The aim of this study was to analyze the promoter methylation patterns of inhibitory killer cell immunoglobulin-like receptor (KIR) which gene expression and the effect of demethylation treatment were studied, and to explore the possible regulation mechanism of inhibitory kir gene expression. The promoter methylation levels of kir2DL1 and kir2DL2/kir2DL3 in NK-92MI cell line were detected by bisulfite sequencing technique. Then NK-92MI cells were treated with 5-azacytidine to induce the demethylation of CpG islands. The levels of gene expression of kir were determined by RT-PCR. The results demonstrated that the methylation frequencies of CpG dinucleotides surrounding the promoter regions of kir2DL1 and kir2DL2/kir2DL3 genes were 25% to 88% and 5% to 80% respectively. DNA-demethylating treatment with 5-azacytidine resulted in re-expression of kir2DL1 gene and increased expressions of kir2DL1, kir2DL2 and kir2DL3 genes in NK-92MI cells. In conclusion, the promoter DNA methylation participates in the regulation of kir gene expression in NK-92MI cells.
Cell Line ; DNA Methylation ; Gene Expression ; Humans ; Killer Cells, Natural ; metabolism ; Receptors, KIR2DL1 ; genetics ; metabolism ; Receptors, KIR2DL2 ; genetics ; metabolism ; Receptors, KIR2DL3 ; genetics ; metabolism

OBJECTIVETo explore the genetic variations of HLA-Cw and 5 KIR2D loci in 2 Chinese Han populations residing at Southern and Northern mainland China, respectively, and to investigate the HLA-Cw polymorphism of a Mongolian Chinese population.

METHODSHLA-Cw genotyping was performed in a total of 293 healthy individuals including 1 Southern Han population living in Hunan Province (n=112), 1 Northern Han population (n=98) and 1 Mongolian Chinese population(n=83) in the Inner Mongolia Autonomous Region, using polymerase chain reaction-sequence specific primer(PCR-SSP) technique. Dimorphism at residue 80 of domain in the HLA-Cw molecule was examined by an additional set of PCR-SSP reactions. PCR-SSP was also used to detect the presence or absence of inhibitory KIR2DL1/2DL2/2DL3 loci and activating KIR2DS1/2DS2 loci for the 2 Han populations.

RESULTSThe main findings were: (1) Very significant frequency difference in the HLA-Cw alleles and dimorphism at codon 80 was detected between Hunan Han and Northern Han population, and between Hunan Han and Mongolian population (P < 0.001),while there was no such difference between the 2 Northern Chinese populations (P> 0.05); (2) There was no significant difference in frequencies of either the 5 individual KIR2D genes or the genotype distributions between the 2 Han populations (P> 0.05); (3) Asn(80)ls/Asn(80), 2DL1+/2DL2-/2DL3+/2DS1-/2DS2- predominated in both Han populations (45/112, 29/98), followed by Asn(80)/Asn(80), 2DL1+/2DL2-/2DL3+/2DS1+/2DS2- (18/112,16/98) and Asn(80)/Lys(80), 2DL1+/2DL2-/2DL3+/2DS1-/2DS2-(11/112,17/98). Among the 12 types of HLA-Cw codon 80 and KIR2D combinations, only Lys(80)/Lys(80), 2DL1+/2DL2-/2DL3+/2DS1-/2DS2- showed marginally significant frequency difference between the 2 Han populations(1/112 vs 8/98; Fisheros P was 0.0134).

CONCLUSIONOur study provided the polymorphism data of HLA-Cw gene for 3 Chinese populations with different geographic and/or ethnic background, we further analyzed the distribution of 5 KIR2D receptor genes in 2 Han populations. Our data suggest that in spite of HLA-Cw heterogeneity, remarkable similarities may exist between the Southern and Northern Chinese Han populations at the combinational level of HLA-Cw and KIR2D, which are characterized by preponderant inhibitory signal pathways.

Asian Continental Ancestry Group ; Genetic Variation ; genetics ; HLA-C Antigens ; genetics ; Humans ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; genetics ; Receptors, KIR ; genetics ; Receptors, KIR2DL1 ; genetics ; Receptors, KIR2DL2 ; genetics ; Receptors, KIR2DL3 ; genetics

OBJECTIVETo investigate the effect of blocking the inhibitory receptors KIR2DL1 and KIR2DL2/2DL3 with monoclonal antibody on cytotoxic activity of human NK cells.

METHODSHuman peripheral blood NK cells were isolated by Rosettesep NK sorting kit. The cytotoxic activity of NK cells against human leukemia NB4, K-562, Raji cells and allogeneic mature or dendritic cells (DCs) was detected before or after KIR2DL1 and KIR2DL2/2DL3 were blocked. The effect of NK cells on T lymphocyte proliferation was detected by mixed lymphocyte reaction and TGF-β1 concentration in culture supernatant was measured.

RESULTSThe cytotoxicity of NK cells to NB4 cells was augmented with increasing concentration of the antibody. Combination of both antibodies enhanced killing activity of NK cells. NK cells had strong cytotoxicity to K-562 cells, but were not enhanced by the blockade of inhibitory receptors. The cytotoxicity to Raji cells was not evidently augmented. The cytotoxicity of NK cells to mature DC was enhanced remarkably with the increase of concentration of the antibodies (2.20% ±1.10% compared with 37.59% ±5.06%, P<0.05). In mixed lymphocyte reaction, the blockade of two antibodies enhanced the inhibition effect of NK cells on T cell proliferation (77.85% ± 8.31% compared with 43.05% ± 5.95%, P<0.05) and the content of TGF-β1 in the supernatant was increased.

CONCLUSIONThe cytotoxic effects of human NK cells against target cells were significantly enhanced with the blockade of inhibitory KIR receptor; and the cytokine TGF-β1 secreted by NK cells further inhibits T cells proliferation.

Antibodies, Monoclonal ; immunology ; pharmacology ; Cell Line ; Cells, Cultured ; Cytotoxicity, Immunologic ; drug effects ; immunology ; Dendritic Cells ; immunology ; Humans ; Killer Cells, Natural ; drug effects ; immunology ; metabolism ; Lymphocyte Culture Test, Mixed ; Receptors, KIR2DL1 ; drug effects ; immunology ; Receptors, KIR2DL2 ; drug effects ; immunology ; Receptors, KIR2DL3 ; drug effects ; immunology ; T-Lymphocytes ; immunology ; Transforming Growth Factor beta1 ; metabolism