脐血CD3AK细胞治疗恶性肿瘤的临床应用研究

目的:研究用脐血单个核细胞制备CD3AK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 CD3Ab诱导LAK和CD3AK细胞,并测其扩增数量和对K562细胞的杀伤活性;测定肿瘤患者用CD3AK细胞治疗前后外周血T淋巴细胞亚群绝对计数的变化情况和外周血单核细胞(PBMC)的NK杀伤活性.结果:脐血LAK细胞和脐血CD3AK细胞均于培养后11天时扩增倍数最高,分别是培养前的18倍、24倍,对K562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CD3AK细胞一疗程后,其外周血T淋巴细胞亚群绝对计数有明显升高:总T升高66.0%,Th升高68.0%,Ts升高58.0%;其PBMC的NK杀伤活性由63.0%升高到81.0%,平均升高28.0%.结论:1)脐血单个核细胞是LAK细胞和CD3AK细胞良好的前体细胞.2)LAK和CD3AK细胞的数量和杀伤能力在培养的11天时达高峰,而且CD3AK细胞数量和杀伤活性明显优于LAK细胞.3)CD3AK细胞输注能明显提高肿瘤患者外周血T淋巴细胞亚群绝对计数和PBMC的NK活性.4)肿瘤患者的外周血T淋巴细胞计数和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.     

CD3AK细胞的生物学特性及其抗肿瘤作用

CD3AK细胞是用抗T细胞表而CD3分子的单克隆抗体激活的新型杀伤细胞，在肿瘤的免疫疗法中愈来愈受到重视。本文用抗CD3单克隆抗体(北医太免疫室提供)和rIL-2(日本盐野义制药株式会社产品)共同刺激人外周血单个核细胞(PBMC)，诱导CD3AK细胞，系统地研究了其生物特性及其体外抗肿瘤活性并与LAK细胞作比较。     

CD_3单克隆抗体激活细胞的体外抗肿瘤作用

目的:观察CD3单克隆抗体激活的杀伤细胞(CD3AK)的体外杀伤肿瘤作用。方法:采用MTT法测定不同时期CD3AK细胞的杀伤活性。结果:培养到第4天的CD3AK细胞已有明显的杀伤活性,第10天达高峰;CD3AK对K562、H7402和MNK45肿瘤细胞的杀伤活性显著高于LAK和NK细胞(P<0.05),对Hela-3肿瘤细胞的杀伤活性略低于LAK细胞。结论:CD3AK是一种广谱、高效的肿瘤杀伤细胞,但它对不同种类的肿瘤细胞的杀伤效应又存在着较大的差异。     

PHA-CD3AK细胞的体外诱导及其生物学特性初步研究

目的:研究PHA-CD3AK细胞的体外诱导方法及其生物学特性,并与LAK细胞进行比较.方法:分离人外周血单个核细胞(PBMC),采用植物血凝素(PHA)、单克隆抗体(anti-CD3McAb)和基因重组人白细胞介素2(rhIL-2)、共同诱导制备PHA-CD3AK细胞;应用流式法分析PHA-CD3AK细胞的免疫表型、乳酸脱氢酶法(LDH)检测PHA-CD3AK细胞的杀伤活性,并观察其形态;吉姆萨染色法观察其核型.结果:微量anti-CD3McAb(0.05μg/ml)辅以少量rhIL-2(300U/ml)和PHA(100μg/ml)共同培养,即能诱导和大量扩增PHA-CD3AK细胞,其扩增倍数显著高于LAK细胞,维持高扩增的时间也远较LAK细胞持久;当效靶细胞比为80:1时,PHA-CD3AK细胞对体外肿瘤细胞(K562)杀伤的百分率为56.5%;免疫表型检测PHA-CDB AK细胞中CD3+、CD4+、CD8+细胞的比率分别为(86.5±5.89)%、(38.20±5.27)%、(42.63±3.50)%;核型为正常二倍体,染色体数目为46条.结论:PHA-CD3AK细胞是以CD3+、CD4+、CD8+细胞为主的异质细胞群,并具有淋巴母细胞样特征,PHA-CD3AK细胞为正常二倍体细胞.PHA-CD3AK细胞是易于体外诱导、扩增能力强,体外存活时间长、杀瘤活性高的一种具有广阔应用前景的肿瘤过继免疫效应细胞.     

增强PHA-LAK细胞诱导激活新途径的研究

本文将新分离的成人外周血单个核细胞(PBMC)分3组培养:(1)用抗CD3单抗(αCD3)、rIL-2共同刺激PBMC,诱生、扩增CD3AK;(2)用PHA预刺激PBMC48小时,再用含rIL-2的全培养基培养,诱生、扩增PHA-LAK细胞;(3)用PHA预刺激PBMC48小时,再用αCD3、rIL-2协同刺激,诱生、扩增新型效应细胞PHA-αCD3LAK.利用活细胞计数、MTT、ELISA、A-PAAP等方法对3组效应细胞的增殖动力学、细胞毒活性、细胞表型、mIL-2Rα的表达及培养上清液中sIL-2R和IL-2的含量作了测定和比较.结果表明:PHA-αCD3LAK、PHA-LAK和CD3AK 3组细胞(1)均为异质性细胞群体,PHA-αCD3LAK以CD3~ 、CD8~ T细胞为主,含有较多的CTL细胞,(2)其增殖曲线及mIL-2Rα的表达均于培养的第6天达到高峰,其峰值和扩增倍数均为PHA-αCD3LAK>PHA-LAK>CD3AK(P<0.05),(3)均可介导MHC非限制的细胞毒性,对K562细胞和     

HSP70-TKD诱导的NK细胞杀伤肝癌细胞的实验研究

目的:探讨从人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中获得足够数量和高细胞毒活性的CD3-CD56 NK细胞的方法,并观察其对肝癌细胞HepG2杀伤效应.方法:以干细胞培养基(SCGM)为基础培养基,在不同浓度的抗CD3单抗、IL-2和PHA的培养条件下,从健康人PBMC中高效扩增获得CD3-CD56 NK细胞;以不同剂量的HSP70-TKD诱导NK细胞的活性,MTT法测定HSP70-TKD诱导的NK细胞对HepG2和榄香烯处理的HepGZ细胞的杀伤活性.结果:建立了从人PBMC体外扩增CD3-CD56 NK细胞体系:以SCGM为基础培养基,在600 U/mL IL-2、500 μg/L抗CD3单抗和50 mg/L PHA联合作用下,培养14 d细胞扩增了116倍,舍有(66.97±8.76)?3-CD56 NK细胞;HSP70-TKD诱导的NK细胞对HepG2杀伤活性低.与未经TKD诱导的NK细胞之间差异无统计学意义,P=0.298;而对税香烯处理过的HepG2杀伤活性高,诱导组与未诱导组之间差异有统计学意义,P=0.008;当TKD浓度为2.0 mg/L时,杀伤活性最高为(58.8±3.4)%.结论:以干细胞生长培养基为基础培养基,在抗CD3单抗和IL-2协同刺激下体外大量扩增NK细胞,HSP70-TKD诱导的NK细胞对细胞膜HSP70阳性的肿瘤细胞杀伤活性高,而对于细胞膜HSP70低表达的肿瘤细胞杀伤活性低.     

CD3抗体激活的杀伤细胞实验性抗喉癌作用的研究

基于CD3抗原广泛存在于成熟T细胞表面的理论基础,我们应用CD3单克隆抗体与IL-2在体外成功诱导出了具有较好增殖活性的CD3抗体激活的杀伤细胞(CD3AK细胞),并对其抗喉癌作用进行了观察和研究.结果表明:与常规培养的LAK细胞相比,以健康人新鲜外周静脉血单个核细胞为前体、经CD3单抗(4μg/ml)和IL-2(1000U/ml)诱导产生的CD3AK细胞具有集落形成率高、体外扩增能力强、存活时间长、抗瘤作用持久稳定等优势.在培养初期,CD3AK细胞的扩增情况与LAK细胞相似,但CD3AK细胞生长后劲明显,于培养8～10天达增殖高峰,平均扩增18.5倍,并可维持体外长期存活达两周之久,而同期培养的LAK细胞1周时平均仅增5.5倍,且多于1周后逐渐死亡,二者相比有非常显著性差异;同时,CD3AK细胞在体     

肝癌患者自体CD3AK细胞与LAK细胞的杀伤活性比较

目的 比较肝癌患者自体CD3AK细胞和LAK细胞的杀伤活性,为临床是供依据。方法 以流式细胞仪检测细胞的杀伤活性。结果 原发性肝癌患者自体的CD3AK细胞的NK活性和LAK活性均高于LAK细胞。结论 CD3AK细胞效果稳定,抗瘤效应明显高于LAK细胞,CD3AK细胞可望作为一种新的生物治疗手段用于肿瘤治疗。     

脐血CD3AK和LAK细胞免疫学特征的比较研究

γIL-2激活的脐血杀伤细胞(LAK)体内外抗肿瘤已有报道,而Anti-CD3McAb作为免疫增强剂,在骨髓、外周血的过继免疫中已取得明显疗效,脐血单个核细胞(MNC)能否被Anti CD3McAb激活为抗肿瘤的杀伤细胞(CD3AK)?是否比其LAK有更多的活性?为探讨这些问题,本实验采用γIL-2+Anti-CD3McAb和单纯γLI-2刺激脐血MNC,观察其表面抗原及细胞因子的变化,并探讨两者对K562和HL-60白血病细胞株的杀伤作用.     

抗人肺癌单克隆抗体3D3 scFv基因构建及其在E.coli中的表达

本研究应用PHA、抗CD3单抗(aCD3)和rIL-2共同刺激人外周血单个核细胞(PBMC),诱生、扩增新型抗肿瘤效应细胞PHA-aCD3LAN,并与PHA-LAk和CD3AK细胞在某些生物学特性方面进行了比较,结果表明,PHA、aCD3和IL-2具有协同增强效应、使PHA-aCD3LAK细胞的增殖能力、细胞毒活性、mIL-2Ra的表达水平及对IL-2的利用均高于PHA-LAK和CD3AK细胞;三组效应细胞均为异质性细胞群体,均以CD3~ CD8~ T细胞为主,而PHA-aCD3LAK的CD8~ 细胞百分率高于其它两组细胞.采用PHA-aCD3LAK可进一步提高LAK细胞的数量和活性,具有重要的临床应用前景     

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.     

荷CEA-rV的DC增强CD3AK对CEA阳性肿瘤特异性杀伤作用的研究

Objective： To survey the special killing activity of CD3AK on anti-CEA-positive tumor enhanced by umbilical cord blood dendritic cell （DC） loaded with CEA recombinant vaccinia virus （CEA-rV）. Methods： Freshly isolated umbilical blood mononuclear calls （UBMC） were cultivated for 3 h. Suspension cells and attached calls were used to induce CD3AK calls and DC separately. DC was loaded with CEA-rV on the 3rd day to prepare CEA-rV＋DC. CD3AK cells were co-cultured with CEA- rV＋DC on the 8th day, to prepare CEA-rV＋DC＋CD3AK. The killing activity of each effector＇s cell, which included UBMC, CD3AK, DC＋CD3AK and CEA-rV＋DC＋CD3AK, was measured respectively by MTT reduction assay. Results： （1） 4 target cells were con- firmed by CEA monoclonal antibody of rabbit anti-human. Lovo and A549 were really CEA positive cell lines, while Bel-7402 and K562 were CEA negative cell lines. （2） It was showed by flow-cytometry that the mature DC cultured at 10th day expressed MHC I, II molecules such as CD86, CDS0, CD83 and CD40 highly, but CD123 lowly. The expression rates of CD86, CDS0, CD83 and CD40 was 82.7%, 51.1%, 57.5% and 69.4%, respectively. The appearances and intra-cellular structures of DC were observed through light and electron microscope. The diameter of mature DC was 15-20 μm presented the irregular morphologic appearanca, much prominences and pseudopodium. There were abundant mitochondria and endoplasmic reticulum in DC endochylema. （3） The rates of CD3, CD4, CD8 and CD28 in CD3AK cells group were 2 folds higher than that in UBMC group by FACS. It was said that the numbers of the mature T lymphocyte in CD3AK cells group were much greater than that in UBMC group. （4） The killing activities to 4 target cells of 3 effector＇s cells, which included CEA-rV＋DC＋CD3AK, DC＋CD3AK and CD3AK, were much greater than that of UBMC （P〈0.01）. Moreover, comparing with the killing activities of 4 effector＇s： CEA-rV＋DC＋CD3AK group 〉 DC＋CD3AK group 〉 CD3AK group 〉 UBMC group. It showed that, cytokine, DC and CEA-rV could efficiently elevate the killing activity of UBMC on broad-spectrum tumor cells. （5） Comparing with the killing activities of CEA-rV＋DC＋CD3AK and CD3AK cells to CEA positive and negative cells, the killing activities of CEA-rV＋DC＋CD3AK to CEA positive tumor calls, Lovo and A549 calls （P〈0.01） were remarkably better than that to CEA negative tumor cells BEL-7402 and K562 cells （P〈0.05）. It was said that the CEA-rV＋DC could obviously enhance the killing activity of CD3AK on CEA positive tumor cells. Comparing with the killing activities of CEA-rV＋DC＋CD3AK and DC＋CD3AK cells, the killing activity of CD3AK on CEA negative tumor cells was no statistical difference （P〉0.05）. However, the killing activity to CEA positive cells of CEA-rV＋DC＋CD3AK group was notably higher than that of DC＋CD3AK group. Namely, CEA-rV could distinctly promote the special killing activity to CEA positive tumor cells of CD3AK, but could not do it to CEA negative tumor cells. Conclusion： CEA-rV＋DC could obviously enhance the special killing activity of CD3AK on CEA positive tumor cell lines, while the DC only couldn＇t. The results indicated that the CEA-rV played an important role during the special killing activity of CD3AK cells to CEA positive tumor cells.     

卵巢癌抗原等诱导杀瘤性免疫细胞的实验研究

为了探讨肿瘤过继细胞免疫治疗和研制肿瘤疫苗的新方法,用卵巢癌细胞（ＣＯＣ１）提取可溶性抗原成份,将其与葡萄球菌超抗原共同诱导外周血单个核细胞,置于含ＩＬ２的培养基中培养,待产生杀瘤性免疫效应细胞后,对这种细胞的某些生物学特性进行初步研究,并与ＴＡＫ,ＣＤ３ＡＫ,ＬＡＫ细胞进行比较。结果显示培养到第１０ｄ时,实验组效应细胞,ＴＡＫ细胞,ＣＤ３ＡＫ细胞和ＬＡＫ细胞的增殖活性分别为４０．２倍,４１．７倍,３２．４倍和２１．５倍;对卵巢癌ＣＯＣ１细胞的细胞毒活性分别为８１．２％,８２．５％,５４．３％和５６．７％。实验组效应细胞对４种卵巢癌细胞ＣＯＣ１,ＣＯＣ２,ＳＫＯＶ３和Ａ２７８０的细胞毒活性分别为８３．１％,５１．４％,３７．６％和４９．７％。结果表明,用ＴＳＡ和超抗原共同诱导产生的免疫效应细胞增殖快,细胞毒活性高,对来源于抗原的肿瘤细胞具有选择性杀伤作用。该实验方法和结果为肿瘤过继细胞免疫治疗和研制肿瘤疫苗提供了新思路     

Membrane-associated Lymphotoxin Expression and Functional Analysis of Lymphokine-activated Killer Cells Derived from Tumor-infiltrating Lymphocytes

The expression of a membrane-associated lymphotoxin molecule (mLT) on lymphokine-activated killer (LAK) cells obtained from 18 patients with malignant tumors and its role in the tumor cell killing mechanisms were investigated. LAK cells from tumor-infiltrating lymphocytes (TIL-LAK cells) were mainly composed of CD3-positive cells, whereas LAK cells from peripheral blood lymphocytes (PBL-LAK cells) were mainly composed of CD16- and CD56-positive cells. However, mLT was found to be expressed on TIL-LAK cells as well as PBL-LAK cells. The degree of mLT expression correlated with the killing activity of LAK cells towards L929 cells (r=0.806, P <0.01, n = 15), but not with that towards Daudi or K562 cells. Although the degree of mLT expression correlated with the amount of secreted lymphotoxin (LT) in the supernatant of LAK cell culture, the secreted LT itself could not account for the tumor cell killing activity of LAK cells. Polyclonal rabbit anti-LT antibody partially inhibited the killing activities of LAK cells towards L929 cells and this inhibition was found in the combination of autologous tumor cells and PBL-LAK cells. These findings suggest the possibility that the mLT-related cytotoxicity is involved in the tumor cell killing mechanisms of TIL-LAK cells as well as PBL-LAK cells.     

Relation of natural killer cell line NK-92-mediated cytolysis (NK-92-lysis) with the surface markers of major histocompatibility complex class I antigens, adhesion molecules, and Fas of target cells.

Natural killer (NK) cell line NK-92 has recently been established by Klingemann et al. In this study, we compared the NK-92-mediated cytolysis (NK-92-lysis) with the killing of healthy volunteers' NK cells and lymphokine-activated killer (LAK) cells. The NK-92-lysis was partially different from the NK- and LAK-lysis. 1) The NK-92 could kill most of major histocompatibility complex (MHC) class I antigen-positive tumor cells. 2) The NK cells killed a myeloid leukemia cell line K562, but the NK-92 showed low killer activity against it. 3) The LAK cells could not kill a CD58-deficient cell line OKM-2T, whereas the NK-92 could kill it sufficiently. 4) The NK-92 could not kill CD54-, CD102-deficient cell lines T98G and U373MG; however, the LAK cells could kill them. Blocking tests using specific antibodies revealed the reason for these differences. The K562 expressed relatively low levels of CD54 and CD102. When the K562 was pretreated with anti-CD54 and anti-CD102, the NK-92 could not kill it at all, whereas the NK cells could still kill it, although the killing level decreased. The NK-92 could not kill the anti-CD54- and anti-CD102-treated OKM-2T. The LAK cells could not kill anti-CD58-treated U373MG and T98G. These findings suggest that NK-92-lysis may require the CD54 and CD102 but that NK-lysis does not require them as much, whereas the LAK-lysis may be rather in relation with the CD58. The NK-92 has high killer activity, and may be applicable for clinical use. However, it should be considered that the NK-92 cannnot kill CD54-, CD102-deficient tumor cells.     

Phenotypical analysis of effector cells on nonspecific cancer cell therapy

In the present study, we examined the contributions of lymphocyte subpopulations in lymphokine activated killer (LAK) activity. LAK cells prepared from peripheral blood mononuclear cells (PBMC) of healthy donors showed highly cytotoxic activities against target tumor cells. When CD16 and CD56 positive cells in LAK cells were depleted by magnetic cell sorting, their cytotoxic activities were dramatically decreased. In contrast, little change was observed by the depletion of CD3 positive cells, suggesting that CD16 and/or CD56 positive populations, but not CD3 positive populations, including natural killer (NK) cells are the major cell types involved in LAK activity. Indeed, NK-enriched LAK cells prepared by culturing PBMC with IL-2 and OK-432 showed a more potent LAK activity than conventional LAK cells and CD3-activated T cells. These results suggest that selective expansion and activation of CD16 and CD56 positive cells in LAK cells is a useful strategies to improve their anti-tumor potential in nonspecific immunotherapy, and possibly in combination therapy with other target immunotherapies as well.     

Preparation of NK-enriched LAK cells--their potential cytotoxic and ADCC activities

We examined several culture methods to induce proliferation of natural killer (NK) cells from peripheral blood mononuclear cells (PBMC). In the presence of IL-2, a remarkable proliferation of NK cells was observed when PBMC were co-cultured with MMC-treated K562, which is known as a highly sensitive in vitro target cell for the NK assay. Addition of OK-432 or TNF-alpha and IL-1 beta also induced marked NK proliferation in a dose dependent manner. These NK-enriched lymphokine activated killer (LAK) cells showed highly cytotoxic activities against various MHC class I positive or negative tumor cells. They also showed potent ADCC activities against Herceptin-coated SK-BR-3, a HER2/neu positive breast cancer cell line. These results indicated that NK-enriched LAK cells are potent effector cells, and suggested novel therapeutic strategies for nonspecific immunotherapy as well as target immunotherapy in combination with anticancer antibodies, such as Herceptin.     

Reversible inhibition of lymphokine-activated killer cell activity by lipoxygenase-pathway inhibitors

Natural killer (NK) cells and lymphokine-activated killer (LAK) cells are anomalous cytotoxic cells which are potentially important in host defense against cancer. Several studies have demonstrated that natural killer (NK) cell activity can be suppressed by chemical inhibitors of the lipoxygenase pathway through inhibition of the production of leukotriene B4 (LTB4). The present study investigated the effects of the lipoxygenase inhibitors BW755C and nordihydroguaiaretic acid (NDGA) on NK and LAK cell activity. NK cell function of fresh peripheral blood mononuclear cells (PBMC) was determined via a standard chromium release assay employing K562 as the tumor target. The LAK cell activity of PBMC which had been stimulated with 10 IU of interleukin-2 for 72 hr was determined against the NK-resistant cell line Daudi. Both BW755C and NDGA inhibited NK and LAK cell function at a variety of concentrations. Indomethacin, a prostaglandin synthesis inhibitor, did not bring about an appreciable diminution in NK or LAK cell activity. Inhibition of NK and LAK cell activities by BW755C and NDGA could be reversed by washing the effector cell suspensions prior to the cytotoxic assay or by adding LTB4 (10(-11)-10(-8) M) directly to the effector:target suspensions. These data indicate that certain arachidonic acid oxidation products of the lipoxygenase pathway are essential for the function of LAK cells.