Study of cytotoxicity of recombinant interleukin-2 (rIL-2) expanded tumor infiltrating lymphocytes (TIL) in renal cell cancer

We studied subsets and cytotoxicity of recombinant interleukin-2 (rIL-2) expanded tumor infiltrating lymphocytes (TIL) from renal cell cancer (RCC) patients. TIL were successfully expanded in 13 of 14 RCC cases using anti-CD3 during initial 48 hours of culture. Percentages of CD8 positive cells among rIL-2 expanded TIL at 1 tp 4 week(s) of culture were 56.2 +/- 15.1% (range 26.2 to 79.8%, N = 13) and not necessarily predominant over CD4 positive cells. NK and LAK activities of TIL at 3 to 6 weeks of culture were 31.6 +/- 15.8% (range 1.4 to 57.4%, N = 9) and 16.6 +/- 11.6% (range 3.8 to 35.6%, N = 6), respectively. Autologous and allogeneic RCC cytotoxicity of TIL at 3 to 4 weeks of culture were 17.9 +/- 19.7% (range 0 to 47.6%, N = 4) and 18.9 +/- 14.8% (range 0 to 47.3%, N = 12), respectively. Since there was no statistical difference between them, autologous specific cytotoxicity was not demonstrated. From these results of present study, it is unlikely that most of effector cells of rIL-2 expanded TIL in autologous RCC lysis are major histocompatibility complex restricted cytotoxic T cells. And we concluded that it is doubtful that TIL is significantly superior over LAK cells in immunotherapy of human RCC.     

肿瘤浸润淋巴细胞及重组白细胞介素2联合化疗在大肠癌？…

目的 研究进展期大肠癌根治术后应用肿瘤浸润淋巴细胞（ＴＩＬ）及重组白细胞介素－２（ｒＩＬ－２）联合化疗的临床治疗效果。方法 将４８例Ｄｕｋｅｓ Ｂ及ＤｕｋｅｓＣ期大肠癌患者于大肠癌根治术后采用前瞻性随机分组的方法分为２组：一组４４例,术后使用自身航ｒＩＬ－２联合－５氟脲嘧淀及丝裂霉素Ｃ治疗;另一组４０例,术后单纯采用相同方案的化疗。随访３年。结果 ＴＩＬ及ｒＩＬ－２联合化疗组患者３年内局部复发率为     

Adriamycin-mediated potentiation of cytotoxicity against freshly isolated bladder cancer cells by autologous non-activated peripheral blood lymphocytes and tumor infiltrating lymphocytes

PURPOSE: Previous studies indicated that cancer patients lack functional anti-tumor cytotoxic lymphocytes. However, anti-tumor cytotoxic lymphocytes may coexist with immunoresistant tumor cells. We reasoned that anti-tumor cytotoxic activity of lymphocytes may be revealed if the tumor cells are sensitized to killing. It has been reported that adriamycin (ADR) exhibits various immunomodulating activities. In the present study, we investigate the effect of ADR on the susceptibility of freshly isolated bladder cancer cells to lysis by autologous non-activated peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). MATERIALS AND METHODS: Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. RESULTS: Treatment of ADR-resistant fresh bladder cancer cells with ADR at 0.1 microg./ml. or more for 3 hours or more enhanced their susceptibility to lysis by autologous PBL. This ADR-induced enhancement of susceptibility of fresh bladder cancer cells to lysis by PBL was also observed when lymphokine activated killer cells, purified natural killer cells and T lymphocytes were used as effector cells. Furthermore, while cytotoxicity of freshly derived TIL against autologous bladder cancer cells was minimal, significant cytotoxicity was observed with ADR-treated bladder cancer cells. The ADR analogs, epirubicin and pirarubicin, also enhanced the susceptibility of bladder cancer cells to lysis by autologous PBL. Treatment of bladder cancer cells with ADR had no effect on the expression of MHC class I on the cancer cells or the frequency of bladder cancer target cell conjugates to autologous PBL. Treatment of bladder cancer cells with ADR augmented their sensitivity to anti-Fas monoclonal antibody and tumor necrosis factor-a. Pretreatment of effector cells with ADR had no effect on their cytotoxic function. CONCLUSIONS: These findings demonstrate that PBL and TIL in patients with bladder cancer exhibit anti-tumor cytotoxic function, but their function is not manifested due to development or acquisition of tumor cell resistance to killing. However, the resistance of bladder cancer cells to killing by cytotoxic lymphocytes is overcome if cancer cells are sensitized by subtoxic concentrations of ADR. These findings suggest that treatment of bladder cancer patients with low doses of ADR may sensitize the cancer cells to killing by autologous circulating and tumor infiltrating lymphocytes and may be a novel immunotherapeutic modality for the treatment of drug-resistant and/or immune-resistant bladder cancer.     

Regulatory effects of interleukin-7 on renal tumor infiltrating lymphocytes

Summary Biological therapy using a combination of lymphokine and tumor infiltrating lymphocytes (TILs) is a new approach to the treatment of patients with advanced cancer. To improve the potency of TILs, new cytokines with T-cell stimulatory effects used alone or in combination with interleukin-2 (IL-2) are currently being investigated. We have studied the effect of interleukin-7 (IL-7) on TILs derived from renal cell carcinoma. Our data demonstrated that five of ten TILs proliferated in response to IL-7 alone. This proliferative response was 73–90% less than that obtained with IL-2 alone. The use of IL-7 plus IL-2 resulted in a 1.2- to 4.7-fold increase in proliferation of six of ten TILs compared with IL-2 alone. IL-7-driven TIL growth was consistently blocked by anti-IL-2, anti-IL-2R and anti-IL-7 antibodies (37.2%, 41.6% and 82.2% suppression, respectively). The expression of IL-2 receptors was also significantly increased in the presence of IL-7 or IL-7 phytohemagglutinin (40.6+3.8 and 72.5+1.5). In comparison with IL-2, IL-7 treatment was associated with a decrease in CD56 (46.3%±19 vs 10%±4.9) and increase in CD3 (29.3%±12 vs 73%±6.4) and CD 4 (19.3%±15 vs 58%±10). These studies suggest that in some renal TILs, IL-7 and IL-2 can have a synergistic proliferative effect. The IL-7 stimulatory effect appears to be mediated via both an IL-2 pathway and an IL-7-independent pathway.This paper was selected for publication in Urological Research from the program of the 1991 meeting of the European Society of Urological Oncology and Endocrinology (ESUOE)     

Augmentation of cytotoxicity of regional lymph node lymphocytes of gastric cancer after intratumoral injection of OK-432

Although regional lymph node lymphocytes (LNL) are thought to be a barrier of the immunological surveillance, their natural cytotoxicity is strongly suppressed. In this study, an immunopotentiator OK-432 (10K.E.) was injected into gastric cancer lesions under endoscopy 1 week before operation, and the effects on the cytotoxicity of LNL were examined by single cell assay. This assay is characterized by assessment of killer cell frequency in preventing the killer cells from recycling by their fixation in agarose, and by direct microscopic observation of both binding and killing phases. NK sensitive cell line K562 was used as target cells. By intratumoral injection of OK-432, the killer cell frequency of LNL in 8 out of 20 cases was elevated to almost equal level as that of peripheral blood lymphocytes. In clinical stages these 8 cases belonged to early stages (stage I 7 cases, stage II 1 case). In LNL subsets of OK-432 injected cases, the percentage of OKT8 positive cells was decreased and the ratio of OKT4 and OKT8 was significantly increased compared with non-injected group.     

肿瘤浸润淋巴细胞在肾细胞癌原位表达的特点

目的:研究肾细胞癌组织中肿瘤浸润淋巴细胞(TIL)的分布特点及其与预后的关系.方法:对52例肾透明细胞癌组织T,辅助T及自然杀伤细胞(NK细胞)进行原位免疫组化研究,并与病人预后作相关性分析.结果:TIL细胞中主要为T细胞,占70%;TIL细胞表达的程度对肾细胞癌患者预后的影响较大(P<0.01);TIL细胞亚群之间的表达为正相关.结论:TIL在对抗肾细胞癌中起较重要的作用,它们的表达程度影响预后.     

Immunology of tumor infiltrating lymphocytes.

Frequently peripheral blood lymphocytes (PBL) do not reflect the tumor host relationship and cell mediated immunity in the PBL does not often correlate with prognosis. The tumor infiltrating lymphocytes (TIL) interact most closely with the tumor cells and are likely to more accurately reflect tumor host interactions. These studies indicate that TIL from pulmonary tumors are similar to PBL so far as their cell surface markers are concerned. The percentage of T-cells, B-cells, helper cells, suppressor cells, and NK cells are similar in the two compartments. However, the TIL are markedly suppressed in their functional capacity as measured by their proliferative and cytotoxic activity. In addition, natural killer (NK) cell activity is markedly diminished in TIL as opposed to the PBL. In addition, the direct injection of BCG into these tumors reverses this phenomenon by significantly increasing T-cell and NK cell functional activity. Thus, the microenvironment of the tumor profoundly affects the immunologic relationship between the tumor and the host.     

胃癌微环境中树突状细胞浸润的临床意义

目的 探讨胃癌微环境中肿瘤浸润性树突状细胞(TIDC)与癌细胞浸润程度、淋巴结转移及患者术后存活时间的关系。方法 采用免疫组织化学方法,运用抗S-100检测100例胃癌组织中的TIDC,并分析其与临床病理参数及生存率的关系。结果 TIDC在胃癌组织中的阳性率为41.0％(41/100),其与癌细胞浸润程度无相关性(P>0.05)。56例有淋巴结转移的患者中,TIDC阳性者淋巴结转移率为56.1％,阴性者为43.9％,两者比较差异无显著性意义(P>0.05);第2站以上淋巴结转移率,TIDC阳性者为29.7％,而阴性者为70.3％,两者比较,差异有显著性意义(P<0.05)。TIDC阳性和阴性患者5年生存率分别为59.0％和37.7％,两者比较.差异有显著性意义(P<0.05)。结论 TIDC阳性与癌细胞浸润程度无相关性,但与第2站以上淋巴结转移和患者术后生存率密切相关。     

原发性肝癌患者肿瘤浸润T淋巴细胞亚群分析

目的:了解原发性肝癌患者肿瘤浸润T淋巴细胞中CD4+及CD8+的表达特点。方法:收集30例原发性肝癌患者(均有乙型肝炎病毒感染背景)的癌组织(乙肝肝癌组)和癌旁组织(乙肝癌旁组)以及30例因良性病变而行肝切除的患者的新鲜肝组织(对照组),分离组织浸润淋巴细胞,用抗CD3、CD4和CD8单克隆抗体同时荧光染色,用流式细胞仪检测各表面标志的表达情况。结果:1)乙肝肝癌组、乙肝癌旁组及对照组CD3+CD4+T细胞占浸润淋巴细胞的比例分别为(22.31±3.68)%、(10.69±2.47)%及(4.21±4.26)%。乙肝肝癌组显著高于乙肝癌旁组及对照组,差异有统计学意义(P=0.000,P=0.001),乙肝癌旁组高于对照组,差异亦有统计学意义(P=0.019)。2)乙肝肝癌组、乙肝癌旁组及对照组CD3+CD8+T细胞占浸润淋巴细胞的比例分别为(26.10±5.82)%、(21.82±2.70)%及(41.31±14.01)%,乙肝肝癌组及乙肝癌旁组显著低于对照组,差异有统计学意义(P=0.014,P=0.004),而乙肝肝癌组与乙肝癌旁组之间差异无统计学意义(P=0.651)。3)乙肝肝癌组组织浸润淋巴细胞中CD3+CD4+T细胞与CD3+CD8+T细胞的比值为0.91±0.30,显著高于乙肝癌旁组(0.47±0.11,P=0.003)及对照组(0.11±0.13,P=0.000),CD3+CD4+与CD3+CD8+T细胞的比值出现失衡。结论:原发性肝癌患者肿瘤浸润淋巴细胞中T细胞亚群失调,表现为CD3+CD4+T细胞所占比例升高,CD3+CD8+T细胞所占的比例下降,CD3+CD4+/CD3+CD8+明显升高。     

Prognostic significance of tumor infiltrating lymphocytes in melanoma

The prognostic significance of tumor infiltrating lymphocyte (TIL) response in cutaneous melanoma is controversial. This analysis of data from a prospective, randomized trial included patients with cutaneous melanoma > or = 1.0 mm Breslow thickness who underwent wide local excision and sentinel lymph node (SLN) biopsy. Univariate and multivariate analyses were performed to determine factors associated with TIL response, disease-free survival (DFS), and overall survival (OS). A total of 515 patients were included; TIL response was classified as "brisk" (n = 100; 19.4%) or "non-brisk" (n = 415; 80.6%). Patients in the nonbrisk TIL group were more likely to have tumor-positive SLN (17.6% vs 7%; P = 0.0087). On multivariate analysis, nonbrisk TIL response, increased tumor thickness, and ulceration were significant independent predictors of tumor-positive SLN. By Kaplan-Meier analysis, 5-year DFS rate was 91 per cent for those with a brisk TIL response compared with 86 per cent in the nonbrisk group (P = 0.41). The 5-year OS rates were 95 per cent versus 84 per cent in the brisk versus nonbrisk TIL groups, respectively (P = 0.0083). However, on multivariate analysis, TIL response was not a significant independent factor predicting DFS or OS. TIL response is a significant predictor of SLN metastasis but is not a major predictor of DFS or OS.     

肿瘤浸润性淋巴细胞治疗原发性肝癌的基础与临床研究

目的 探讨肿瘤浸润性淋巴细胞（TIL）对原发性肝癌（HCC）患者术后细胞免疫功能的影响和临床疗效及半乳糖抗CD3单克隆体－TIL复合物（Gal-Anti-CD3-McAb-TIL)体外及体内的趋肝性。方法 （1）对30例HCC患者术后进行了TIL和重组白介素－2（rIL-2)治疗。（2）制备半乳糖基抗CD3单克隆抗体－TIL（McAb-TIL)复合物。将TIL和McAb－TIL分别与小鼠肝细胞置CO2孵箱中孵育,用倒置相差显微镜观察其趋肝性。将^125I－TIL和^125I-McAb-TIL分别经小鼠尾静脉注入,观察示踪剂在脏器中的分布。结果 TIL治疗后,30例患者外周rIL-2、T细胞亚群均有上升。24例肝癌根治性切除术患者随访60－48个月无复发。McAb－TIL与细胞有明显粘附,给小鼠静脉注射后,TIL和McAb-TIL主要在肺中积聚,在肝内浓度较低。结论 TIL治疗可能提高HCC患者术后抗钟瘤细胞免疫功能。TIL与半乳糖抗CD3单克隆抗体结合物体内肝靶向性不明显。     

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.     

Augmentation of the lymphokine-activated killer cell response in head and neck cancer patients by combination interleukin-2 and interferon-alpha

The majority of patients with head and neck cancer present with advanced disease (stage III and IV), for which current chemotherapeutic regimens offer dismal results. Although known to have defects in their cell-mediated immunity, their poor performance status makes them unlikely candidates for aggressive immunotherapeutic protocols because of associated severe toxicities. This study evaluates the effect of subthreshold recombinant interferon-alpha (rIFN-alpha) and interleukin-2 (rIL-2) on the generation of lymphokine-activated killer (LAK) cells from the peripheral blood of patients with head and neck cancers. In vitro treatment of patients' lymphocytes consisted of incubation in 1,000 U/mL rIL-2, 100 U/mL rIL-2, 100 U/mL rIFN-alpha, and 100 U/mL rIFN-alpha plus 100 U/mL rIL-2 for 4 to 5 days. Cytotoxicity was measured using a standard 4-hour chromium-51 (51Cr)-release assay with Raji (B lymphoblastoid) tumor target cells. LAK activity was arbitrarily defined as greater than 20% cytolysis of Raji target cells. LAK activity was generated in a smaller percentage of the head and neck cancer patients by 1,000 U/mL rIL-2 compared with normal adult donors: 54% versus 100%, p less than 0.05; IFN-alpha (100 U/mL) induced LAK activity in approximately 50% of the cancer patients. The addition of rIFN-alpha (100 U/mL) to rIL-2 (100 U/mL) resulted in LAK generation in a higher percentage of patients (83% versus 54%), as well as increased levels of cytotoxicity, p = 0.05. This combination also resulted in cytotoxicity levels equivalent to high-dose (1,000 rIL-2 U/mL). These in vitro data support a clinical trial to assess the therapeutic efficacy of combined low-dose rIL-2 and rIFN-alpha in vivo in head and neck cancer patients.     

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。     

Expansion of CD3+CD56+ lymphocytes correlates with induction of cytotoxicity by interleukin-2 gene transfer in human breast tumor cultures

Background: Mice immunized with murine mammary carcinoma cells genetically engineered to secrete interleukin-2 (IL-2) are rendered resistant to subsequent challenge with unmodified tumor cells, and in the case of mice bearing established tumors, the rate of development of pulmonary metastases is reduced. Despite these encouraging animal results, little is known about the induction of antitumor immunity by IL-2 gene transfer in human breast cancer. Methods: Adenovirally mediated IL-2 gene transfer was performed in 12 tumor fragment cultures established from seven primary breast cancers. Autologous tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) were cocultured with transduced tumor fragments, and changes in phenotype and cytotoxicity were measured. Results: IL-2 was never detectable in the untransduced cultures, but it peaked at 5.0—1,324.8 ng/ml in the transduced cultures. Lymphocyte counts declined in all untransduced cultures, but they increased two- to sevenfold in four transduced cultures. CD4:CD8 ratios decreased from a mean of 2.11 at baseline to 1.27 after stimulation in coculture (p=0.03). Expansion of lymphocytes expressing the natural killer cell phenotype (CD3⁻CD56⁺) occurred in only one culture, but the CD3⁺CD56⁺ population increased in four of six cultures. Lymphocytes from four of 10 cocultures generated significant cytotoxicity against allogeneic breast cancer cells. Induction of cytotoxicity correlated with expansion of the CD3⁺CD56⁺ phenotype (R²=0.805, p=0.02). Conclusions: IL-2 gene expression by human breast cancer causes expansion of CD3⁺CD56⁺ cytotoxic lymphocytes. This phenotype is consistent with that of a non-major histocompatibility complex (MHC)-restricted cytokine induced killer cell population previously described. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the U.S. Army. Presented at the 49th Annual Cancer Symposium of The Society of Surgical Oncology, Atlanta, Georgia, March 21–24, 1996.     

BCG激活膀胱肿瘤浸润淋巴细胞的抗肿瘤作用研究

为了探讨ＢＣＧ的抗肿瘤机理，从１５例手术切除的膀胱移行细胞癌新鲜组织标本中制备肿瘤浸润淋巴细胞（ＴＩＬ），分别在含ＢＣＧ或ＩＬ－２的全培养基中培养扩增，测定不同培养时间的抗瘤活性。结果：用活ＢＣＧ培养的ＴＩＬ第１２天对自体肿瘤细胞的杀伤活性达高峰，杀伤率为４８．３％；用ＩＬ－２培养的ＴＩＬ第１４天达杀伤高峰，杀伤率为４３．８％，用死ＢＣＧ培养的ＴＩＬ，其扩增结果和抗瘤活性均明显低于活ＢＣＧ及ＩＬ－２。提示ＢＣＧ对ＴＩＬ的直接激活作用可能是其抗肿瘤机理之一。     

肿瘤浸润淋巴细胞对结肠癌细胞的杀伤作用及机制

目的探讨肿瘤浸润淋巴细胞在结肠癌细胞杀伤中的作用及机制。方法从6例结肠癌患者转移淋巴结中分离TIL细胞,采用^51Cr释放法测定其杀伤结肠癌SW480细胞的作用;采用流式细胞术测定结肠癌细胞的凋亡;采用固定化蛋白印迹法（Western blot）测定TIL细胞和结肠癌细胞Fas、FasL蛋白的表达。结果TIL细胞能有效杀伤结肠癌SW480细胞,其杀伤作用明显强于淋巴因子活化的杀伤细胞（100：1,P=0.004;50：1,P=0.002;10：1,P=0.006）和细胞因子诱导的杀伤细胞（100：1,P=0.001;50：1,P=0.002;10：1,P=0.008）,差异具有统计学意义（P〈0.01）;TIL细胞能促进结肠癌细胞的早期凋亡;TTL细胞高表达FasL分子,而SW480细胞高表达Fas分子,不表达FasL分子。结论TIL细胞能有效杀伤不表达FasL分子的结肠癌细胞,其机制可能通过Fas/FasL通路诱导结肠癌细胞的凋亡。     

Correlation between DNA ploidy patterns and tumor progression, prognosis, and tumor infiltrating lymphocytes in human esophageal cancer

Cytophotometric DNA analyses were performed on 35 primary esophageal cancer. Histograms of DNA measurement were classified into three patterns (diploid pattern, aneuploid pattern and mosaic pattern) and were compared with histological findings, prognosis, and degree of lymphocytes infiltration around the tumor. Survival rate was worse in patients with mosaic pattern than the others, and 4-year survival rates of each patterns were 66.7% (diploid), 53.6% (aneuploid) and 25.4% (mosaic). Diploid cell line was observed frequently in the superficial cancers, and as the cancers infiltrated more deeply, mosaic cell line increased. Mosaic cell line appeared more frequently in well differentiated squamous cell carcinomas. In patients with mosaic pattern, there was high frequency of lymphnode metastasis and vascular invasion, as compared with diploid pattern. The rate of vascular invasion tended to increase in the following order; diploid, aneuploid and mosaic types. Furthermore in the diploid tumors, the degree of lymphocyte infiltration around tumor tended to increase. These findings suggest that the change of DNA content may occur frequently during tumor progression and may be affected by tumor infiltrating lymphocytes. So the DNA ploid pattern will be also to be one of the conjecturable factors of the prognosis of esophageal cancer.