Suppression by Human Placental Protein 14 of Natural Killer Cell Activity

ABSTRACT: Human decidua of early pregnancy contains considerable numbers of CD3^? CD56⁺ natural killer (NK) cells. In this study, two major protein products of the decidua, placental protein 14 (PP14) and placental protein 12 (PP12), were tested for the ability to regulate human NK cell activity. In vitro overnight exposure to PP14 of blood lymphocytes or purified large granular lymphocytes (LGL) resulted in suppression of cytotoxicity against K562 target cells in a 4-h ⁵¹Cr release assay. The NK inhibition was dependent on concentrations of PP14, being detectable at 5 μg/ml and reaching maximum at 50 μg/ml. Manifestation of PP14-induced NK suppression required 18-h contact with NK cells. The suppression of NK activity by PP14 was not abolished by indomethacin. In a target binding assay the number of PP14-treated LGL binding to K562 was comparable to that of untreated ones. By contrast with PP14, PP12 produced no effects on NK cells. These results indicate that PP14 suppresses the function of NK cells, which might be involved in prevention of maternal immune rejection of fetus at the fetomaternal interface.     

The molecular basis of Natural Killer (NK) cell recognition and function

Natural Killer cells are likely to play an important role in the host defenses because they kill virally infected or tumor cells but spare normal self-cells. The molecular mechanism that explains why NK cells do not kill indiscriminately has recently been elucidated. It is due to several specialized receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA class I alleles leads to NK-mediated target cell lysis. Different types of receptors specific for groups of HLA-C, HLA-B, and, very recently, HLA-A alleles have been identified. While in most instances, they function as inhibitory receptors, an activatory form of the HLA-C-specific receptors has been identified in some donors. Molecular cloning of HLA-C-, HLA-B- or HLA-A-specific receptors has revealed new members of the immunoglobulin superfamily with two or three Ig-like domains, respectively, in their extracellular portion. While the inhibitory form is characterized by a long cytoplasmic tail associated with a non-polar transmembrane portion, the activatory one has a short tail asociated with a Lys-containing transmembrane portion. Thus, these human NK receptors are different from the murine Ly49, that is a type II transmembrane protein characterized by a C-type lectin domain. A subset of activated T lymphocytes expresses NK-type class I-specific receptors. These receptors exert an inhibiting activity on T cell receptor-mediated functions and may provide an important mechanism of downregulation of T cell responses.     

Cytolytic and Cytotoxic Activity of a Human Natural Killer Cell Line Genetically Modified to Specifically Recognize HER-2/neu Overexpressing Tumor Cells

NK92 cells genetically engineered to recognize the HER-2/neu oncoprotein have been previously reported to lyse HER-2/neu positive tumor cell lines through direct cell to cell contact. In the present study we have transduced NK92 cells with a chimeric receptor gene composed of the HER-/neu specific scFv (FRP5) antibody fragment, joined to the peptide CD8 hinge region and the signaling CD3 ζ chain. NK92 cells expressing this chimeric receptor (NK92.HER-2/neu/ζ) specifically recognized and lysed HER-2/neu overexpressing tumor cell lines both in vitro and in preclinical tumor models in vivo. More important we demonstrate that NK92.HER-2/neu/ζ cells constitutively secrete high levels of soluble scFv which mediate strong tumor cytostatic effects by directly binding on cell surface HER-2/neu. Our data uncover an additional mechanism through which NK92.HER-2/neu/ζ cells mediate antitumor effects and further support their use in cell based therapeutics for the treatment of HER-2/neu expressing cancers.     

TRAIL is Involved in the Tumoricidal Activity of Mouse Natural Killer Cells Stimulated by Newcastle Disease Virus in Vitro

Newcastle disease virus (NDV) is a potential antitumor agent, and its antitumor effect has been evaluated in preclinical tests. However, the mechanisms of NDV‐based antitumor therapy are still not completely clear. In the present study we found that NDV‐stimulation enhanced the killing ability of mouse spleen natural killer (NK) cells towards mouse hepatoma cell lines, and tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) plays an important role in this tumoricidal activity. NDV stimulation induced up‐regulation of TRAIL both at the mRNA and protein levels in NK cells. Blocking TRAIL by antibody (Ab) almost completely eliminated the killing effect of NK cells on hepatoma cell lines. Furthermore, neutralizing interferon (IFN)‐γ by Ab could inhibit TRAIL expression and tumoricidal activity of NDV‐stimulated NK cells. These results indicated a substantial role of TRAIL as an effector molecule in NDV‐induced NK cells mediated tumoricidal activity. The NDV stimulation triggered TRAIL expression in mouse spleen NK cells could be mediated by IFN‐γ induction. Anat Rec, 296:1552–1560, 2013. © 2013 Wiley Periodicals, Inc.     

长托宁治疗有机磷农药中毒的临床研究

目的探讨长托宁在治疗有机磷农药中毒中的作用。方法将急性有机磷农药中毒患者随机分为长托宁试验组和阿托品对照组，采用推荐剂量按轻、中、重组治疗，严密观察用药后M样症状、N样症状、中枢神经系统症状持续时间，阿托品和长托宁用量的总量和次数的变化以及阿托品化时间及治愈时间的变化。结果长托宁试验组，重度患者M样症状与中枢神经系统症状消失时间明显缩短（P〈0．01），用药总量和用药次数明显减少（P〈0．01），阿托品化时间及治愈时间明显缩短（P〈0．01）。结论长托宁治疗有机磷农药中毒明显优于阿托品。     

Role of TNF Family Ligands in Antitumor Activity of Natural Killer Cells

NIC cells have the ability to destroy tumor cells by two main cytotoxic pathways, the well-known perforin/granzyme-mediated secretory/necrotic killing and the newly defined TNF family ligand-mediated apoptotic killing. The former mechanism is operative mainly against a few cultured leukemia cell targets, while the latter mediates substantial activity against most tumor cell targets. It also appears from emerging data that the apoptotic mechanism is the main antitumor pathway in vivo. This review is focused on the apoptotic mechanism of killing, the molecules and cell signaling pathways involved in this process, and its potential biologic significance along with its relation to the secretory/necrotic cytolytic pathway.     

Phenotypic and Functional Heterogeneity of Natural Killer Cells from Umbilical Cord Blood Mononuclear Cells

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-γ and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the naïve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56^hiCD16^? NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-γ expression by CD56^hiCD16^? and CD56^loCD16^? subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.     

Suppression of Natural Killer Cell Activity and Interleukin-2 Concentration of Serum Obtained from In Vitro Fertilization-Embryo Transfer Patients

PROBLEM: The effect of serum obtained from in vitro fertilization-embryo transfer (IVF-ET) patients on healthy volunteers' natural killer (NK) cell activity was evaluated. We also measured interleukin (IL)-2 concentration with IVF-ET patients' serum and clarified the relationship between IL-2 levels and the suppressive effect on NK cell activity. METHOD OF STUDY: A retrospective nonrandomized clinical study was performed. The suppressive effect on NK cell activity and IL-2 concentrations was measured with serum obtained from 30 pregnant and 30 nonpregnant women during an IVF-ET procedure. The suppressive effect of the serum on NK cell activity was evaluated by the formula that we defined in our previous study. RESULTS: The suppression of NK cell activity was significantly higher in the nonpregnant women than in the pregnant women (P < 0.05); however, IL-2 concentration did not differ. There was a positive correlation between the suppression of NK cell activity and IL-2 levels in the pregnant women, but no significant correlation in the nonpregnant women. CONCLUSIONS: These results suggest that the suppression of NK cell activity may be one of the prognostic factors for IVF-ET. In addition, we speculate that an unidentified humoral factor other than IL-2, which could increase NK cell activity, might exist in the serum of the nonpregnant patient.     

Recipient T Cell Exhaustion and Successful Adoptive Transfer of Haploidentical Natural Killer Cells

Natural killer (NK) cells mediate surveillance for malignancy. In some chemotherapy refractory myeloid leukemia patients, adoptive transfer of NK cells from haploidentical donors can induce remission. We have previously shown that remission induction is linked to NK cell persistence at day +14, but the factors influencing NK cell persistence are unknown. To address this question, patient samples from a phase I trial of National Cancer Institute (NCI) IL-15 in whom either did or did not show NK cell expansion were compared with healthy donor control subjects. Before lymphodepleting chemotherapy, high absolute CD3⁺ count was predictive of patients who failed to expand their haploidentical NK cell graft. Interestingly, both groups had elevated expression of inhibitory receptors and decreased cytokine production compared with control subjects, suggestive of T cell exhaustion among all patients before haploidentical NK cell infusion. At day +14, however, haploidentical NK cell expanders had persistence of recipient CD8⁺ T cells with the most exhausted inhibitory phenotype (either PD-1^high or dual PD-1⁺Tim-3⁺) and elevated expression of T-bet and Eomes compared with NK cell nonexpanders and control subjects. This suggested that maintenance of an exhausted T cell state at day +14 permits haploidentical NK cell expansion and supports further efforts to selectively deplete recipient T cells or modulate their dysfunction.     

应激对小鼠T、B细胞分裂原反应性及天然杀伤细胞活性的影响

自Jerne提出免疫网络学说,对免疫调节的研究日益深入。但这些工作多限于研究免疫系统内部的相互作用,而忽略了免疫系统外的因素对免疫应答的影响。近年来有些作者注意到神经-内分泌系统对免疫反应的调控作用,应激的研究应为其中之一。应激往往引起神经-内分泌系统的一系列变化,并导致免疫功能的改变。手术、创伤、疼痛引起的应激反应,往往造成患者的抗感染、抗肿瘤的能力下降,认为与应激介导的免疫抑制效应有关。但未见有关手术应激的动物实验报告。本文重点研究了手术应激动物模型的建立,证明此种应激可明显抑制小鼠的分裂原反应性及NK活性。比较不同分裂原反应性受抑的动力变化,发现不同分裂原反应性对手术应激的抑制效应的敏感性不     

Ontogeny and Expansion of Human Natural Killer Cells: Clinical Implications

Our knowledge of NK cells and their critical role in the innate immune system has increased enormously since their discovery several decades ago. However, it is only within the last 10 years that rational cytokine therapies, such as those utilizing low doses of IL-2, have been successful in expanding NK cells in patients with cancer and/or immunodeficiency. Such experiences in vivo have highlighted the importance of basing immunotherapeutic strategies on the known cellular and molecular properties of the targeted cell population. Recent advances in our understanding of the physiologic factors and events that orchestrate NK cell ontogeny, including IL-15 and receptor tyrosine kinase ligands to c-kit and fit3, provide novel therapeutic possibilities for cytokine therapy. This review summarizes our current understanding of human NK cell ontogeny, and links this knowledge to ongoing and future clinical strategies for the endogenous expansion of NK cells in patients with cancer and/or immunodeficiency.     

Ultrastructure of IL2-stimulated Tumor-infiltrating Lymphocytes Showing Cytolytic Activity Against Tumor Cells

Tumor-infiltrating lymphocytes (TIL) obtained from tumor tissue and pleural effusion of breast carcinoma were cultured with interleukin-2 (IL2) and thus activated. The ultrastructure of TIL stimulated by IL2 to kill various breast carcinoma cells was then investigated. Freshly isolated TIL cultured with autologous tumor cells for 48 h without IL2 were small, round and showed neither binding to nor killing of tumor cells. TIL stimulated to proliferate by IL2 became effector cells and showed cytotoxicity against tumor cells. Ultrastructurally, the effector TIL resembled large granular lymphocytes, and adhered to tumor cells through interdigitation or close apposition of the two plasma membranes accompanied by spot-like close membrane contacts. At the site of each spot-like contact, there was a 5-nm intercellular space. The morphology of the TIL processes did not differ from those of LAK and other CTL or NK cell processes during contact, invagination or the killing of target cells. The granules in TIL were considered to participate in the cytotoxic effect. Phenotypically heterogeneous TIL, CD87CD57- and CD8⁺/CD57⁺, adhered to autologous tumor cells and MCF7 (human breast carcinoma cell line). However, it was unclear which cell or cells acted as the effector for tumor-cell killing. Acta Pathol Jpn 41: 94-105, 1991.     

Effects of Serum from Pregnant Versus Nonpregnant Women on Natural Killer Cell Activity

PROBLEM: To test the effect of serum obtained from in vitro fertilization-embryo transfer (IVF-ET) patients on the healthy volunteers natural killer (NK) cell activity. METHOD OF STUDY: Retrospective non-randomized clinical study. Suppressive effect on NK cell activity was measured with serum obtained from 25 pregnant and 24 non-pregnant women during IVF-ET procedure. RESULTS: Suppressive serum effect of volunteers' NK cell activity was significantly higher in pregnant than in non-pregnant women (P < 0.01). CONCLUSIONS: The suppressive activity of serum from pregnant women on NK cell suppression activity was useful in predicting the establishment of a successful pregnancy.     

人早孕期蜕膜基质细胞下调蜕膜NK细胞杀伤活性

通过分析人早孕期蜕膜基质细胞(decidual stromal cell,DSC)对蜕膜NK细胞(dNK)表面趋化因子受体CXCR4与细胞内颗粒酶B表达水平的影响,研究早孕蜕膜基质细胞对局部NK细胞的训导作用。收集早孕蜕膜组织,分离DSC及蜕膜免疫活性细胞,进一步通过磁珠分选蜕膜CD3-CD56bright NK细胞,将分离的蜕膜NK细胞与DSC按1∶1比例共培养24h,收集蜕膜NK细胞,流式细胞仪检测其表面趋化因子受体CXCR4和细胞内颗粒酶B(granzyme B)的表达水平。结果显示,与对照组相比,在与DSC细胞共培养之后,趋化因子受体CXCR4+NK细胞的百分率明显上升,而蜕膜NK细胞内颗粒酶B阳性率显著下降(P<0.05)。结果表明,人早孕母-胎界面DSC细胞上调蜕膜NK细胞表面趋化因子受体CXCR4的表达,下调NK细胞内颗粒酶B的表达水平,可能抑制其杀伤活性。     

CTLA4Ig诱导细胞无能的作用机制探讨

IL 2受体α链 (又称CD2 5 )是T淋巴细胞活化的标志分子 ,CTLA4Ig所诱导的细胞无能CD2 5的表达下降。用FACS方法检测CTLA4Ig诱导的细胞无能的凋亡现象 ,结果发现未处理组与处理组间无差异 ;CTLA4Ig处理后的细胞无能Fas和FasL的表达 ,与未处理对照组无差异。用免疫印迹方法 ,检测发现CTLA4Ig诱导的细胞无能与未处理组相比P2 1Ras没有明显变化。用RT PCR方法检测一些细胞因子mRNA的表达水平 ,发现CTLA4Ig所诱导的细胞无能不能表达IL 2mRNA ,IFN γmR NA的表达下降 ,而IL 4mRNA、IL 10mRNA仍可表达 ,表明细胞无能的基因格局显示Th1基因受抑 ,有向Th2偏移倾向     

粗江蓠多糖对辐射损伤小鼠NK细胞的影响

目的：研究粗江蓠多糖（Gracilaria Gigas Harvey Polysaccharides,GHPS）对辐射损伤小鼠NK细胞的影响。方法：采用60Coγ射线5Gy全身照射小鼠制造辐射损伤模型,通过乳酸脱氢酶释放法检测不同剂量（10mg/kg,20mg/kg,40mg/kg）的GHPS对辐射损伤小鼠NK细胞杀伤靶细胞能力的影响。结果：辐射对照组小鼠接受60Coγ射线5Gy照射后,NK细胞的活性显著低于正常对照组（P〈0.01）。经腹腔注射（10、20、40）mg/kg GHPS,再接受γ射线5Gy照射后,小鼠NK细胞活性明显高于辐射对照组（P〈0.01）,并有剂量依赖性。结论：5Gyγ射线可以抑制小鼠NK细胞杀伤靶细胞的活性,而GHPS对辐射损伤小鼠的NK细胞有保护作用。     

Natural Killer Cytotoxicity and Antibody-Dependent Cell Cytotoxicity to Herpes Simplex Virus-Infected Target Cells in Murine Pregnancy

ABSTRACT: In vitro natural killer cytotoxicity (NKC) and antibody-dependent cell cytotoxicity (ADCC) activity against herpes simplex virus (HSV)-infected cells were evaluated in a pregnant murine model (C₅₇B1₆inbred strain). Virgin (n = 16) and pregnant (late gestation) mice (n = 15) were infected intraperitoneally with HSV, type 1. After 18 hr, a 0.5-ml aliquot of the peritoneal wash was frozen for virus plaque assay, and the cells were cultured in the ⁵¹chromium release assay for NKC and ADCC. %NKC (mean ± S.E.) to HSV-infected targets was significantly suppressed (P < 0.05) in pregnant mice, 10.3% ± 1.9, compared to that of virgin mice, 32.5% ± 2.5. This suppression was abrogated with HSV-specific antisera (%ADCC); 53.9% ± 4.4 (pregnant) compared to 49.1% ± 3.6 (virgin). The diminished NKC activity in pregnant mice was reflected in an increased mean number of virus particles in the peritoneal wash, 266 + 66 PFU/ml, compared to 38 ± 11 PFU/ml in virgin mice (P < 0.05). We concluded that NKC, but not ADCC, to HSV-infected targets was suppressed and that HSV elimination was impaired in pregnant mice.     

Lymphomatous features of aggressive NK cell leukaemia/lymphoma with massive necrosis, haemophagocytosis and EB virus infection

AIMS : Aggressive natural killer (NK) cell leukaemia will be categorized as a distinct entity in the new WHO classification of malignant lymphomas. However, its non-leukaemic features remain unclear. We therefore investigated the morphological and immunophenotypic features of this lymphoma. METHODS AND RESULTS : Four cases with aggressive NK cell lymphoma were morphologically and immunohistochemically studied. All cases followed an aggressive course with death occurring within about 3 months of initial presentation. In these cases, the neoplastic cells disseminated throughout systemic lymph nodes and invaded various tissues and organs. The lymphoma cells were large cells showing nuclear irregularity and a pattern of sinusoidal invasion in lymph nodes. Apoptosis and coagulation necrosis were both frequently observed. Haemophagocytosis was observed in all cases. Neoplastic cells in paraffin-embedded tissue specimens from these patients had CD3(CD3epsilon)+ CD56(123C3)+ granzyme+ TIA-1+ EBERT+ CD43(MT1)- CD45RO(UCHL-1)- CD57(Leu7)- CD20(L26)- phenotypes. In the two cases where tissue was available for immunohistochemical study in frozen sections, neoplastic cells showed CD56(Leu19)+ perforin+ Fas ligand(FasL)+ CD2(Leu5b)- CD3(Leu4)- CD4(Leu3)- CD5(Leu1)- CD7(Leu9)- CD8(Leu2)- betaF1- TCRdelta1- phenotypes. CD16(Leu11b) was positive in one case. CONCLUSIONS: : Natural killer cell lymphomas appear to represent a non-leukaemic counterpart of aggressive natural killer cell leukaemia, a relationship similar to that in adult T-cell leukaemia/lymphoma. Awareness and diagnosis of this aggressive lymphoma is important because of its fulminant course.