淋巴结细胞毒性自然杀伤/T细胞淋巴瘤

目的 探讨淋巴结细胞毒性自然杀伤（NK/T）细胞淋巴瘤的临床病理学特征。方法 对5例淋巴结细胞毒性NK/T细胞淋巴瘤作临床病理观察及随访、用ISAB法做免疫表型分析（CD35RO、CD8、CD56、CD30、CD20、TIA-1）及EBER1/2原位杂交检测。结果 淋巴结细胞毒性NK/T细胞淋巴瘤的瘤 理组织学特点为：（1）淋巴结结构明显破坏并被瘤细胞所取代：（2）瘤细胞呈多形性;（3）我数肿瘤细胞表达淋巴细胞分化抗原。5例中CD45RO阳性的有4例,其中3例瘤细胞同时呈CD56阳性;1例为无标记细胞性;所有病例的TIA-1和EBER均为阳性。结论 淋巴结细胞毒性NK/T细胞淋巴瘤有特征性的形态改变和免疫表型。提示肿瘤进展及预后不良。     

人外周血自然杀伤T细胞体外扩增及其功能的初步研究

为了建立人自然杀伤T(NKT)细胞在体外扩增的方法并对其功能进行初步的研究,通过不同的方法从人外周血单个核细胞(MNC)和纯化T淋巴细胞中扩增TCRVα24+/Vβ11+NKT细胞,并采用流式细胞术测定NKT细胞中IL-4、IFN-γ、TNF-α分泌水平。采用CD4/CD8免疫磁珠去除CD4+和CD8+细胞进一步纯化NKT细胞,并用DIOC18染色及流式细胞术测定NKT细胞杀伤活性。α半乳糖神经酰胺(α-Galcer)和IL-2可以使NKT细胞在体外扩增。扩增19d后,TCRVα24+/Vβ11+细胞比率最高上升到25.5%±7.2%,NKT细胞最大扩增倍数达到(1.51±0.91)×104倍。体外扩增的NKT细胞高表达TCRVα24、Vβ11、CD3、CD161,低表达CD56。在CD3单克隆抗体和IL-2刺激下,TCRVβ11+细胞分泌IL-4和IFN-γ的细胞比例均高于TCRVβ11-细胞(P<0.05)。去除CD4+和CD8+细胞后NKT细胞含量上升到80%。NKT细胞对肿瘤细胞株U937和HL60以及树突状细胞具有较强的细胞毒效应。NKT细胞可以通过α-Galcer直接从人外周血MNC扩增获得,从而简化实验步骤。     

小鼠小肠上皮内淋巴细胞的提取与抗原表型测定和功能的初步研究

目的建立一种提取小肠上皮内淋巴细胞的方法,并对其功能进行初步研究。方法采用机械分离法和Percoll非连续密度梯度离心法提取小鼠小肠上皮内淋巴细胞,经间接荧光染色后用流式细胞仪检测表型。应用乳酸脱氢酶法检测其自然杀伤功能。结果应用该法可获得小鼠小肠上皮内淋巴细胞2×10     

简易自然杀伤试验——LDH释放改良法

<正> 自然杀伤试验作为体外细胞介导细胞毒试验,已从基础免疫学研究逐渐应用于对肿瘤、免疫性疾病及病毒感染性疾病的动态研究。为便于在临床上普及应用,一个简易、快速、稳定的常规检测方法是尚待解决的问题。 自然杀伤试验测定方法较多,常用者有形态学法、同位素法、酶释放法等。同位素法有客观指标、微量、敏感,是一般研     

鼻型自然杀伤/T细胞淋巴瘤肿瘤相关基因的研究进展

自然杀伤(NK)／T细胞淋巴瘤是原发于结外的淋巴瘤,亚洲和南美地区高发。男性多见。最常见的部位是面部中线部位。临床上呈进行性的过程。同其他恶性肿瘤一样,该肿瘤的发生是一个多基因多步骤突变的过程。基因的突变包括点突变,缺失突变,甲基化等。由于NK／T细胞淋巴瘤最常发生于鼻腔和副鼻窦,该部位送检组织一般较少,加上肿瘤组织本身坏死比较严重,故对该肿瘤进行基因方面的研究比较困难。     

黄芪苷对人外周血自然杀伤活性影响因素的研究

应用~(51)Cr释放法研究黄芪苷ASI和SK对人外周血NK活性的影响。结果表明,高浓度(250 μg/ml)有抑制作用,在适当浓度范围内(5μg/～0.05μg/ml)有较显著的促进作用(P<0.05),当与PHA、rIFN α或rIL-2联合作用时对NK活性有明显的增强作用。本文还证明了黄芪苷对地塞米松抑制NK活性有一定的恢复作用。本研究证实黄芪苷是增强机体免疫功能的一种有效成分。它是一种免疫调变剂,其作用强弱与机体的免疫水平有关。本研究也为临床使用黄芪制剂提供一些依据。     

PHA-CIK细胞的免疫表型和细胞毒活性

目的：探讨PHA-CIK细胞的免疫表型和细胞毒活性。方法：分离健康人外周血单个核细胞，用PHA先刺激单个核细胞24小时，然后按培养CIK细胞的传统方法继续培养至第15天。用流式细胞术检测免疫表型和MTT法检测细胞毒作用。结果：培养体系中CD3^ 、CD8^ 、CD3^ CD8^ 、CD3^ CD56^ 细胞较多，PHA-CIK细胞有较强的细胞毒活性。结论：PHA-CIK(细胞有较强的抗肿瘤活性，可作为生物治疗应用于临床。     

淋巴因子激活的LAK／TIL细胞的表型

为研究肿瘤生物疗法中淋巴因子激活的杀伤细胞及其功能和特征与表型之间的关系，用流式细胞术（ＦＣＭ）结合抗体双荧光标记法对培养的淋巴因子激活杀伤细胞作连续的多项表型监测，发现存在着ＣＤ３－ＣＤ１６＋ＣＤ５６＋型，ＣＤ４＋型，ＣＤ８＋ＣＤ２８＋型，ＣＤ８＋ＣＤ２８－型的淋巴因子激活杀伤细胞的亚型。研究了ＣＤ３－ＣＤ１６＋ＣＤ５６＋亚型、ＣＤ８＋ＣＤ２８＋亚型的杀伤活性，显示了对不同的肿瘤靶细胞有不同的敏感性。     

CD4~+T细胞表型与功能上的异质性

<正> 根据能识别大鼠白细胞共同抗原(L-CA)家族中某些抗原决定簇的小鼠 McAb(MRCOX-22)与大鼠外周血 CD4~+细胞的反应格局,大鼠的CD4~+细胞可从表型上分为不同的亚群。本文综合分析了作者和其他实验室有关大鼠T诱导/辅助细胞的体内外功能。 大鼠T细胞L-CA的表达:L-CA首先     

慢性乙型肝炎患者树突状细胞形态、表型和功能的变化

目的 观察慢性乙型肝炎病毒（hepatitis B virus,HBV)感染者树突状细胞（dendritic cells,DC)形态、表型和功能的改变。方法 从13例慢性乙肝患者和11例健康人外周血中分离和培养DC,观察DC的形态。用流式细胞仪检测DC的表面标志,HLA－DR、CD1a,CD80和CD86的表达。用^3H-TdR掺入法,检测DC诱导混合性淋巴细胞反应（mixed leukocytes reaction,MLR)的能力。结果 正常人的DC较慢乙肝患者的DC在形态上更为典型。前者的DC不规则,细胞可表达较多的HLA－DR、CD80和CD86分子（P＜0．05）,诱导MLR的能力也较强（P＜0．05）。结论 慢乙肝患者外周血DC处于不完全成熟状态,其免疫刺激能力较低。     

Selective cross-talk among natural cytotoxicity receptors in human natural killer cells

The cytolytic activity of human natural killer cells is induced by several triggering cell surface receptors upon interaction with specific cellular ligands. These receptors include NKp46, NKp30 and NKp44, collectively termed natural cytotoxicity receptors (NCR). Co-operation among NCR has been shown to occur for optimal recognition and killing of most tumor target cells. In this study, we show that the mAb-mediated engagement and clustering of one or another NCR results in the activation of an identical set of tyrosine kinases. These kinases are included in the signaling cascade leading to tyrosine phosphorylation of different receptor-associated signal transducing molecules i.e. CD3 zeta (associated with NKp46 and NKp30) and KARAP/DAP12 (associated with NKp44). In line with the notion that the engagement of inhibitory receptors prevents NCR-mediated responses, we show that the engagement of CD94/NKG2A virtually abrogates the tyrosine phosphorylation of the NCR-associated signaling molecules, i.e. it acts at the very early steps of the signaling cascade. Importantly, the engagement of a single NCR resulted in the activation of the signaling cascades associated with the other NCR. This "cross-talk" is confined to NKp46, NKp30 and NKp44 since neither CD16-nor KIR2DS4-associated signaling polypeptides were phosphorylated following the NCR engagement. These results suggest that a functional cross-talk specifically occurs among different NCR, possibly resulting in the amplification of the activating signals.     

Lactoferrin-inducible monocyte cytotoxicity for K562 cells and decay of natural killer lymphocyte cytotoxicity.

Monocyte-enriched and lymphocyte-enriched fractions of peripheral blood from three healthy volunteers were obtained by percoll density gradient centrifugation. The cytotoxic activity of each fraction against 51Cr-labelled K562 cells was quantified in a 2-h assay using freshly isolated cells of each fraction and cells of each fraction which had been incubated with and without lactoferrin in complete medium for 18 h before performing the assay. We have thereby shown that cytotoxicity was not demonstrable in the lymphocyte fraction (containing 7.3 +/- 2% large granular lymphocytes) after 18 h in medium, whereas the cytotoxicity of the monocyte fraction (containing 3 +/- 0.4% large granular lymphocytes) was still significantly increased (P less than or equal to 0.01) and that lactoferrin had no effect on lymphocyte fraction cytotoxicity while producing an 11-fold increase in the cytotoxicity of the monocyte fraction. It is therefore possible to perform a relatively simple test of monocyte cytotoxicity using lactoferrin as a stimulant in a 2-h 51Cr-labelled K562 assay system by allowing 18 h to elapse for lymphocyte natural killer cytotoxicity to decay.     

Subepithelial B cells in the human palatine tonsil. II. Functional characterization

This study investigates the main functional features of subepithelial (SE) B cells and compares them with those of purified germinal center (GC) and follicular mantle (FM) B cells isolated from the same tonsils. Unlike GC B cells, SE B cells failed to produce polyspecific antibodies in vitro; unlike GC B cells, SE B cells expressed high levels of Bcl-2 and failed to undergo spontaneous apoptosis in vitro. The most striking function of SE B cells was their ability to produce IgM antibodies to T cell-independent type-2 (TI-2) (but not to TI-1) antigens (Ag). These antibodies could not be detected when both FM and GC B cells were stimulated with TI-2 Ag in vitro. Moreover, B cells isolated from peripheral blood were unable to mount a response to TI-2 Ag. The latter finding is consistent with the observation that B cells with the phenotypic features of SE B cells were virtually absent in the peripheral blood and emphasizes the notion that SE B cells belong to a subset of non-recirculating B cells. SE B cells were by far superior to FM B cells in mixed lymphocyte reaction (MLR) stimulation of allogeneic T cells in vitro, although they were not as efficient as dendritic cells (DC). In order to stimulate T cells efficiently, SE B cells had to be exposed to anti-μ antibody, a treatment which induced expression of activation markers such as CD80, CD86, CD69 and CD39, usually absent in resting SE B cells. CD80 and CD86 molecules expressed by SE B cells participated in the chain of events required to promote the proliferation of allogeneic T cells as demonstrated by inhibition tests with the appropriate mAb. The expression of CD80 and CD86 by anti-μ-treated SE B cells was not, however, the sole explanation for their good antigen presenting capacities since the exposure of FM B cells to anti-μ antibody also induced expression of these surface structures. Nevertheless, these cells failed to become good MLR stimulators. Collectively, the above data contribute further to the characterization of a distinct subset of tonsillar B cells which resemble, both phenotypically and functionally, the B cells of the splenic marginal zone.     

Increased natural killer cell cytotoxicity and IL-2 production in recurrent spontaneous abortion

PROBLEM: To determine whether natural killer (NK) cells cytotoxicity in peripheral blood is altered in patients with a history of recurrent spontaneous abortion (RSA); also, if there is any correlation between cytokine production and NK cytotoxicity. METHOD OF STUDY: In this case-control study, 21 patients with RSA within 24 hr of the last abortion (group I), and 32 pregnants with no history of abortion (group II) were surveyed. NK cell cytotoxicity was evaluated by flow cytometry, and IL-2, IL-10, transforming growth factor beta1 were measured in cell culture supernatant by ELISA method. RESULTS: Group I showed higher NK cytotoxicity than group II at all of effector to target (E:T) ratios (P < or = 0.045).The correlation between production of IL-2 and NK cytotoxicity was positively significant (R = 0.350, P = 0.001). Group I had significantly higher levels of IL-2 than group II (P = 0.001). In group II, the production of IL-10 by peripheral blood mononuclear cells was higher than group I (P = 0.002). CONCLUSION: Increased NK cell cytotoxicity and high level of IL-2 may be considered as a risk factor for RSA.     

Subepithelial B cells in the human palatine tonsil. I. Morphologic,cytochemical and phenotypic characterization

This study describes the purification of a subset of tonsillar B cells which share phenotypic, morphologic and cytochemical features with subepithelial (SE) B cells. These cells, which represented the 5–10% of the total tonsillar B cells, were found in the Percoll gradient fraction of highest density, together with resting follicular mantle (FM) B cells. The latter B cells, however, expressed surface CD5 and could be removed by an immune rosetting procedure. The remaining small CD5⁻ B cells had a surface phenotype (IgM⁺, IgD⁺, CD23⁻, CD38^±, CD10⁻, CD44⁺) that was different from that of FM (IgM⁺, IgD⁺, CD23⁺, CD39⁺, CD38⁻, CD10⁻, CD44^±) and of germinal center (GC) (CD23⁻, CD39⁻, CD38⁺, CD10⁺, CD44^±, IgG⁺) B cells isolated from the same cell suspensions. Furthermore, the absence of surface activation markers (CD71 and CD69) and of surface IgG allowed us to distinguish small CD5⁻ B cells from activated and memory cells migrating within Percoll fractions of lower density. In situ immunohistochemical studies revealed that B cells with an identical phenotype as that of small CD5⁻ B cells could be detected predominantly in the SE region (lamina propria) of the tonsil, and also within the epithelium lining the cryptae. This area was also comprised of a relatively minor proportion of activated B cells, not found in the small CD5⁻ B cell fraction owing to the separation procedure used. Consistent with the notion that the SE area could be a site of B cell activation was also the presence of activated macrophages and of plasma cells. Thirty to forty percent of small CD5⁻ B cells isolated in suspension were positive for the endogeneous alkaline phosphatase (ALP) activity. In contrast, only a few FM B cells were ALP⁺, while GC cells were consistently ALP⁻. In situ studies also demonstrated a prevalent expression of ALP activity by the B cells in the SE area. At the ultrastructural level, small CD5⁻ B cells were clearly different from both FM and GC B cells. They displayed a cytoplasm more extended than that of FM B cells with abundant endosomes and plasma membrane projections, and a speckled pattern of nuclear heterochromatin distribution. When fixed tissue sections were examined, cells with identical ultrastructural features could be demonstrated in the tonsillar lamina propria. Collectively, the above data demonstrate an identity of features between the small CD5⁻ B cells isolated in suspension and SE B cells analyzed in situ. Since tonsillar SE B cells are generally thought to represent the homolog of the extrafollicular B cells (including those of the splenic marginal zone), these studies may provide new opportunities for functional studies on this so far incompletely characterized B cell subset.     

Cellular cytotoxicity of human natural killer cells and lymphokine-activated killer cells against bladder carcinoma cell lines

The cytotoxicity of natural killer (NK) and lymphokine activated killer (LAK) cells against two human bladder tumor cell lines (BT-A and BT-B) was investigated using a fluorometric assay by labeling tumor cell DNA with Hoechst dye No. 33342. Our results demonstrate that BT-A and BT-B cells have low sensitivity to the cytotoxic activity of mononuclear cells (MNC) and NK cells. Cytotoxicity of MNC or NK cells against both tumor cell lines is enhanced during co-culture of the effector cells with the target cells, which suggests that BT-A and BT-B cells provide the signals which could activate MNC to exert cytotoxicity. In contrast to NK cells, IL-2-generated LAK cells showed profound cytotoxicity to BT-A and BT-B within 24 h. In addition to cellular cytotoxicity to bladder tumor cells, we also tested the effect of recombinant interleukin 1 beta (rIL-1 beta), recombinant tumor necrosis factor (rTNF), and the supernatants of co-culture of MNC or LAK cells with bladder tumor cells. The results show no cytotoxic or growth-promoting activity of rIL-1, rTNF, or the crude culture supernatants on bladder tumor cells. We found that LAK cells, but not macrophages or NK cells, may play a major role in cellular cytotoxicity against the two bladder tumor cell lines tested. From this finding we conclude that activation of LAK cells may be one important mechanism induced by adjuvant bacillus Calmette-Guérin (BCG) therapy leading to effective prevention of urothelial bladder carcinoma reappearance.