Expression of murine killer immunoglobulin-like receptor KIRL1 on CD1d-independent NK1.1<Superscript>+</Superscript> T cells

Three mouse killer immunoglobulin-like receptors (KIRs), namely, KIR3DL1, KIRL1, and KIRL2, have recently been identified in C56BL/6 (B6) mice. However, only two Kir genes are found in the B6 mouse genome sequence data base. To clarify this discrepancy, we cloned Kir cDNAs from multiple strains of mice. Sequencing of the cDNA clones showed that the Kir3dl1 gene is found in C3H/HeJ and CBA/J but not in B6 mice. Analysis of the single nucleotide polymorphism data base suggested that Kir3dl1 is the C3H/HeJ and CBA/J allele of Kirl1. We generated mAb to the recombinant KIRL1 protein to investigate its expression pattern. The anti-KIRL1 mAb bound to NK1.1⁺ T cells but only very weakly or at undetectable levels to other lymphocytes including natural killer (NK) cells and conventional T cells. Among NK1.1⁺ T cells, conventional NK T cells stained with CD1d tetramer did not significantly bind anti-KIRL1 mAb, whereas CD1d-tetramer-negative subset was KIRL1-positive. Furthermore, the expression of KIRL1 is readily detected on NK1.1⁺ T cells from β₂-microglobulin-deficient B6 mice. Thus, KIRL1 is predominantly expressed on CD1d-independent NK1.1⁺ T cells.     

自然杀伤细胞对妊娠期糖尿病患者并发高脂血症的影响

目的：观察自然杀伤细胞（NK 细胞）对妊娠期糖尿病患者并发高脂血症的影响。方法：选择2013 年2月至2015 年4 月在我 院确诊的妊娠期糖尿病高脂血症患者95 例,作为研究组,同期选择体检健康孕妇100 例,作为对照组,对比两组的血糖、糖化血 红蛋白、身体质量指数（BMI）、总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）、白 细胞介素-2（IL-2）、IL-6、干扰素-r（IFN-r）、NK 细胞及细胞亚型,并采用Person相关性分析NK细胞与血脂的相关性。结果：①研 究组空腹血糖、餐后2 h 血糖、糖化血红蛋白、BMI均明显高于对照组,差异具有统计学意义（P<0.05）;②研究组TC、TG、LDL-C 均明显高于对照组,HDL-C明显低于对照组,差异具有统计学意义（P<0.05）;③研究组IL-2、IFN-r明显低于对照组,差异具有统 计学意义（P<0.05）;IL-6 明显高于对照组,差异具有统计学意义（P<0.05）;④研究组的NK 细胞数量、CD4+/ CD3+、CD4+/ CD8+、 CD56++CD4+/ CD3+均低于对照组的（P<0.05）;CD8+/ CD3+、CD18+/ CD8+、CD19+/ CD3+均高于对照组的（P<0.05）;两组CD28+/ CD8+差异无统计学意义（P＞0.05）。⑤NK 细胞数量与TC、TG、LDL-C水平均呈负相关（r=-0.527、-0.598、-0.485）,NK 细胞数量与 HDL-C 水平呈正相关（r=0.528）。结论：NK 细胞在妊娠期糖尿病孕妇中数量减少,活性也降低,可能与参与免疫反应的NK细胞 不同表型细胞含量发生变化,导致血脂调节失衡,引发高血脂症的过程有关。     

Anti-cancer activity and mechanistic features of a NK cell activating molecule

Natural cytotoxicity receptors (NCRs) are major activating receptors involved in NK cytotoxicity. NCR expression varies with the activation state of NK cells, and the expression level correlates with NK cells’ natural cytotoxicity. In this study, we found that Gö6983, a PKC inhibitor, induced a remarkable increase of NCR expression on primary NK cells, but other PKC inhibitors and NK cell stimulators such as IL-2 and PMA, did not. Gö6983 increased the expression of NCR in a time- and concentration-dependent manner. Furthermore, Gö6983 strongly upregulated the surface expression of death ligands FasL and TRAIL, but not cytotoxic molecules perforin and granzyme B. Unlike two other NK stimulating molecules, IL-2, and PMA, Gö6983 did not induce NK cell proliferation. Up-regulation of NCRs and death ligands on NK cells by Gö6983 resulted in a significant enhancement of NK cytotoxicity against various cancer cell lines. Most importantly, administration of Gö6983 effectively inhibited pulmonary tumor metastasis in mice in a dose-dependent manner. These results suggest that Gö6983 functions as an NK cell activating molecule (NKAM); this NKAM is a novel anti-cancer and anti-metastasis drug candidate because it enhances NK cytotoxicity against cancer cells in vivo as well as in vitro.     

Increased activating killer immunoglobulin-like receptor genes and decreased specific HLA-C alleles in couples with recurrent spontaneous abortion

Accumulating evidence indicates natural killer (NK) cells play crucial roles in successful pregnancy. To investigate whether the killer cell immunoglobulin-like receptor (KIR) gene polymorphism and the corresponding specific HLA ligands in parent couples possessing a susceptibility to unexplained recurrent spontaneous abortion (RSA), we searched 73 pairs of childless couples with three or more abortions characterized as unexplained RSA and 68 pairs of healthy control couples. Peripheral blood was drawn to obtain genomic DNA which was used for a polymerase chain reaction using sequence-specific primers (PCR-SSP) in order to determine whether 15 selected KIR genes and two groups of HLA-C alleles were present. Our result showed that gene frequency of KIR2DS1 was higher in patients with RSA compared to that of control subjects (P =0.029). Increased numbers of activating KIR genes was observed in patients (P =0.041). Women who possessed more than two activating KIR genes were found more frequently in patients than those in control subjects (P =0.018). From a cohort of husband and wife couples, the women with a KIR2DS1 gene, and with a decreased group 2 HLA-C allele for the homologous inhibitory receptor KIR2DL1, had a tendency to fall into the RSA group (P =0.004). The results suggest that a genetic variation at the KIR locus influences the susceptibility to unexplained RSA in the Chinese Han population. Moreover, decreased ligands for inhibitory KIRs could potentially lower the threshold for NK cell activation, mediated through activating receptors, thereby contributing to pathogenesis of RSA.     

PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab/IPH4301探针的初步研究

摘要 目的：制备PEG-G5.NH2-FITC-DOTA(Gd)- Monalizumab/IPH4301纳米探针,并研究其与NK-92MI细胞的结合能力、毒性、细胞体外MRI成像能力、促进NK-92MI细胞对MDA-MB-231三阴性乳腺癌细胞的杀伤能力。方法：制备PEG-G5.NH2-FITC-DOTA(Gd)- Monalizumab/IPH4301纳米探针,利用透射电子显微镜（Transmission Electron Microscope,TEM）进行表征,并使用ImageJ对其粒径进行统计;通过流式细胞术分析纳米材料和NK-92MI细胞的结合能力;应用MRI对纳米探针标记的NK-92MI细胞体外成像并分析其T1加权（T1WI）的信号改变。利用蛋白印迹（Western blot）检测经过与NK-92MI细胞共培养后三阴性乳腺癌MDA-MB-231细胞凋亡相关蛋白的表达以及Elisa法检测培养体系内的IFN-γ的表达量。结果：制备的纳米探针颗粒粒径分布均匀,形态近似球形。其与NK-92MI细胞结合的量以及T1信号随着纳米探针浓度增加而逐渐增加。使用Monalizumab和IPH4301两种抗体修饰的纳米探针增强了NK-92MI细胞释放IFN-γ并促进MDA-MB-231乳腺癌细胞的凋亡。结论：PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab/IPH4301纳米探针在结合NK-92MI细胞后,可以在3.0T磁共振下进行体外成像,并增强了NK-92MI细胞对MDA-MB-231细胞的杀伤能力。     

Molecular cloning of KLRI1 and KLRI2, a novel pair of lectin-like natural killer-cell receptors with opposing signalling motifs

We here report the molecular cloning of a novel family of killer-cell lectin-like (KLR) receptors in the rat and the mouse, termed KLRI. In both species, there are two members, KLRI1 and KLRI2. While the extracellular lectin-like domains of KLRI1 and KLRI2 are similar [74% (rat) and 83% (mouse) amino acid identity], they differ intracellularly. KLRI1 has two immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic domain, suggesting an inhibitory function. KLRI2 has no ITIM, but a positively charged lysine residue in the transmembrane region, suggesting association with activating adapter molecules. Klri1 and Klri2 are localized within the natural killer (NK) cell gene complex on rat chromosome 4 and mouse chromosome 6. By RT-PCR and Northern blot analysis KLRI1 and KLRI2 were selectively expressed by NK cells in both rat and mouse. Epitope-tagged expression constructs of rat KLRI1 and rat KLRI2 induced surface expression of a nondisulphide-linked protein of M_r 36,000/39,000 and M_r 34,000, respectively.     

Natural killer activity against human K562 tumour cells during Plasmodium cynomolgi malarial infection of rhesus monkeys

Abstract This study assessed the natural killer (NK) cell activity profile during Plasmodium cynomologi infection in rhesus monkeys. There was a significant decrease in the NK cell activity in the peripheral blood leukocytes of infected monkeys during the early, asending phase of infection. However, as the parasite load decreased, NK cell activity returned to normal levels. This could be correlated with the peak increase in lymphocyte counts. This indicated that a decrease in NK cell activity observed at an earlier stage during an active P. vivax malarial infection was a temporary phenomenon.     

Drug resistance in rat colon cancer cell lines is associated with minor changes in susceptibility to cytotoxic cells

The development of resistance to anticancer drugs urges the search for different treatment modalities. Several investigators have reported the concomitant development of drug resistance and resistance to natural killer (NK), lymphokine-activated killer (LAK) or monocyte/macrophage cell lysis, while others described unchanged or even increased susceptibility. We investigated this subject in the rat colon carcinoma cell line, CC531-PAR, which is intrinsically multidrug-resistant (MDR), and in three sublines derived from this parental cell line: a cell line with an increased MDR phenotype (CC531-COL), a revertant line from CC531-COL (CC531-REV), which demonstrates enhanced sensitivity to anticancer drugs of the MDR phenotype, and an independently developed cisplatin-resistant line (CC531-CIS). In a 4-h⁵¹Cr-release assay we found no difference in susceptibility to NK cell lysis. No significant differences in lysability by adherent LAK (aLAK) cells were observed in a 4-h assay. In a prolonged 20-h⁵¹Cr-release assay an enhanced sensitivity to aLAK-cell-mediated lysis was observed in the revertant, P-glycoprotein-negative cell line and in the cisplatin-resistant cell line (CC531-CIS). None of the cell lines was completely resistant to lysis by aLAK cells. Therefore, a role for immunotherapy in the treatment of drug-resistant tumors remains a realistic option.     

NK细胞中过表达Livin的实验研究

目的:将携带Livin的质粒pIRES2-EGFP-Livin进行扩增,转染自然杀伤细胞(NK)及胃癌细胞株SGC-7901,并检测其在NK及胃癌细胞株SGC-7901中的表达。方法:将携带Livin基因的质粒p IRES2-EGFP-Livin进行扩增,鉴定质粒纯度与浓度;从健康人外周血中获得NK细胞,应用HP转染试剂将质粒pIRES2-EGFP-Livin转染体外培养的NK及胃癌细胞,对比分析NK及胃癌细胞株SGC-7901中基因转染效率及目的基因的表达情况。结果:用无血清培养基在体外成功的扩增大量的NK细胞;质粒提取试剂盒抽提得到大量无内毒素的质粒,质粒DNA基因序列并未发生突变,浓度和纯度较高。胃癌细胞株SGC-7901中观察到明显的质粒pIRES2-EGFP-Livin绿色荧光表达;而NK中未观察到绿色荧光表达。结论:质粒pIRES2-EGFP-Livin能使Lvin蛋白表达于胃癌细胞株SGC-7901中,而在NK中未表达。     

A single major chromosomal region controls natural killer cell-like activity in rainbow trout

We report the identification of a single major chromosomal region controlling natural killer (NK) cell-like activity in rainbow trout (Oncorhynchus mykiss). A genetic map based on 484 AFLP and 39 microsatellite genotypes from 106 doubled haploid fish was constructed. These fish were produced by androgenesis from a hybrid of two clonal lines divergent in NK-like activity. NK-like activities for 75 of the doubled haploids were quantified by an in vitro chromium release assay utilizing ⁵¹Cr-labeled YAC-1 target cells. Composite interval mapping revealed a single major quantitative trait locus (QTL) associated with NK-like activity in this rainbow trout model. Genetic mapping revealed this QTL to also be unlinked to: fragmented MHC class I and MHC class II regions, the leukocyte receptor cluster, the natural killer cell enhancement factor (NKEF) gene, the RAG-1 gene, and two QTL associated with resistance to infectious pancreatic necrosis virus in rainbow trout. Collectively, these results extend the utility of rainbow trout as an immunological model and are consistent with the idea that a single chromosomal region homologous to the natural killer cell complex (NKC) located on syntenic portions of mouse chromosome (Chr) 6, human Chr 12, and rat Chr 4 may exist in a lower vertebrate model.     

自然杀伤细胞参与肝脏恶性肿瘤免疫逃逸研究进展

自然杀伤细胞是机体固有免疫系统重要组成部分,在肝脏等免疫器官中含量丰富,而且免疫表型、功能等表现出器官特异性。在正常情况下,靶细胞表面的配体与自然杀伤细胞表面的活化性受体直接结合并释放细胞毒性物质,诱导活化靶细胞凋亡程序,从而发挥抗感染、抗肿瘤作用。然而肿瘤细胞仍能够通过多种途径逃逸机体的免疫监视功能,研究认为肿瘤细胞抗原异常表达、肿瘤微环境中细胞因子及其他免疫细胞相互作用等因素所引起的自然杀伤细胞活性降低对于诱导肿瘤免疫逃逸起重要作用。本文综述了自然杀伤细胞在肝脏恶性肿瘤发生过程中参与免疫逃逸的机制及研究进展,以期为临床抗肿瘤免疫治疗的研究提供参考。     

The relationship between structural properties and activation of RAW264.7 and natural killer (NK) cells by sulfated polysaccharides extracted from Astragalus membranaceus roots

The aim of the present study to isolate the water-soluble polysaccharide from Astragalus membranaceus roots (AMP) and investigate the structural effects on RAW 264.7 murine macrophage and natural killer (NK) cells. AMP mainly consists of carbohydrates (66.2 %), proteins (11.8 %), and sulfates (18.0 %) with minor level of uronic acid (2.0 %). The structural modification was carried out by removal of protein and sulfate from AMP through the deproteination and desulfation. After deproteination (DP), the protein content was decreased from 11.8 % to 5.4 %. Similarly, the sulfate content of desulfated AMP (DS) was decreased from 18.0%–8.1%. AMP and DP could stimulate RAW264.7 cells to produce nitric oxide (NO) and up-regulate mRNA expression through NF-κB and MAPKs pathways. However, DS showed a considerably lower level of NO production than AMP and DP, suggesting that DS could not stimulate RAW264.7 cells. AMP and its derivatives significantly increased the natural killer cells (NK cell) proliferation (113.1%–128.7%) and cytotoxicity against HeLa cells (37.4%–55.5%). However, DS showed the lowest level of NK cells activation through the expression of IFN-γ, TNF-α, Granzyme-B, and NKp44. These results suggest that sulfate groups of AMP might play a crucial role in the RAW264.7 cells and NK cells activation.     

NK1.1+ cells mediate the antitumor effects of a dual Toll-like receptor 7/8 agonist in the disseminated B16-F10 melanoma model

Innate immune stimulation with Toll-like receptor (TLR) agonists is a proposed modality for immunotherapy of melanoma. Here, a TLR7/8 agonist, 3M-011, was used effectively as a single systemic agent against disseminated mouse B16-F10 melanoma. The investigation of the mechanism of antitumor action revealed that the agonist had no direct cytotoxic effects on tumor cells tested in vitro. In addition, 3M-011 retained its effectiveness in scid/B6 mice and scid/NOD mice, eliminating the requirement for T and B cells, but lost its activity in beige (bg/bg) and NK1.1-immunodepleted mice, suggesting a critical role for natural killer (NK) cells in the antitumor response. NK cytotoxicity was enhanced in vivo by the TLR7/8 agonist; this activation was long lasting, as determined by sustained expression of the activation marker CD69. Also, in human in vitro studies, 3M-011 potentiated NK cytotoxicity. TLR7/8-mediated NK-dependent antitumor activity was retained in IFN-α/β receptor-deficient as well as perforin-deficient mice, while depletion of IFN-γ significantly decreased the ability of 3M-011 to delay tumor growth. Thus, IFN-γ-dependent functions of NK cell populations appear essential for cancer immunotherapy with TLR7/8 agonists. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. All authors are or were employed by 3M while this work was being conducted.     

Expression of chemokine receptors on natural killer cells in HIV-infected individuals

Chemokine receptors CCR5 and CXCR4 are of major importance in the pathogenesis of HIV-1 infection because they are co-receptors for human immunodeficiency virus (HIV) entry. We examined the frequency of CD3⁻CD56⁺CCR5⁺ and CD3⁻CD56⁺CXCR4⁺ in HIV-infected long-term slow progressors (SPs), HIV typical progressors (TPs) with or without highly active antiretroviral therapy (HAART), and HIV-seronegative controls. The results showed that the frequency of CD3⁻CD56⁺CCR5⁺ was up-regulated, and frequency of CD3⁻CD56⁺CXCR4⁺ was down-regulated in HAART-naïve HIV TPs group compared with HIV SPs group and HIV-seronegative controls (P < 0.05). The frequency of CD3⁻CD56⁺CCR5⁺ was down-regulated by HAART therapy (P < 0.05). The frequency of CD3⁻CD56⁺CCR5⁺ was lower in HIV SPs compared with controls (P < 0.05). Lower frequency of CD3⁻CD56⁺CXCR4⁺ and higher frequency of CD3⁻CD56⁺CCR5⁺ positively correlated with the level of HIV viral loads and negatively correlated with CD4 T cell counts (P < 0.05). These results indicated that the expression of chemokine receptors on NK cells correlated with HIV disease progression.     

自然杀伤细胞受体的研究进展

自然杀伤细胞（ＮＫ细胞）可表达两类功能相悖的识别受体,即活化受体（ＫＡＲ）和抑制受体（ＫＩＲ）。ＫＩＲ能识别自身细胞上的ＭＨＣⅠ类分子与自身或外来肽形成２的复合物,所产生的抑制信号可阴断ＫＡＲ的活化,以此抑制ＮＫ细胞的细胞毒作用。如果靶细胞失去ＫＩＲ所识别的配体,ＮＫ细胞即可通过ＫＡＲ对靶细胞进行攻击。本文将介绍此类受体的结构及基识别与信号转导机制的研究进展。     

Chondrocyte-specific phenotype confers susceptibility of rat chondrocytes to lysis by NK cells

Normal chondrocytes are targets for natural killer (NK) cells. Since the mechanism of this phenomenon remains unknown, the present study was aimed at testing whether it is associated with chondrocyte-specific phenotype defined as ability of cartilage cells to produce sulfated glycosaminoglycans (GAG) and express collagen II and aggrecan mRNA. Lysis of rat epiphyseal chondrocytes by syngeneic spleen mononuclear cells (SMCs) was evaluated by ⁵¹Cr-release assay. Loss of chondrocyte phenotype following long-term culture resulted in their decreased susceptibility to lysis. Similar effect was also observed after suppression of chondrocyte phenotype by TNF. On the other hand, stimulation of cartilage-specific matrix component synthesis by IGF-1 resulted in increased chondrocyte killing and exogenous chondroitin sulfate A stimulated NK cell-mediated cytotoxicity against chondrocytes and human K562 cells. This suggests that chondrocyte susceptibility to lysis by NK cells depends on chondrocyte-specific phenotype, especially sulfated GAG production.     

Natural killer,natural killer T,helper and cytotoxic T cells in the decidua from recurrent spontaneous abortion with normal and abnormal chromosome karyotypes

Problem

Recurrent spontaneous abortion (RSA) is associated with immune imbalance at the maternal–fetal interface. Decidual immune cells and cytokines expressed at this interface regulate the response of the maternal immune system to the fetus. However, the populations and cytokine expression levels of these lymphocytes in miscarriage with normal and abnormal chromosome karyotypes remain unclear.

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7 days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2–activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2–activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-2.