OBJECTIVETo explore the mechanism of NK cell dysfunction in patients with multiple myeloma (MM).

METHODSThe expression of inhibitory receptors (CD158a and CD158b) and activating receptors NKG2D and NCRs (NKp30, NKp44 and NKp46) on CD3-CD56+NK cell of 13 MM patients and 30 healthy controls were analyzed by flow cytometry. The concentration of soluble NKG2D ligands (MICA, MICB, ULBP1, ULBP2 and ULBP3) in serum was detected by enzyme- linked immunosorbent assay (ELISA), and the cytotoxicity of NK cell against MM cell line by flow cytometry.

RESULTSThere are no significant differences of percentage and absolute number of NK cells, and the expression level of CD158a and CD158b between MM patients and healthy individuals (P>0.05). No NKp44 expression was detected on fresh isolated NK cells from both groups. There is no difference in inhibitor receptors expression between MM patients and healthy individuals but the expression of NKG2D, NKp30 and NKp46 on NK cells were higher in MM patients as compared with that in healthy individuals. The concentration of soluble NKG2D ligands in serum was higher in MM patients as compared with that in healthy individuals (P<0.05). Cultured healthy individual's NK cells with MM patient's serum could significantly decrease its cytotoxicity against MM cell line U266 cells [(38.5 ± 6.5) % vs (25.4 ± 5.9)%, P=0.044］.

CONCLUSIONThe higher level of soluble NKG2D ligands in serum may be the mechanism of NK cell dysfunction in MM patient.

Cells, Cultured ; Flow Cytometry ; Humans ; Killer Cells, Natural ; metabolism ; pathology ; Multiple Myeloma ; immunology ; metabolism ; NK Cell Lectin-Like Receptor Subfamily K ; metabolism ; Natural Cytotoxicity Triggering Receptor 1 ; metabolism ; Natural Cytotoxicity Triggering Receptor 2 ; metabolism ; Natural Cytotoxicity Triggering Receptor 3 ; metabolism ; Receptors, KIR2DL1 ; metabolism ; Receptors, KIR2DL3 ; metabolism

The objective of this study was to elucidate the correlation of killer immunoglobulin-like receptor (KIR) gene diversity with nasopharyngeal carcinoma (NPC) in the Chinese southern Han population. KIR genotyping of peripheral blood samples from 67 patients with NPC and 77 randomly-selected healthy controls was performed by PCR-SSP, the relative risk (RR) value was calculated by means of Wolf method. The results showed that the KIR2DL3 gene frequency in NPC patient group was significantly lower than that in healthy controls (χ²>3.84, p < 0.05, RR = 0.08), whereas the KIR2DS5 and KIR2DL5B gene frequencies in patient group were significantly higher than those in healthy controls (χ²>3.84, p < 0.05, RR > 1), the other KIR gene frequencies were no statistically different between two groups. It is concluded that the KIR2DL3, KIR2DS5 and KIR2DL5B genes may be correlated with pathogenesis of NPC in the Chinese southern Han population.
Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; genetics ; Receptors, KIR ; genetics ; Receptors, KIR2DL3 ; genetics ; Receptors, KIR2DL5 ; 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

In order to investigate the effect of demethylating treatment on the expression of inhibitory KIR and the cytolytic activity of NK-92MI cells, and to study the possible relationship between the demethylation of inhibitory kir gene and the function of NK cells. NK-92MI cells were treated with 5-azacytidine to induce DNA demethylation. The expression of KIR3DL1, KIR2DL2/KIR2DL3, KIR2DL1 and the viability of NK-92MI cells were detected by flow cytometry. The KIR3DL1 positive and the KIR3DL1 negative NK-92MI cells were also sorted by flow cytometry. The cytotoxicity of NK-92MI against K562 cells was detected by the LDH release assay. The results demonstrated that the expressions of KIR3DL1, KIR2DL2/KIR2DL3 and KIR2DL1 in NK-92MI cells all increased after treating with 1.0, 2.5 and 5 micromol/L of 5-azacytidine for 72 hours. And the cytotoxicity of NK-92MI against K562 cells decreased. In these dose range, 5-azacytidine did not influence the viability of NK-92MI cells. Additionally, the cytotoxicity of KIR3DL1 positive NK-92MI cells was lower than that of the KIR3DL1 negative cells. It is concluded that the demethylating treatment suppresses the cytotoxicity of NK-92MI cells through increasing the expression of inhibitory KIR.
Cytotoxicity, Immunologic ; immunology ; DNA Methylation ; Flow Cytometry ; Gene Expression Regulation ; Humans ; K562 Cells ; Killer Cells, Natural ; immunology ; metabolism ; Receptors, KIR2DL1 ; metabolism ; Receptors, KIR2DL3 ; metabolism ; Receptors, KIR3DL1 ; metabolism ; Receptors, KIR3DL2 ; metabolism

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 study the effects of small interfering RNA (siRNA)-mediated silencing of CD158b expression on the efficiency of the natural killer (NK) cells in killing allogeneic dendritic cells.

METHODSAfter the knockdown of CD158b by CD158b -SiRNA, the CD158b mRNA expression in natural killer cells was examined by qRT-PCR and the CD158b protein expression by flow cytometer. The cytotoxic activity of RNAi-NK cells and normal NK cells against the allogeneic dendritic cells was detected by LDH release assay.

RESULTSThe CD158b mRNA expression and its protein expression were decreased significantly in the NK cells by CD158b siRNA (P/0.05). The cytotoxic activities of alloreactive NK cells generated by RNAi CD158b expression against allogeneic dendritic cells were increased significantly.

CONCLUSIONSilencing CD158b gene can inhibit the NK cell CD158B mRNA and protein expression. Alloreactive NK cells generated by RNAi CD158b expression have the potential for use in interventions of GVHD.

Bone Marrow Transplantation ; immunology ; methods ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; Flow Cytometry ; Gene Silencing ; Graft vs Host Disease ; immunology ; Humans ; Killer Cells, Natural ; cytology ; immunology ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Receptors, KIR ; metabolism ; Receptors, KIR2DL3 ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

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

The study was purposed to investigate the polymorphism of killer cell immunoglobulin-like receptor (KIR) gene of the patients with leukemia and to explore the correlation between the KIR gene and susceptibility of leukemia. The KIR genotype of 50 patients with leukemia and 60 healthy controls in northern. Hans were analyzed by PCR-SSP. The results indicated that the present known 18 KIR genes were detected and identified. The frequencies of KIR 3DL3, 3DL2 and 2DL4 were 100% in all subjects, with the most frequent genotype KIR 3DP1 (0.86) followed by 2DP1, 2DL3, 3DL1, 2DL1, 3DS1, 2DL5, 2DS4, 2DS2, 1D, 2DS5, 2DL2, 2DS1, 2DS3 and 3DP1v in leukemia successively. Compared with the control, the KIR 3DL1 (0.60) and 2DL1 (0.57) were significantly lower in the leukemia patient group than that in the control group (1.00) (P < 0.01). It is concluded that the polymorphism of KIR gene is associated with susceptibility of leukemia in Hans. There may be a negative correlation between pathogenesis of leukemia and KIR 3DL1, KIR 3DS1, KIR 2DL1, KIR 2DL5 genes.
Adolescent ; Adult ; Child, Preschool ; Female ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Male ; Middle Aged ; Polymorphism, Genetic ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Receptors, Immunologic ; genetics ; Receptors, KIR ; Receptors, KIR2DL1 ; Receptors, KIR2DL3 ; Receptors, KIR2DL4 ; Receptors, KIR3DL1 ; Receptors, KIR3DL2 ; Receptors, KIR3DS1

OBJECTIVETo analyze the allelic polymorphism of killer cell immunoglobulin-like receptor 2DL3 (KIR2DL3) gene in Zhejiang Han population.

METHODSAllelic of KIR2DL3 was detected by PCR sequence based typing (PCR-SBT) method. Two specific fragments of KIR2DL3 were amplified using different primers and sequenced for exons 4,5,7,8,9 of KIR2DL3. All the samples would be assigned to KIR2DL3 allele according to the polymorphism site patterns.

RESULTSTwo KIR2DL3 alleles were observed, with KIR2DL3*001 having higher frequency, 0.77.

CONCLUSIONThe PCR-SBT method is reliable and there are distinctive frequencies of KIR2DL3 alleles in Zhejiang Han population.

Alleles ; Base Sequence ; China ; Gene Frequency ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; genetics ; Receptors, KIR2DL3 ; genetics ; Sequence Analysis, DNA

OBJECTIVETo analyze the changes of inhibitory killer cell immunoglobulin-like receptors (KIRs), NKG2D receptor and the cytotoxicity of natural killer (NK) cells induced by persistent exposure to CNE2 cells.

METHODSThe HLA-class I genotypes of CNE2 cells and KIR genotypes were determined by PCR with sequence-specific primers (PCR-SSP). The expressions of KIR2DL1, KIR2DL3, KIR3DL1, and NKG2D by the NK cells (freshly isolated NK cells, NK cells cocultured with 100 U/ml IL2 or with 100 U/ml IL2 and CNE2 cells as the control, IL2 and CNE2 groups, respectively) were analyzed by flow cytometry. Cytotoxicity of NK cells against CNE2 cells were detected by LDH releasing assay.

RESULTSThe HLA genotypes of CNE2 cells were A2, 24, B18, 35, Cw4, 7. NK cells isolated from 3 healthy donors expressed KIR2DL1, KIR2DL3, and KIR3DL1. After 4, 24 and 48 h of culture, NK cells in CNE2 group displayed higher KIR2DL1, KIR2DL3 but lower NKG2D expression than those in the control and IL2 groups (P<0.01), whereas the latter two groups showed no significant difference in KIR2DL1, KIR2DL3, and NKG2D expressions (P>0.05), and no difference in KIR3DL1 expression was found between the 3 groups (P>0.05). After 24 h of culture, the cytotoxicity against CNE2 cells mediated by the NK cells in IL2 and CNE2 groups were (26.96-/+1.47) % and (2.74-/+1.64) % at E:T ratios of 10:1, and (35.74-/+3.59)% and (4.57-/+2.41) % at E:T ratio of 20:1, respectively. NK cells in CNE2 group displayed lower cytotoxicity than those in IL2 group (P<0.01).

CONCLUSIONSPersistent exposure to tumor cells expressing NKG2D ligands can lead to downregulated expression of NKG2D receptor, increased expression of KIRs and reduction of NK-mediated cytolysis. These results elucidate the molecular mechanism of reduced cytotoxicity mediated by the edited NK cells and indicate that blocking HLA-class I-bound KIRs or enhancing the expression of NKG2D may promote NK cell-mediated cytolysis.

Cell Line, Tumor ; Cell Survival ; immunology ; Cytotoxicity, Immunologic ; immunology ; Flow Cytometry ; Humans ; Killer Cells, Natural ; cytology ; immunology ; metabolism ; NK Cell Lectin-Like Receptor Subfamily K ; Nasopharyngeal Neoplasms ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; metabolism ; Receptors, KIR ; metabolism ; Receptors, KIR2DL1 ; metabolism ; Receptors, KIR2DL3 ; metabolism ; Receptors, Natural Killer Cell