MiRNA Regulate Of Programmed Cell Death-1+ Exhausted T Cells

Chapter 1 Introduction There have a lots of reasons to induce tumor immune tolerance in the tumor microenvironment, such as the change of negative regulator in T cells, the regulate T cells, cytokines and the disorder of metabolic function. T cells extraction is a state of T cell functional defect, those exhausted T cells which express negative regulator have faintly effection. The most important feature of those T cells is the up regulation of those negative regulator and decrease of cytokine. Programmed cell death-1/ programmed cell death ligand-1(PD-1/PD-L1) pathway is important to regulate the T cell activation. It is associated with tumor immune tolerance and tumor progress. micro RNAs(mi RNAs) can effect the process of tumor development. mi RNAs regulate the chromosome abnormality, gene mutation, gene polymorphism and the change of epigenetics. Our research focus on the relation of mi RNA and the exhausted T cells in tumor immune tolerance. The purpose is finding a new diagnosis marker and treatment to tumor.Chapter 2 the differential expression profiling of mi RNA between CD4+PD-1+ and CD4+PD-1- T cells Objective: Compare with the differential expression of mi RNA between CD4+PD-1+ and CD4+PD-1- T cells, find the PD-1 associated mi RNA. Methods: Establish the mice molanoma tumor model, separate the lymphocyte from the lypho node of tumor bearing mice. Flow Cytometer separate the CD4+PD-1+ and CD4+PD-1- T cells. mi RNA was extracted from those different T cells, Affymetrix mi RNA 3.0 micro Array were used to detected the expression of mi RNAs, RT-q PCR was used to vertify the results. mi Randa was used to forecast the binding site of mi RNAs. Dual-Luciferase Reporter Assay System was used to detect firefly and renilla luciferase from the PD-1 3’UTR pmir GLO plasmid. Results: 19 mi RNAs are differential expression between CD4+PD-1+ and CD4+PD-1- T cells, 8 of mi RNAs are up regulated and 11 of them are down regulated in CD4+PD-1+ T cells. RT-q PCR results show that 8 of mi RNAs (mi R-let-7e, mi R-103, mi R-107, mi R-27 a, mi R-23 a, mi R-21, mi R-155, mi R-146a) are up regulated in CD4+PD-1+ T cells and 3 mi RNAs(mi R-150, mi R-28, mi R-151-5p) are down regulated. The mi R-150, mi R-28, mi R-let-7e, mi R-103, mi R-107, mi R-27 a have the binding site with PD-1 3’UTR. Dual-Luciferase Reporter Assay System show that the mi R-28 and mi R-107 can silence the PD-1 through binding to the PD-1 3’UTR. Conclusions: 8 of mi RNAs(mi R-let-7e, mi R-103, mi R-107, mi R-27 a, mi R-23 a, mi R-21, mi R-155, mi R-146a) are up regulated and 3 mi RNAs(mi R-150, mi R-28, mi R-151-5p) are down regulated in CD4+PD-1+ T cells. mi R-28 and mi R-107 can silence the PD-1 through binding to the PD-1 3’UTR.Chapter 3 The expression of exhausted T cells in tumor bearing mice and the exhausted T cells model in vitro Objective: Compare with the exhausted T cells phenotype in tumor bearing mice and normal mice, establish the inducing exhausted T cells model in vitro. Methods: Melanoma cells were injected to the C57BL/6 mice, separate the T cells and tumor infiltrating cells from lymph node, spleen and tumor tissue. Flow Cytometer detected the phenotype of exhausted T cells. T cells were cultured with anti-CD3 e antibody combined with or not interferon-a(IFN- ?) or CD28 in vitro. Flow Cytometer detected the phenotype, RT-q PCR detected the gene expression. Results: Both the CD4+ and CD8+ exhausted T cells phenotype are higher in the tumor bearing mice than in the na?ve mice. Anti-CD3 e antibody can induce the exhausted T cell phenotypes in vitro, the expression is associated with the concentration of anti-CD3 e antibody. There is no big difference between CD4+ and CD8+ T cells. The mi RNAs of those T cells were decrease when cultured in vitro. Conclusions: tumor microenvironment can stimulate the exhausted T cells increasing in the process of tumor growth. Coating anti-CD3 e antibody can induce the exhausted T cells in vitro, the mi RNA will also change when cultured in vitro.Chapter 4 mi R-28 regulate the cellular biological characteristic of exhausted T cells Objective: Detected the effection of mi R-28 to the exhausted T cells phenotype, and the change of T cell proliferation, apoptosis, Treg, cytokines and tumor-specific cytotoxic T lymphocyte(CTL). Methods: Transfected with mi R-28 mimic or inhibitor to the tumor bearing mice lymphocytes, RT-q PCR detected the gene expression of PD-1, TIM-3 and BTLA. Flow Cytometer detected the phenotype of exhausted T cells and the Treg, Foxp3+PD-1+, Foxp3+TIM-3+ T cells. CFSE detected the cell proliferation, Annexin V/PI detected the cell apoptosis, ELISA detected the cytokines; MACs separate the CD8+ T cells, then culture with the antigen-loaded dendritic cells, Cyto Tox ? 96 non radioactive cytotoxicity analysis kit was used to detect the CTLs after the transfection of mi R-28 mimic or inhibitor. Results: mi R-28 can regulate the gene expression of PD-1, TIM-3 and BTLA. mi R-28 mimic can down regulate the PD-1+ T cells, increase the secretion of interleukin-2(IL-2) and the tumor necrosis factor-a ?(TNF- ?), enhance the ability of CTLs to the melanoma tumor. mi R-28 inhibitor can increase the expression of PD-1, TIM-3, increase Foxp3+PD-1+ and Foxp3+TIM-3+ T cells expression, and also restrain the secretion of TNF- ?. Conclusions: mi R-28 can down regulate the expression of PD-1 on the T cells, increase the secretion of IL-2 and TNF- ?, effect the expression of Foxp3+PD-1+ and Foxp3+TIM-3+ T cells. mi R-28 also enhance the ability of CTLs to the melanoma tumor. mi R-28 may be used to treatment to the melanoma.