卡介苗激活杀伤细胞抗膀胱肿瘤作用研究

为了进一步探讨ＢＣＧ抗膀胱肿瘤的作用机理，采取１５例膀胱肿瘤患者外周血单个核细胞（ＰＢＭＣ），置于含ＢＣＧ或ＩＬ２的培养基中培养，计算扩增倍数，检测培养细胞抗自体及异体膀胱癌细胞活性。结果：卡介苗激活杀伤细胞（ＢＡＫ）与淋巴因子激活杀伤细胞（ＬＡＫ）分别于培养第７和第３天达增殖高峰，对自体瘤杀伤率分别为３６．２％和３１．４％，差异有显著性（Ｐ＜０．０５）；对异体瘤杀伤率分别为２５．２％和２８．３％，差异无显著性。结果表明：ＢＡＫ细胞抗自体瘤活性高于ＬＡＫ细胞，死ＢＣＧ对ＰＢＭＣ无激活作用，ＢＡＫ细胞抗肿瘤效应可能是ＢＣＧ抗膀胱肿瘤重要作用机理之一。     

膀胱肿瘤浸润性淋巴细胞与LAK细胞抗肿瘤作用的比较研究

对１６例手术切除的膀胱移行细胞癌新鲜组织标本，用淋巴细胞分离液经不连续密度梯度离心获得ＴＩＬ，同时收集分离获取同一患者外周血淋巴细胞（ＰＢＬ）。分别于含ＩＬ－２的ＲＰＭＩ１６４０全培养基中培养扩增。用ＬＤＨ释放试验测定培养不同时间的ＴＩＬ及ＬＡＫ细胞的抗瘤活性。结果：ＬＡＫ细胞培养第３～７天对自体肿瘤细胞的杀伤活性可达高峰，杀伤率为２８％～３２％；ＴＩＬ培养第１４～２１天为杀伤高峰，杀伤率为４０％～４４％。结果还显示，ＴＩＬ的抗肿瘤作用有一定的选择性，而ＬＡＫ细胞的作用属非特异性。     

白细胞介素(IL)-2与IL-4对小鼠膀胱癌肿瘤浸润性淋巴细胞增殖及细胞毒性的协同调节作用

目的 探讨白细胞介素（ＩＬ）－２与ＩＬ－４对膀胱癌肿瘤浸润性淋巴细胞（ＴＩＬ）体外增殖及细胞毒性免疫调控的协同作用。方法 分离膀胱癌ＴＩＬ,置于含ＩＬ－２和（或）ＩＬ－２的完全培养基因中培养４周,定期计数ＴＩＬ增殖数量。四甲基偶氮唑蓝（ＭＴＴ）比色法检测ＴＩＬ细胞毒性。结果 对比单纯ＩＬ－２的培养条件,ＩＬ－２联合ＩＬ－４后４周时ＴＩＬ扩增数量是前者的１．６５倍（Ｐ〈０．０５）。在交靶比为１０：１时,ＴＩＬ对自体膀胱癌细胞（ＢＴＴ７３９）表现出高水平的杀伤活性（Ｐ〈０．０５）。联合培养的ＴＩＬ抗ＢＴＴ３９或小鼠淋巴瘤瘤株（ＹＡＣ－１）的活性与在单纯ＩＬ－２培养的条件下相比无显著改变（Ｐ均〉０．０５）。结论 ＩＬ－４对ＩＬ－２活化的膀胱癌ＴＩＬ增殖具有较强的正向调节效应,而对ＴＩＬ细胞毒性未见明显影响。     

瘤体原位G—CSF基因治疗对化疗后结肠癌小鼠瘤体免疫功能？…

探讨瘤体原位脂质体介导的Ｇ－ＣＳＦ基因治疗抗肿瘤作用的机制。方法 将脂质体包裹的Ｇ－ＣＳＦ基因注射至结肠癌小鼠瘤体内，观察肿瘤浸润性淋巴细胞信全身免疫功能的变化。结果 治疗后，新鲜分离的ＴＩＬ的ＮＫ活性及经ＩＬ－２体外培养的ＴＩＬ杀伤Ｃ－２６细胞的活性均有明显提高；胸细胞与新鲜分离的Ｃ－２６细胞培养后诱导的ＣＴＬ活性以及外周血中性粒细胞杀伤Ｃ－２６细胞的活性显著高于对照组。     

CD_3－TIL抗肿瘤作用的实验研究

作者研究了人原发性肝癌浸润淋巴细胞（ＴＩＬ）在体外经ＣＤ３单抗和重组人ＩＬ－２（Ｉｎｔｅｒ－ｌｅｕｋｉｎ－２）刺激诱导成为ＣＤ３－ＴＩＬ，并且ＣＤ３－ＴＩＬ与单纯ＩＬ－２诱导的ＴＩＬ进行了体外增殖能力及体内外抗肿瘤作用的比较。结果发现，ＣＤ３单抗浓度为１００ｎｇ／ｍｌ时，为ＴＩＬ体外刺激增殖的最适剂量；ＣＤ３－ＴＩＬ在体外的扩增能力显著高于ＴＩＬ，并且在体外杀伤肿瘤细胞的活性显著地高于ＴＩＬ。体内试验发现ＣＤ３－ＴＩＬ可使裸鼠成瘤期显著地延长、瘤体缩小、荷瘤裸鼠生存期延长，表明ＣＤ３－ＴＩＬ细胞具有较强的体外增殖能力及有效地杀伤肿瘤细胞的活性，可望该实验为临床上治疗原发性肝癌提供实验依据。     

卡介苗联合白细胞介素-2膀胱灌注预防膀胱癌复发机理的研究

目的探讨卡介苗（ＢＣＧ）联合白细胞介素－２（ＩＬ－２）膀胱灌注预防膀胱癌复发的机理。方法对３５例膀胱移行细胞癌术后患者分别行ＢＣＧ和ＢＣＧ加ＩＬ－２膀胱灌注,随访１４～２２个月,复发率分别为３１．２５％和２１．０５％。结果应用ＩＬ－２加ＢＣＧ膀胱灌注６周后,外周血天然杀伤细胞激活因子（ＮＫＣＦ）活性明显增强,在ＮＫＣＦ活性与ＩＬ－２活性间有显著正相关变化。结论ＩＬ－２加ＢＣＧ膀胱灌注预防膀胱癌复发作用明显优于单纯用ＢＣＧ,ＩＬ－２和ＢＣＧ两者间有免疫促进和协同作用。     

肾癌TIL的分离培养和其表型分析

对两例原发性肾癌患者手术切除肿瘤组织中肿瘤浸润性淋巴细胞（ＴＩＬ）进行了体外分离与培养。结果表明：ＴＩＬ体外扩增倍数分别达３２～２０３倍，对自体肿瘤靶细胞的最高杀伤活性达５３％和６４％，且呈现一定的靶细胞特异性。免疫组化分析结果：未经激活的ＴＩＬ细胞其膜抗原（ＣＤ３，ＣＤ４，ＣＤ８）的表达动态变化不大，但经ＩＬ－２激活的ＴＩＬ细胞随着培养无数的增加，其ＣＤ３细胞数比例及ＣＤ４／ＣＤ８比值上升明显，在培养至３２天时分别达９５％和１．６５。     

活卡介菌激活杀伤细胞(BAK)与LAK细胞抗膀胱肿瘤作用比较研究

从15例膀胱移行细胞癌患者外周血制备PBMC,置于含BCG或IL-2的培养基中培养,计算扩增倍数,测不同培养时间细胞抗自体及异体膀胱癌活性。BAK与LAK细胞分别于培养第7天和第3天达其增殖高峰,对自体瘤杀伤率分别为36.2％和31.4％,差异有显著性(P<0.05);对异体瘤杀伤率分别为25.2％和28.3％,差异无显著性。BAK细胞抗自体瘤活性高于LAK细胞,死BCG对PBMC无激活作用。BAK细胞抗肿瘤效应可能是BCG抗膀胱肿瘤重要机理之一。     

联合应用TIL和IL－2治疗肾癌

联合应用ＴＩＬ和ＩＬ－２治疗肾细胞癌１７例，检测了治疗前后外周血淋巴细胞（ＰＢＬ）对ｋ５６２、Ｒａｊｉ细胞的杀伤活性及ＣＤ_３、ＣＤ_４、ＣＤ_８水平，结果治疗前对Ｋ５６２、Ｒａｊｉ细胞杀伤活性分别为２８．２３±１４．１８％及２２．３９±８．８４％，而治疗后的杀伤活性分别为３６．１８±１３．０８％及２６．４７±５．２７％，治疗前后比较，ＰＢＬ对Ｋ５６２及Ｒａｊｉ细胞的杀伤活性均有显著提高（Ｐ＜０．０５）。治疗前后ＰＢＬ中的ＣＤ_３分别为５６．５８±４．０３％及５８．４３±３．４９％，ＣＤ_４分别为３９．８８±１．５１％及４１．１９±１．９９％，ＣＤ_８分别为３０．２０±１．５２％及３３．５４±３．３３％，治疗前后有显著性差异（Ｐ＜０．０５）。随访３～１５个月，平均９．７个月，１３例无转移者均无瘤存活，有转移的４例中完全缓解１例，部分缓解１例，死亡２例。提示联合应用ＴＩＬ和ＩＬ－２可以提高病人免疫力，在质和量两方面提高ＰＢＬ中的ＣＤ_３、ＣＤ_４和ＣＤ_８水平，近期疗效较好。     

人肝癌TIL体外抗癌活性及其表型特征的研究

体外分离、扩增培养１０例肝癌ＴＩＬ细胞，用ＭＴＴ法检测８例对靶细胞的杀伤活性，结果８例ＴＩＬ显示对自体肿瘤细胞、ＳＭＭＣ—７７２１和Ｋ５６２细胞株的明显杀伤活性，并在培养３０ｄ内抗瘤活性呈现逐渐增强趋势。新鲜分离的ＴＩＬ表型主要呈现ＣＤ３，培养２０天后ＣＤ４＋ＴＩＬ细胞增加，与ＣＤ８＋ＴＩＬ比例为１．０５。本文结果将为ＴＩＬ的临床应用提供实验依据。     

The effects of interleukin-6 on tumor-infiltrating lymphocytes derived from human renal cell cancer

Tumor-infiltrating lymphocytes (TIL) are a heterogeneous population of T cells with potent antitumor activity against a wide variety of tumors. TIL from renal cell cancer (RCC) typically exhibit diminished growth and antitumor activity after four weeks in vitro. We have therefore investigated effects of varying doses of interleukin-6 (IL-6) (0, 25, 100 units/ml.) on in vitro expansion, proliferation, cytotoxicity, and expression of cell surface phenotypes of long term renal TIL cultures from three RCC patients. Among the various conditions tested, three of three TIL cultures displayed a mild increase in cell expansion when grown in IL-2 with the addition of 100 units/ml. of IL-6. Two of three TIL cultures grown in IL-2 and 100 U/ml. of IL-6 demonstrated enhanced proliferation as determined by 3H-thymidine uptake. TIL could not be isolated or maintained in vitro when grown in the presence of IL-6 alone without IL-2. IL-6 was also found to enhance the long term non-specific cytotoxicity against an allogeneic nonrenal tumor target. No consistent effect on autologous tumor-specific cytotoxicity was demonstrated. We conclude that IL-6, when used in combination with IL-2, may modestly enhance the long-term growth of RCC-derived TIL.     

肿瘤浸润性淋巴细胞在白细胞介素-2、-4协同下局部过继免疫治疗膀胱癌的实验研究

目的　观察膀胱癌肿瘤浸润性淋巴细胞 (TIL)联合不同细胞因子瘤灶内过继免疫抗癌的效应及对机体全身抗肿瘤免疫机制的影响。方法　建立BTT73 9动物模型 ,分离、培养TIL。采用正交设计实验方法 ,将TIL、白细胞介素 (IL) 2、 4及三因素交互组合悬液分别直接注射至瘤体内 ,定期测量肿瘤体积 ,免疫治疗 2周后检测NK细胞活性、T淋巴细胞转刺激指数 ,观察组织学及超微结构变化。结果　比较对照组 ,治疗 2周时各TIL相关组均不同程度抑制了膀胱肿瘤体积的增长 ,且NK细胞活性及T淋巴细胞转化增殖能力得以提高 (P <0 .0 5 )。TIL/IL 2疗法明显抑制了瘤体的增长 ,免疫治疗 1周后即表现出协同增强效应 (P <0 .0 5 ) ,而NK细胞活性及T淋巴细胞转刺激指数也显著提高 (P <0 .0 5 )。TIL/IL 2 /IL 4组获得了较强的抗癌功效 ,但与TIL/IL 2组差异无显著性 (P >0 .0 5 )。超微结构变化显示出TIL强烈的溶癌现象。结论　TIL在细胞因子特别是IL 2协同下瘤灶内注射的局部免疫疗法 ,具有较强的抗膀胱癌效应 ,并显著提高了机体全身抗瘤免疫功能。     

Adoptive immunotherapy and metastasized cancer of the kidney. Regulator effects of interleukin-4 on tumor infiltrating lymphocytes (TIL)]

Adoptive immunotherapy is a new therapeutic approach of the treatment of advanced renal cell cancer. Experimental studies have shown that the cells with the highest cytolytic activity are tumor infiltrating lymphocytes (TIL). The effects of interleukin-4 (IL-4) on the expansion, proliferation, phenotype and antitumor activity of TIL were studied. Cultures were obtained from three primary renal tumors and one group of tumor invaded, regional lymph nodes. IL-4 induced a significant increase in lymphocytes expansion and proliferation, but the response was dependent of the concurrent dose of IL-2 in culture. TIL grown in the presence of IL-4 significantly reduced the level of non specific, non MHC restricted antitumor activity while exhibiting no effect on the level of autologous killing. The effects of irradiated autologous tumor stimulation on TIL cultures were also evaluated. Addition of autologous tumor increased expansion and proliferation of all cultures and significantly enhanced levels of autologous killing. IL-4 and autologous tumor stimulation are effective growth factors when used in combination with a lose dose IL-2 regimen and may be of significant benefit in the expansion of TIL for clinical trials.     

人白细胞介素-15与18协同增强肿瘤浸润淋巴细胞体内对结肠癌细胞的杀伤功能

目的 观察白细胞介素(IL)-15与IL-18体内增强肿瘤浸润淋巴细胞(TIL)杀伤功能的效果.方法 用IL-15与IL-18共同刺激从结肠癌患者分离得到的TIL,通过测定其分泌转化生长因子(TGF)-β、IL-4、干扰素(IFN)-γ水平以及TIL的杀伤活性.用IL-15与IL-18共同刺激的TIL与结肠癌细胞SW480共孵育后,接种到SCID鼠皮下,测定肿瘤生长的大小、肝脏转移的肿瘤结节多少、血清中IL-4、IFN-γ水平以及腹腔巨噬细胞的吞噬能力.结果 IL-15与IL-18共同作用于TIL,能够抑制TGF-β、IL-4的分泌(P＜0.05),促进IFN-γ的分泌(P＜0.05).IL-15与IL-18共同作用于TIL后,能明显提高TIL对结肠癌细胞SW480的杀伤活性(P＜0.05).肝脏转移的肿瘤结节明显减少,血清中IFN-γ水平明显升高,腹腔巨噬细胞的吞噬能力增强.结论 IL-15与IL-18协同刺激体内能增强TIL对结肠癌细胞的杀伤功能.     

Augmentation of cytotoxicity of tumor infiltrating lymphocytes by interleukin-2 in gastric cancer patients

Lymphocyte infiltration into tumor has been regarded as an expression of host reaction against tumor, but the natural cytotoxicity of tumor infiltrating lymphocytes (TLL) is often very low. In order to augment this low cytotoxicity, TIL of gastric cancer patients were cultured with interleukin-2 (IL-2) in vitro. On the other hand, immunopotentiators (OK432, PSK) were injected into gastric cancer intralesionally under endoscopy. By the in-vitro culture with IL-2, the cytotoxicity of TIL was augmented against both targets of K562 and MNN28 (gastric carcinoma cell line). In particular, the augmentation of cytotoxicity against MKN28 was more obvious in TLL than PBL (peripheral blood lymphocytes). In the ascitic lymphocytes, the in-vitro culture with IL-2 induced autologous tumor cell killing. Intralesional injection of OK432 or PSK augmented the natural cytotoxicity of TIL, and the ratio of OKT8 and Leu7 cells increased in the TIL of OK432-injected group.     

Functional analysis of interleukin-2 in immune surveillance against brain tumors

We studied the capacity of peripheral blood lymphocytes (PBLs) from patients with malignant brain tumors to produce interleukin-2 (IL-2) and to respond to IL-2. The role of IL-2 in the generation of T cells cytotoxic against tumor cells was also studied. PBLs from the patients with malignant brain tumors tended to produce a level of IL-2 lower than that in normal controls because of the decreased number of IL-2-producing T cells. Phytohemagglutinin activated PBLs from normal controls and the patients, however, responded equally well to IL-2. This indicates that the expression of IL-2 receptors is abundant in PBLs of these patients, although IL-2 production may be depressed. Furthermore, after incubation with IL-2, PBLs from the patients with malignant glioma exhibited higher natural killer activity and strong cytotoxicity against glioma cells. This increased cytotoxicity was evident by Day 3 of culture in IL-2 and remained effective for at least 2 days. These observations of antitumor cytotoxicity make IL-2 a likely candidate for use in adoptive immunotherapy.     

Surface antigen expression on bladder tumor cells induced by bacillus Calmette-Guérin (BCG): A role of BCG internalization into tumor cells

BACKGROUND: The antitumor mechanisms of bacillus Calmette-Guérin (BCG) against bladder cancer is still unclear. We previously reported that BCG was internalized by and survived within murine bladder tumor cells (MBT-2) for at least 40 days. In the present study, we investigated the effect of BCG on the surface antigen expression of bladder tumor cells and the characteristics of these cells as antigen-presenting cells in vitro. METHODS: Surface antigen (major histocompatibility complex (MHC) Class II, CD1, CD80 and intercellular adhesion molecule-1 (ICAM-1)) expression on BCG-treated murine (MBT-2) and human (T-24, J82) bladder tumor cells were analyzed using flow cytometry. The production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) from murine lymphocytes sensitized with BCG or BCG-treated tumor cells were also investigated. RESULTS: The expressions of MHC Class II, CD1, CD80 and ICAM-1 were augmented in all of the bladder tumor cell lines used; however, they were augmented to varying degrees among the cell lines that were treated with live BCG. Heat-killed BCG had little or no effect. When murine lymph node cells sensitized with BCG or BCG-treated MBT-2 cells were cocultured with BCG-treated MBT-2 cells, significant amounts of IL-2 and IFN-gamma were produced in the culture medium. CONCLUSIONS: BCG induced the augmented expression of surface antigens, such as MHC Class II, CD1, CD80 and ICAM-1, of bladder tumor cells. Furthermore, BCG-treated MBT-2 cells could stimulate BCG-sensitized lymphocytes to produce IL-2 and IFN-gamma. These results strongly suggest that bladder tumor cells gained the characteristics and functions of antigen-presenting cells (APC).