NKG2D Research Articles

Arsenic trioxide upregulates UL16 binding protein 1 expression in KG1a cells through ataxia telangiectasia mutated and RAD3-related kinase pathway

Objective To observe the effect of arsenic trioxide (ATO) on the expression of NKG2D ligand UL16 binding protein 1(ULBP1) in acute myeloid leukemia KG1a cells, and explore the molecular mechanism for its regulation of ULBP1 expression. Methods KG1a cells were cultured in vitro.Then, the inhibition of KG1a cell proli-feration by different concentrations of ATO was detected by cell counting kit-8(CCK8) assay, and the expression of ULBP1 mRNA and surface protein in KG1a cells were examined by real-time RT-PCR and flow cytometry, respectively.After that, the blocking effects of ataxia telangiectasia mutated and RAD3-related kinase (ATM/ATR) inhibitor caffeine on ATO-upregulated expression of ULBP1 mRNA and surface protein expressions were investigated, and the effects of ATO on the expression of CHK1 and CHK2 proteins and their phosphorylation in KG1a cells were observed by Western blot method. Results Different concentrations (1, 2, 3, 4, 5 μmol/L) of ATO could inhibit the proliferation of KG1a cells, which was concentration dependent, and the half inhibitory (IC50) concentration to KG1a cells was 2.7 μmol/L.The expression of ULBP1 mRNA on KG1a cells were increased when incubated with ATO at concentration 1, 2, 3, 4, 5 μmol/L, compared without ATO group, ULBP1 mRNA expression level relatively increased respectively to (1.86±0.30) times, (3.02±0.71) times, (3.16±0.75) times, (4.80±0.70) times and (3.70±0.89) times, and the differences were statistically significant (all P<0.05). Furthermore, ATO (1, 2, 3, 4 and 5 μmol/L) upregulated ULBP1 protein expression on KG1a cells compared with that in the group without caffeine, and the differences were statistically significant (all P<0.05). After caffeine pretreat KG1a cell 2 h and ATO incubate KG1a cell 24 h, ULBP1 mRNA and protein expression levels were significantly reduced.When caffeine concentration was 8 mmol/L, ULBP1 mRNA expression level relatively reduces from (9.55±0.38) times to (6.36±0.93) times compared with that in the group without caffeine, and the difference was statistically significant (P<0.05). When caffeine concentration was 2, 4 and 8 mmol/L respectively, the expression of ULBP1 protein was reduced from that in the group without caffein treatment (3.50±0.08) times to (2.17±0.07) times, (2.02±0.06) times and (1.75±0.06) times, respectively, and the differences were statistically significant (all P<0.05). The expression of CHK1 and CHK2 proteins decreased with the increase of ATO concentration, while p-CHK1 and p-CHK2 are increased as ATO. Conclusions ATO upregulate the expression of ULBP1 mRNA and protein in KG1a cells, and the ATM/ATR-CHK1/CHK2 pathway may be involved in it. Key words: Arsenic trioxide; KG1a cells; Natural killer cell; UL16 binding protein 1; Ataxia telangiectasia mutated and RAD3-related kinase pathway

Research Advances on the Biological Characteristics of Hematological Malignant Cells Immunologically Regulated by Exosome--Review

Abstract The minimal residual disease (MRD) is the origin element that caused the relapse and drug resistance of hematological malignancies, the immune cells play a great role to clear MRD. A variety of immune cells have anti-tumor effects. However, tumor cells antagonize anti-tumor effects by reprogramming of constituents associated with tumor environment. Many different cell types, including immune cells, mesenchymal cells and tumor cells in tumor microenvironment release exosomes. The latest researches indicate that "cargo" and surface ligands carried by exosomes secreted by hematological malignant cells not only can affect the function of natural killer cell (migration, activation, proliferation, secretion and NKG2D expression), macrophage (migration and secretion) and dendritic cell (maturation and presentation), but also regulate the expression of PD-L1 and CCR2, CCL2 secretion and transformation of monocytes. The altered function of immune cells will eventually have effect on the progression of hematological malignancies.

NKG2D-IL-15 fusion protein encapsulated in N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride retards melanoma growth in mice

Background: Chitosan can be modified to increase the efficiency of the delivery of chemical drugs, nucleic acids, or proteins. Sodium tripolyphosphate (TPP) is a noncytotoxic and polyanionic crosslinker that binds with the positively charged ions of chitosan. DsNKG2D-IL-15 is a fusion protein that exerts promising antitumor effects via lymphocyte activation. The extracellular domains of double NKG2D is linked to IL-15. Methods: To increase the stability and efficiency of dsNKG2D-IL-15 protein, the fusion protein was encapsulated in nanoparticles based on chitosan pre-modified with N-(2-hydroxy) propyl-3-trimethyl ammonium (HTCC). Moreover, the biological activity of protein nanoparticle was evaluated on the mouse lymphocyte ex vivo and mouse tumor model in vivo . Results: TPP sharply promoted the HTCC chitosan encapsulating efficiency (85–95%) with dsNKG2D-IL-15. The protein nanoparticle displayed a spherical shape with a diameter of 200–400 nm and zeta-potential value of 15.6±4.82 mV. DsNKG2D-IL-15 could be released from the nanogel within 72 h. In addition, the protein biological activity for lymphocyte activation was maintained. Natural killer (NK) and CD8 + T cells increased the activity of IFN-γ production and degranulation after incubation with the dsNKG2D-IL-15-HTCC-TPP nanoparticle ex vivo . Treatment with dsNKG2D-IL-15 nanoparticles exhibited better effects of inhibiting tumor growth and prolonging the life span of B16BL6-MICA tumor-bearing mice in vivo than by using the dsNKG2D-IL-15 protein alone. Conclusions: The dsNKG2D-IL-15 protein nanoparticle exhibited notable effects of lymphocyte activation and tumor inhibition. The protein nanoparticle could be developed further for tumor therapy in clinical practice.

Astragalus polysaccharide improves anti-tumor immunity mediated by macrophages and natural killer cells in mice

Objective To evaluate the regulatory effects of Astragalus polysaccharide (APS) on macrophage polarization and NK cell-mediated anti-tumor responses in mice. Methods C57BL/6 mice were injected intraperitoneally with APS once a day for seven consecutive days. Activation of immune cells was then induced by intraperitoneal injection of polyinosinic-polycytidylic acid (Poly I∶C) 24 h after the APS intervention. Peritoneal macrophages were collected 24 h after induction to analyze the status of polarization and the production of nitric oxide (NO). Cytotoxicity and exocytosis of activated NK cells were measured to assess the effector functions of these cells. NK cell activities induced by NKG2D were studied in the absence of the whole JNK or JNK2 signaling pathway. Results Intraperitoneal injection of APS promoted the polarization of macrophages induced by tumor cells in mice, and enhanced the cytotoxicity of NK cells to tumor cells. However, APS was in need of the involvement of appropriate stimulatory factors to have regulatory effects. Complete inhibition of JNK signaling pathway dramatically reduced the effector functions of NK cells, which could not be recovered by APS administration. Conclusions APS was involved in the regulation of anti-tumor innate immunity through enhancing the M1-polarization of macrophages and improving the effector functions of NK cells. This study might to some extent elucidate the mechanism of APS in immune regulation and anti-tumor immunity. Key words: Astragalus polysaccharide; Innate immunity; Anti-tumor immunity; Immune regulation

Immunoreceptor NKG2D and its ligand-mediated cellular immunity and its relationship with diseases

Natural killer (NK) cells play an important role in host defense.NKG2D is expressed as a homodimer on the surface of NK cells and is an important lymphocyte activating receptor.NKG2D ligands are negatively or poorly expressed on the surface of normal cells, but when the cells are in a state of oxidative stress, malignant transformation, autoimmune or inflammatory stimulation, these ligands will be induced on the cell surface.The NKG2D receptor and its ligand-mediated cellular immune response play an important role in immune surveillance of tumor cells, and at the same time it is also closely related to certain autoimmune diseases. Key words: NKG2D; NKG2D ligand; Cellular immunity; Autoimmune diseases

Regulatory effect of decitabine on human acute myeloid leukemia cell line HL-60 and its mechanism

Objective To investigate the effect of decitabine (DAC) on human acute myeloid leukemia (AML) cell line HL-60 and the regulating of natural killer (NK) cell activating receptor (NKG2D) ligands (NKG2DL), and to detect the molecular mechanism of JAK-STAT3-SOCS signaling pathway. Methods The effect of DAC on the proliferation of HL-60 was detected by using CCK-8 assay. The cell apoptosis was analyzed by using Annexin-V/PI double standard method. The expressions of receptor NKG2DL including MICA/B and ULBPs in HL-60 cells were detected by using flow cytometry (FCM). The killing activity of NK cells was analyzed by using carboxy fluorescein diacetate succinimidyl ester (CFSE). The expressions of JAK/STAT3 signaling pathway or molecules including STAT3, its upstream kinases JAK1, JAK2 and the negative regulator of STAT3, SOCS-1, SOCS-3 were examined by Western blot. Methylation level of the SOCS-1, SOCS-3 gene after the treatment of DAC was analyzed by using methylation-sensitive high resolution melting (MS-HRM). Results There was an obvious inhibitory effect of DAC on HL-60 cells. The cell viability of HL-60 treated with 0.2, 0.5, and 1.0 μmol/L DAC for 48 h was decreased by (25±11) %, (39±8) % and (50±7) % (P < 0.01) respectively compared with those cells without DAC treatment. The incidence of apoptosis was (24.77±7.50) %, (27.10±4.48) % and (30.53±3.93) % after DAC treatment for 48h respectively, which were higher than that of untreated cells [(3.11±0.50) %] (P < 0.01). DAC induced a significant up-regulation of MICA/B, ULBP-1, ULBP-3 in HL-60 cells, and enhanced the sensitivity of HL-60 cells to NK cytotoxicity. Western blot results showed that a down-regulating expression of STAT3 and JAK1, JAK2 protein was detected, in addition to the phosphor-STAT3 and phosphor-JAKs in HL-60 cells after DAC treatment, but the expressions of SOCS-1 and SOCS-3 protein were increased. HRM results showed that DAC could inhibit the methylation of SOCS-3 gene. Conclusion DAC can inhibit the proliferation of HL-60 cells, upregulate the expression of NKG2DL and enhance the cytotoxicity of NK targeted to HL-60 cells, which might be related to the activity regulation of intracellular JAK-STAT3-SOCS signaling pathway. Key words: Leukemia, myeloid, acute; Cell proliferation; Apoptosis; Molecular mechanisms of action; Decitabine

Role of NKG2D ligand in murine heterotopic tracheal transplantation model

Objective To observe the changes of expression of NKG2D ligand Rae-1 and H60 at different time points after heterotopic tracheal transplantation in mice, and to explore its important role in bronchiolitis obliterans syndrome (BOS) after lung transplantation. Methods The model of heterotopic tracheal transplantation in mice was established and drawn at 7, 14 and 28 days after operation. Hematoxylin and eosin (HE) staining and Mallory staining were used to observe the postoperative tracheal occlusion rate. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression of Rae-1 and H60 at different time points. The expression of Rae-1 and H60 protein was detected by immunohistochemistry correspondingly. Results After transplantation, HE staining showed that BOS pathology continued to progress, and luminal occlusion rate at 14 d and 28 d in tracheal grafts was increased [(30.82±7.61)% and (83.52±13.12)% vs. (3.75±1.78)%, P=0.012, 0.007]. Mallory staining and α-smooth muscle actin (α-SMA) expression was increased. The results of RT-qPCR showed that the expression of H60 mRNA (14 d: 12.712 2±2.828 0 vs. 0.739 6±0.042 8, P=0.014) and Rae-1 mRNA (28 d: 10.464 9±2.109 1 vs. 0.617 6±0.041 3, P=0.022) was increased after transplantation as compared with the control group. Immunohistochemistry showed that the expression of H60 and Rae-1 proteins was significantly higher than that in the control group at 14 d (207.97±25.87 vs. 31.63±9.42, P=0.023) and 28 d (185.34±24.06 vs. 32.84±7.85, P=0.026). Conclusion NKG2D ligand may be involved in the development of BOS after lung transplantation. Strengthening the major histocompatibility complex (MHC) class Ⅰ chain related (MIC) typing or specifically blocking NKG2D pathway may delay and reduce BOS after lung transplantation. Key words: Lung transplantation; Bronchiolitis obliterans syndrome; NKG2D; Rae-1; H60

Effect of ionizing radiation on the NKG2D ligand expression on the tumor cell SCC25 surface and the killing effect on tumor cells

Objective To investigate the effect of ionizing radiation on the expression of NKG2D ligand on the surface of oral squamous cell carcinoma cell line SCC25 and its cytotoxicity to tumor cells. Methods When SCC25 cells were cultured into logarithmic growth phase, they were randomly designed as control (without treatment) and experimental group (2 Gy ionizing radiation treatment) by drawing lots. Flow cytometry was used to detect the expressions of NKG2D ligands major histocompatibility complex class Ⅰ chain-related molecule (MIC)A, MICB, UL16 binding protein (ULBP)1 on the surface of SCC25 in the control group and the experimental group cultured for 24 h. Real-time fluorescence quantification polymerase chain reaction (RT-PCR) was used to detect the changes of NKG2D ligand mRNA expression on SCC25 cell surface after 24 h culture in the experimental group and the control group. The cells were prepared and divided into blank control group (NC), 2 Gy ionizing radiation group (R), NK1 group (target ratio was 5∶1), NK2 group (target ratio was 20∶1), NK1+ R group (target ratio was 5∶1, 2 Gy ionizing radiation), NK2+ R group (target ratio was 20∶1, 2 Gy ionizing radiation). After each group was cultured for 24 h, the killing abilities of ionizing radiation and natural killer (NK) cells to oral squamous cell carcinoma SCC25 cells were detected by CCK8. Results Flow cytometry experiment showed that, among the NKG2D ligands, the MICA fluorescence values of experimental group and control group were respectively 21.04±0.39, 22.90±0.40 (t=2.465, P=0.069), MICB fluorescence values were 27.58±0.50, 29.83±1.05 (t=1.936, P=0.125), and ULBP1 fluorescence values were 21.04±0.40, 21.78±0.50 (t=1.154, P=0.313). This indicated that after ionizing radiation on SCC25, the NKG2D ligand MICA, MICB, ULBP1 expression increased slightly, but the differences were not statistically significant. RT-PCR indicated that mRNA expressions of MICB, ULBP1 were significantly different between the control group and the experimental group (t=18.334, P=0.000; t=6.381, P=0.008). The expressions of the experimental group were respectively 6.49, 1.64 times as those of the control group. The results of CCK8 showed that, there was a significant difference in cell killing ability among NK1 group, NK2 group and NC group (F=344.600, P=0.000), suggesting that NK cells could kill tumor cells, and the higher ratio of NK cells and SCC25, the stronger killing effect. The comparison between R group and NC group showed that the difference in cell killing ability was not statistically significant (P=0.567). NK1+ R group and NK1 group were compared and the difference was not statistically significant (P=0.915). There was no significant difference between NK2+ R group and NK2 group (P=0.678). This showed that the killing effect of ionizing radiation was weak. Conclusion Ionizing radiation can increase the mRNA expression of NKG2D ligands MICB and ULBP1. This may provide a new way for tumor immunotherapy. The killing effect of ionizing radiation on cells is not obvious. It may be related to low radiation dose and only 24 h for cell culture. Key words: Radiation, ionizing; Killer cells, natural; NKG2D ligands

KIR3DS1-mediated recognition of HLA-*B51: modulation of KIR3DS1 responsiveness by self HLA-B allotypes and effect on NK cell licensing

Several studies described an association between killer-cell immunoglobulin-like receptor (KIR)/HLA gene combinations and clinical outcomes in various diseases [1-2]. Here, we show that KIR3DS1 mediates positive signals upon recognition of HLA-B*51 (Bw4-I80) surface molecules on target cells and that this activation occurs only in Bw4-I80neg individuals, including those carrying particular KIR/HLA combination settings. In addition, killing of HLA-B*51 transfected target cells mediated by KIR3DS1+/NKG2A+ NK cell clones from Bw4-I80neg donors could be partially inhibited by antibody-mediated masking of KIR3DS1. Interestingly, KIR3DS1-mediated recognition of HLA-B*51 could be better appreciated under experimental conditions in which the function of NKG2D was reduced by mAb-mediated blocking. This experimental approach may mimic the compromised function of NKG2D occurring in certain viral infections. We also show that, in KIR3DS1+/NKG2A+ NK cell clones derived from an HLA-B Bw4-T80 donor carrying 2 KIR3DS1 gene copy numbers, the positive signal generated by the engagement of KIR3DS1 by HLA-B*51 resulted in a more efficient killing of HLA-B*51-transfected target cells. Finally, we analyzed KIR3DS1+/NKG2A+ NK cell clones from a HLA-B Bw4neg donor carrying cytoplasmic KIR3DL1. Although these clones expressed lower levels of surface KIR3DS1, they displayed responses comparable to those of NK cell clones derived from HLA-B Bw4neg donors that expressed surface KIR3DL1. Altogether these data suggest that, in particular KIR/HLA combinations, KIR3DS1 may play a role in the process of human NK cell education.

Antitumor effects and mechanisms of DC-CIK biotherapy combined with sorafenib against lung adenocarcinoma cells

Objective To investigate the antitumor effects and the possible mechanisms of dendritic cells co-cultured with cytokine-induced killer cells (DC-CIK) in combination with sorafenib on two lung adenocarcinoma cell lines, A549 cells (harboring KRAS gene mutation) and PC-9 cells (harboring EGFR gene mutation). Methods DC and CIK cells were routinely generated in vitro by stimulating PBMCs isolated from lung cancer patients with different cytokines and then co-cultured after a week of culturing. Flow cytometry analysis (FCM) was used to analyze the phenotype of DC-CIK cells after 7 days of co-culturing. The 50% inhibitory concentration (IC50) of sorafenib against tumor cells was detected by MTT assay. The tumor cells were treated with DC-CIK cells alone or in combination with sorafenib. The proliferation of tumor cells was tested by CCK-8 kit and dynamically monitored by real-time cellular analysis (RTCA). Annexin-V/PI staining was used to examine the apoptosis rates in each group. Real-time fluorescent quantitative PCR and FCM were respectively performed to detect the expression of natural killer group 2 member D ligands(NKG2DLs) at mRNA and protein levels after the treatment with sorafenib for 24 h. Results There was no significant difference between the IC50 of sorafenib against A549 and PC-9 cells after a 24-hour exposure (P>0.05). Compared with the DC-CIK biotherapy, treating the tumor cells with DC-CIK cells in combination with sorafenib significantly inhibited the cell proliferation and increased the total apoptosis rates of tumor cells (P<0.05). Moreover, the inhibition rates to tumor cell proliferation were enhanced along with the increase of effect-to-target ratio (E/T). Compared with the single-factor treatment groups, the normalized cell index (NCI) in the combined treatment group was significantly decreased. Blocking NKG2D could abate the inhibitory effect of DC-CIK cells on tumor cell proliferation (P<0.05). The expression of NKG2DLs (including ULBP1, UBLP2 and ULBP3) on tumor cells at mRNA and protein levels were increased to different extent after treating with 5 μmol/L of sorafenib for 24 h. Conclusion There was no significant different between the inhibitory effects of sorafenib on the proliferation of lung adenocarcinoma cancer cells harboring KRAS or EGFR gene mutation. The antitumor effects of DC-CIK cells combined with sorafenib on lung adenocarcinoma cells might be induced by regulating the NKG2D-NKG2DLs pathway and enhancing apoptosis. Moreover, the antitumor effects of the combined treatment were better than those of single-factor treatments. Key words: Lung adenocarcinoma; Sorafenib; Cytokine-induced killer cell; Dendritic cell; Immunotherapy

Natural killer cell receptors and human cytomegalovirus infection

Human cytomegalovirus(HCMV)infection is quite prevalent in population.HCMV triggers important disorders in pregnant women, immune-compromised individuals and organ-transplant patients.For decades, more and more scholars believe that NK cells are important immune cells against HCMV.The activity of NK cells largely depends on the balance between the signals transducted by inhibitory receptors and activatory receptors, therefore the study of NK receptors is of great importance.It may be helpful to the basic research and clinical treatment of HCMV infection.Here, this paper reviews the changes and the molecular mechanism of NK receptors in the control of HCMV infection. Key words: Human cytomegalovirus; Natural killer cell; Receptors; NKG2A; NKG2C; NKG2D

Analysis of the phenotypes and the function of CD56+ T cells during primary HIV-1 infection

Objective To investigate the changes of phenotypes and function of CD56+ T cells during primary HIV-1 infection and their relationship with disease progression. Methods Peripheral blood mononuclear cells (PBMCs) were collected from 53 subjects with primary HIV-1 infection and 31 HIV-1-negative healthy subjects. The percentages of CD56+ T cells and the expression of several phenotypic markers on CD56+ T cells including CD16, CD161, NKB1, NKG2A, NKp46, NKG2D, NKG2C and CD158a were analyzed by flow cytometry. IFN-γ and TNF-α released by CD56+ T cells with and without K562 stimulation and the levels of cytotoxic molecular CD107a were measured. Results The percentages of CD56+ T cells in patients with primary HIV-1 infection were significantly lower than those of healthy subjects (P=0.025). The levels of CD56+ T cells were negatively related to the viral loads in plasma samples (P=0.021, r=-0.316). Compared with healthy subjects, the expression of CD16 (P=0.003), CD161 (P=0.023), NKB1 (P=0.023) and NKp46 (P=0.021) on CD56+ T cells were decreased in patients with primary HIV-1 infection. The levels of NKB1 were positively related to the CD4+ T cell counts (P=0.007, r=0.364), but were negatively related to the viral loads in plasma samples (P=0.030, r=-0.299). Spontaneous secretion of IFN-γ and TNF-α by CD56+ T cells and the expression of CD107a were dramatically inhibited in patients with primary HIV-1 infection as compared with healthy subjects (all P<0.001). Moreover, the killing ability of CD56+ T cells against K562 target cells was weakened in patients with primary HIV-1 infection as the levels of IFN-γ-, TNF-α- and CD107a-producting CD56+ T cells were significantly decreased (P<0.001 for IFN-γ and TNF-α, P=0.016 for CD107a). Conclusion Inhibited expression and altered phenotypes of CD56+ T cells were identified during primary HIV-1 infection. Lower levels of cytokines and cytotoxic molecular were also detected, indicating the dysfunction of CD56+ T cells appeared during early stage of HIV-1 infection and was associated with disease progression. Key words: Human immunodeficiency virus; CD56+ T cells; Phenotype; Cytotoxicity

JNK2 enhances the NKG2D-mediated natural killer cell cytotoxicity

Objective To investigate the effects of c-Jun N-terminal kinase （JNK） 2 on NKG2D-mediated natural killer （ NK） cell cytotoxicity .Me thods NK cells were activated by polyinosinic-polycyti-dylic acid （ Poly I∶C ） .The activation status of JNK signaling pathway was detected .The cytotoxicity of ac-tivated NK cells and the level of IFN-γproduced were measured to determine the function of NK cells in the absence of JNK2.Tumor growth in wild type and JNK2-knockout （JNK2-/-） mice with lymphoma xenograft were measured to evaluate immune surveillance of NK cells .Results The phosphorylation of JNK and up-stream kinases were observed in the early stage of cell activation after treatment of Poly I ∶C.The expressed ligands of the activating receptors NKG2 D significantly increased NK cell cytotoxicity to lymphoma cells . JNK2 deficiency impaired the antitumor effects of NK cells , and then resulted in enhanced tumor growth in JNK2-/-mice.Conclusion JNK2 signaling pathway is involved in the NKG2 D-mediated activation of NK cells and regulates immune surveillance of NK cells against tumor . Key words: JNK; NKG2D; Tumor immunity; Cell killing; c-Jun N-terminal kinase; Natural killer cell; NKG2D

Changes of natural kill cell in peripheral blood of patients with myelodysplastic syndrome

To analyze the percentage and functional changes of natural kill (NK) cell in peripheral blood of myelodysplastic syndrome (MDS) patients so as to evaluate the relationships between these changes and hematopoietic functions and explore the role of NK cell in the pathogenesis of MDS. By flow cytometry, the percentage of NK cell (CD3⁻CD56⁺CD16⁺) in peripheral blood lymphocytes was detected in 20 MDS patients, 15 acute myelogenous leukemic (AML) and 15 normal controls from March 2013 to November 2013 at our hospital.NK cell activation receptors (NKG2D, NKp44), inhibitory receptors (CD158a, CD158b), perforin and granzyme-β of patients and normal controls were also detected. The correlation between these changes and hematopoietic functions, including the percentages of neutrophil granulocyte (ANC%), lymphocyte (Lym%), reticulocyte (RET%) and hemoglobin, thrombocyte in peripheral blood and the hematopoietic function in bone marrow (CD34⁺%) were evaluated. (1) The percentage of NK cell (3.87% ± 0.97%) in MDS patients was significantly lower than that of normal controls (6.08% ± 1.37%, P < 0.05) and higher than that of AML patients (2.58% ± 0.78%, P < 0.05).(2) The expression of NKG2D in MDS patients (52.83%) was significantly lower than that of normal controls (86.36%, P < 0.05) and higher than that of AML patients (42.00%, P < 0.05). The expression of NKp44 in MDS patients (2.41%) was significantly higher than that of normal controls (0.62%) and AML patients (0.92%) (both P < 0.05). (3) The expression of CD158a in MDS and AML patients (5.46% ± 2.40%, 4.05% ± 1.89%) were significantly both lower than those of normal controls (7.97% ± 2.85%, both P < 0.05). The expression of CD158b had no significant difference among the 3 groups. (4) The expression of perforin in MDS patients (17.83%) was significantly lower than that of normal controls (59.79%, P < 0.05) and higher than that of AML patients (13.06%, P < 0.05). The expressions of granzyme-β in MDS and AML patients (21.54%, 30.65%) were significantly both lower than those of normal controls (83.42%, both P < 0.05). (5) The percentage of NK cell and the expression of NKG2D and NKp44 in MDS patients with low-risk group were in turn higher than those of high-risk group (4.06% ± 0.56% vs 3.59% ± 1.37%, 53.42% ± 6.85% vs 47.29% ± 8.08%, 2.46% vs 2.07%, all P < 0.05) , the expression of CD158a and CD158b were in turn lower than those of high-risk group, but both P > 0.05.(6) The percentage of NK was positively correlated with ANC% (r = 0.780, P < 0.05), but negatively with Lym% and the CD34⁺% in bone marrow (r = -0.543, -0.610, both P < 0.05). The expression of CD158a was positively correlated with CD34⁺% in bone marrow (r = 0.612, P < 0.05). The expressions of NKp44, CD158a, perforin and granzyme-β of NK cells had no correlation with hematopoiesis (all P > 0.05). The lowered percentage and function of NK may cause the immunological dysfunction and lead to underkill of pathological hematopoietic cells in MDS.

Correlation between vasoactive intestinal peptide and expression of NKG2D, adaptor protein 10 and extracellular signal-regulated kinase in inflammatory cells of gastric adenocarcinoma

Objective To investigate the expression of NKG2D and related signaling molecules adaptor protein(DAP10) and extracellular signal-regulated kinase(ERK) in the inflammatory cells of gastric adenocarcinoma and para-carcinoma tissues,the effects of vasoactive intestinal peptide(VIP) on the expression of NKG2D,DAP10 and ERK in natural killer(NK) cells of healthy volunteers.Methods The expression of NKG2D,DAP10 and ERK in the inflammatory cells of 36 gastric adenocarcinoma and para-carcinoma tissues were detected by immunohistochemistry.The peripheral blood NK cells of healthy volunteers were isolated and purified with CDC method.The expressions of NKG2D,DAP10 and ERK in NK cells were determined by immunocytochemistry and reverse transcription-polymerase chain reaction(RT-PCR) methods before and after VIP intervention.The ttest was used for comparison between two groups and rank transformation nonparametric test (Kruskal-Wallis H test) for count data analysis.Results The expression levels of NKG2D,DAP10 and ERK in the inflammatory cells of gastric adenocarcinoma were significantly lower than those of para-carcinoma tissues (H=30.640,24.910,20.320,all P＜0.01).The expression levels of NKG2D,DAP10 and ERK were much lower in poorly differentiated gastric adenocarcinoma (H =4.049,11.830,8.118),at stage Ⅲ-Ⅳ (H=4.275,11.560,7.686),and the expression levels of NKG2D,ERK were lower with lymph nodes metastasis (H=6.513,6.064,all P＜0.05).The expression levels of NKG2D,DAP10 and ERK in gastric adenocarcinoma tissues and inflammatory cells were not correlated with gender,age and the location of lesions (all P＞0.05).The expression levels of NKG2D,DAP10 and ERK in NK cells was decreased after VIP intervention (protein:H=12.438,4.798,13.745,mRNA:F=337.640,638.579,1055.015,all P＜0.05).Conclusion Gastric adenocarcinoma may downregulate the expression of NKG2D,DAP10 and ERK in NK cells through VIP secretion,which may be involved in the immune escape of gastric cancer. Key words: Vasoactive intestinal peptide; Killer cells, natural; Stomach neoplasms; Adenocarcinoma; DAP10 protein; Mitogen-activated protein kinases ; Tumor escape

Expression of ligands of DNAM-1 and NKG2D in colonic cancer

Objective To investigate the expression of ligands of DNAM-1 and NKG2D in the colonic cancer.Methods The colonic cancer tissue and adjacent normal colonic tissues were collected from 42 colonic cancer patients who were admitted to the Tangdu Hospital of Fourth Military Medical University from June 2010 to January 2011 were retrospectively analyzed.The expressions of CD155,CD112 and MICA/B in the colonic cancer tissues and the normal colonic tissues were detected by immunohistochemistry.The expressions of CD155,CD112 and MICA/B in the colonic cell line SWll6,SW480,SW620 and Colo205 in the Duke's A,B,C and D phases were detected by cell cytometry.The relationship of the expressions of the 3 ligands and the clinicopathological parameters was analyzed using the Mann-Whitney U test,chi-square test and Fisher exact probobility.Results Week expression of CD155 was found in the normal colonic tissues,while the expressions of CD112 and MICA/B were not found.In the colonic cancer tissues,the expressions of CD155,CD112 and MICA/B were 81.0％,52.4％ and 47.6％,which were significantly increased.The expressions of CD155,CD112 and MICA/B were not correlated with the gender,tumor differentiation,lymph node metastasis and Duke's staging (P ＞ 0.05).The overall expression rates of CD155,CD112 and MICA/B in the colonic cancer cell line SWll6,SW480,SW620 and Colo205 were 88.9％,67.4％ and 42.3％,respectively.The overall expression of CD155 was significantly higher than CD112 and MICA/B (F =23.17,P ＜ 0.05).Conclusion CD155,CD112 and MICA/B express in the colonic cancer tissues and colonic cancer cell line SW116,SW480,SW620 and Colo205,and the expression of CD155 is the highest. Key words: Colonic neoplasms ; Natural killer cells ; Receptor; Immunotherapy

Preliminary study on the changes of NKG2D expression and its role in children with infectious mononucleosis

Objective To investigate the mechanism of disturbed immunological function induced by Epstein-Barr virus(EBV) infection through evaluating NKG2D expressions in NK cells and CD8+ T lymphocytes from children with infectious mononucleosis.Methods Twenty-nine children with infectious mononucleosis (IM) and twenty-five age-matched healthy children were enrolled in the study.NKG2D/NKG2A expressions in NK cells and CD8+T lymphocytes,and NKG2D ligand MHC Ⅰ chain-related molecules A(MICA) and UL-16binding proteins (ULBP-1) expressions on CD14+ mononuclear cells (MC) were analyzed by flow cytometry.The concentrations of cytokines such as IL-7,IL-12,IL-15,IFN-γ,TGF-β and soluble MICA(sMICA) in plasma were determined by enzyme-linked immunosorbent assay (ELISA).Results (1) Compared with the control group,NKG2D expression was significantly decreased in both NK cells and CD8+T lymphocytes from children with IM (P＜0.05),especially in the three cases of suspected EVB-HLH children.(2)There was no difference in the expression of MICA and ULBP-1 in CD14+ MC between the children with IM and the normal control(P＞0.05).(3)The concentrations of IL-15 and TGF-β in plasma were lower than those of the control group (P＜0.05),while the levels of IL-7,IL-12,IFN-γ and sMICA were up-regulated(P＜0.05) in children with IM.(4)NKG2A expression in NKcells in children with IM was significantly increased as compared with healthy controls(P＜0.05),however,there was no differences in the expression of NKG2A in CD8+ T lymphocytes between the two groups (P＞0.05).Conclusion Remarkable down-regulated expressions of NKG2D in NK cells and CD8+T lymphocytes might be one of the factors causing disturbed immunological function in children with EBV infection,which might have a close relationship with abnormal cytokine milieu of IL-15 and IL-12or high concentration of sMICA. Key words: Epstein-Barr virus; Infectious mononucleosis; NKG2D

Correlation and its clinical significance of regulatory T cells and NKG2D-expressing NK cells in lung cancer patients

Objective To detect the levels of CD+4 CDHi25 CDLo127 regulatory T (Treg) cells and NKG2D-expressing NK cells in peripheral blood of lung cancer patients and investigate their relationship,and explore their roles in the immune escape of lung cancer and clinical significance.Methods The levels of CD+4 CDHi25 CDLo127 Treg cells,NK cells and NKG2D-expressing NK cells were measured in peripheral blood of 70 lung cancer patients and 50 healthy controls.Results Compared with control group,the levels of CD+4 CDHi25 CDLo127 Treg cells enormously increased [(8.4±4.1) ％,(6.7±1.7) ％] (t =3.09,P ＜ 0.05),while the value of NKG2D obviously decreased [(83.3±4.9) ％,(87.4±2.9) ％] (t =3.16,P ＜ 0.05); And the group of NK cells is not different [(15.6±8.3) ％,(17.2±4.2) ％] (t =-1.33,P ＞ 0.05); NKG2D-expressing NK cells had a perfect negative correlation with CD+4 CDLo25 CDLo127 Treg cells (r =-0.302,P ＜ 0.05).Conclusion Accumulation of Treg cells in serum may lead to the down-modulation of NKG2D-expressing NK which participates the tumor immune escape.They may be potential indicators evaluating immune functions and prognosis of lung cancers. Key words: Lung neoplasms; Treg; NK; NKG2D; Immune escape

Effect and mechanism of RetroNectin on the proliferation and cytotoxic activity of acute leukemia natural killer cells

To explore the effects of precoated RetroNectin on the proliferation and cytotoxicity of natural killer cells (NK) from acute leukemia (AL). Mononuclear cells (MNCs) were isolated from peripheral blood of complete remission AL patients. The MNCs were cultured in vitro precoated with 50 µg/ml RetroNectin (group I), 25 µg/ml RetroNectin (group II) and by the traditional method (control group) to generate NK. The changes of growth rate, phenotypic characterization, secretion of cytokines of NK, cell cycle, apoptosis and cytotoxicity of NK were determined. The amplification of NK in group I ((51 ± 9)×10(6)) and II ((79 ± 16) ×10(6)) were higher than that in control group ((37 ± 11)×10(6)) (both P < 0.05), and the amplification of NK in groupII was higher than that in group I (P < 0.05). There were no differences among three groups with regards to the phenotypic characterization of CD3(-)CD56(+). The secretions of interleukin 2, interleukin 12, tumor necrosis factor alpha and interferon gamma in group I and II were higher than that in control group (all P < 0.05). The expression of CD25 positive cells in groups I (38.2% ± 4.1%) and II (37.5% ± 5.1%) were higher than that in control group (24.2% ± 5.8%) (both P < 0.05). The expression of NKG2D positive cells in groups I (81.6% ± 17.9%) and II (85.7% ± 20.1%) were higher than that in control group (63.6% ± 21.9%) (both P < 0.05). The percentages of G(1) stage cells in groups I (50% ± 10%) and II (49% ± 11%) were lower than that in control group (71% ± 15%) while the percentages of S stage cells in groups I (36% ± 14%) and II (37% ± 8%) were higher than that in control group (19% ± 10%) (all P < 0.05). But no difference existed between two groups (all P > 0.05). The expression of cell cycle regulatory genes p21 and p27 were reduced in groups I and II. The percentages of apoptotic cells in groups I (10.7% ± 2.1%) and II (9.4% ± 3.1%) were lower than that in control group (29.6% ± 10.3%) (both P < 0.05). The cytotoxicity to AL cells in groups I (82% ± 21%) and II (80% ± 18%) were higher than that in control group (61% ± 14%) (both P < 0.05) at the E/T scope 20:1. But no difference existed between two groups (all P > 0.05). The proliferation and cytotoxicity of NK cells may be boosted by precoated RetroNectin.

Protective effect of breviscapine on human hepatocytes under hypoxia/re-oxygenation and its possible mechanism

To explore the protective effect of breviscapine on human hepatocytes (L-02) under hypoxia/re-oxygenation (H/R) and elucidate its possible mechanism. A in vitro model of H/R was employed to mimic H/R injury of graft organ. L-02 cells were randomly divided into 3 groups: control, H/R and breviscapine treatment (pre-treated with breviscapine and H/R). After a 10 h hypoxic culturing under 1% O(2), 94% N(2) and 5% CO(2), L-02 cells received oxygen for 1, 2, 4, 8, 16 and 24 h respectively. MICA mRNA and protein levels were detected by quantitative real-time polymerase chain reaction (PCR) and Western blot. And the activity of natural killer (NK) cell cytotoxicity for L-02 was measured by lactate dehydrogenase (LDH) release assay. After blocking I/R treatment with NKG2D antibody, the activity of NK cell cytotoxicity for L-02 was detected. After 10 h hypoxia, MICA mRNA and protein levels significantly increased from 1 h, stayed up-regulated until 8 h and then went back to normal level after reoxygenation versus the control group. The activity of NK cell cytotoxicity for L-02 under the treatment of H/R increased markedly from 1 h post-reoxygenation and stayed up-regulated from 1 h to 8 h versus the control group. After hypoxia, L-02 cells were blocked with NKG2D antibody and the NK cell cytotoxicity for L-02 significantly decreased versus the I/R group. The administration of breviscapine significantly lowered the mRNA and protein levels of MICA in L-02 under I/R and then significantly decreased the NK cell cytotoxicity. The process of I/R may mediate the NK cell cytotoxicity activity toward to L-02 by inducing a strong increase of MICA at mRNA and protein levels in L-02 cells. And the administration of Breviscapine significantly reduces the NK cell cytotoxicity for L-02 under I/R through the down-regulated expression of MICA.