Foxp3+Treg, Treg) in the human DLBCL tissue and peritumoral normal lymphatic tissue by flow cytometry analysis.Results:1. The data of Western Blot indicated that B7-H1molecule can be detected in all of patients with DLBCL and the expression level is significantly higher in DLBCL than in its adjacent normal lymphatic tissue.2. The results of flow cytometry1) The expression level of B7-H1on the surface of HLA-DR+CD14+macrophages cell is significantly higher in DLBCL tissue compared to its adjacent normal lymphatic tissue.The results showed that the proportion of B7-H1on the surface of HLA-DR+CD14+macrophages cell in peritumoral normal lymphatic tissue is (8.36±0.52)%, in DLBCL tissue of IPI<2points is (12.85±0.82)%, and in DLBCL tissue of IPI>2points is (30.57±0.76)%. The expression level of B7-H1is significantly higher in DLBCL tissue compared to its adjacent normal lymphatic tissue, P<0.05.It is significantly increased in DLBCL tissue of IPI>2points than DLBCL tissue of IPI<2points, P<0.01.2) It is significantly higher of Treg, CD4+T/CD8+T and pDC in DLBCL tissue compared to its peritumoral normal lymphatic tissue.The percentage of Treg in peritumoral normal lymphatic tissue is (1.58±0.14)%, in DLBCL tissue of IPI<2points is (4.68±0.30)%, and in DLBCL tissue of IPI>2points is (5.55±0.50)%. The expression level of Treg is significantly higher in DLBCL tissue compared to its adjacent normal lymphatic tissue, P<0.01. And it is no significant in DLBCL tissue of IPI>2points than DLBCL tissue of IPI<2points, P>0.05. The data indicate that DLBCL tissue infiltrated high levels of Treg. The percentage of CD4+T/CD8+T in peritumoral normal lymphatic tissue is (0.44±0.03)%, inDLBCL tissue of IPI<2points is (1.04±0.56)%, and in DLBCL tissue of IPI>2points is (1.78±0.06)%. The percentage of CD4+T/CD8+T is significantly higher in DLBCL tissue compared to its adjacent normal lymphatic tissue, P<0.01. And it is significantly increased in DLBCL tissue of IPI>2points than DLBCL tissue of IPI<2points, P<0.01. It is indicate that DLBCL tissue have high levels of CD4+T/CD8+T.The percentage of mDC in peritumoral normal lymphatic tissue is (57.00±1.53)%, in DLBCL tissue of IPI<2points is (65.50±2.16)%, and in DLBCL tissue of IPI>2points is (60.60±1.05)%. The percentage of mDC is no significant in DLBCL tissue compared to its adjacent normal lymphatic tissue, P>0.05.The percentage of pDC in peritumoral normal lymphatic tissue is (64.54±1.97)%, in DLBCL tissue of IPI<2points is (86.66±1.75)%, and in DLBCL tissue of IPI>2points is (87.33±1.45)%. The percentage of pDC is significantly higher in DLBCL tissue compared to its adjacent normal lymphatic tissue, P<0.01.And it is no significant in DLBCL tissue of IPI>2points than DLBCL tissue of IPI<2points, P>0.05. The data indicate that DLBCL tissue infiltrated high levels of pDC.Conclusions:It is significantly higher of B7-H1, pDC, CD4+T/CD8+T and Treg in DLBCL tissue compared to its adjacent normal lymphatic tissue, and suggest an association beween them. The data indicates that the cellular immune imbalance in DLBCL microenvironment may be associated with the immune escape of lymphoma. Part Ⅱ The Study of PD-1Expression and Relationship to the functions of the T Cells in Human DLBCLObjective:To analyze the he expression of PD-1on the surface of infiltrating T cell in DLBCL, and that impact to the functions of the T Cells.Methods:54human DLBCL tumor tissue (DLBCL tissue of IPI<2points have31cases and DLBCL tissue of IPI>2points have23cases) and14peritumoral normal lymphatic tissue were got from patients with DLBCL after operation. And it is analyzed through the following methods:1. Western Blot was used to investigate the expression of PD-1in DLBCL and its adjacent normal tissue, in which the expression of PD-1molecules on the surface of T cell was measured by using flow cytometry as well.2. Make a comparison of the expression of the PD-1molecules on the surface of CD8+T cell and to impact the functions of the CD8+T Cells to the secretion of IFN-y and IL-2in human DLBCL and its adjacent normal tissue.Results:1. The data of Western Blot indicated that PD-1molecule can be detected in all of patients with DLBCL and the expression level is significantly higher in DLBCL than in its adjacent normal lymphatic tissue.2. The results of flow cytometry1) The results showed that the proportion of PD-1+CD8+T cell in DLBCL tissue of IPI<2points is (2.40±0.85)%is significantly higher than peritumoral normal lymphatic tissue (1.16±0.41)%, P<0.01.It is significantly increased in DLBCL of IPI>2points (2.46±0.87)%than DLBCL tissue of IPI<2points (2.40±0.85)%, P<0.01.2) The percentage of IFN-γ+IL-2+PD-1+CD8+T cell of peritumoral normal lymphatic tissue is(8.70±3.08)%, IFN-γ+IL-2+PD-1"CD8+T cell of peritumoral normal lymphatic tissue is(5.11±1.8)%, and no differences between the two, P>0.05. The percentage of IFN-γ+IL-2+PD-1+CD8+T cell in DLBCL tissue of IPI<2points is(35.16±1.23)%is significantly lower of compared to IFN-γ+IL-2+PD-1-CD8+T cell(67.68±1.74)%in DLBCL tissue of IPI<2points, P<0.01. In DLBCL tissue of IPI>2points, the percentage of IFN-γ+IL-2+PD-1+CD8+T cell is (11.78±1.57)%, that is significantly lower than the percentage of IFN-γ+IL-2+PD-1-CD8+T cell (59.45±1.43)%,P<0.01.Conclusions:CD8+T cells expressed more PD-1molecule in DLBCL, and secreted less anti-tumor cytokines, so that PD-1of CD8+T cells was highly expressed affect its ability to resist cancer.