T-AK细胞的细胞毒活性研究

探讨抗ＣＤ３单抗和ｒＩＬ－２共同激活诱生的Ｔ－ＡＫ细胞的细胞毒活性本质。方法：采用ＭＴＴ法分别检测Ｔ－ＡＫ细胞杀伤白血病细胞的活性及其构成。结论Ｔ－ＡＫ细胞为异质性细胞群体,其杀伤活性主体为ＣＤ３ＡＫ活性和ＬＡＫ活性。     

树突状细胞对LAK细胞抗人鼻咽癌细胞的促进作用

本研究对人血树突状细胞联合ＬＡＫ细胞和ＩＬ－２对人鼻咽癌细胞株Ｈｅｐ－２的抗肿瘤活性进行了体外观察。实验分为ＬＡＫ组，ＬＡＫ＋ＤＣ组和ＬＡＫ＋ＤＣ＋ＩＬ－２组；效靶比例分别采用１０：１和２０：１二种。３７℃，５％ＣＯ２，饱湿条件下培养４８ｈ后，用中性红摄入比色法检测细胞毒活性。     

CD3AK细胞的研究进展

ＣＤ３ＡＫ细胞是ＣＤ３单抗起始激活的杀伤细胞，不同的诱导方法可导致两种不同的细胞亚类，即ＣＤ３ＡＫ＾＋和ＣＤ３ＡＫ＾－，前者介导ＰＫＣ非依赖性快溶解细胞毒作用；后者介导ＰＫＣ依赖性慢溶解胞毒作用，本文归纳了在两种ＣＤ３ＡＫ细胞的诱导过程中，单核巨噬细胞，各种细胞因子以及谷胱苷肽等因素对其活化增殖，细胞毒活性的免疫调节作用。     

PHA－LAK与常规LAK细胞生物学特性的比较

本文对来自正常人Ｏ型外周血单个核细胞（ＰＢＭＣ）,经ＰＨＡ预刺激４８小时,然后用ｒＩＬ－２诱导的ＬＡＫ细胞（ＰＨＡ－ＬＡＫ）和直接用ｒＩＬ－２诱导的常规制备的ＬＡＫ细胞的生物学特性进行了比较。结果表明ＰＨＡ－ＬＡＫ和常规ＬＡＫ在细胞形态、核型和细胞化学等方面均相似;而在增殖能力、体外存活时间、细胞毒活性方面ＰＨＡ－ＬＡＫ明显优于常规ＬＡＫ。ＦＡＣＳ表型分析表明：ＰＨＡ－ＬＡＫ的表型以ＣＤ３＋ＣＤ８＋为主,而常规ＬＡＫ则以ＣＤ３＋ＮＫＨ￣＋＿１为主,且ＰＨＡ－ＬＡＫＩＬ－２Ｒ的表达水平高于常规ＬＡＫ。采用ＰＨＡ－ＬＡＫ可较好地解决ＬＡＫ细胞的数量和活性问题,这在ＩＬ－２／ＬＡＫ疗法中有重要的实用价值。     

PHA－LAK与常规LAK细胞生物学特性的比较

本文对来自正常人Ｏ型外周血单个核细胞（ＰＢＭＣ）,经ＰＨＡ预刺激４８小时,然后用ｒＩＬ－２诱导的ＬＡＫ细胞（ＰＨＡ－ＬＡＫ）和直接用ｒＩＬ－２诱导的常规制备的ＬＡＫ细胞的生物学特性进行了比较。结果表明ＰＨＡ－ＬＡＫ和常规ＬＡＫ在细胞形态、核型和细胞化学等方面均相似;而在增殖能力、体外存活时间、细胞毒活性方面ＰＨＡ－ＬＡＫ明显优于常规ＬＡＫ。ＦＡＣＳ表型分析表明：ＰＨＡ－ＬＡＫ的表型以ＣＤ３＋ＣＤ８＋为主,而常规ＬＡＫ则以ＣＤ３＋ＮＫＨ￣＋＿１为主,且ＰＨＡ－ＬＡＫＩＬ－２Ｒ的表达水平高于常规ＬＡＫ。采用ＰＨＡ－ＬＡＫ可较好地解决ＬＡＫ细胞的数量和活性问题,这在ＩＬ－２／ＬＡＫ疗法中有重要的实用价值。     

T—AK细胞的形态学和免疫表型研究

Ｔ－ＡＫ（Ｔ－ａｃｔｉｖａｔｅｄ ｋｉｌｌｅｒ，Ｔ－ＡＫ）细胞是由抗ＣＤ３单克隆抗体和ｒＩＬ－２共同激活诱生的具有高度溶细胞活性的肿瘤杀伤效应细胞。为探讨Ｔ－ＡＫ细胞的免疫生物学特征，研究了Ｔ－ＡＫ细胞的形态学和细胞表型。结果表明，Ｔ－ＡＫ细胞体积较大，核／浆比例小，胞浆内含有大量颗粒，具有活化淋巴母细胞的形态学特征；９０％以上的Ｔ－ＡＫ细胞表达Ｔ淋巴细胞表型（ＣＤ３＾＋，ＣＤ８＾＋），２０％￣５     

抗人黑素瘤双特异性抗体增强LAK细胞毒效应的实验研究

抗肿瘤单克隆抗体的高度特异性与杀伤细胞对肿瘤的细胞毒效应相互结合起来，是增强ＬＡＫ细胞抗肿瘤作用的重要途径。本研究用化学连接法制备了既可识别淋巴细胞表面ＣＤ３抗原又可识别ＬｉＢｒ黑素瘤细胞表面肿瘤相关抗原的双特异性抗体ＣＤ３－ＨＢ８７５９。ＦＡＣＳ分析证实，ＣＤ３－ＨＢ８７５９具有结合两种抗原的活性。４ｈ５１Ｃｒ释放细胞毒实验结果表明，ＣＤ３－ＨＢ８７５９在诱导相可显著增强ＬＡＫ对ＬｉＢｒ细胞的细胞毒效应，也能使非ＬＡＫ细胞获得细胞毒性；在杀伤相加入ＣＤ３－ＨＢ８７５９对ＬＡＫ细胞的细胞毒作用也具有促进作用。上述结果表明，ＣＤ３－ＨＢ８７５９是一种抗人黑素瘤的双特异性抗体，为体内应用提供了良好的实验基础。     

抗CD_3激活的杀伤细胞的研究进展（文献综述）

ＣＤ３ＡＫ细胞是ＣＤ３单抗起始激活的杀伤细胞。不同的诱导方法可诱导出两类不同杀伤效应的细胞，即活性状态的ＣＤ３ＡＫ和非活性状态的ＣＤ３ＡＫ细胞。前者介导ＰＫＣ非依赖性快溶解细胞毒作用，后者介导ＰＫＣ依赖性慢溶解细胞毒作用。本文尚归纳了在ＣＤ３ＡＫ细胞的诱导过程中，单核－巨噬细胞、各种细胞因子以及谷胱苷肽等因素对其活化增殖、细胞毒活性的免疫调节作用。     

可溶性肿瘤抗原和CD3单克隆抗体共同诱导的杀瘤细…

用可溶性肿瘤抗原和ＣＤ３单克隆抗体共同刺激外周血单个核细胞产生ＣＤ８＾＋细胞为主的杀瘤细胞，称其为Ｔ－ＡＫ细胞。与ＬＡＫ细胞和ＣＤ３－ＡＫ细胞比较，Ｔ－ＡＫ细胞扩增快、低依赖ＩＬ－２，培养１４天细胞数扩增２４倍，比ＣＤ３－ＡＫ细胞高８倍，比ＬＡＫ细胞高１５倍，并能持续生长２１天。     

OKT3＋rhIL－2激活胎儿脾细胞的研究

观察了１８～２８周龄胎儿脾单个核细胞（ＦＳＭＣ）在体外对ＯＫＴ３＋ｒｈＩＬ－２联合刺激的反应性，结果发现：ＯＫＴ３单独能活化ＦＳＭＣ，最适浓度ＯＫＴ３与ｒｈＩＬ－２联合对ＦＳＭＣ有强协同刺激作用。ＯＫＴ３刺激ＦＳＭＣ后明显促进ＩＬ－２Ｒ表达，表明ＯＫＴ３对ＦＳＭＣ的括化作用与ＩＬ－２／ＩＬ－２Ｒ途径相关。ＯＫＴ３＋ｒｈＩＬ－２协同诱导的ＦＳＭｃ能产生ＮＫ和ＬＡＫ活性，且ＬＭ活住较单用ｒｈＩＬ－２诱导者强，间接免疫荧光染色ＦＡＣＳ分析显示，ＯＫＴ３＋ｒｈｌＬ－２协同激活的ＦＳＭＣ主要是ＣＤ８￣＋Ｔ细胞。结果表明ＦＳＭＣ与成人ＰＢＭＣ－样能被ＯＫＴ３活化。     

Human recombinant IL-4 suppresses the induction of human IL-2 induced lymphokine activated killer (LAK) activity.

The effect of recombinant human interleukin 4 (rhIL-4) on the induction in vitro of human lymphokine activated killer cell (LAK) activity was investigated. Peripheral blood mononuclear cells (PBMC) from normal healthy donors were incubated for 4 days with or without recombinant human interleukin-2 (rhIL-2) in the presence or absence of rhIL-4. LAK activity was measured against the NK-resistant colon adenocarcinoma cell line SW742, and NK mediated cytotoxicity was determined using NK sensitive K562 cells. Unlike previous reports using mouse effector cells, rhIL-4 neither induced LAK activity nor augmented the cytotoxic response induced by rhIL-2. In four out of six experiments there was a significant reduction of rhIL-2 induced LAK in the presence of rhIL-4, accompanied by a reduction of Tac antigen expression by rhIL-2 activated cells. Recombinant hIL-4 failed to influence the effector phase of the activated PBMC against SW742 or K562 targets.     

Long-term cultures of human peripheral blood lymphocytes with recombinant human interleukin-2 generate a population of virtually pure CD3+ CD16- CD56- large granular lymphocyte LAK cells.

It has been reported that lymphocytes from peripheral blood (PBL) cultured with interleukin-2 (IL-2) produce predominantly CD16+ lymphokine-activated killer (LAK) cells. We developed a two-step method to generate LAK cells from human PBL in long-term cultures (10-12 days) with recombinant human IL-2 (rhIL-2) and characterized the evolving LAK cell population by testing its phenotype and cytotoxic activity as a function of time. The starting PBL displayed some natural killer (NK) cytotoxicity but no LAK activity. At day 6, the cells were a mixed population of about 80% CD3+ and 6% CD16+ cells. Little proliferation was evident but strong LAK activity was detected. After 10-12 days, major cell expansion had occurred and they were essentially a pure (greater than 90%) CD3+ CD16- CD56- cell population large granular lymphocyte (LGL) by morphology that displayed strong non-MHC-restricted killing activity (greater than 200 lytic units). Over the same period of time, the CD16+ cells had almost completely regressed in these cultures. This preferential induction of CD+ LAK cells was not an effect of IL-2 concentration as 10 U/ml was as effective as 500 U/ml. Further characterization revealed a major population of CD4+ (60%) and CD8+ (30%) with a smaller fraction (less than 9%) of gamma delta + cells. These results indicate that a virtually pure CD3+ LAK cells population was produced with long-term cultures of lymphocytes from peripheral blood in rhIL-2, in which active proliferation of the CD3+ but not CD16+ cells occurred.     

Age-associated decline in IL-2 and IL-12 induction of LAK cell activity of human PBMC samples

Previously we reported that young and elderly natural killer (NK) cell activity against the standard NK sensitive K562 cell line can be augmented to the same degree by IL-2 and IFN-. We have extended these studies to include IL-12. Similar to IL-2 and IFN-, IL-12 can enhance NK cytotoxicity to the same degree in both young and elderly samples over a wide range of doses and incubation times when K562 cells are used as targets. However, in contrast to our findings with the NK system, we have observed that induction of lymphokine activated killer (LAK) cell activity, as defined by the ability of peripheral blood mononuclear cells (PBMC) samples to lyse the normally NK resistant Daudi cell line, was significantly decreased in the elderly samples compared to young samples. Comparable age-associated differences were observed in LAK activity after induction with IL-2, IL-12, and IFN- at varying doses and incubation times. We hypothesize an age-associated deficiency either in the mechanism of LAK induction or in target cell recognition.     

Lower concentrations of methyl-β-cyclodextrin combined with interleukin-2 can preferentially induce activation and proliferation of natural killer cells in human peripheral blood

Previous studies have demonstrated that high concentrations of methyl-β-cyclodextrin (MβCD, 10-15 mM) can interfere with the formation of lipid rafts and inhibit activation of lymphocytes. In this report, we determined that lower concentrations of MβCD (1-4 mM) could accelerate the proliferation of lymphocytes in human peripheral blood mononuclear cells (PBMCs). In the expanded cells, CD3(-)CD56(+) natural killer (NK) cells were the dominant subpopulation, and a significant dose-effect relationship existed between the proportion of NK cells and the concentration of MβCD. In the groups treated with 3-4 mM MβCD, the proportions of NK cells reached a level of more than 60%. When PBMCs were treated with MβCD, CD69 was more preferentially expressed on CD3(-)CD56(+) cells than on CD3(+) cells at 48 and 72 hours. The expression of CD25 had no distinct difference at 48 hours, but when recombinant human interleukin-2 (IL-2) was added for a further 24 hours, it was also preferentially expressed on NK cells. MβCD and IL-2 synergistically could also induce interferon-γ (IFN-γ) production in CD56(+) human PBMCs. Mechanistic studies revealed that IFN-γ production in response to MβCD plus IL-2 was IL-12 independent but depended on endogenous IL-18 and IL-1β, and CD56(+)CD14(+) dendritic cell-like cells and B cells might mediate the ability of MβCD to activate NK cells. The MβCD-activated NK cells also had high cytotoxicity against the natural killer cell-sensitive K562 cells or lymphokine-activated killer cell-sensitive DAUDI cells in vitro. These studies indicated that lower concentrations of MβCD combined with IL-2 can preferentially induce activation and proliferation of NK cells in PBMCs.     

Human recombinant IL-4 decreases the emergence of non-specific cytolytic cells and favours the appearance of memory cells (CD4+CD45RO+) in the IL-2-driven development of cytotoxic T lymphocytes against autologous ovarian tumour cells.

As IL-4 and IL-6 have also been reported to promote the development of T lymphocytes such as IL-2, we investigated their role in the development of specific cytotoxic T lymphocytes (CTL) against autologous ovarian tumours in mixed lymphocyte tumour cultures (MLTC). Peripheral blood lymphocytes (PBL) from five ovarian carcinoma (OC) patients were incubated with autologous OC cells at a PBL:OC cell ratio of 20:1 in IL-2 alone (50 U/ml for the first week and 200 U/ml thereafter) or with IL-4 (100 U/ml) and/or IL-6 (5 U/ml). Neither IL-4 nor IL-6 improved lymphocyte proliferation consistently. In contrast, IL-4 reduced significantly the development of LAK activity as assayed against Daudi cell line, and decreased modestly the emergence of natural killer (NK) activity as assayed against K562. This property was not shared by IL-6. The prevention of the development of non-specific cytolytic activity (LAK and NK activities) was much stronger when the MLTC was started with IL-4 in the absence of IL-2 during the first week in culture. A concomitant drop in NKH-1 expression (CD56) was observed. By inhibiting the emergence of non-specific cytotoxicity, IL-4 provided better evidence of the specific cytolytic activity directed at ovarian cells. In parallel, a significant increase in the generation of memory cells (CD4+CD45RO+) was observed with IL-4. In conclusion, in this model, IL-4 added before IL-2 decreases significantly the emergence of non-specific cytotoxic cells, and promotes the generation of memory cells. These properties may be of interest in the design of strategies aimed at obtaining tumour-specific cells for investigational and immunotherapeutic purposes.     

Peripheral blood lymphocytes resistant to Epstein-Barr virus immortalization manifest high natural killer (NK) type activity against NK-resistant target cells

Epstein-Barr virus (EBV) readily immortalizes human peripheral blood lymphocytes (PBL) in vitro. We found recently that PBL from two EBV-seropositive healthy adults were exceptionally resistant to immortalization by EBV. In contrast to PBL from other EBV-seropositive donors sensitive to immortalization by EBV (S-PBL), the "resistant" PBL (R-PBL) respond to EBV infection with an early interleukin-2 (IL-2) synthesis and high interferon gamma (IFN gamma) production. In order to determine whether these differences in cytokine responses between R-PBL and S-PBL could be associated with a detectable difference in lymphocyte cytotoxicity, we compared the natural killer (NK) activity of R-PBL and S-PBL effectors by using both NK-sensitive (i.e. K562) and NK-resistant (i.e. Raji) targets. We found that, while effectors from EBV-infected R-PBL and S-PBL cultures exhibited comparable NK activity against the K562 targets, they differed remarkably in their cytolytic activity against Raji cells. At days 3 and 5 of culture, effectors from EBV-infected R-PBL showed a significantly higher lytic activity against Raji targets, whereas S-PBL did not. Culture of EBV-infected R-PBL and S-PBL effectors in the presence of recombinant IL-2 (rIL-2) for 5 days resulted in increases of their lytic activity against Raji cells, whereas pretreatment of these effectors with recombinant IFN gamma (rIFN gamma) was found to increase only R-PBL cytotoxicity. These results suggest that the resistance of R-PBL to EBV immortalization could be associated with a lymphokine-mediated early cellular cytotoxic response of the NK/LAK (lymphokine-activated killer cell) type against EBV-infected cells.     

Binding of Leukaemic Blasts to Various Subpopulations of Lymphokine-Activated Killer Cells

Conjugate formation by natural killer (NK)-resistant and N K-sensitive leukaemic cell lines with fresh and IL-2-stimulated (lymphokine-activated killer, LAK) peripheral blood lymphocytes (PBL) was studied by a flow cytofluorometry method with double staining, A significant difference in binding of NK-resistant T-cell lymphoma (HuT 78) and NK-sensitive myeloid (K562) blasts to fresh PBL was observed ( P <0.01). Activation of lymphocytes with IL-2 resulted in a significant increase of binding and killing of both K562 and HuT 78. However, in the case of blasts from NK-resistant cell line Daudi a similar conjugate formation with fresh PBL and LAK effectors was observed, despite a significant increase in killing. Various subpopulalions of LAK effectors (CD3, CD16 and CD56 positive) displayed similar binding activity towards myeloid (K562) and lymphoid (Raji) blasts, which shows that conjugate formation occurs not only with NK-cell-derived. but also with T-cell-derived LAK cells. The blocking of CD71 antigen (transferrin receptor) on K562 blasts inhibited binding of cytotoxic lymphocytes, which was mostly due to the blocking of binding of CD56⁺ subpopulation.
Our results indicate that the resistance of leukaemic blasts to cell-mediated cytotoxicity may depend on different (and probably several) steps of this process.     

Exposure to cigarette smoke suppresses IL-15 generation and its regulatory NK cell functions in poly I:C-augmented human PBMCs

Both NK cells and IL-15 play crucial roles in innate immunity against viral infections and cancer. Cigarette smoke is known to increase susceptibility to infections and certain cancers. Interleukin (IL)-15 plays an important role in immune responses by regulating proliferation, survival and functions of NK cells. Here, we examined the impact of cigarette smoke on IL-15 production and IL-15 mediated NK cell functions in human PBMCs. We report that cigarette smoke significantly suppresses the induction of IL-15 by poly I:C in human PBMCs. Serum IL-15 levels among smokers was significantly lower than non-smokers. In contrast to a profound increases in intracellular IL-15/IL-15Rα in poly I:C-treated PBMCs, exposure of PBMCs to smoke-conditioned media (SCM) diminished the IL-15/IL-15Rα production. We examined if inhibition of IL-15 production could lead to less NK cell activation. Interestingly, SCM-treated PBMCs had diminished up-regulation of NK cell activation marker, CD69, but not NKG2D compared with controls after poly I:C stimulation. We then confirmed by using IL-15 neutralizing antibody as well as exogenous IL-15 that the ploy I:C-induced NK cells activation was IL-15 mediated. More importantly, cigarette smoke significantly impaired NK cell cytolytic potential to kill K562 cancer cells which was found to be IL-15 mediated. The inhibition of IL-15 and its regulatory NK cell activities were linked to attenuated STAT3 and STAT5, but not ERK1/2 phosphorylations. We demonstrate, for the first time, that cigarette smoke compromises IL-15 production and as a result NK cell function which could link to the higher incidence of cancers or viral infections observed among smokers.     

NK 细胞的高效扩增及其对体外肿瘤模型的杀伤效果研究

目的:高效扩增NK 细胞,并确定其对不同肿瘤的杀伤作用,为临床应用提供参考。方法:从成人外周血中分离PBMCs 并利用表面表达4-1BBL、IL-15 和IL-21 的K562 滋养细胞对其进行共刺激培养,15 d 后计数并检测CD3- CD56+细胞纯度;利用间接免疫荧光及Real-time PCR 方法检测NK 细胞Granzyme B 及Perforin 表达变化的同时检测其对肺癌、胃癌、肝癌、卵巢癌、胰腺癌、宫颈癌的杀伤作用,确定其对不同肿瘤的杀伤效果。结果:扩增15 d 后,细胞数量高于110 ‘,CD3-CD56+比例达95%以上;培养后的NK 细胞高表达Granzyme B、Perforin,且对A549 的杀伤效果(E:T=10 :1)约为90%,同时对其他肿瘤细胞(E:T=10:1)的杀伤效果由强到弱的顺序为:胃癌、胰腺癌、宫颈癌、卵巢癌、肾癌、肝癌(P<0.05);NK 细胞对宫颈癌、肝癌、胰腺癌的杀伤效果呈现一定的时间依赖性。结论:此方法可以扩增出高数量、高纯度的NK 细胞,同时该NK 细胞具有高效杀伤多种肿瘤细胞的能力。