PURGING OF BONE MARROWS CONTAMINATED WITH MYELOID LEUKEMIC CELLS BY INTERLEUKIN-2 AND LYMPHOKINE-ACTIVATED KILLER CELLS

The capability of recombinant human interleukin-2 ( rhIL-2) and lymphokine-activated killer (LAK) cells in the purging of normal human bone marrows contaminated with human myeloid leukemic cell lines was evaluated. Mixtures of normal human bone marrow mononuclear cells ( BMC) and K562 cells or HL-60 cells (at the BMCK562 ratio of 200:1, 100:1 or 20:1) were incubated with IL-2 with or without LAK cells at the BMC:LAK ratio of 1:1 for one or three days. The nubmers of residual K562 cells, BFU-E and CFU-GM were examined by clonogenic assays. In 200:1 mixture groups without LAK cells, the number of K562 colonies reduced by 50% with no loss of BFU-E and CFU-GM in one-day cultures, and no K562 colonies formed in three-day cultures with about 20% loss of BFU-E and CFU-GM. If the BMC.K562 ratios were 100:1 or 20:1 in the mktures, the leukemic cells could not be eliminated. When the mixtures were incubated with IL-2 and LAK cells, no leukemic cell colonies were detected in the 20:1 group following one-day

BIOLOGICAL FEATURES OF HUMAN T-ACTIVATED KILLER CELLS

Objective: To investigate the immunobiological essence of T-activated killer (T-AK) cells induced by anti-CD3 monoclonal antibody (CD3McAb) and recombinant interleukin-2 (rIL-2) co-stimulation. Methods: The cytomorphology, phenotype and cytotoxicity of T-AK cells generated from human peripheral blood mononuclear cells (PBMC) were determined. Results: T-AK cells were similar to activated lymphoblasts in morphology, more than 90% of T-AK cells expressed the phenotypes of T-lymphocytes (CD3 +, CD8 +, and 20%～50% of the cells were NK-like phenotype (CD16 +, CD56 +, some of them expressed IL-2 receptor (CD25 +), CD38 antigen (CD38 +) and MHC-II antigen (HLA-DR+) characteristic marks for the activated T lymphocytes. T-AK cells attacking targets were morphologically large volumes with granules and mainly contained CD8 + and CD56 + cells. T-AK cells possessed high tumoricidal activities against NK-sensitive K562 cells and NK-resistant Raji cells, the cytotoxicity was composed of mainly CD3McAb-activated CD3AK activity (～50%), IL-2 induced LAK activity (～30%), NK activity (～10%) and the activities of inhibitory factors in T-AK supernatant (～10%). Conclusion: T-AK cells are a heterogeneous cell population consisting of mainly activated T lymphocytes and NK-like cells, the main part of T-AK cytotoxicity is the common activities of CD3AK cells and LAK cells.

Research on stress-induced apoptosis of natural killer cells and the alteration of their killing activity in mouse liver

AIM:To investigate the stress-induced apoptosis of natural killer(NK)cells and the changes in their killing activity in mouse livers.METHODS:A restraint stress model was established in mice.Flow cytometry was employed to measure the percentage of NK cells and the changes in their absolute number in mouse liver.The cytotoxicity of hepatic and splenic NK cells was assessed against YAC-1 target cells via a 4 h 51Cr-release assay.RESULTS:The restraint stress stimulation induced the apoptosis of NK cells in the liver and the spleen,which decreased the cell number.The number and percentage of NK cells in the spleen decreased.However,the number of NK cells in the liver decreased,whereas the percentage of NK cells was significantly increased.The apoptosis of NK cells increased gradually with prolonged stress time,and the macrophage-1(Mac-1)+NK cells were more susceptible to apoptosis than Mac-1-NK cells.Large numbers of Mac-1-NK cells in the liver,which are more resistant to stress-induced apoptosis,were observed than the Mac-1-NK cells in the spleen.The stress stimulation diminished the killing activity of NK cells in the spleen was significantly decreased,but the retention of numerous Mac-1-NK cells in the liver maintained the killing ability.CONCLUSION:Significant stress-induced apoptosis was observed among Mac-1+NK cells,but not Mac-1-NK cells in the mouse liver.Stress stimulation markedly decreased the killing activity of NK cells in the spleen but remained unchanged in the liver.

Effect of anti-asthma Chinese medicine Chuankezhi on the anti-tumor activity of cytokine-induced killer cells

Chuankezhi(CKZ),a new Chinese medicine,plays an important role in immunoregulation.Cytokineinduced killer(CIK)cells have been commonly used for immunotherapy in recent years.In this study,we aimed to investigate the immunoregulatory effect of CKZ on CIK cells.Peripheral blood monocytes were isolated from healthy donors,and CIK cells were generated by culturing monocytes with interferon-gamma(IFN-γ)and interleukin 2.Different concentrations of CKZ were added on day 2.After incubation for 14days in culture,the antitumor effects of CIK cells were measured by cytotoxicity assay.Flow cytometry was used to explore the effect of CKZ on CIK cell immunophenotype,intracellular cytokine production,and apoptosis.The effect of CKZ on the antitumor activity of CIK cells in nude mice was also investigated.CKZ increased the percentage of CD3+CD56+CIK cells but did not significantly change the percentage of CD4+,CD8+,or CD4+CD25+CIK cells.CKZ-conditioned CIK cells showed a greater ability to kill tumor cells,as well as a higher frequency of IFN-γand TNF-αproduction,compared with the CIK cells in the control group.CKZ also suppressed the apoptosis of CIK cells in vitro.Furthermore,CKZ combined with CIK cells had a stronger suppressive effect on tumor growth in vivo than the CIK,CKZ,or normal saline control groups.Our results indicate that CKZ enhances the antitumor activity of CIK cells and is a potential medicine for tumor immunotherapy.

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

Double potentialities of tumoricide and hematopoiesis of human bone marrow cells activated by IL-2 and IL-3

The biomodulative and hematopoietic potentialities of IL-2 and IL-3 activatedbone marrow(ABM)cells from patients with lung adenocarcinoma were studied in vitro.Human bone marrow(BM)cells could be activated by IL-2 in culture for 7d.TheseIL-2 ABM cells had higher cytolytic activities against cells of H 7402 cell line and freshautologous adenocarcinoma cells and maintained the cytotoxicities longer than IL-2 acti-vated peripheral blood lymphocytes(APBLs),a point of possible importance in adoptiveimmunotherapy for cancer patients.The IL-2 ABM cells also had similar number ofBFU-E and CFU-GM to that had fresh BM cells if 1L-3 was added 48h alter IL-2 inculture.The IL-2 and IL-3 ABM cells might be used to eliminate tumor cells and tosupply reconstitutive elements of BM for autologous bone marrow transplantation.

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

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THE EFFECT OF PHENYLACETATE ON THE EXPANSION AND CYTOTOXIC ACTIVITY OF ADHERENT LAK CELLS FROM PATIENTS WITH HEPATOCELLULAR CARCINOMA

Objective: To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and study the synergistic anti-tumor effect of phenylacetate (PA) and A-LAK cells. Methods: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. The proliferation of SMMC7721 cell line treated with PA was studied. A-LAK cells were treated with the supernatant of SMMC7721 cells which had been pretreated with PA and the changes of the proliferation and anti-tumor activity of A-LAK cells were investigated. Results: The expansion of A-LAK cells was significantly higher than that of non-adherent LAK (NA-LAK) cells as well as regular LAK cells. The growth of SMMC7721 cells was significantly suppressed by PA. The supernatant of cultured tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of supernatant treated with PA previously was decreased. Conclusion: A-LAK cells could be simply prepared by using PME, and showed a synergistic anti-tumor effect with the combination of PA.

THE ULTRASTRUCTURAL OBSERVATION OF TARGET CELLS ATTACKED BY HUMAN LAK

In order to explore the killing mechanism of LAK, we observed the morphological change of K562 and Raji attacked by human LAK with transmission electron microscope, The result showed that 1 hour after coculture of LAK and target cell, target cell was significantly damaged.Part of target cells died via necrosis, and part via apoptosis. Our findings show that human LAK can kill target cells via necrosis and apoptosis simultaneously

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

客观：象杀死活动到胃的癌症房间火车在试管内的变化一样学习脐带血 mononuclearcells 的多方向性的推导到 CD_3AK， LAK 和 CIK 房间。方法：CD_3mAb 和 IL-2 被用来导致 CD_3AK 房间，并且 IL-2 被用来导致 LAK 房间；IFN- γ在开始被使用，然后， IL-1， CD_3mAb 和 IL-2 为观察扩大并且分析他们的关系在 24 h 以后习惯于 induceCIK 房间。有教养的 CIK 房间的显型被流动血细胞计数分析。由使用胃的癌症房间作为目标房间拉紧的 MGC-803，随后， CD_3AK， LAK 和 CIK 房间的杀死的活动被使用 MTT 方法评估。结果：CD_3AK 和 CIK 房间的扩大活动都比 LAK 房间高远(P 【 0.05 ) 。扩大活动没在前期有 CIK 房间和 CD_3AK 房间之间的明显的差别，但是那比在大约 20thday 的 CD_3AK 房间在 CIK 房间更高是远的(P 【 0.05 ) 。流动血细胞计数表明 CD_3^+CD_(56 )^+ 房间的数量，在是有教养的 CIK 房间以后的主要受动器房间显著地被增加(P 【 0.05 ) ，而且， CD_8^+ 房间的数量显著地也被增加(P 【 0.05 ) 。到 MGC-803 胃的癌症房间紧张的杀死的活动 ofCD_3AK 和 CIK 房间都是显著地更高的 thanLAK 房间，当 CIK 房间的杀死的活动比 CD_3AK 房间强壮时(P 【 0.05 ).Conclusion：CIK 房间举办更强壮的扩大活动和杀死的活动，并且当更有效的杀死房间把采纳免疫疗法用于肿瘤，能被拿。

莫诺苯宗对肺腺癌细胞自噬和凋亡的分子机制研究

目的:探究莫诺苯宗和自然杀伤细胞激活受体基因(KLRC3)对肺腺癌(LUAD)细胞凋亡和自噬的影响,为LUAD的治疗提供理论基础。方法:分别使用不同浓度莫诺苯宗处理LUAD细胞,并采用CCK-8法检测莫诺苯宗对LUAD细胞活力的影响;随后,使用80μmol/L的莫诺苯宗、50 nmol/L的KLRC3干扰质粒(si-KLRC3)、和100 nmol/L自噬抑制剂3-甲基腺嘌呤(3-MA)1.5μg/ml KLRC3过表达载体(pcDNA-KLRC3)处理细胞后采用流式细胞术和Western blotting检测细胞凋亡以及凋亡相关蛋白和自噬相关蛋白的表达量。结果:莫诺苯宗浓度大于20μmol/L时可有效减弱LUAD细胞活力,且以剂量依赖性地促进了LUAD细胞的凋亡和自噬(均P<0.05)。KLRC3在LUAD细胞中低表达,干扰KLRC3减弱了莫诺苯宗对LUAD细胞自噬和凋亡的促进作用,比较差异有统计学意义(均P<0.05)。此外,过表达KLRC3也促进了LUAD细胞的凋亡和自噬,而KLRC3和莫诺苯宗对LUAD细胞凋亡和自噬的促进作用均可被3-MA所抵消,比较差异有统计学意义(均P<0.05)。结论:莫诺苯宗可能通过促进KLRC3的表达诱导自噬进而促进LUAD细胞的凋亡。

石斛多糖增强脐带血和肿瘤病人外周血LAK细胞体外杀伤作用的研究

目的：探讨石斛多糖（ dendrobium candidum polysaccharide,DCP）与 rIL- 2联合应用对脐带血 LAK细胞（ CB-LAK）和肿瘤病人外周血 LAK细胞（ PB-LAK）体外杀伤肿瘤细胞作用的影响。方法：应用 3H-TdR释放法测定 CB-LAK和 PB-LAK细胞的杀伤活性。结果：① rIL-2(500 u/ml)能明显提高 CB-LAK对 Raji细胞的杀伤活性 ,0 h的 CB-LAK活性为 13.63％ ,rIL-2(500 u/ml)诱导 48、 72、 96、 120 h后 ,CB-LAK活性分别提高至 22.28％、 26.23％、 28.55％和 25.76％ (P0.05）；③ DCP（ 200 mg/L）与 rIL-2（ 500 u/ml）联合培养 96 h，显著增强 PB-LAK杀伤活性。 10例恶性肿瘤病人外周血（ DCP＋ rIL-2）组与单纯 rIL-2组诱导的 PB-LAK平均活性分别为 34.78％和 23.24％ 。

氧化苦参碱对LAK细胞活性的影响

ＬＡＫ细胞具有很强的广谱杀瘤作用，而氧化苦参碱具有较强的免疫抑制作用。本文研究了氧化苦参碱对ＬＡＫ细胞活力的影响，结果表明：氧化苦参碱可抑制ＩＬ－２对小鼠脾细胞的促增殖作用。并且对ＩＬ－２活化ＬＡＫ细胞杀伤Ｐ８１５的能力也有抑制作用。当ＩＬ－２（５００ｕ／ｍｌ）与２００μｇ／ｍｌ的氧化苦参碱共同孵育４ｄ后，可使ＬＡＫ细胞杀瘤能力（在效靶比为１００：１时）的８２．５％被抑制。同时氧化苦参碱本身对Ｐ８１５的增殖无任何效应。

参麦注射液对晚期癌症患者sIL－2R、LAK及NK细胞活性影响

本研究测定了６０例晚期癌症患者用参麦注射液治疗前后血清可溶性白细胞介素－２受体（ｓＩＬ－２Ｒ）、淋巴因子活化的杀伤细胞（ＬＡＫ）和天然杀伤细胞（ＮＫ）的活性，并以４０例正常人为对照。结果显示：治疗前癌症患者ｓＩＬ－２Ｒ显著升高（Ｐ＜０．０５），ＬＡＫ细胞与ＮＫ细胞活性显著减低（Ｐ＜０．０５）。而上述变化在胃癌、肠癌与肺癌之间无显著差异（Ｐ＞０．０５）。治疗后３种癌症患者的ｓＩＬ－２Ｒ均有显著下降（Ｐ＜０．０５），ＬＡＫ与ＮＫ细胞活性均有显著增强（Ｐ＜０．０５）。而ｓＩＬ－２Ｒ与ＬＫＡ及ＮＫ细胞活性之间无相关性。提示晚期癌症患者免疫功能低下，参麦注射液对癌症患者的免疫功能有广泛的调节作用。

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。

人LAK细胞免疫效应分子HMGN2的鉴定

为分离纯化人淋巴因子激活的杀伤细胞(LAK)小分子抗菌多肽,应用制备尿素-聚丙烯酰胺凝胶电泳技术和反向高效液相色谱技术分离纯化人LAK细胞酸溶性提取物,纯化出一个具抗菌活性的多肽HLP-3p21.蛋白质N端氨基酸测序、质谱精确分子质量测定、蛋白质印迹分析证明HLP-3p21为HMGN2.最小抑菌浓度(MIC)和最小杀菌浓度(MBC)试验证明HMGN2有抗大肠杆菌ML-35p氨苄青霉素耐药株、铜绿假单胞菌ATCC27853、白色念珠菌ATCC10231活性,无抗金黄色葡萄球菌ATCC25923活性.制备HMGN2多克隆抗体,应用免疫荧光化学、酶联免疫吸附测定和蛋白质印迹方法对HMGN2进行定位分析,证明单个核细胞经IL-2刺激成为LAK细胞时部分HMGN2由胞核转移至胞浆,进而分泌到胞外.提示HMGN2是LAK细胞一个新的免疫效应分子.

抗肿瘤多价转移因子对肿瘤患者NK及LAK活性的影响

目的 制备并观察抗肿瘤多价转移因子 (M TF )对肿瘤患者NK及LAK活性的影响。方法 分别用肝癌细胞悬液、胃癌细胞悬液免疫山羊 ,取其淋巴组织经匀浆透析法制备肝癌特异性转移因子 (HCC STF)和胃癌特异性转移因子 (GC STF) ,用肝癌、胃癌及大肠癌混合细胞悬液免疫山羊 ,制备M TF。比较其理化性质及对肿瘤患者NK及LAK活性的影响。结果 三种转移因子理化性质十分相似 ,肽类、核苷酸类物质及氨基酸含量相近。HCC STF能明显增加肝癌患者NK、LAK活性及LAK细胞对肝癌 772 1细胞的特异细胞毒作用 ,GC STF能明显增加胃癌和大肠癌患者NK、LAK活性及LAK细胞对胃癌 790 1细胞的特异细胞毒作用 ,M TF能显著增加肝癌、胃癌及大肠癌的NK、LAK活性及LAK细胞对 772 1和 790 1细胞的细胞毒作用 ,其增加效果与HCC STF及GC STF比无明显差异。结论 M TF具有免疫调节性强、抗肿瘤谱广的特性 。

Th-LAK细胞对肺癌试验治疗的观察

利用Th-LAK细胞对46例肺癌患者进行了临床试验治疗的观察。结果表明,71.7%的病例原有症状和体征都有不同程度的改善;各期综合的一年生存率为82.6%,高于其它疗法的生存率;部分病例的肿瘤及转移淋巴结有不同程度的缩小。

白细胞介素2快速诱导人LAK细胞的实验研究

本文采用^(125)IUdR释放法测定了大剂量白细胞介素2(IL-2)体外短期诱导人脐血,正常人、良性瘤及恶性瘤病人外周血淋巴细胞的抗肿瘤活性,发现四种来源的淋巴细胞在大剂量IL-3作用15分钟时均可诱导出一定水平的LAK活性,最适IL-2用量为6×10~3u/1.2×10~7细胞/ml,最适体外培养时间为3天。

小鼠LAK细胞MHC表达水平对其体外杀伤肿瘤活性的影响

目的探讨LAK细胞表面MHC的表达水平与体外肿瘤细胞杀伤活性的关系。方法使用siRNA沉默小鼠LAK细胞MHC-Ⅰ(H-2Kd)分子的表达,同时设空转染组和无关序列组,流式细胞仪检测不同组别靶蛋白的表达,LDH释放试验检测H-2Kd表达降低的LAK细胞对K562和H22肿瘤细胞株的杀伤活性。结果流式细胞检测证实siRNA能够抑制靶蛋白的表达,H-2Kd表达降低组对K562和H22肿瘤细胞的杀伤活性与对照组相比明显降低,空转染组和无关序列组与对照组无显著性差异。结论小鼠脾LAK细胞表面H-2Kd的表达水平与其体外杀伤活性相关。