冷冻瘤苗刺激的肿瘤浸润性淋巴细胞体外增殖及抗肿瘤活性研究

目的 研究两种不同肿瘤组织中分离的肿瘤浸润性淋巴细胞在冷冻瘤菌刺激下的增殖反应和抗肿瘤作用。方法 利用冷冻瘤苗刺激从肝癌组织、头颅恶性肿瘤组织手术标本中分离得到的ＴＩＬ，在培养３０天后，观察ＴＩＬ的增殖反应及抗肿瘤活性。结果 冷冻瘤苗刺激后的ＴＩＬ与未经冷冻瘤苗刺激的ＴＩＬ相比，扩增倍数无显著差异。但抗肿瘤活性显著提高。结论 冷冻瘤苗刺激可使ＴＩＬ的肿瘤杀伤活性大为增强，但并不能使ＴＩＬ细胞更有效地扩增。     

经瘤苗刺激的肝癌TIL细胞的诱导及其体外抗肿瘤活性

目的　研究sTIL细胞的体外增殖动力学和细胞生物学特性及体外抗肿瘤活性。　方法　采用冷冻瘤苗刺激肝癌TIL细胞,研制具有较强的抗瘤活性的由瘤苗激活的杀伤细胞(sTIL),并与TIL比较。　结果　冷冻肝癌瘤苗在TIL的培养扩增过程中不断刺激,对保持TIL较长时期的增殖力和杀伤肿瘤细胞活性等生物学特性明显增强。sTIL细胞培养至d5即有较强的杀瘤活性。持续时间可>60d,而最佳杀瘤活性时间为20～40d,37℃和5%CO2条件下细胞存活率为98%以上。　结论　sTIL可能通过特异性的T细胞的增殖且分泌细胞因子来增强其杀瘤活性,具有较强的抗瘤作用     

恶性黑色素瘤冷冻瘤苗激活的TIL在体外抗肿瘤活性的研究

目的 寻求提高肿瘤浸润淋巴细胞(TIL)的抗肿瘤活性。方法 通过应用恶性黑色素瘤冷冻瘤苗来激活TIL，观察其抗肿瘤活性的改变。结果 经冷冻瘤苗激活的TIL在培养扩增10、20、30和40d的体外对自体恶性黑色素瘤细胞的杀伤率均有明显提高，其中以培养扩增30d时为最高。结论 经恶性黑色素瘤冷冻瘤苗激活的TIL具有更高的体外杀伤自体恶性黑色素瘤细胞的能力。     

肺癌浸润淋巴细胞的分离及增殖研究

目的　从肺癌患者手术切除的肿瘤组织中分离出肺癌浸润淋巴细胞 (TIL) ,经rIL 2体外激活培养 ,研究TIL体外分离、增殖的条件和特点。方法　应用机械、酶消化和不连续密度梯度离心法从 67例肺癌患者手术切除的实体瘤中分离得到TIL ,其中 3 4例TIL放入 10 %人AB血清RPMI 164 0液中培养。结果　本组 67例标本中 ,每克瘤组织获得的TIL平均为 6.84× 10 6个。在培养的 3 4例TIL中 ,第 7～ 2 8天之间增殖最旺盛 ,以后增殖活性降低 ,数量减少。增殖达到 10 9以上有 17例 ,可回输率为 5 0 %。Ⅰ期患者分离获取TIL数明显多于Ⅲ、Ⅳ期 ,但在增殖倍数上无显著性差异 ;鳞癌与腺癌分离获取TIL数无显著性差异 ,但腺癌TIL扩增明显比鳞癌好。结论　TIL能有效地分离和增殖 ,并已具备了应用于临床治疗肺癌的条件。     

小剂量X射线照后TIL细胞体外扩增及抗癌作用的变化

目的:为了解小剂量射线对肿瘤浸润淋巴细胞体外扩增及抗癌细胞作用的影响.方法:从肝癌肿瘤模型小鼠腹水中分离取得肿瘤浸润淋巴细胞(TIL),给予不同剂量的X线照射,后培养,观察其对TIL细胞扩增及抗肿瘤活性的影响.结果:小剂量电离辐射可增加TIL细胞的扩增量,增加对肿瘤细胞的杀伤活性.结论:小剂量射线对肿瘤浸润淋巴细胞体外扩增及抗肿瘤均有显著作用.     

三种肿瘤浸润淋巴细胞的体外增殖和抗癌作用

目的：研究三种不同肿瘤组织在相同条件下获得TIL的抗瘤活性及增殖作用。方法：采用新鲜标本经消化液分离得到TIL、再经高浓度的rIL-2激活后制备成细胞悬液，测定其抗瘤活性和扩增倍数。结果：高浓度的rIL-2能有效激活TIL细胞并达到治疗数量，而肝癌、胃癌的TIL细胞毒性均高于肉瘤。结论：采用体外培养方法所获得三种肿瘤TIL具有不同的杀伤毒性。     

肿瘤浸润淋巴细胞治疗非霍奇金淋巴瘤的临床疗效分析

20世纪80年代肿瘤生物免疫疗法创立以来,为肿瘤的临床治疗开辟了新途经,其中,免疫活性细胞的过继免疫疗法是引人注目的手段之一.肿瘤浸润淋巴细胞(tumor 1nfiltratinglymphocytes,TIL)是一种肿瘤局部组织的免疫反应细胞,是继LAK细胞之后的第2代新型抗肿瘤效应细胞.TIL经rIL-2刺激后,在一般条件下可扩增上百倍、甚至上千倍,对自体肿瘤具有杀瘤活性、特异、高效.鉴于目前难治性NHL的治疗困难、效果不好,我院1977～1999年对人TIL的分离、培养、扩增及其中晚期NHL的临床疗效进行了初步研究,报道如下.     

恶性胸水中IL-2水平与TIL细胞体外扩增及活性的关系

目的　通过对恶性胸水中内源性白介素 2的水平进行检测及对胸水中肿瘤浸润淋巴细胞体外增殖能力及体外抗肿瘤活性的观察 ,了解内源性IL 2水平对胸水TIL细胞体外扩增及抗肿瘤活性的影响。方法　用MTT法检测TIL细胞体外杀瘤活性 ,用ELISA法检测IL 2的水平。将IL 2水平较高的2 4例患者分为一组 ,IL 2水平较低的 2 4例患者分为另一组。比较两组患者胸水TIL细胞体外增殖速度及杀瘤活性。结果　恶性胸水中内源性IL 2水平较低组 ,TIL细胞体外扩增倍数明显高于内源性IL 2水平较高组。IL 2水平较低组TIL细胞体外杀瘤率明显高于IL 2水平较高组。结论　恶性胸水中内源性IL 2水平高低与TIL细胞体外增殖的速度及其杀瘤活性明显相关 ,原有IL 2水平较低的患者胸水中TIL细胞体外增殖的速度较快 ,杀瘤活性较强。胸水中IL 2水平可望作为恶性胸水IL 2 /TIL治疗选择的指标。     

粒─-巨细胞集落刺激因子对人胃腺癌TIL增殖及体外杀瘤活性的影响

本研究观察粒-巨细胞集落刺激因子（GM－CSF）对胃腺癌TIL增殖及体外杀瘤活性的影响。结果发现，单独GM－CSF刺激不诱导TIL增殖扩增，而在400U／ml的IL－2培养条件下，各剂量GM－CSF均可促进TIL增殖，其中，100μg／mlGM－CSF协同IL－2促增殖效应显著。体外杀瘤活性试验（MTT法）证实100ug／mlGM－CSF协同IL－2诱导TIL细胞增强了其杀瘤活性，无论是对同种异体细胞，亦或是自体肿瘤细胞。本文探讨了GM－CSF促TIL增殖及增强杀瘤活性的可能机制。     

粒─-巨细胞集落刺激因子对人胃腺癌TIL增殖及体外杀瘤活性的影响

本研究观察粒-巨细胞集落刺激因子（GM－CSF）对胃腺癌TIL增殖及体外杀瘤活性的影响。结果发现，单独GM－CSF刺激不诱导TIL增殖扩增，而在400U／ml的IL－2培养条件下，各剂量GM－CSF均可促进TIL增殖，其中，100μg／mlGM－CSF协同IL－2促增殖效应显著。体外杀瘤活性试验（MTT法）证实100ug／mlGM－CSF协同IL－2诱导TIL细胞增强了其杀瘤活性，无论是对同种异体细胞，亦或是自体肿瘤细胞。本文探讨了GM－CSF促TIL增殖及增强杀瘤活性的可能机制。     

肝癌瘤苗激活的TIL的临床应用研究

目的：探讨经肝癌瘤苗激活的ＴＩＬ治疗原发性肝癌的作用。方法：使用冷冻方法制成肝癌瘤苗，用于激活ＴＩＬ。对３６例原发性肝癌患者使用经肝癌瘤苗激活的ＴＩＬ治疗，并检测治疗前后其ＣＤ３、ＣＤ４、ＣＤ８、ＣＤ４／ＣＤ８比例和ＮＫ活性的变化，计算其１ａ、２ａ、３ａ生存率，并与对照组相比较。结果：应用经肝癌瘤苗激活的ＴＩＬ患者，其ＣＤ３、ＣＤ４、ＣＤ４／ＣＤ８比值和ＮＫ活性明显升高，ＣＤ８则明显下降，其３ａ生存率明显升高。结论：应用经肝癌瘤苗激活的ＴＩＬ治疗，能明显地提高原发性肝癌患者的免疫功能和远期疗效。     

Modulation of tumor-infiltrating lymphocyte cytolytic activity against human non-small cell lung cancer

The cytokines expressed in tumor microenvironments are thought to be important mediators of both the host immune response and tumor survival. The source of these cytokines includes tumor cells, infiltrating leukocytes, fibroblasts, and other stromal elements. We previously reported that tumor-infiltrating lymphocytes (TIL) from human non-small cell lung cancer (NSCLC) express predominantly type 1 cytokines, which are known to enhance cell-mediated immunity. The purpose of this study is to assess the cytokine mRNA expression of human NSCLC primary cell lines and the capacity of the tumor-associated cytokines to modulate the development of TIL cytolytic activity against the autologous tumor. Cytokine mRNA expression was determined by RT-PCR and the capacity of TIL to kill autologous lung tumor cells was measured by the chromium-51 (51Cr) release assay. All NSCLC primary cell lines expressed mRNA for IL-4, IL-6, and transforming growth factor-beta1 (TGFbeta1), whereas IL-10 was expressed in only 1/7 cell lines. When added to TIL cultures stimulated with anti-CD3+IL-2, IL-4 and IL-10 enhanced and TGF-beta1 suppressed the development of TIL cytolytic activity against autologous tumor cells. The effects of IL-6 were inconsistent and for the group, were not statistically significant. These results demonstrate that human NSCLC cells express cytokines with the capacity to regulate the in situ anti-tumor immune response. However, the effects of tumor-derived cytokines varied qualitatively and quantitatively suggesting the balance between specific type 2 cytokines or TGF-beta1 within tumor microenvironments may influence prognosis or response to immunotherapy.     

Solid-phase anti-CD3 antibody activation and cryopreservation of human tumor-infiltrating lymphocytes derived from epithelial ovarian cancer.

The effect of solid-phase anti-CD3 antibody activation and cryopreservation was evaluated on thirteen samples of tumor-infiltrating lymphocytes (TILs) derived from epithelial ovarian cancer. Seven preparations of TILs were cultured with or without solid-phase anti-CD3 antibody in addition to 100 units/ml of recombinant interleukin-2 (rIL-2). The proliferation rate of all of the seven TIL preparations stimulated by anti-CD3 antibody on the fourth or fifth day of culture was 3.4 to 9.8 times greater than that of lymphocytes cultured with rIL-2 alone. Furthermore, in an experiment with five TIL samples activated with anti-CD3 antibody, three of them showed augmented cytotoxic activity against autologous fresh tumor cells. The population of CD3+/CD8+ TILs was increased after 4-5 weeks of cultivation and CD8+ lymphocytes amounted to over 70% in all of seven preparations tested, whereas two of seven preparations not activated by anti-CD3 antibody were CD3+/CD4(+)-dominant. In addition, nine preparations of TILs cultured with rIL-2 were cryopreserved for several weeks; after recovery from cryopreservation, no major change was observed in cell surface markers, in growth rate or in cytotoxic activity. These results suggest that cryopreserved and/or anti-CD3 antibody-activated lymphocytes could conveniently be employed in a clinical trial of adoptive immunotherapy employing TIL.     

Clinical application of adoptive immunotherapy by cytotoxic T lymphocytes induced from tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TIL) were cultured with interleukin 2 (IL-2) to induce the cytotoxic T lymphocytes possessing autologous tumor-killing activity from 21 cancer patients (11 with solid tumor and 10 with malignant peritoneal or pleural effusions), and transferred into 7 patients as IL-2-activated TIL adoptively. The clinical application of activated TIL by adoptive transfer could result the complete regression of malignant pleural effusions in a patient with pancreatic cancer, and the nearly complete regression of malignant ascites in a patient with gastric cancer. The autologous tumor cells were isolated at the purity of more than 90% by Ficoll-Hypaque and Percoll discontinuous gradients, and then the TIL were cultured with IL-2 until 4 weeks. The optimal concentration of IL-2 was 1,500 IU/ml to obtain maximum proliferation and autologous tumor killing activity. The cytotoxic activities of activated TIL at 3 weeks-incubation was 72 +/- 15, 42 +/- 26, 27 +/- 21 and 22 +/- 15% against K562, Daudi, KATO-III and autologous tumor, respectively. By negative selection method, it was clarified that the killer cells recognizing autologous tumor consisted of CD4 or CD8 positive T lymphocyte in 43% of patients. The CD8 positive cells and CD56 positive cells increased, the CD4 positive cells and CD16 positive cells decreased by flow cytometry. The activated TIL could lyse not only cultured tumor cell lines, also other autologous tumor cells. The CD56+ cells were isolated by the Panning method, these cells could not lyse autologous tumor cells. Thus, it was indicated that the cytotoxic T lymphocytes recognizing autologous tumor could be generated from TIL and the adoptive immunotherapy of activated TIL was effective in cancer therapy.     

Solid-phase Anti-CD3 Antibody Activation and Cryopreservation of Human Tumor-infiltrating Lymphocytes Derived from Epithelial Ovarian Cancer

The effect of solid-phase anti-CD3 antibody activation and cryopreservation was evaluated on thirteen samples of tumor-infiltrating lymphocytes (TILs) derived from epithelial ovarian cancer. Seven preparations of TILs were cultured with or without solid-phase anti-CD3 antibody in addition to 100 units/ml of recombinant interleukin-2 (rIL-2). The proliferation rate of all of the seven TIL preparations stimulated by anti-CD3 antibody on the fourth or fifth day of culture was 3.4 to 9.8 times greater than that of lymphocytes cultured with rIL-2 alone. Furthermore, in an experiment with five TIL samples activated with anti-CD3 antibody, three of them showed augmented cytotoxic activity against autologous fresh tumor cells. The population of CD3⁺/CD8⁺ TILs was increased after 4–5 weeks of cultivation and CD8⁺ lymphocytes amounted to over 70% in all of seven preparations tested, whereas two of seven preparations not activated by anti-CD3 antibody were CD3⁺/CD4⁺-dominant. In addition, nine preparations of TILs cultured with rIL-2 were cryopreserved for several weeks; after recovery from cryopreservation, no major change was observed in cell surface markers, in growth rate or in cytotoxic activity. These results suggest that cryopreserved and/or anti-CD3 antibody-activated lymphocytes could conveniently be employed in a clinical trial of adoptive immunotherapy employing TIL.     

胃肿瘤浸润淋巴细胞的基础研究与临床应用

对胃肿瘤浸润淋巴细胞（ＴＩＬ）的分离、制备、扩增、杀瘤活性、表型变化进行研究，对２３例胃癌患者进行生物治疗。结果显示：ＴＩＬ经１５天培养后，杀伤活性达７９％以上，ＴＩＬ的扩增可达９００倍。其收获率、活性与所取瘤组织及转移淋巴结的大小及新鲜程度相关。ＴＩＬ生物治疗晚期胃癌，可使患者瘤体部分缩小，存活期延长，临床症状体征明显改善，免疫功能显著提高，抗癌消瘤能力增强。本研究认为，对于晚期胃癌患者的综合治疗，ＴＩＬ生物治疗是有效方法之一。     

Different immune functions of peripheral blood, regional lymph node, and tumor infiltrating lymphocytes in lung cancer patients

Immune functions of peripheral blood (PBL), regional lymph node (RLNL), and tumor infiltrating lymphocytes (TIL) were evaluated in lung cancer patients. PBL had many natural killer (NK) cells and the highest NK activity, and it showed the highest augmentation of NK activity by interferon-gamma (IFN-gamma) + recombinant interleukin-2 (rIL-2) among the three groups of lymphocytes. PBL had high lymphokine-activated killer (LAK) activity of against a broad spectrum of cell lines and moderate activity against autologous tumor cells by increased effector to target (ET) ratio but the lowest ability of IL-2 production of the three groups of lymphocytes. The RLNL not associated with tumor metastasis had a few NK cells and lower NK activity than PBL, but its LAK activity was almost the same but not greater than that of PBL. RLNL had the highest ability of IL-2 production among the three groups of lymphocytes. All activities of RLNL associated with tumor metastasis were lower than those not associated with tumor metastasis. TIL exclusively consisted of T-cells, especially cytotoxic/suppressor T-lymphocytes. NK activity and lymphocyte blastogenesis of TIL were lower than those of other groups. The LAK activity of TIL differed greatly with the case, and it was the highest against autologous tumor cells among the three groups of lymphocytes in three of eight cases. These findings showed that PBL, RLNL, and TIL had characteristic subpopulations of lymphocytes and different functions of host immune responses in lung cancer. Efficient augmentation of the characteristic immune responses will lead to a more effective total cancer therapy.     

过继免疫疗法(AIT)与羟基喜树碱(HCPT)序贯应用治疗膀胱癌的实验研究

目的了解过继免疫疗法(AIT)与羟基喜树碱(HCPT)序贯应用对膀胱肿瘤的作用.方法体外实验测定TIL及TIL与HCPT联合应用对不同靶细胞的杀伤活性,体内实验了解不同治疗方法对小鼠膀胱肿瘤的治疗效果.结果TIL对同种异体膀胱肿瘤细胞有强大的杀伤活性,对正常组织细胞无毒性,TIL与HCPT联合应用对肿瘤细胞的杀伤毒性低于单独应用时的杀伤活性.体内实验说明同时应用TIL及HCPT无明显疗效,而合理联合应用即序贯疗法有强大的抗癌作用.结论通过本实验说明过继免疫疗法与HCPT序贯应用才能起到抗癌作用,其可能机理是序贯疗法充分发挥HCPT化疗作用又不影响TIL的杀伤活性,并且有促进TIL聚积于肿瘤周围的效果.     

Interleukin-12 Augments the Generation of Autologous Tumor-reactive CD8+ Cytotoxic T Lymphocytes from Tumor-infiltrating Lymphocytes

Human tumor-infiltrating lymphocytes (TIL) were obtained from breast cancer, renal cancer or neuroblastoma to investigate the generation of autologous tumor-reactive CD8⁺ cytotoxic T lymphocytes (CTL). When TIL were cultured with interleukin (IL)-2 (100 U/ml), the growth of TIL peaked around 8–10 days after the initiation of culture. In contrast, the proliferation of TIL cultured with IL-2 plus IL-12 peaked around 4–5 days after culture and tumor cells rapidly disappeared from the culture. To determine the generation of autologous tumor-reactive CD8⁺ CTL, TIL-derived CD8⁺ T cells were separated by FACStar. Both IL-2-activated and IL-2 plus IL-12-activated TIL-CD8⁺ T cells showed the same level of lymphokine-activated killer activity against a variety of tumor cells. However, TIL-CD8⁺ T cells activated with IL-2 plus IL-12 revealed greatly augmented cytotoxicity against autologous tumor cells compared with that induced by IL-2 alone. The autologous tumor cell-killing activity of TIL-CD8⁺ CTL was significantly inhibited by the addition of F(ab)₂ anti-CD3 monoclonal antibody, indicating that these CTL recognize autologous tumor antigen through T cell receptor. These results imply that IL-12 is a novel cytokine which facilitates the generation of autologous tumor-reactive CD8⁺ CTL from TIL.     

MTT法应用于肝癌化学免疫治疗敏感性研究

目的 探讨MTT法应用于肝癌细胞化疗敏感性检测及化疗与免疫治疗的合理应用。方法 应用MTT法检测肝癌细胞的化疗敏感性和化疗对肿瘤浸润淋巴细胞（TIL）的毒性。结果 肝癌细胞和TIL细胞数增多与生成的甲Xun（formazan)的光密度（OD）值呈线性正相关。TIL在体外对自体肝癌细胞显示高活性的细胞毒作用。肝癌细胞对化疗药物的敏感性存在较大的个体差异。化疗药物对TIL的毒性作用大于对肝癌细胞的杀伤作用。结论 以MTT法作肝癌细胞化疗敏感性检测,既可指导临床选用肿瘤敏感性化疗药物,又可避免盲目选用肿瘤非敏感性化疗药物对机体抗肿瘤免疫的毒性;TIL过继免疫治疗与化疗不宜同时应用。