The Studying Of Disparity In Cytotoxicity Of Allo-NK Cells Against Umbilical Vein Endothelial Cells And Its Potential Mechanisms

Background and objectivesAlloreactive natural killer (NK) cell is a special kind of lymphocyte killer cells.Its cytotoxicyt need not be primed by either antigenic stimulation or antibody participation,with the function of rejecting nonself cells. Cytotoxicity against target cells by NK cells is closely associated with the balance between activitory receptors and inhibitory receptors. KIR conduct inhibition signals, whose ligand are HLA class I molecule. NKG2D conducts activation signals, it's ligand consist of Human MHC class I chain-related molecule A (MICA) or B (MICB)and ULBPs( human cytomegalovirus glycoprotein UL16 binding proteins, including ULBP1, ULBP2, ULBP3). Whether HLA class I molecule expressed by cells of donor organ can be recognized by receptor KIR may effect cytotoxicity of allo-NK cells against target cells. Cytotoxicity against target cells by NK cells relate with veno-occlusive disease of the liver in bone marrow transplantation and organ-graft refection.When donor organ recovers blood flow, endothelial cell is the first barrier 'between donor and receptor. Circulatory lymph cell of host origintation can directly discriminate allo-vascular endothelial cell to launch immunity attack or interact with antigen presenting cells on blood vessel endothelium to impaire endothelial cell. It results in dysaemia of transplant dysaemia and induce graft rejection finally.In thsis study, Human ECV304 cell line is allocated as target cells, so as to study wether allo-NK cells could mediate cytotoxicity against ECV304 in vitro. If thecytotoxicity is confirmed, we will further investigate the potential molecularmechanism participating of activitory and inhibitory signal system.MethodsChapterâ… Genotype of HLA -class I molecules and NKG2D ligands in ECV304cellsECV304 cells were gathered and DNA was extracted with QIAamp DNA extracting kit. The HLA-class I genotypes of ECV304 cells were determined by sequence specific primer polymerase chain reproduction (PCR-SSP, Biotest). mRNA expressions of NKG2D ligands (MICA, MICB, ULBP1, ULBP2, ULBP3) were analyzed by RT-PCR.Chapterâ…¡Detection of KIR2DL1 on NK cells and NKG2D ligands and HLA -class I molecules on ECV304 cells and K562 cellsThe KIR genotypes of the 8 healthy persons were determined by PCR-SSP. Expressions of KIR2DL1 on natural killer cells from 8 healthy donors is measured by flow cytometry. Expressions of HLA class I molecules and NKG2D ligands on ECV304 cells and K562 cells and ECV304 cells after incubation for 24 hours with CSA (1.63ug/ml) and LPS (2ug/ml) were analyzed by flow cytometry.Chapterâ…¢Cytotoxicity assays of NK Cells against ECV304 cells inVitrowith anti-KIR2DLl monoclonal antibody (mAb) influenced cytotoxicity of the NK cellsNK cells is isolated from 8 healthy persons by magnetic bead selection. Normal peripheral blood(PB) were obtained with informed consent. Expressions of KIR2DL1 on peripheral blood cells is measured by flow cytometry. Cytotoxicity of NK cells against ECV304 cells were detected by LDH- releasing method at effect-to-target cell ratios (E:T) of 20:1. In blocking experiments, anti-KIR2DL1 monoclonal antibody (mAb) was added to the NK cells before lysis experiment performed.ResultsChapter I Genotype of HLA -class I molecules and NKG2D ligands in ECV304 cellsHLA genotypes of ECV304 cells were A1,-, B18, -, Cw5,-. Expression of MICA, MICB, ULBP1, ULBP2, ULBP3 were confirmed by RT-PCR.Chapterâ…¡Detection of KIR2DL1 on NK cells and NKG2D ligands and HLA -class I molecules on ECV304 cells1. Expressions of KIR2DL1 on peripheral blood cells is detected by flow cytometry. Expressions of HLA class I molecules and NKG2D ligands on the surface of ECV304 cells were analyzed by flow cytometry .HLA genotypes indicated that, the ligand of KIR2DL1 but not KIR2DL2/3 or KIR3DL1 expressed on ECV304. The eight healthy donors experssed KIR2DL1. Expressions of KIR2DL1 on peripheral blood cells among eight donors are different from 6.2% to 46.2% . Therefore, KIR2DL2/3,KIR3DL1 mismatch existed between ECV304 cells and donor NK cells .MICA, MICB, ULBP1 , ULBP2 and ULBP3 were not detected on the surface of ECV304 cells by FACS. Expression of HLA class I molecules on ECV304 cellsis 97.5%.2. MICA, MICB, ULBP1 , ULBP2 and ULBP3 were not detected on the surface of ECV304 cells after incubation for 24 hours with CSA (1.63ug/ml) and LPS (2ug/ml) by FACS.3. K562 cells expressed all the NKG2D ligands: MICA, MICB, ULBP1, ULBP2, ULBP3 but not expressed HLA class I molecules.Chapterâ…¢Cytotoxicity assays of NK Cells against ECV304 cells inVitro and anti-KIR2DLl monoclonal antibody (mAb) influenced cytotoxicity of the NK cellsPurity of CD3 - CD16+CD56+ cells isolated from 8 healthy persons by magnetic bead selection is above 90.0%.The different expression of KIR2DL1 from eight individuals influence the cytotoxicity of NK cells against ECV304 cells at E:T ratios of 20:1. Expressions of KIR2DL1 on peripheral blood cells among eight donors are different from 6.2% to 46.2 %.Cytotoxicity of NK cells against ECV304 cells were vary from 1.2%--28.0%. Paired-Samples T Test show that NK cells displayed higher cytotoxicity against ECV304 when blocked with KIR2DL1 mAb(t=-4.860, P=0.002) . Spearman correlation analysis show that KIR2DL1 expression was negatively correlated withcytotoxicity of NK cell against ECV304 (rs= -0.994, P=0.000).Summary1. MICA/B, ULBP1-3 were expressed at mRNA level, but not on the surface of ECV304 detected by FACS. MICA, MICB, ULBP1 , ULBP2 and ULBP3 were not detected on the surface of ECV304 cells after 24 hours incubation with CSA (1.63ug/ml) and LPS (2ug/ml) by FACS. HLA genotypes indicated that, the ligand of KIR2DL1 but not KIR2DL2/3 or KIR3DL1 expressed on ECV304.2. The different expression of KIR2DL1 from eight individuals influence the cytotoxicity of NK cells against ECV304. Spearman correlation analysis show that KIR2DL1 expression was negatively correlated with cytotoxicity of NK cell against ECV304(rS=-0.994, P=0.000). Paired-Samples T Test show that NK cells displayed higher cytotoxicity against ECV304 blocked with KIR2DL1 mAb (t=-4.860, P=0.002) .3. The different expression of KIR2DL1 from individuals influence the cytotoxicity of NK cells against ECV304. Known ligands of NKG2D as MICA/B,ULBP1-3 do not participate in the cytotoxicity, suggesting for other activation signals.Innovation of our researchWe discovered that KIR-HLA mismatch signal system play a main role in NK cells-mediated cytotoxicity against the ECV304 cells. Value of our researchOur findings provide that, accordingly to guide more suitable donor under the guidance of expression and matching degree of KIR-HLA signal system .