小鼠大颗粒淋巴细胞的研究Ⅰ.作为NK及K细胞毒性效应细胞的鉴定

作者认为存在于小鼠脾及其它淋巴样器官中的大颗粒淋巴细胞(LGL),具有自然杀伤(NK)活性及杀伤(K)活性。作者用2次不连续Percoll密度梯度离心法,于低密度部分发现富含NK及K活性的LGL,其形态学特征是在着色浅的胞浆中具有嗜天青颗粒和一个肾形核。LGL表面携带asialoGM_1抗原(一种以高浓度表达于小鼠NK细胞上的糖脂),用抗asialo GM_1 抗体可灭活NK及K活性,同时伴随着LGL的破坏。LGL表面缺乏可检出的Thy 1抗原,用抗Thy 1.2单克隆抗体处理后,NK活性虽有     

带有自然杀伤样活性和T细胞样表面标志的一种人大颗粒性淋巴细胞克隆

自然杀伤细胞(NK)是单个核白细胞,它能杀伤某些其他细胞,尤其是某些肿瘤细胞,因而它可构成机体防御肿瘤的前线。根据全部或绝大多数NK细胞的活性,认为它是一个同T和B淋巴细胞完全不同的大颗粒淋巴细胞群(LGL)。虽然最近几年对NK的活性进行了广泛地研究,但仍然难以描述NK细胞的特点。从表面标志来看,NK细胞和LGL细胞似乎是异源的。多数LGL都具有FcrR、Ia抗原以及用单克降抗体OKM1检出的抗原。为了查清这种可能     

人大颗粒淋巴细胞能产生IL-1

人大颗粒淋巴细胞(LGL)具有NK细胞活性,表面有T细胞和髓单核细胞的标志,可有类似T细胞的作用,如产生IL 2和α、γ干扰素等.本文报导人LGL能产生髓单核细胞因子IL 1.作者取健康人外周血淋巴细胞(经贴壁和尼龙柱法除去粘附细胞)用不连续Percoll密度梯度离心法获取不同组分的细胞,然后测定NK细胞活性和IL 1的产量,组分1和组分2分别含有30～40%和5～10%的单核细胞,其余为LGL;组分3主要是LGL(80～85%),大多细胞有NK细胞标志(67%OKM1~+,62%OKT10+,75%B     

妊高征患者围产期大颗粒淋巴细胞变化的临床意义

近年来有人认为大颗粒淋巴细胞(LGL)与NK细胞是同一细胞的论述。由于LGL在病理性妊娠与正常妊娠期的活性表现不一，为此我们对妊高征患者48例和正常孕妇45例进行分娩前、分娩时及产后2周的LGL含量监测，以期用比较简单和实用方法了解LGL在围产期保护正常妊娠和导致病理妊娠中的作用。     

大鼠自然杀伤细胞毒因子(NKCF)分析:作用机制及与其它细胞毒/细胞抑制因子的相互关系

属于大颗粒淋巴细胞(LGL)的自然杀伤(NK)细胞可能具有第一线防御肿瘤的能力。NK 的杀伤被认为是一种刺激—分泌机制,LGL 受到特异性刺激后分泌致密颗粒,颗粒中含有细胞毒性蛋白,它使靶细胞膜上的脂质双层出现环状结构的管道,分子可从此进入靶细胞并使之溶解;被刺激的LGL 还可直接分泌可溶性细胞毒因子,NKCF 即其中之一。已在小鼠、大鼠和人的NK 细胞与NK 敏感的肿瘤靶细胞共育时得     

人大颗粒淋巴细胞的功能研究进展

人大颗粒淋巴细胞(简称LGL)是具特殊形态和功能的免疫活性细胞,在免疫监视、免疫防御中起重要作用.最近发现,LGL执行全部NK/K细胞效应,分泌多种淋巴因子,参与调节T、B细胞功能.因此本文就LGL的功能研究近况作一文献复习.     

外周血NK 细胞动态监测与遗传性易栓症患者妊娠结局的相关性研究

目的:探讨反复妊娠失败合并遗传性易栓症患者外周血NK细胞百分数及其杀伤活性对妊娠结局的影响。方法:采用单中心、回顾性队列研究方法,纳入2009年1月至2013年12月期间于Chicago Medical School at Rosalind Franklin University of Medicine and Science就诊的178例反复妊娠失败合并遗传性易栓症患者,使用流式细胞技术测定孕前及孕期外周血NK细胞百分数及杀伤活性,分析比较其与患者妊娠结局的关系。结果:外周血NK细胞百分数与其杀伤活性呈正相关;妊娠结局为流产组的患者各孕周NK细胞百分数及杀伤活性均高于活产组,其中孕前及孕6周时差异存在统计学意义。在调整包括年龄、BMI、NK细胞杀伤活性在内的协变量后,多元回归方程提示:孕前外周血NK细胞百分数≥12%的患者发生流产风险是NK细胞百分数12%的患者的4.75倍(OR 4.75,95%CI 1.57～14.32,P=0.01);而在孕6周时,外周血NK百分数≥12%的患者发生流产风险是NK细胞百分数12%患者的6.77倍(OR 6.77,95%CI 1.06～43.11,P=0.04)。结论:外周血NK细胞百分数及其杀伤活性均与遗传性易栓症患者妊娠结局相关,孕前和孕6周时NK细胞百分数≥12%是不良妊娠结局的独立危险因素。     

养血安胎颗粒对反复自然流产患者非孕期NK及Ts细胞活性的影响

目的观察养血安胎颗粒对反复早期自然流产患者非孕期外周血中自然杀伤细胞（NK）及抑制性T细胞（Ts）活性的影响.方法检测42例反复早期自然流产非孕期患者（治疗组）的NK及Ts细胞活性变化,给予养血安胎颗粒干预治疗,40例同样条件患者未经任何治疗,3个月为1个疗程,观察NK及Ts细胞活性变化.结果治疗组NK细胞活性治疗后较治疗前明显下降,差异有极显著性意义,P〈0.001;与对照组治疗后比较差异有显著意义,P〈0.05.Ts细胞活性较治疗前明显提高,差异有显著性意义,P〈0.01;与对照组治疗后比较,差异有显著性意义,P〈0.05.结论养血安胎颗粒可以通过调节NK、Ts细胞活性从而调节机体免疫功能.     

淋巴细胞亚群与肿瘤免疫的初探

应用淋巴细胞单克隆抗体分离主要的淋巴细胞亚群,用间接免疫荧光法测定了癌病人外周血CD_4~+细胞的数量,显著少于正常人。分离了CD_4~+和CD_4~-细胞,分别用PHA激活及~3H-TdR掺入反映其功能变化,发现CD_4~+和CD_4~-细胞的掺入活性均比正常人显著降低。观察了正常人NK、CD_4~+、CD_8~+和B细胞对K_(562)肿瘤细胞的协同杀伤活性,以NK+CD_4~+细胞的活性最高。本文提示癌病人的CD_4~+细胞数量和功能下降,正常人CD_4~+细胞在协同杀伤肿瘤细胞中具有重要作用,说明CD_4~+细胞在肿瘤免疫中的重要意义。     

人大颗粒的淋巴细胞(LGL)是IL-1的强有力的产生细胞

IL-1是由很多种细胞如单核细胞、角质细胞和人树枝状细胞产生的分子量为1.2-1.8万的一种淋巴因子,它在免疫调节和炎症反应中超重要作用.近年来已有报告IL-1和IL-2或干扰素在增强LGL介入的NK活性中起协同作用.本文报告高度纯化的具有NK活性的人LGL能被刺激分泌IL-1.     

Large granular lymphocyte leukemia. A heterogeneous lymphocytic leukemia in F344 rats.

The morphology, histochemistry, cell surface antigens, and natural killer cell (NK) activity of 10 primary and 10 transplantable large granular lymphocyte (LGL) leukemias of aging F344 rats were studied. The LGL leukemia is the major cause of death of aging F344 rats. Morphologically, the LGL leukemias were composed of cells with either pleomorphic nuclei with many intracytoplasmic granules or round nuclei with few intracytoplasmic granules. The granules appeared to be lysosomes containing beta-glucuronidase and acid phosphatase and ultrastructurally developed in association with vesicles in the Golgi apparatus. Splenic natural killer cell activity against YAC-1 cells varied from case to case, and it appeared to be associated with LGL leukemia cells. Some transplantable leukemias had stable NK activity. Fluorescence-activated cell sorter (FACS) analysis of surface antigens revealed the LGL leukemias to be heterogeneous, and there was no correlation between cytotoxic activity and cell surface antigens. Although the morphologic features of cells in LGL leukemias resemble those of normal rat LGLs, differences in cytotoxic activity and surface antigens suggest that LGL tumors represent a heterogeneous group of leukemias which may serve as a model for the study of origin and lineage of normal LGL and NK cells.     

Inhibition of NK and ADCC activity by antibodies against purified cytoplasmic granules from rat LGL tumors

Highly purified preparations of cytoplasmic granules from transplantable rat large granular lymphocyte (LGL) tumor lines (rat natural killer (RNK) tumors) were used to immunize rabbits. Antibodies from these animals gave two precipitin lines with granule extracts in Ouchterlony experiments. They reacted with at least four different bands on nitrocellulose blots of SDS gels of LGL granule proteins. By immunofluorescence, specifically adsorbed antigranule antibodies did not recognize LGL or T cell surface antigens but reacted with the cytoplasmic granules in permeabilized RNK tumor cells as well as with normal rat LGL. These same antisera showed little or no reactivity with a panel of other cells, including peripheral blood T cells, thymocytes, macrophages, and EL-4 tumor cells. F(ab')2 preparations of these antigranule antibodies completely blocked granule-mediated lysis of both SRBC and nucleated targets, while control F(ab')2 preparations from rabbits immunized with EL-4 granules or TNP-KLH showed no significant inhibition of this cytolytic activity at the same antibody concentration. Anti-granule F(ab')2 preparations specifically inhibited (greater than 75%) rat natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in a dose-dependent manner but did not effect cytotoxic T cell activity. Pretreatment of either effectors or targets by these antibodies had no effect. Anti-granule F(ab')2 preparations, at concentrations showing strong inhibition of lysis, did not inhibit the binding of LGL to YAC-1 or Ab-coated P815 targets. These results demonstrate that a granule component(s) is necessary for the lytic activity of LGL in both NK and ADCC and provide the first direct evidence that a secretory event involving these granules is part of the lytic process.     

Resistance of mouse cytolytic cells to pore-forming protein-mediated cytolysis

Pore-forming protein (perforin, PFP) was isolated from a mouse large granular lymphocyte (LGL) [natural killer (NK-like)] cell line. Purified PFP lysed a variety of mouse tumor cell lines and helper T lymphocyte cell lines. However, LGL and cytotoxic T lymphocyte cell lines were resistant to PFP-mediated cell lysis. The presence of hemolytic activity in the granule was examined in these resistant cell lines. Four out of five of these resistant cell lines had hemolytically active granules. We determined whether NK cells freshly isolated from BALB/c nude mouse spleens were resistant to PFP-mediated cytolysis. Nylon column-passed spleen cells with an enriched content of NK cells exhibited more resistance than whole spleen cells. Moreover, when spleen cells were treated with PFP the remaining live cells showed enriched NK activity suggesting that normal peripheral cells with NK activity are resistant to PFP. These results indicate that cytolytic cells containing PFP have developed defense mechanisms to inhibit PEP-mediated cell lysis.     

Serum factors which suppress natural cytotoxicity in cancer patients

Spontaneous cytotoxicity mediated by natural killer (NK) cells is potentially an important mechanism of immunosurveillance against tumor or virus-infected cells. NK activity is impaired in many cancer patients. This study investigated the possibility that humoral factors are responsible for depressed NK activity in cancer patients and examined whether these factors were anti-lymphocyte antibodies (ALA) or immune complexes. The degree of NK suppression induced by 20 cancer patients' sera was determined by preincubating normal peripheral blood mononuclear cells with cancer sera. Eight of the 20 serum samples from cancer patients had NK-suppressive humoral factors. Elimination of immune aggregates by ultracentrifugation did not remove the inhibitory factors. The degree of NK suppression induced by cancer sera correlated with the extent of NK impairment in the serum donors (p less than 0.05). The cancer sera was examined for the presence of ALAs using flow cytometry. All cancer sera tested contained only low ALA reactivity to NK cell-enriched suspensions (less than 15%). This is in marked contrast to previous reports regarding NK-inhibitory systemic lupus erythematosus sera which contained large amounts of large granular lymphocyte (LGL) reactive ALAs. This study demonstrates that certain cancer sera suppress NK cell function. These inhibitory serum factors do not appear to be LGL-reactive ALAs or immune aggregates.     

Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neoformans

Murine natural killer (NK) cell-mediated inhibition of growth of a yeast-like target cell, Cryptococcus neoformans, was completely abrogated by blocking the effector cell secretory process with monensin. Therefore, further studies were performed to determine the ability of various cytoplasmic fractions of NK cells to mediate inhibition of cryptococcal growth. Percoll-fractionated homogenates of rat LGL tumor cells demonstrated that the granule-containing fractions plus three additional sets of less dense cytoplasmic fractions displayed anti-cryptococcal activity; whereas only the cytoplasmic granule-containing fractions had cytotoxic activity against YAC-1 tumor cell and sheep erythrocyte targets. Maximal cryptococcal growth inhibition induced by LGL granules occurred after a 1 h incubation, required the presence of Ca2+ (1.0 mM) or Mg2+ (0.5 mM or 5.0 mM), and was completely abrogated in the presence of rabbit anti-LGL granule IgG. Cytolysin, the granule component which mediates tumor cell and sheep erythrocyte lysis, effectively limited the growth of cryptococci. Since Percoll gradient fractionation of the LGL homogenates demonstrated three separate peaks of anti-cryptococcal activity other than the granule peak, it is possible that the cytolysin-containing granules are not the only subcellular component of NK cells playing a role in inhibition of C. neoformans growth.     

Involvement of HLA-DR+ large granular lymphocytes in the induction of tumor necrosis factor by Mycobacterium avium-intracellulare complex.

This study shows that normal human large granular lymphocytes (LGL) secrete tumor necrosis factor (TNF) in response to Mycobacterium avium-intracellulare complex (MAI). Percoll density gradient fractionation of peripheral mononuclear cells showed TNF activity in the fractions corresponding to LGL and not T cells, even when 5% monocytes were added to the T lymphocytes for accessory function. TNF release was not abrogated by treatment of the crude LGL preparations with anti-Leu M3, -CD4, and -CD8 antibodies (Ab) plus complement (C), but was abrogated by anti-CD16 and -CD2 Ab, as expected. Interestingly, anti-HLA-DR monoclonal antibody (mAb) treatment significantly diminished TNF activity from LGL, but maintained natural killer (NK) cell function unmodified as opposed to CD2+ and CD16+ cell depletion. Panning studies demonstrated that TNF secretion upon MAI stimulation resided only in the HLA-DR+ LGL and not the DR- LGL population. These results indicate that normal fresh HLA-DR+ LGL, as well as monocytes, are also responsible for rapid TNF secretion during early MAI infection. These DR+ cells appear to be distinct from those expressing NK function.     

Human Schistosomiasis: Deficiency of Large Granular Lymphocytes and Indomethacin-Sensitive Suppression of Natural Killing

Twenty-eight children infected with Schistosoma haematobium and S. mansoni were tested for natural killer (NK) cell activity in vitro using the myeloid/erythroid cell line K562 as target. In addition, the frequency of large granular lymphocytes (LGL) and the number of HNK-1+ lymphocytes were examined in peripheral blood. NK cell activity was found to be markedly reduced in most patients when compared with a group of healthy Caucasian individuals (P less than 0.005). Moreover, the impairment of NK activity clearly correlated with the intensity of infection, which was quantified by parasite ova excretion in stool and urine. Within the lymphocyte compartment the percentages of cells with the NK phenotype (HNK-1+) were found to be normal, although the majority of patients exhibited decreased numbers of LGL (P less than 0.005). The absolute and relative frequencies of LGL and HNK-1+ lymphocytes by no means correlated with the parasite load. In vitro results suggest an at least partly prostaglandin-mediated and interferon-resistant functional defect of NK cells.     

Complement Receptor Distinguishes between Two Subsets of Large Granular Lymphocytes with Different Natural Killer Activity and Cytochemical and Ultrastructural Features

Human peripheral blood large granular lymphocytes (LGL)--that is, cells with intracytoplasmic azurophilic (electron-dense) granules, with a positivity for the cytochemical localization of certain acid hydrolases, and with avid surface receptors for the Fc portion of IgG--have been purified on Percoll density gradients. Approximately 30% of these cells expressed receptors for the third complement component (C3R). They were separated into C3R-positive and C3R-negative cells. C3R+ cells had a significantly greater natural killer (NK) activity against K562 target cells than C3R+ cells. This difference was unrelated to the presence in the C3R+ cells of a contaminant cell type incapable of NK activity, since cytochemical and ultrastructural analysis revealed that C3R+ and C3R- fractions contained comparable LGL numbers. Agarose cytotoxicity assays at the single-cell level demonstrated that C3R+ LGL contained a large number of cells that bound to but did not lyse the target. The remaining fully cytotoxic C3R+ LGL had, however, the same killing and recycling properties as the cells from the C3R fraction. Electron microscopy and cytochemical studies showed that C3R+ cells had fewer electron-dense granules than C3R cells and stained more faintly for the localization of alpha-naphtyl acetate esterase. In contrast to C3R cells, C3R+ LGL displayed morphological features suggesting that an active process of granule formation was taking place. Taken together, the data indicate that C3R+ cells represent a discrete subset or a maturational stage of LGL.     

Morphological heterogeneity of Leu7, Leu11 and OKM1 positive lymphocyte subsets: an ultrastructural study with the immunogold method.

The morphological features of normal peripheral blood lymphocytes reactive with three monoclonal antibodies (MoAb) against natural killer (NK) cells, Leu7, OKM1 (CD11b) and Leu11 (CD16) and with two anti-T cell MoAb, CD4 and CD8, have been analysed at ultrastructural level by an indirect immunogold method. Cells having the features of large granular lymphocytes (LGL) but also lymphocytes displaying different morphological characteristics (non LGL; e.g. high nucleo-cytoplasmic ratio and few cytoplasmic organelles) were seen reactive with each of the MoAb investigated. Leu7 identified a higher proportion of LGL (60-80%) than OKM1 (10-95%) and Leu11 (20-48%), and with a stronger binding. A distinct granular structure, recognized as parallel tubular arrays, was more characteristic of the Leu7+, CD8+ LGL and was less frequently seen in the OKM1 and Leu11 positive LGL subpopulation in four out of the five donors investigated. It is of interest that the Leu11 and OKM1 positive subsets, which correspond functionally to cells with greater NK function, had relatively less LGL than the Leu7 positive subsets, raising the issue of the true morphology of NK cells in man. The existence of a minority of CD4 positive LGL was confirmed. Our findings demonstrate that there is a degree of morphological heterogeneity within the normal NK lymphoid population as defined by the membrane phenotype and that certain variability among normal individuals regarding the proportion and structural features of the NK subpopulations may be present.     

The role of human spleen interferon on natural killer activity of peripheral blood lymphocytes enriched in large granular lymphocytes

The elimination of monocytes as well as B- and T-lymphocytes by forming rosettes with high affinity for sheep red blood cells yielded an enriched population of both natural killer (NK) activity (cytotoxicity: 65.4 +/- 9.9% with an E/T ratio of 12:1, P less than 0.005) and large granular lymphocytes (LGL: 76 +/- 13%) compared to the untreated lymphocyte population where NK activity is 35.7 +/- 17.3% (E/T 12:1) and the percentage of LGL of 26 +/- 6%. We studied the action of type I interferon (IFN) obtained from human spleens, on NK activity of 9 peripheral blood lymphocyte populations and 9 enriched in LGL. NK activity of the total lymphocyte population is significantly increased (P less than or equal to 0.05) in 6 out of 9 cases after treatment by interferon. Cell populations enriched in LGL showed increased NK activity in only one case after treatment by interferon, but no increased activity was found in the other cases. These results are compatible with the notion of cellular cooperation in increased NK activity by interferon.