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。     

CD3单克隆抗体激光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地ＬＡＫ和ＮＫ细胞,对Ｈｅｌａ－３肿瘤细胞的杀伤活性略低于ＬＡＫ细胞。     

CD3AK细胞及其抗肿瘤研究进展

抗ＣＤ３抗体激活的杀伤细胞－ＣＤ３ＡＫ是一种新型抗瘤免疫活性细胞。Ｔ细胞经抗ＣＤ３单克隆抗体激活后，采用极低剂量ＩＬ－２维持即可长期活性增殖，发挥明显的抗肿瘤细胞效应。依诱导培养过程中ＣＤ３单抗持续存在与否，形成ＣＤ３ＡＫ＾＋和ＣＤ３ＡＫ＾－两种亚类，其作用及方式亦有所差异。ＣＤ３ＡＫ的体内外广谱、高效抗肿瘤作用明显优于ＬＡＫ细胞，临床初步应用亦显示出良好疗效。     

可溶性胃癌抗原和抗CD3单抗共同诱导杀瘤细胞的实验

用盐析法提取胃癌可溶性抗原（ＴＳＡ），并用抗ＣＤ３单抗和ＩＬ－２共同刺激正常人的外周血单核细胞，培养１０天后，经流式细胞仪表型分析，表明其免疫效应细胞以ＣＤ８Ｔ细胞为主，其细胞增殖速度、增殖水平与ＣＤ３ＡＫ和ＬＡＫ相比明显升高，且对其抗原来源的胃癌细胞具有极强的杀伤活性（９８．５％），高于ＣＤ３ＡＫ（８２．１％）和ＬＡＫ（６２．０％）。     

粘附性淋巴因子活化的杀伤细胞抗自体白血病细胞作用的研究

建立了一套培养和处理粘附ＬＡＫ细胞（Ａ－ＬＡＫ）的系统，将１２例缓解期急性髓系白血病（ＡＭＬ）患者（ＲＰＳ）的Ａ－ＬＡＫ细胞与常规制备ＬＡＫ细胞（ＲＴ－ＬＡＫ）进行了对照研究，结果显示ＲＰＳ－Ａ－ＬＡＫ细胞于培养第１０天时其扩增指数（２０．１±１３．９）较ＲＴ－ＬＡＫ细胞的扩增指数（７．５±２．１）明显提高（Ｐ＜０．０５）。形态学研究显示Ａ－ＬＡＫ细胞主要由大颗粒淋巴细胞组成，免疫表型分析显示其主要由ＣＤ１６＋的ＮＫ细胞组成，ＲＴ－ＬＡＫ细胞主要由ＣＤ３＋的Ｔ细胞组成。其杀伤活性与ＣＤ１６＋ＮＫ细胞之间有很好的相关性（ｒ＝０．８２，Ｐ＜０．０５）。这提示ＣＤ１６＋ＮＫ细胞是Ａ－ＬＡＫ细胞的主要组成细胞，代表了杀伤功能最强的亚群。     

癌性胸水LAK细胞的特性和抗肿瘤作用的观察

１７例癌性胸水经不连续密度梯度离心分离淋巴细胞，在ｒＩＬ－２诱导下体外培养，观察不同时期的ＬＡＫ细胞增殖速度、细胞表型和细胞毒活性，结果显示，胸水ＬＡＫ细胞在培养第１４天细胞扩增至原代的２０倍～８０倍，２１天后生长速度逐渐减慢。培养７天时对Ｋ５６２细胞和Ｒａｊｉ细胞的杀伤活性分别为７２％和６５％，以后随培养天数增加均呈逐渐下降趋势，但均高于同期培养的外周血ＬＡＫ细胞杀伤活性。培养７天～２１天间的细胞表型分析提示，ＣＤ＋３细胞百分率无明显改变，ＣＤ＋４细胞略增加，ＣＤ＋８细胞略有下降。ｒＩＬ－２／ＬＡＫ联合治疗肺癌胸水和肾癌胸水总有效率分别为５０％和６７％。     

CD3AK细胞的体外诱导及免疫生物学特征的实验研究

目的：研究ＣＤ３ＡＫ细胞的体外诱导方法及其免疫生物学特征。方法：采用抗单克隆抗体（ａｎｔｉ－ＣＤ３ＭｃＡｂ）和基因重组人白细胞介素２（ｒＩＬ－２）、植物血凝素（ＰＨＡ０共同诱导人外周血单核细胞（ＰＢＭＣｓ）制备ＣＤ３ＡＫ细胞,应用ＡＰＡＡＰ法、吉姆萨染色法分析ＣＤ３ＡＫ细胞的表型、核型等免疫生物学特征。结果：微量的ａｎ－ｔｉ－ＣＤ３ＭｃＡｂ（终浓度３０～５０００ｎｇ／ｍｌ）辅以少量的ｒＩＬ－２（１     

低剂量IL-2诱导的人胚胎脾脏CD_3AK细胞治疗晚期恶性肿瘤效果及实验研究

用ＣＤ３ＭｃＡｂ和低剂量ＩＬ－２（５００Ｕ／ｍｌ）诱导的人胎脾ＣＤ３ＡＫ细胞治疗４３例晚期恶性肿瘤患者，取得疗效。经２～８疗程治疗，多数患者症状缓解，生活质量改善，ＰＲ＋ＭＲ１８例（４１．９％），Ｓ＋Ｐ２５例（５８．１％），死亡９例（２０．９％）。全组无严重毒副反应发生。同时比较ＣＤ３ＡＫ细胞和常规ＬＡＫ细胞体外增殖及其杀伤活性，表明前者１４４ｈ增殖力高于后者（Ｐ＜０．０１），靶细胞抑制率二者无显著差异（Ｐ＞０．０５）。初步表明ＣＤ３ＡＫ／ＩＬ－２疗法效果肯定，毒副作用小，可能成为继ＬＡＫ／ＩＬ－２之后更为有效的肿瘤辅助治疗。     

可溶性胃癌抗原和抗CD_3单抗共同诱导杀瘤细胞的实验研究

用盐析法提取胃癌可溶性抗原（ＴＳＡ），并用抗ＣＤ３单抗和ＩＬ－２共同刺激正常人的外周血单核细胞，培养１０天后，经流式细胞仪表型分析，表明其免疫效应细胞以ＣＤ８Ｔ细胞为主，其细胞增殖速度、增殖水平与ＣＤ３ＡＫ和ＬＡＫ相比明显升高，且对其抗原来源的胃癌细胞具有极强的杀伤活性（９８．５％），高于ＣＤ３ＡＫ（８２．１％）和ＬＡＫ（６２．０％）。     

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。     

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     

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.     

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.     

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     

Lymphokine-activated killer-cell function of lymphocytes from peripheral blood, regional lymph nodes and tumor tissues of patients with oral cancer

Lymphokine-activated killer (LAK) cells, generated from peripheral blood lymphocytes (PBL) from patients with oral cancer or oral leukoplakia and from healthy donors showed comparable lysis of 6 target tumor cell lines, including 3 derived from head and neck and oral cancers. The tumor burden of the host did not appear to influence the systemic LAK activity. LAK activity of lymphocytes infiltrating the tumor tissues (TIL) was also comparable to that of the PBL. Both TIL and PBL showed a parallel increase in proportion of HNK-I+ and CD-25+ cells upon activation with IL-2. The lymph-node lymphocytes (LNL) from metastatic (met) and non-metastatic (non-met) draining lymph nodes, however, showed reduced LAK activity and an increase in CD8+ cells, in addition to CD25+ and HNK-I+ cells, when cultured with IL-2. When IL-2-activated LNL were co-cultured with autologous PBL during IL-2 activation of the latter, a strong suppressive effect was exerted by LNL. In contrast, IL-2-activated PBL did not suppress autologous LAK generation in spite of an increase in CD8+ cells seen after activation with IL-2. Frequency distribution of LAK precursors was significantly lower in LNL than in PBL from oral cancer patients. LAK precursor frequency in TIL was comparable to that of PBL. The results show that, in oral cancer, regional lymph nodes may not have adequate IL-2-inducible cytotoxic potential, due to a reduced number of LAK progenitors and possible activation of suppressor cells. Alternatively, TIL can be a potential source for LAK cell function.     

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／m1）辅以少量rhIL～2（300U／ml）和PHA（100μg／m1）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—CD3 AK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—CD3 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—CD3 AK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。     

Natural killer and lymphokine-activated killer activities in stomach cancer patients with special emphasis on the effect of 5-fluorouracil, adriamycin and mitomycin-C chemotherapy

The cytotoxicities of peripheral blood lymphocytes (PBL) and lymphokine-activated killer (LAK) cells were studied to evaluate the effect of chemotherapy on cellular immunity, in 18 patients with unresectable stomach cancer before and after chemotherapy with 5-fluorouracil, adriamycin and mitomycin-C (FAM), and in 21 healthy volunteers. LAK cells were generated in vitro by culturing PBL with 100 U recombinant human interleukin-2 (rH-IL-2)/ml for 72 h. K562 (human myelogenous leukemia), MKN-45 (human stomach adenocarcinoma) and PC-14 (human pulmonary adenocarcinoma) were used as target cells. The cytotoxicity of PBL to K562 and MKN-45 was suppressed in patients with stomach cancer before chemotherapy, compared with that in healthy volunteers (P less than 0.05). The cytotoxicity of LAK cells was significantly higher to all three cell lines tested than that of PBL in both the healthy volunteers and stomach cancer patients (P less than 0.01); however, a lower level of LAK activity was generated in patients with cancer compared to that in the healthy volunteers. FAM therapy did not suppress the cytotoxicities of PBL and LAK cells. The surface markers of PBL and LAK cells were measured, demonstrating that there was no significant change in the percentage of lymphocytes with CD3+, CD4+, CD8+, CD16+ or CD19+ after chemotherapy. The ratios of CD4+ to CD8+ cells in PBL and LAK cells were also not significantly changed after chemotherapy. In the present study, we have demonstrated that the PBL of stomach cancer were defective in generating LAK activity compared to those of controls, but the LAK activity generated from PBL receiving chemotherapy was similar to that from PBL without chemotherapy in stomach cancer patients.     

Phenotypic and functional analysis of human CD3+ and CD3- clones with "lymphokine-activated killer" (LAK) activity. Frequent occurrence of CD3+ LAK clones which produce interleukin-2

Clones capable of lysing fresh, uncultured tumor cells ("lymphokine-activated killer": "LAK" activity) were selected from microcultures derived from either E-rosette-positive or E-rosette-negative cell populations. All the selected clones displayed a strong cytolytic activity against the NK-sensitive K562 cell line. Two major phenotypic groups of clones could be identified: a first group expressed the CD3 differentiation antigen, present exclusively on mature T lymphocytes; however, in contrast to typical cytolytic T lymphocytes, the majority of these clones expressed the unusual CD4- CD8- phenotype, whereas the remainder were CD4- CD8+. A second group was represented by CD3- clones which, in most instances, expressed the T-cell-lineage-specific CD2 antigen. Following stimulation with phytohemagglutinin (PHA), most of the CD3+ LAK clones produced Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) whereas those expressing the CD3- phenotype did not. Since previous studies indicated that PHA may be inefficient in inducing lymphokine production by T-cell variants lacking the CD3/T cell receptor complex (TCR), CD3- clones were further stimulated with the calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA). Only 2/11 CD3- LAK clones produced small amounts of IL-2, whereas the majority released IFN-gamma. Given the peculiar phenotypic and functional properties of many CD3 + LAK clones, we suggest that they may belong to a T-cell subset distinct from typical CTLs.     

LAK1 antigen defines two distinct subsets among human tumour infiltrating lymphocytes.

Both lymphokine activated killer (LAK) cells and specific cytotoxic T lymphocytes appear to play a role in tumour immunity. Tumour infiltrating lymphocytes (TIL) which display a CD56+ phenotype (both CD3+ and CD3-) are also likely to possess anti-tumour activity. We have previously described a 120 kDa surface antigen, termed LAK1, expressed on a subset of human peripheral blood lymphocytes (20-50%) with both NK and LAK activity. The aim of the present study was to determine whether LAK1 antigen is able to distinguish among TIL two populations of effector cells displaying either specific or non MHC-restricted (NK/LAK) activity. We showed that about 25% of freshly derived TIL were weakly stained with anti-LAK1 monoclonal antibody and most of them were also CD3+ CD56-. After culture in recombinant interleukin-2 the majority of TIL were CD3+ CD56- and the percentage of LAK1+ cells increased up to 50%. Among cloned TIL, only those lacking LAK1 antigen displayed a specific cytotoxicity against the autologous tumour, whereas the non-lytic clones were able to produce both tumour necrosis factor and gamma-interferon. Moreover, when TIL from a renal cell carcinoma were fractionated into LAK1- and LAK1+ populations, the specific lytic activity was mainly evident when LAK1- lymphocytes were used as effector cells. Conversely, LAK activity was confined to the LAK1+ subset.     

Differential activation of lymphokine-activated killer cells with different surface phenotypes by cultivation with recombinant interleukin 2 or T-cell growth factor in gastric cancer patients

Fourteen days' culture of human peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL 2) or T cell growth factor (TCGF) results in the generation of lymphokine-activated killer (LAK) effector cells which have the unique property of lysing natural killer (NK)-resistant human tumor cells, Daudi, as well as NK-sensitive, K562 cells. LAK cells were generated from both normal and gastric cancer patients' PBL. However, LAK cell activities induced by rIL 2 or TCGF decreased with the progress of the tumor growth. In addition, TCGF-induced LAK cell activities were found to be lower than the rIL 2-induced LAK cell activities. Different mechanisms may be involved in the decreases of the rIL 2-induced and TCGF-induced LAK cell activities. This study further demonstrates that the cell types involved are also heterogeneous, as determined by phenotypic characteristics. The LAK-effector cell type was analyzed by two-color flow cytometry. RIL 2-induced LAK cells showed increased proportions of CD4+Leu 8- and Leu 7+CD16-, and a decreased proportion of CD8+CD11- cells, which are believed to be associated with killer T cell functions. In contrast, TCGF-induced LAK cells revealed significantly increased proportions of CD8+CD11- and CD4+Leu8- cells, and a decreased proportion of Leu 7+CD16- cells. Thus, LAK cells with different surface phenotypes were induced by the cultivations with rIL 2 and with TCGF.