应用LAK细胞和IL－2局部过继免疫治疗表浅膀胱癌的临床研究

应用ＬＡＫ细胞和ＩＬ－２局部过继免疫治疗表浅膀胱癌的临床研究欧阳骏，陈赐龄，郭震华，温端改我们在体外实验ＬＡＫ／ＩＬ－２抗人新鲜膀胱癌细胞取得肯定结果之基础上，进行了ＬＡＫ／ＩＬ－２局部注输治疗表浅膀胱癌，现报告如下。材料与方法（１）临床资料：自１９...     

免疫杀伤中LAK细胞的坏死和凋亡及黄芪多糖的影响

目的：进一步探讨ＬＡＫ细胞攻击Ｈｅｌａ细胞后的归宿及黄芪多糖（ＡＰＳ）的影响。方法：分别以ＩＬ－２和ＩＬ－２、ＡＰＳ激活的ＬＡＫ细胞与Ｈｅｌａ细胞共育，用电镜和电脑图像分析系统观察和测定ＬＡＫ细胞的存活、坏死、凋亡细胞及各期凋亡细胞不同时段的体密度。结果：对照组１６小时Ｈｅｌａ细胞大部分死亡，ＬＡＫ细胞本身死亡５７％。８、１６小时实验组ＬＡＫ细胞存活、坏死、凋亡细胞各自的体密度与对照组比均有差异（Ｐ＜０．０５）或明显差异（Ｐ＜０．０１），各期凋亡细胞的体密度也有差异或明显差异。结论：ＡＰＳ可降低ＬＡＫ细胞坏死和凋亡细胞的体密度，尤其是典型凋亡细胞的体密度，为抗肿瘤应用ＡＰＳ提高ＩＬ－２／ＬＡＫ细胞的活性提供实验根据。     

CD3单克隆抗体活化的杀伤细胞对人胃癌细胞株MNK45体外杀伤及…

用人的外周血单个核细胞（ＰＢＭＣ）与抗ＣＤ３单克隆抗体和基因重组的人ＩＬ－２，共同培养制备ＣＤ３ＡＫ细胞，ＰＢＭＣ与ｒＩＬ－２共同培养制备ＬＡＫ细胞，用单纯ＰＢＭＣ作为对照细胞。对这三种细胞的增殖及在体外和裸鼠体内的抗人胃癌细胞株ＭＮＫ４５作用进行了实验研究。体外试验结果表明，ＣＤ３ＡＫ对ＭＮＫ４５有明显杀伤作用，而ＬＡＫ细胞杀伤率较低，二者差异有非常显著性意义（Ｐ＜０．０１）；用ＭＴＴ法和台盼蓝     

CD_3单克隆抗体活化的杀伤细胞对人胃癌细胞株MNK_（45）体外杀伤及裸鼠体

用人的外周血单个核细胞（ＰＢＭＣ）与抗ＣＤ３单克隆抗体和基因重组的人ＩＬ－２，共同培养制备ＣＤ３ＡＫ细胞（ＣＤ３ＭｃＡｂＡｃｔｉｖａｔｅｄＫｉｌｌｅｒＣｅｌｌｓ），ＰＢＭＣ与ｒＩＬ－２共同培养制备ＬＡＫ细胞，用单纯ＰＢＭＣ作为对照细胞。对这三种细胞的增殖及在体外和裸鼠体内的抗人胃癌细胞株ＭＮＫ４５作用进行了实验研究。体外试验结果表明，ＣＤ３ＡＫ对ＭＮＫ４５有明显杀伤作用，而ＬＡＫ细胞杀伤率较低，二者差异有非常显著性意义（Ｐ＜０．０１）；用的ＭＴＴ法和台盼蓝活细胞计数法测定这三种细胞的增殖能力和细胞数量的增加，表明ＣＤ３ＡＫ的增殖能力和扩增数量均明显高于ＬＡＫ细胞（Ｐ＜０．０１）。裸鼠体内试验结果显示，对照组及ＬＡＫ组全部长出瘤结节，而ＣＤ３ＡＫ组无一例长出皮下结节，且生存期明显长于前者。     

IL—2／LAK疗法对肿瘤患者免疫功能的影响

我们观察了２０例肿瘤患者应用ＩＬ－２／ＬＡＫ疗法前后其免疫指标的改善，包括Ｔ淋巴细胞亚群，ＬＡＫ细胞活性，白介素Ⅱ分泌细胞及血清白介素Ⅱ受体。通过治疗显示：ＣＤ４／ＣＤ８比值，ＬＡＫ活性，白介素Ⅱ膜受体及白介素Ⅱ分泌细胞的水平均显著升高，而可溶性白介素Ⅱ受体减少，提示ＩＬ－２／ＬＡＫ可以改善机体免疫，功能。     

脐血T淋巴细胞亚群、LAK细胞表型和杀伤活性研究

应用单克隆抗体技术活细胞间接免疫荧光法及乳酸脱氢酶释放法分别检测了３０例脐血和３０例成人外周血Ｔ淋巴细胞亚群、ＬＡＫ细胞表型及杀伤活性，结果表明：脐血ＣＤ３、ＣＤ２及ＨＬＡ－ＤＲ的表达均明显低于成人外周血，而ＣＤ４、ＣＤ８、ＣＤ４／ＣＤ８、ＣＤ１６、ＣＤ２５及ＣＤ５６的表达与成人外周血相似。经ＩＬ－２诱导后，脐血ＬＡＫ细胞表型较诱导前ＣＤ８、ＣＤ１６、ＣＤ２５及ＨＬＡ－ＤＲ表达均明显增加，脐血ＬＡＫ细胞杀伤活性较成人外周血ＬＡＫ细胞低。     

CD3AK细胞和LAK细胞治疗晚期恶性肿瘤的临床和实验研究

将５１例晚期恶性肿瘤患者（男性２３例，女性２８例）分成两组，其中一组（３１例）以ＣＤ３ＭｃＡｂ（ＣＤ３单克隆抗体）和小剂量ＩＬ－２（５００ｕ／ｍｌ）共同诱导的ＣＤ３ＡＫ细胞治疗，另一组（２０例）输注大剂量ＩＬ－２（１０００ｕ／ｍｌ）诱导的常规ＬＡＫ细胞治疗，以探讨降低ＩＬ－２用量、提高杀伤细胞细胞毒活性的可能性。结果显示ＣＤ３ＡＫ组患者生活质量改善、症状缓解均优于ＬＡＫ组。ＣＤ３ＡＫ组ＰＲ＋ＭＲ率较ＬＡＫ组高２９．０％，Ｓ＋Ｐ率和死亡率分别较ＬＡＫ组低１２．４％和９．６％。同时比较了ＣＤ３ＡＫ细胞与ＬＡＫ细胞的体外增殖和细胞毒活性，结果表明ＣＤ３ＡＫ细胞增殖率高于ＬＡＫ细胞（Ｐ＜０．０１），靶细胞抑制率二者在０．０５水平无显著差异。提示ＣＤ３ＭｃＡｂ在刺激杀伤细胞活性，尤其在提高其增殖能力方面，具有显著的作用，ＣＤ３ＡＫ／ＩＬ－２能更有效地治疗晚期恶性肿瘤。     

白血病中IL－2受体基因的表达及其意义

利用聚合酶链反应（ＰＣＲ）方法检测１１６例各种白血病细胞ＩＬ－２受体基因的表达情况。该基因在Ｔ－ＡＬＬ、ＣＬＬ、Ｃ－ＡＬＬ和ＡＮＬＬ都有相当高的表达率，分别为１００％、１００％、７１．４３％和６５％。该方法较ＡＰＰＡＰ和免疫荧光法发现更高的阳性率，提示其敏感性及准确性更高。本文结果显示了大部分的白血病细胞都表达了ＩＬ－２受体基因，推测ＩＬ－２受体可能是白血病细胞所具有的一种普遍特性，而ＩＬ－２则可能在白血病细胞的增殖中起一定的作用。故提出临床上应重新评价ＬＡＫ细胞和ＩＬ－２在白血病中的治疗价值。     

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。     

多抗甲素对LAK细胞增殖及杀伤活性的影响

将Ｃ５７ＢＬ／６小鼠脾淋巴细胞分成四组：①单纯培养液组；②单纯ＰＡ组；③ｒＩＬ－２组；④ｒＩＬ－２＋ＰＡ组。采用ＭＴＴ比色方法及间接免疫荧光法测定并比较各组淋巴细胞增殖反应，淋巴细胞对ｐ８１５肿瘤细胞的细胞毒作用以及淋巴细胞表面ＩＬ－２表达的情况，以此观察ＰＡ体外对小鼠脾ＬＡＫ细胞增殖反应、杀瘤活性及ＩＬ－２Ｒ表达的影响。结果表明，ＰＡ单独对静止淋巴细胞增殖反应无作用，也不能诱导出ＬＡＫ活性，但能显著增强ＩＬ－２诱导的淋巴细胞增殖反应，杀瘤活性及其表面ＩＬ－２Ｒ表达。当浓度为１０μｇ／ｍ１的ＰＡ与低剂量（２００ｕ／ｍ１）ＩＬ－２联合应用时，诱导出的ＬＡＫ活性强度相当于浓度为１０００ｕ／ｍ１ＩＬ－２水平。提示，ＰＡ与ＩＬ－２合用能共同诱导ＬＡＫ活性，降低ＩＬ－２用量，ＰＡ可望作为一种新的免疫调节剂用于肿瘤的ＬＡＫ／ＩＬ－２生物治疗。     

Impact of IL-2 and IL-2R SNPs on Proliferation and Tumorkilling Activity of Lymphokine-Activated Killer Cells from Healthy Chinese Blood Donors

One of the goals of tumor immunotherapy is to generate immune cells with potent anti-tumor activity throughin vitro techniques using peripheral blood collected from patients. However, cancer patients generally havepoor immunological function. Thus using patient T cells, which have reduced in vitro proliferative capabilitiesand less tumor cell killing activity to generate lymphokine-activated killer (LAK) cells, fails to achieve optimalclinical efficacy. Interleukin-2 (IL-2) is a potent activating cytokine for both T cells and natural killer cells.Thus, this study aimed to identify optimal donors for allogeneic LAK cell immunotherapy based on singlenucleotide polymorphisms (SNP) in the IL-2 and IL-2R genes. IL-2 and IL-2R SNPs were analyzed using HRMPCR.LAK cells were derived from peripheral blood mononuclear cells by culturing with IL-2. The frequencyand tumor-killing activity of LAK cells in each group were analyzed by flow cytometry and tumor cell killingassays, respectively. Regarding polymorphisms at IL-2-330 (rs2069762) T/G, LAK cells from GG donors hadsignificantly greater proliferation, tumor-killing activity, and IFN-γ production than LAK cells from TT donors(P<0.05). Regarding polymorphisms at IL-2R rs2104286 A/G, LAK cell proliferation and tumor cell killing weresignificantly greater in LAK cells from AA donors than GG donors (P<0.05). These data suggest that either IL-2-330(rs2069762)T/G GG donors or IL-2R rs2104286 A/G AA donors are excellent candidates for allogeneicLAK cell immunotherapy.     

Adhesion Molecules on Murine Lymphokine-activated Killer Cells Responsible for Target Cell Killing: A Role of CD2

Lymphokine-activated killer (LAK) cells were induced from C57BL/6 mouse spleen cells and the effects of culture time on the expression of cell surface phenotypes and cytotoxic activity of LAK cells were determined. The expression of CD2 remarkably decreased after culture of LAK cells for 30 days, while LFA-1, a principal adhesion molecule in LAK cells, and CD3 were not changed by the culture. LAK cells cultured for 90 days completely lost CD2. In accordance with the decrease of CD2, the cytotoxic activity of LAK cells declined but a certain leven was retained even after the complete loss of CD2. The established LAK cell clones were also strongly positive for the expression of LFA-1 but negative for CD2. When the LAK cell clones were transfected with the CD2 cDNA, they started to express CD2 on their cell surface and to show greater binding ability and stronger cytotoxicity to target tumor cells. These results indicated that CD2 plays a role as an adhesion molecule responsible for target cell killing in murine LAK cells.     

RESEARCHES ON THE ANTI-AUTOLOGOUS TUMOR EFFECTS OF HUMAN CANCER-KILLING CELLS IN NUDE MICE

An investigation of human splenic LAK cells killing autologous tumor in vivo has been performed in this study. Briefly, an ovary embryo carcinoma (OEC) removed surgically from patient was transplanted Into nude mice (s. c. ) and cultured in vitro, that would be used as targets. Lymphokine-activated killer (LAK) cells were generated from splenic lymphocytes of the OEC patient, who died two and half months after operation, by co- culture with recombinant human interleukin- 2 ( rIL- 2 ) in vitro. The results from Winn' s test in nude mice suggested that these LAK cells could effectively inhibit the tumorlgenicity of autologous tumor m vitro.     

Cell Cycle Assessment of Adoptive Transferred Lymphokine Activated Killer Cells

The use of lymphokine activated killer (LAK) cells for adoptivetransfer therapy has been reported from number of clinical trials.To our knowledge, however, there has been no report concerningthe cell cycle progression of LAK cells. Thus, for the presentstudy we have attempted to examine the LAK cell cycle eitherbefore or after transfer. In vitro and in vivo analyses of LAKcells labeled with bromodeoxyuridine (BrdU) were carried outusing two-parameter flow cytometries of their nuclear stainingusing fluorescein isothiocyanate(FITC)- conjugated anti-BrdUantibody and propidium iodide (PI). The in vitro growth of BrdU-positivecells showed the cells to divide once in the S phase, continueto the G₂-M phase and return to the G₀-G₁ phase with a similarpattern after 48 or 72 h culture. They formed a definite subpopulationand were of phenotypes, thy-1.2 (+), Lyt-1.1 ( – ), Lyt-2.1(+), L3T4 ( – ) and AGM1 (+). The percentage of BrdU-positivecells decreased significantly (P< 0.05) when treated withcomplement plus anti-thy-1.2, anti-Lyt-2.1 or anti-AGM1 anti-bodies, demonstrating BrdU-labeled LAK cells to have the samephenotype as control LAK cells. As in vitro, the in vito cellcycle of LAK cells 48 and 72 h after transfer had a similarpattern, and the LAK cells also continued on to the S phaseafter the first division. The in vivo growth of the LAK cellstreated with interleukin-2 (IL-2) was promoted 24 and 48 h aftertransfer. In conclusion, the present study showed LAK cellsto be capable of dividing following transfer and to keep theirown phenotypic characteristics; also, that treatment with IL-2may promote their division. Adoptive transfer therapy seemsto be a viable therapeutic method.     

Preparation and Bioactivity Assay of CD3AK Cell from Lmbilical Cord Blood：—Clinic Report of 10 Tumor Cases

Objective To study the anti-tumor effect of CD3AK cell prepared from umbilical blood,to explore the short-term curative effect on tumor cases and seek better immune index for biotherapy.Methods IL-2 and IL-2 CD3Ab were used to induce LAK cells and CD3AK cells isolated from umbilical blood mononuclear cells(UBMC).The expanding number and bioactivity of LAK cells and CD3AK cells were examined at different time points after culture,the NK activity of peripheral blood mononuclear cells(PBMC)of 10 cases of malignant tumor were determined before and after CD3AK cell adopting immune therapy as well.Results The number and bioactivity(NK killing K562 cell)of LAK and CD3AK cells reached their peaks on 11 th day.The number of LAK and CD3AK cells were 18 folds and 24 folds of that before culture;The NK activities of LAK and CD3AK against K562 were 2.6 folds and 3.2 folds of those before culture respectively.The nK activity for killing K562 cells of malignant tumor patient‘s PBMC was increased from 63%-81% by CD3AK cell transfusing,rising mean 28%.Conclusion (1)The UBMC is a potential and better source of predecessor for LAK and CD3AK;(2)The NK activity of LAK and CD3AK cells from UBMC reached their peaks at 11 th day after culture,and the NK aftivity of CD3AK cells in much greater than that of LAK cells;(3)The NK activity of malignant tumor patient‘s PBMC can be obviously elevated by transfusing CD3AK cell(4)The test of NK activity of PBMC of malignant tumor patient may become an objective immune index for tumor biotherapy.     

健康人和骨肉瘤病人来源的LAK细胞体外抗瘤作用

采用５１Ｃｒ释放试验对健康人和骨肉瘤病人外周血单个核细胞（ＰＢＭ）在重组白细胞介素－２（ｒＩＬ－２）条件下，ＬＡＫ细胞的诱导形成及对４种传代瘤细胞的体外杀伤活性进行探讨。实验结果表明：２种来源ＬＡＫ细胞对Ｋ５６２（人慢性髓样红白血病细胞系）、ＳＭＭＣ７７２１（人肝癌细胞系）、ＬＡＸ（人肺腺癌细胞系）的杀伤活性均在５５％以上（按效靶比例５０：１），而对ＯＳ细胞（人骨肉瘤细胞系）的活性普遍低下，杀伤率低于３５％。结果证实：１）健康人和骨肉瘤病人的ＰＢＭ均能在ｒＩＬ－２条件下诱导形成具有广谱抗瘤活性的ＬＡＫ细胞群，二者的杀伤格局和效力相近。２）骨肉瘤细胞本身对ＬＡＫ细胞的杀伤作用存在强烈抗性。     

脐血CIK细胞的体外增生及其抗肿瘤效应

 【摘要】　目的　研究脐血CIK细胞体外增生活性及对肿瘤细胞的杀伤活性，为CIK细胞在肿瘤过继免疫治疗中的应用提供实验依据。方法　脐血经密度梯度离心获得单个核细胞，以CD3单抗、重组人白细胞介素-2（rhIL-2）、重组人白细胞介素-1（rhIL-1）和重组人干扰素-γ（rhIFN-γ）为诱导剂制备多种细胞因子诱导的杀伤细胞（CIK细胞）。并设只加IL-2诱导成的LAK细胞和未加细胞因子的脐血单个核细胞（CBMNC）作为对照。培养前后分别用显微镜观察细胞形态，流式细胞术测定细胞表型的改变，锥虫蓝拒染法测定细胞增生水平，四甲基偶氮唑盐（MTT）法测定对肺癌细胞的杀伤活性。结果　实验发现，通过细胞因子的联合，可大量诱导出高活性的CIK细胞，CIK细胞在培养的第5天开始增生，第14天达到高峰，增生的细胞表型以CD+3 CD+56细胞为主。而LAK细胞的增生高峰出现在第7天，随后增生不明显；CBMNC表型无明显变化，增生不明显。CIK细胞针对肿瘤细胞的体外杀伤活性高于LAK细胞和CBMNC。结论　在体外细胞因子的联合作用下脐血能成功地诱导出大量的CIK细胞。脐血CIK细胞体外扩增快，杀伤活性强，对肿瘤细胞的作用优于传统的LAK细胞，为肿瘤的过继性免疫治疗提供了一个新的方向。     

Interleukin 2 and lymphokine-activated killer cell therapy: analysis of a bolus interleukin 2 and a continuous infusion interleukin 2 regimen

Several groups have described the efficacy of interleukin 2 (IL-2) plus lymphokine-activated killer (LAK) cells in the treatment of cancer patients with significant response rates noted in patients with renal cell cancer and malignant melanoma; however, the optimum regimen remains undefined. The Biological Response Modifiers Program of the National Cancer Institute conducted two consecutive Phase I/II studies evaluating the toxicity and clinical efficacy of different methods of IL-2 and LAK cell therapy. In the first trial, we modified the standard Rosenberg regimen by decreasing the duration of priming in an attempt to reduce the toxicity related to this phase of the therapy and thereby administer more IL-2 doses with the LAK cells. In the second trial, we used a continuous i.v. infusion IL-2 regimen and altered both the leukapheresis procedure and the LAK cell culture techniques based on our in vitro and preclinical studies suggesting that 2-day LAK cells were superior. Thirty cancer patients received i.v. bolus IL-2 at 100,000 units/kg every 8 h for 3 days during priming and for 5 days during LAK cell administration. A second group of 22 cancer patients received IL-2 by continuous i.v. infusion at 3 x 10(6) units/m2 for 5 days during priming and an additional 5 days of IL-2 with the LAK cell phase of the treatment. The timing of the start of the leukapheresis procedures, their duration and number, and the LAK cell culture techniques differed in the two trials. Overall, 52 patients with various cancers were treated. The toxicities associated with each regimen were similar to those seen in other IL-2 plus LAK cell trials. Four patients (one each with melanoma and diffuse large cell lymphoma and two with renal cell cancer) exhibited partial responses lasting 2, 4, 10, and 15+ mo. Serial tumor biopsies from treated patients demonstrated that therapy can produce a marked mononuclear cell infiltrate and an increase in HLA-DR expression on tumor cells. There was no difference in the overall response rate between the two regimens, but toxicity was less with continuous i.v. infusion IL-2. The 5-day continuous i.v. infusion regimen resulted in significantly higher rebound lymphocytosis, cell yield from leukapheresis, and number of LAK cells harvested from culture.     

脐血CD_3AK细胞的制备、生物活性测定及临床应用初探

 目的　研究用脐血单个核细胞制备CD3 AK细胞的抗肿瘤作用 ,探讨肿瘤生物治疗近期疗效的免疫指标。方法　分离脐血单个核细胞 ,分别用IL 2和IL 2 +CD3 Ab诱导LAK和CD3AK细胞 ,并测其扩增数量和对K5 6 2细胞的杀伤活性 ;测定肿瘤患者用CD3 AK细胞治疗前后外周血单核细胞(PBMC)的NK杀伤活性。结果　脐血LAK细胞和脐血CD3 AK细胞均于培养后第 11天时扩增倍数最高 ,分别是培养前的 18倍、2 4倍 ,对K5 6 2的杀伤活性分别是培养前的 2 .6倍和 3.2倍 ;肿瘤患者输注CD3 AK细胞一疗程后 ,其PBMC的NK杀伤活性由 6 2 %升高到 82 % ,平均升高 32 %。结论　 (1)脐血单个核细胞是LAK细胞和CD3 AK细胞良好的前体细胞。 (2 )LAK和CD3 AK细胞的数量和杀伤能力在培养的第 11天时达高峰 ,而且CD3 AK细胞数量和杀伤活性明显优于LAK细胞。 (3)CD3 AK细胞输注能明显提高肿瘤患者PBMC的NK活性。 (4 )肿瘤病人PBMC的NK活性测定可望成为肿瘤生物治疗的一个近期疗效参数