THE IN VITRO POTENTIATION OF LAK CELL CYTOTOXICITY IN CANCER AND AIDS PATIENTS INDUCED BY F3 A FRACTIONATED EXTRACT OF ASTRAGALUS MEMBRANACEUS

The in vitro induction of LAK cell activity was studied in cancer and AIDS patients. F3, an immuno regulatory component of Astragalus membranaceus was shown capable of potentiating LAK cell activity induced by rIL-2. The LAK cells killing activity against Hs294T melanoma cell line induced by 50 U/ml rIL-2 in the Presence of F3 (55 μg/ml) reached 64%, which was comparablc to that (60%) induced by 500 U/ml of rIL-2alone. With F3 and rIL-2, the effcctor to target ratio could be reduced to one-half in order to obtain an equivalent level of cytotoxicity induced by rIL-2 alone.In some patients whose Peripheral blood Iymphocytes were relatively inert of rIL-2, F3 could make them responsive to rIL-2 induction. These results imply that F3 may be useful to potentiate LAk cell activity, reduce the dosage of rIL-2 and thus minimize the later's toxic side effects when used in vivo.

ANTI-HUMAN LUNG GIANT CELL CANCER (PG) EFFECT OF HUMAN LAK CELLS IN VITRO AND IN NUDE MICE

Human LAK cells were prepared by culturing normal human peripheral blood mononuclear cells (PBMC) with or without rIL-2 and assayed for T cell surface markers as well as anti-tumor activity against PC in vitro and in nude mice. Although the percentages of T3, T4, and T8 positive cells in rIL-2-activated cells did not differ significantly from those of control cells in vitro, the former showed stronger cytotoxicity than control cells to PG tumor cells in vitro. In vivo, LAK cells completely inhibited the growth of PG tumor in nude mice, whereas PBMC control cells were to be of no effect. The anti-tumor effect of human LAK cells in nude mice may offer a useful model to study the role of human LAK cells against human tumor in vivo.

THE EFFECT OF THE TRANSFER OF LAK CELLS, COMBINED WITH ALLOIMMUNIZATION, ON THE PULMONARY METASTASES OF RAT MAMMARY CARCINOMA

Alloimmunization was combined with lympho-kine activated killer (LAK) cells to assess its effect on mammary carcinoma in rats. The animals were injected with both irradiated allosplenocytes and syngeneic LAK cells. Metastatic lung nodules were markedly reduced using combined therapy when compared with the transfer of LAK cells or alloimmuni-zation alone. IL-2 activity in the serum of alloim-munized rats could be detected. This activity, maintained in vivo for one week, may be responsible for enhancing the antitumor effect of transferred LAK cells.

TREATMENT OF PATIENTS WITH MALIGNANT PLEURAL EFFUSIONS DUE TO ADVANCED LUNG CANCER BY TRANSFER OF AUTOLOGOUS LAK CELLS COMBINED WITH rIL-2 OR rIL-2 ALONE

Experimental study both in vitro and in vivotogether with clinical trials showed that LAKcells have antitumor and antimetastatic effects(1-5)and that these effects are closely related tothe number of LAK cells transferred and the ad-ministration of rIL-2(1,6-8).Usually,autologousPBL’s are used as the source of LAK precursorsin the adoptive immunotherapy of cancer patients.But this not only puts an added burden on thecancer patient,it can cause serious side effectsas well(9).Although TIL’s may provide a solu-tion to this problem(10,11),their isolation fromsolid tumors is complex and consumes many rea-gents.We have reported that the isolation oflymphocytes from malignant ascites or from ma-lignant pleural effusions is not only simple

Quantitative observation by enzyme cytochemistry of human rectal adenocarcinoma cells killed by LAK cells

Activity of succinic dehydrogenase(SDH)and acid phosphatase(AcPase)of effector-target cells during the process of LAK cells killing HR8348 cells was estimated by enzyme cyto-chemistry technique.SHD positive granules and AePase gray level were assayed with MIAS-300image analyser.The results showed:(1)After cocultivation of effector and target cells for vari-ous times,the activity of AcPase of HR8348 cells was apparently higher than that of the controlgroup,and it increased following prolonged coincubation.SDH activity of target cells increasedmarkedly within 30 and 60 rain cocultivation,but became low after 90 min treatment.(2)Ac-Pase content within LAK cells at 60,90,120,180 and 240 rain cocultivation was significantlyhigher than that of control group(P【0.01).The phenomenon of high AcPase and SDH activitywithin effector-target cells indicates that the function of the two types of cells was in an activestate.At the early stage of effector-target combining,the increase of SDH with HR8348 cellsmay be related to defensive function of the target cells.Higher AcPase activity of target cells in-dicates the activation of lysosomal enzyme which serves as the material basis for autolysis of thecells.

An immunofluorescence study on the changes of microtubulin in human rectal adenocarcinoma cells killed by LAK cells in vitro

The human rectal adenocarcinoma cell line(HR8348)was treated withlympholdne activated killer(LAK)cells in vitro and the changes of the microtubulin inboth the effector and target cells were investigated with the aid of immunofluorescencemicroscopy.It was revealed that after the attachment of LAK cells to the tumor cells,theeffector-target conjugates formed and distribution of the microtubulin in both the effectorand target cells changed.In the LAK cells,the microtubulin concentrated mainly in thecontact region,forming a crescent-like structure,while in the target cells,themicrotubulin condensed into patches and fused with the crescent-like structure of the LAKcells,Eventually,the target cells degenerated and died.It was suggested that the lysis ofthe target cells may be related to the redistribution of the microtubulin in both theeffector and target cells.

EXPERIMENTAL STUDY ON INTERLEUKIN-6 ACTIVITY AGAINST TUMOR: REGULATION OF INTERLEUKIN-2- INDUCED LAK CELL ACTIVITY

The ability of human recombinant interleukin-6 (IL-6) to regulate the induction and cytotoxic function of lymphokine activated killer (LAK) cells from human fetal spleen was studied. The results showed that IL-6 alone was unably to Induce fetal splenic mononuclear cells (FSMC) to develop functional LAK cells, nor was it able to affect the number of IL-2-induced LAK cells and to alter the lytic activity of LAK cells against tumor cells in effector phase. However, when IL-6 was used in combination with IL-2 during the induction phase , the resulting LAK cells displayed considerably greater lytic, activity than that with IL-2 alone (P<0. 01), and this effect was IL-6 dose-dependent. The possible machanism of the synergistic effect of IL-2 and IL-6 in LAK cell Induction from human fetal spleen is discussed.

A STUDY OF THE CYTOTOXICITY OF MALIGNANT PLEURAL EFFUSION LYMPHOCYTES AND LAK CELLS AGAINST AUTOLOGOUS TUMOR CELLS

The cytotoxicity of malignant pleural effusion lymphocytes (MPEL) against autologous tumor cells (ATC) were compared with that of peripheral blood lymphocytes (PBL). It was demonstracted that the cytotoxicity of PBL was higher than that of MPEL (P< 0.001), but the cytotoxicity and expansion of MPEL-activated by rIL-2 was much higher than that of PBL activated by rIL-2 (LAK cells) (P< 0.001). This shows that local immune reaction of the pleural cavity of patients with malignant pleural effusion was in the state of suppression. MPEL activated are better effector cells than LAK cells in tumor adoptive immunotherapy.

TEM observation on LAK cells killing the human rectal adenocarcinoma cells in vitro

Substructural changes during interaction between lymphokine activated killer (LAK) cells and HR8348 cells were observed under transmission electron microscope (TEM). The results showed that: (1)after binding,the membranes of both LAK and HR8348 cells were thickened and condensed to various degrees,and their boundaries became unclear. Adherent plagues or junction-like strutures were formed,which were the morphological basis of effector-target stable binding; (2) some LAK cells inserted their protrusions into the depth of the target cells,and a few LAK cells penetrated into the taraet cells,which means LAK cells have the same'tumor-penetrating'function as natural killer (NK) and cytotoxic 1 lymphocytes (CTL) cells; (3) upon contact,the cytoplasmic organelles and granules of both cells were oriented toward the contact region,numerous microfilaments,microtubules,as well as lysosomes were aggregated at the site of contact,which plays some role in the lysis of the target cells; (4)upon binding,blebbing and ring punctures were apparent in the effector cells,whereas the target cells showed apoptotic or lytic necrosis. Apoptotic necrosis might be the result that non-perforin substances secreted by thet LAK cells triggered the action of endogeneous nucleotidase of the target cells by way of the receptors on the membrane, leading to the cleavage of DNA. The role of perforin,the entrance of calacium and magnesium ions,the edema of cells and the autolysis by lysosomes might be responsible for the lytic necrosis.

SEM observation on LAK cells killing the human rectal adenocarcinoma cells in vitro

Morphological changes of lymphokine activated killer (LAK) cells and human rectal adenocarcinoma (HR8348) cells were observed under scanning electron microscope (SEM) during the interaction of the two kinds of the cells in vitro. It was showed that the effector and target cells could actively and chemotactically move toward each other. LAK and HR8348 cells contacted with each other through cellular projections and microvilli. The LAK cells simply attached to the HR8348 cells at the early stagr, followed by jigsaw-like binding with them. After conjunction, the target cells' membrane appeared to blister and became perforated. The conjunction of microvilli and projection may act as a physical microbridge in the lysis of the target cells, and killing factors may mediate the lysis by the microbridge. 'Closed chamber' was formed between the conjunctive microvilli, which may play an important role in maintaining local concentration of the killing substances.

Study of Multi-directional Derivation of Cord Blood Mononuclear Cells and Observation of Killing Activity to MGC-803 Gastric Cancer Cell Strain In Vitro

Objective： To study multi-directional derivation of cord blood mononuclear cells to CD3AK, LAK and CIK cells as well as changes of killing activity to gastric cancer cell strain in vitro. Methods： CD3mAb and IL-2 were used to induce CD3AK cells, and IL-2 was used to induce LAK cells; IFN-γ was used in the beginning, then IL-1, CD3mAb and IL-2 were used to induce CIK cells after 24 h for observing amplification and analyzing their relationship. The phenotypes of the cultured CIK cells were analyzed by flow cytometry. Subsequently, by using MGC-803 gastric cancer cell strain as target cells, the killing activity of CD3AK, LAK and CIK cells was evaluated by using MTT method. Results： The amplification activity of CD3AK and CIK cells was all far higher than LAK cells （P〈0.05）. The amplification activity had no obvious difference between CIK cells and CD3AK cells at prophase, but that was far higher in CIK cells than CD3AK cells at about 20^th day （P〈0.05）. The flow cytometry revealed that the amount of CD3^＋ CD56^＋ cells, major effector cells after CIK cells being cultured was significantly increased （P〈0.05）, moreover, the amount of CD8^＋ cells was significantly increased as well （P〈0.05）. The killing activities of CD3AK and CIK cells to the MGC-803 gastric cancer cell strain were all significantly higher than LAK cells, while the killing activity of CIK cells was stronger than CD3AK cells （P〈0.05）. Conclusion： CIK cells have stronger amplification activity and killing activity, and can be taken as more effective killing cells applied to the tumor adoptive immunotherapy.

黄芪多糖对IL－2／LAK抗肿瘤增强作用的动物实验研究

采用Ｃ５７ＢＬ／６鼠荷其自发产生的黑色素瘤Ｂ＿１６细胞，以生存期为指标，建立了黄茂多糖（ＡＰＳ）增强ＩＬ－２／ＬＡＫ抗肿瘤作用的动物模型，结果表明：ＡＰＳ自身具有一定的抗肿瘤作用，ＡＰＳ（５ｍｇ／ｋｇ）与ＩＬ－２／ＬＡＫ（５×１０￣３Ｕ／ｋｇ、５×１０￣７／ｋｇ）的抗肿瘤作用相似，荷瘤鼠生存期分别为２１．５７±１．８１天和２０．８６±１．８６天（荷瘤对照鼠为１５．７１±０．４９天）；ＡＰＳ对ＩＬ－２／ＬＡＫ的抗肿瘤效应有明显的增强作用，二者在上述剂量下联合应用，荷瘤鼠生存期为２４．８６±２．７３天（Ｐ＜０．０１）。并对荷瘤鼠进行动态细胞免疫功能观察，提示：ＡＰＳ和ＩＬ－２／ＬＡＭ均具有抵抗鼠脾ＮＫ细胞活性，ＩＬ－２产生能力和腹腔Ｍψ吞噬能力下降的作用，ＡＰＳ对ＩＬ－２／ＬＡＫ仍有显著增强效应。

乌贼墨诱生小鼠IFN-γ及调节LAK细胞活性的研究

为进一步研究乌贼墨对免疫功能的刺激作用 ,本文采用 ELISA技术和生物活性测定技术分别检测了经乌贼墨灌胃后小鼠血清中的 IFN- γ的水平和 L AK细胞活性的变化。结果显示 ,实验组小鼠 IFN- γ的水平于乌贼墨灌胃后 2 4 h开始升高 ,4 8h达高峰 ,72 h降至正常水平。L AK细胞杀伤活性也有明显增强 ,与对照组小鼠相比 ,差异显著 (P<0 .0 1)。

抗NKG2D多克隆抗体抑制NK和LAK细胞细胞毒效应的研究

目的 :分析抗NKG2D多克隆抗体 ( pAb)对NK和LAK细胞毒作用的影响。方法 :应用密度梯度离心法分离外周血单个核细胞 (PBMC) ,经 10mg/LPHA和 1× 10 6U/LrhIL 2诱导LAK细胞产生 ,再应用流式细胞术 (FCM)分选NK细胞并进行表型检测。加入抗NKG2DpAb封闭NK和LAK细胞表面的NKG2D分子后 ,用MTT比色法检测其细胞毒效应。结果 :经FCM分析证实 ,获得高纯度、高活性的NK细胞。抗NKG2DpAb能显著抑制NK和LAK细胞对K5 6 2、HepG2细胞的细胞毒效应。NK细胞对两种靶细胞的细胞毒效应分别下降了 82 .9%和 75 .6 % ;LAK细胞对两种靶细胞的细胞毒效应分别下降了 5 2 .8%和 5 0 .2 %。但抗NKG2DpAb不能显著抑制两种效应细胞对人鼻咽癌细胞系CNE的细胞毒效应。结论 :抗NKG2DpAb可通过封闭NK和LAK细胞表面的NKG2D分子 。

病毒性心肌炎NK和LAK细胞活性与心肌损伤的关系

目的：系统地观察病毒性心肌炎，尤其是柯萨奇Ｂ组病毒（ＣＶＢ）心肌炎患儿自然杀伤细胞（ＮＫ）和淋巴因子激活的杀伤细胞（ＬＡＫ）活性，并分析两者的内在关系，以及与心肌损伤的关系。方法：５６例病毒性心肌炎患儿，年龄３～１２岁，男２６例，女３０例；另设对照组３０例，年龄３～１２岁，男１６例，女１４例。采集静脉血，应用３Ｈ－ＴｄＲ标记法测定ＮＫ和ＬＡＫ细胞活性。结果：（１）心肌炎时ＮＫ和ＬＡＫ细胞活性均明显下降；（２）心肌炎时ＮＫ和ＬＡＫ细胞活性间存在明显的正相关（ｒ＝０．７２，Ｐ＜０．０１）；（３）ＣＶＢ心肌炎患儿的ＮＫ和ＬＡＫ细胞活性下降更显著，且心脏增大和心电图明显异常者发生率亦高于非ＣＶＢ心肌炎者。结论：病毒性心肌炎，尤其是ＣＶＢ心肌炎对ＮＫ及ＬＡＫ细胞活性有明显影响。在病毒性心肌炎诊疗中应同时检测ＮＫ和ＬＡＫ细胞活性。

γδT细胞、NK细胞及LAK的部分抗肿瘤生物特性比较

目的 :通过体外分离、增殖培养获取γδT细胞、NK细胞和LAK细胞 ,并比较了 3种细胞的抗肿瘤的生物学特性。方法 :收集用不同单抗分别包装被粘附的细胞 ,然后通过MACS细胞分选仪的分选 ,获取的细胞进行细胞增殖动力学、细胞表型、细胞杀伤活性的测定以单抗阻断效应的分析。结果 :经MACS分离得到的γδT细胞 ,培养 2w后细胞数扩散 6 0 0～ 80 0倍 ,CD3、CD8和γδ细胞表达阳性率分别是 72 2 9%、5 8 0 2 %和 6 5 98%。γδT细胞对NK敏感细胞K5 6 2以及NK不敏感细胞Raji和XG 7这 3种不同靶细胞均有较高的杀伤率 ,分别为 35 98%、42 2 7%和 6 9 0 8% ;NK细胞对此 3种细胞的杀伤率分别是45 12 %、12 34%和 2 9 2 7% ;LAK细胞的杀伤率分别为 44 0 1%、2 9 2 7%和 2 5 6 8%。γδT细胞对经MHC Ⅰ类单抗阻断前后的K5 6 2、Raji和XG 73种靶细胞的杀伤率无明显改变。结论 :γδT细胞、NK细胞和LAK细胞都具有一定的非特异性杀伤肿瘤细胞的作用 ;γδT细胞对MHC Ⅰ类单抗阻断后的肿瘤细胞的杀伤无明显变化 。

红芪多糖增强LAK细胞对膀胱肿瘤细胞的杀伤作用

用ＭＴＴ法观察了红芪多糖（ＨＰＳ）对ＬＡＫ细胞的影响，结果表明ＨＰＳ无明显的促进ＬＡＫ细胞扩增的作用，但与ＬＡＫ细胞或ＰＢＭＣ合用可显著增强对膀胱肿瘤细胞株ＥＪ和原代肿瘤细胞的杀伤作用。

腹腔内注射TNF基因转染的LAK细胞对于腹水型肝癌小鼠的疗效

在逆转录病毒介导下将TNF基因转染入鼠LAK细胞中,结果发现,转染TNF基因的LAK细胞比正常LAK细胞和转染对照基因的LAK细胞分泌更高水平的TNF,其体外生长能力和杀伤活性均显著升高,抗TNF单抗可显著抑制其体外杀伤活性,表明其杀伤活性升高是由基因转染后所表达的TNF介导的.将较低数量的TNF基因转染的LAK细胞与IL～2联合腹腔内注射后,对腹水型肝癌小鼠有显著的治疗效果.

人外周血中LAK细胞的克隆化

本文报道首次采用半固体-液体两步法克隆正常人外周血单个核细胞(PBMNC)中的淋巴因子激活的杀伤细胞(LAK)获得成功。先用含重组人白细胞介素2(rhIL-2)的软琼脂半固体克隆外周血中的T细胞,再将T细胞克隆转移至96孔板中继续在含rhIL-2的液体中培养。^(51)Cr释放的结果表明,约10～30％的克隆对NK敏感的K562细胞和NK不敏感的H7402、Anip-1肿瘤细胞均有细胞毒性,即为LAK细胞克隆。LAK细胞克隆能在体外含rhIL-2培养液中增殖1.5～3.0个月,每个克隆可扩增至10~9～10^(10)个细胞,仍然维持LAK细胞活性。表型分析的结果表明,克隆的LAK细胞CD3(+)、CD8(+),属T细胞系统。有增殖能力的LAK前体细胞在PBMNC中的频率约为1～3×10^(-4)。用有限稀释法将LAK细胞克隆进一步亚克隆,98％以上的亚克隆均有LAK活性,表型也和原克隆相同,证实原克隆具有克隆源性。本文的两步法克隆LAK细胞程序可在较短时间内获得大量均一的LAK细胞,极大地有助于LAK细胞的深入研究和广泛应用。

LAK细胞和IL－2联合化疗治疗晚期肺癌的近期疗效观察

ＬＡＫ细胞和ＩＬ－２是目前常用的肿瘤生物制剂。自１９９２年以来，对１８例晚期肺癌，男１５例，女３例，年龄２９～６４岁，进行ＬＡＫ细胞和重组ＩＬ－２联合化疗治疗。选择胎脾、胸腺的淋巴细胞做前体细胞，体外用重组ＩＬ－２诱导制备ＬＡＫ细胞，每输３次ＬＡＫ细胞为一疗程、每次输入细胞数０．５～１．０×１０９。化疗采用环磷酰胺，长春新碱，阿霉素为主的方案。治疗结果：完全缓解（ＣＲ）５例，部分缓解（ＰＲ）７例，无效（ＮＲ）３例，病情平稳３例，有效率ＣＲ＋ＰＲ达６６％。采用本疗法后病人精神及饮食好转，能有效缓解胸痛、减轻病人痛苦。提示ＬＡＫ细胞和重组ＩＬ－２联合化疗对晚期肺癌是一种可行的有效治疗，但在临床应用中要注意毒性反应。