人肿瘤浸润淋巴细胞的体外抗瘤活性及其表型特征

通过体外分离、扩增培养１０例肝癌、３例胃癌和１例肾癌肿瘤浸润淋巴细胞（ＴＩＬ细胞），用ＭＴＴ法检测８例对靶细胞的杀伤作用，结果发现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３０天内抗瘤活性呈现逐渐增强趋势。新鲜分离的ＴＩＬ表型主要呈ＣＤ３，培养２０天后肝癌和胃癌ＣＤ＾＋４ＴＩＬ细胞增加，而肾癌ＣＤ＾＋３ＴＩＬ细胞增加明显。本研究结果将为ＴＩＬ的应用提     

顺铂对结直肠癌肿瘤浸润淋巴细胞杀伤活性的影响

为在临床选择有效的免疫化疗方案提供一定的理论依据，作者以结直肠癌肿瘤浸润淋巴细胞（ＴＩＬ）和顺铂（ＣＤＤＰ）为研究对象，对１６名手术治疗的结直肠癌患者，分别观察ＣＤＤＰ体内注射及体外预处理ＴＩＬ和Ｒａｊｉ细胞对ＴＩＬ表面标志和杀伤活性的影响。流式细胞仪检测结果显示，静脉注射ＣＤＤＰ能增加结直肠癌ＴＩＬ中ＣＤ３＋／ＣＤ４＋和ＣＤ３＋／ＣＤ８＋细胞含量，同时增强ＴＩＬ体外杀伤Ｒａｊｉ细胞的活性；而体外以ＣＤＤＰ处理Ｒａｊｉ细胞能增强其对结直肠癌ＴＩＬ杀伤的敏感性。作者认为，对于联合应用ＴＩＬ和ＣＤＤＰ治疗结直肠癌的临床效果有必要进一步研究。     

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。     

肾细胞癌浸润淋巴细胞的杀伤肿瘤活性测定及表型分析

采用Ｗｈｉｔｅｓｉｄｅ方法从１９例ＲＣＣ中成功地分离到１７例ＴＩＬ，经１９天培养，ＴＩＬ数量平均达１．０４×１０~（１１）个细胞。杀伤自体肿瘤活性为５７．８７±５．８８％，对Ｒａｊｉ细胞的杀伤活性为３７．３９±１０．８８％，两组间比较差异有非常显著性（Ｐ＜０．０１）。明显高于外周血淋巴细胞（ＰＢＬ）的杀伤肿瘤活性（２２．３９±８．８４％）。ＴＩＬ中ＣＤ_３和ＣＤ８细胞分别为６９．９３±６．８０％及４０．２１±６．８０％，均明显高于ＰＢＬ中的ＣＤ３和ＣＤ_８细胞（Ｐ＜０．０１）。ＴＩＬ对Ｋ５６２细胞的杀伤活性为２６．８９±１２．７９％，提示活化的ＴＩＬ有显著的杀伤肿瘤活性，且对自体肿瘤细胞的杀伤活性更为显著，其杀伤活性主要由Ｔ淋巴细胞介导。     

消化系肿瘤浸润淋巴细胞不同输注途径对免疫功能的影响

从肿瘤组织中分离出ＴＩＬ，经ｒＩＴ－２培养扩增后进行自体回输，输注途径包括：（１）外周静脉；（２）局部（肿瘤供血动脉或腹腔）；（３）静脉加局部。ＴＩＬ输注总量为１．５×１０＾９以上。治疗前后抽取外周静脉血检测Ｔ细胞亚群和ＮＫ细胞活性。１８例患者（胃癌９例、大肠癌５例、肝癌４例）经ＴＩＬ治疗后，８８．９％的患者ＮＫ活性增强，７７．７％￣９４．４％的患者ＣＤ３＋、ＣＤ４＋和ＣＤ８＋细胞比例升高，不同输     

肿瘤浸润淋巴细胞治疗消化道肿瘤的初步探讨

本文初步探讨了肿瘤浸润淋巴细胞（ＴＩＬ）治疗消化系癌肿的临床疗效。从肿瘤组织中分离出力ＴＩＬ，体外经重组白细胞介素－２（ｒＩＬ－２）培养扩增，扩增后ＴＩＬ的ＣＤ细胞亚群比例升高，细胞毒活性增强。ＴＩＬ输注总量１．５×１０９～３×１０９，治疗有效者外周血Ｔ细胞亚群比例明显升高，自然杀伤细胞（ＮＫ）、淋巴因子激活的杀伤细胞（ＬＡＫ）细胞活性明显增强。５例行根治切除者随访１１～１９个月无复发，４例姑息切除者复发２例，９例未切除者总缓解率达６６．７％。证明ＴＩＬ治疗消化系肿瘤有一定价值。     

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。     

肝癌肿瘤浸润淋巴细胞的研究与应用

作者研究了肝癌患者肿瘤浸润淋巴细胞（ＴＩＬ）的体外分离、扩增培养和临床治疗，其目的是寻求癌症术后综合治疗更为有效的方法。结果表明，１２例用于ＴＩＬ培养的肿瘤组织重量平均４．８ｇ，细胞数５．８×１０￣７，培养时间３１．８天，最大扩增倍数１０００倍。ＬＡＫ上清液能显著促进ＴＩＬ的扩增培养。本组对１０例患者进行了ＴＩＬ的临床治疗，采用了经肝动脉的输注途径，回输细胞量为４×１０￣８～１．１×１０￣（１０）肝癌ＴＩＬ还显示对靶细胞有明显杀伤活性。     

膀胱癌TIL的分离培养及其表型分析

对３例膀胱移行细胞病患者手术切除的肿瘤组织中肿瘤浸浸润性淋巴细胞进行了体外分离与培养，结果２例获得成功，ＴＩＬ体外扩增培数分别达５８－１７０，在培养２４ｄ时对自体肿瘤靶细胞的最高杀伤活性达４１％以上，且呈现一定的靶细胞特异性。     

TIL辅助治疗肾细胞癌9例

肾癌（ＲＣＣ）有着独特的生物学特性，其肿瘤及转移灶对化疗不敏感，而对生物学治疗反应明显，近年来，有人提出肿瘤浸润淋巴细胞（ＴＩＬ），具有较强的抗肿瘤效应。本文通过９例肾细胞癌根治术后ＴＩＬ治疗前后患者外周血淋细胞表型的测定以验证其抗肿瘤效应。结果显示治疗后患者上周血中ＣＤ３、ＣＤ４、ＣＤ８细胞及ＣＤ５６（ＮＫ）细胞增殖；ＮＫ活性得到增强。     

Changes on distribution of CD4+/CD45RA- and CD8+/CD11- cells in tumor-infiltrating lymphocytes of renal cell carcinoma associated with tumor progression.

To study the distribution of subsets of T cells in renal cell carcinoma, peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were analyzed in 43 untreated patients using two-color flow cytometry. An increase in the relative number of CD4+/CD45RA-, CD8+/CD11- and HLA-DR+/CD3+ cells was shown in TIL when compared with PBL. When the influence of various tumor factors on subsets of TIL was examined, a decrease in CD4, CD4+/CD45RA- and CD16+/CD57- cells and an increase in CD8+ and CD8+/CD11- cells was observed along with the aggravation of tumor stage and grade. In TIL of stage III/IV and grade III/IV disease, most patients showed an increase in CD8+/CD11- associated with a decrease in CD4+/CD45RA- cells, or the reverse, resulting in changes of the CD4+/CD45RA- to CD8+/CD11- ratio. The prognosis for these patients was poor, suggesting that changes in the ratio were a sign of the impairment of local immune status associated with disease progression.     

Effect of tumor-infiltrating lymphocyte subsets on prognosis and susceptibility to interferon therapy in patients with renal cell carcinoma

INTRODUCTION: Immunotherapy effectively treats advanced renal cell carcinoma in only a limited number of patients. However, the predicted prognosis for each patient in relation to immune status and response to immunotherapy remains problematic. We analyzed tumor-infiltrating lymphocyte (TIL) subsets to determine whether these correlated with the prognoses for the patients and the response to alpha-interferon therapy. MATERIALS AND METHODS: TIL subsets from resected specimens of 79 patients were analyzed by two-color flow cytometry and then compared with the patients' long-term clinical courses and responses to interferon therapy. RESULTS: In patients with stages III and IV, an increased infiltration of CD4+ cells and decreased CD8+ cells constituted a fair prognostic factor. In 17 patients with metastatic lesions, 8 of 10 patients who had disease progression after interferon therapy showed an increase in CD8+ cells above 25%, whereas 2 responders and 5 patients who had stable disease showed infiltration of CD8+ cells below 25%. CONCLUSIONS: The TIL subset is a prognostic factor for advanced renal cell carcinoma, and its analysis provides a method to predict the susceptibility to interferon therapy.     

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.     

Lymphocytic subsets of tumor tissue, non tumorous kidney, and the peripheral blood in primary renal cell carcinoma]

Lymphocyte subsets were examined in renal cell carcinoma (TILs), adjacent non-tumor renal tissue and peripheral blood (PBLs) by flow cytometry and histochemistry in eighteen patients with renal cell carcinoma. CD5-positive cells were predominant in the TILs in 14 patients. In the renal cell carcinoma tissue, CD8-positive cells were predominant over CD4-positive cells, resulting in a less than unity ratio of CD4/CF8-positive cells. The lymphocyte number was significantly in adjacent normal renal tissue than in renal cell carcinoma. However, lymphocyte subsets ratios were not significantly different between these two tissues. PBLs showed the same proportions (CD4/CD8 mean 1.9 +/- 0.8) as the previously published healthy controlled data. The proportions of CD8-positive cells were significantly increased (p less than 0.05) and those of CD4-positive cells were also significantly decreased (p less than 0.01) in the TILs. The infiltrating pattern of TILs in 17 patients was divided histochemically into cluster (N = 7), single (N = 4), and mixed types (N = 6). The cluster and mixed types were significantly more common in grade 1 tumors and the single type was more common in the grade 2 tumors (p less than 0.05). The pT3 tumors showed the single type of TIL infiltration pattern, but showed no significant difference. In the cluster pattern of TILs, CD8-positive cells were surrounded by CD4-positive cells. Non-tumorous kidneys showed no infiltration of lymphocytes, except in 2 patients of pyelonephritis. These results suggest that cytotoxic T-cells stained as CD8 play an immunoreactive role against renal cell carcinoma.     

Tim-3 expression represents dysfunctional tumor infiltrating T cells in renal cell carcinoma

Purpose

Renal cell carcinoma (RCC) is the most common cancer of kidney. Evidences have shown that RCC is sensitive to various immunotherapies. Tim-3 plays a role in suppressing Th1-mediated immune responses. However, no study has yet examined the effect of Tim-3 on tumor infiltrating lymphocytes (TILs) in RCC.

Methods

We investigated the expression and function of Tim-3 on TIL CD4+ T cells and TIL CD8+ T cells from 30 RCC patients.

Results

Levels of Tim-3 were significantly increased on both TIL CD4+ T cells and TIL CD8+ T cells and were associated with higher stages of the cancer. Also, GATA-3 and interferon gamma (IFN-γ) were down-regulated, whereas T-bet was up-regulated in TIL Tim-3+ T cells, indicating that Tim-3 expression defined a population of dysfunctional TIL Th1/Tc1 cells. Mechanism analyses showed that TIL Tim-3-expressing CD8+ T cells exhibited impaired Stat5 and p38 signaling pathway. Blocking the Tim-3 pathway restored cell proliferation and increased IFN-γ production in TIL CD4+ and CD8+ T cells of RCC.

Conclusions

These results suggest that Tim-3 may be used as a novel target for increasing immune responses in RCC tumor microenvironment.

     

Immunological assessment of tumour infiltrating lymphocyte subsets in renal cell carcinoma patients. An analysis of immunological reaction to immuno-competent lymphocytes by interferon-gamma

Twenty-two patients with renal cell carcinoma subjected to radical nephrectomy were divided into 2 groups. The first group consisted of patients who received nephrectomy alone, and the second of 10 patients who received pre-operative IFN-gamma. Using monoclonal anti-bodies of each subset of lymphocytes, immunological distributions of tumour infiltrating lymphocytes (TIL) were evaluated by means of immunohistochemical staining and the peripheral blood lymphocytes (PBL) were evaluated by flow-cytometry. Furthermore, immunological effects of IFN-gamma on these immuno-competent cells were investigated. The effect of IFN-gamma on TIL was an increase in CD3 (pan-T cells), in particular an increase in CD8 (suppressor/cytotoxic-T cells). On the other hand, a significant increase in CD16 alone was found on PBL. As to the TIL according to stages, all subset ratios tended to be higher in high stage patients without administration of IFN-gamma. With pre-administration of IFN-gamma, a marked increase in CD3 was found among low stage patients. In examining these cells according to grades, no specific trend was observed between low and high grade patients without having IFN-gamma administration. With pre-administration of IFN-gamma, an increase in CD3 and CD8 was observed in low grade patients. As to PBL, a significant increase in CD16 alone was observed with pre-administration of IFN-gamma, and no correlation was found between stage and grade. We draw the conclusions that the subsets of TIL were quite different from those of PBL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Robust method for isolation of tumor infiltrating lymphocytes with a high vital cell yield from small samples of renal cell carcinomas by a new collagenase-free mechanical procedure

Background

Tumor-infiltrating lymphocytes (TIL) play an important role in the pathogenesis of renal cell carcinoma. Characterization of TIL requires efficient isolation procedures, especially in early stage disease when the tumor is of small in size. Conventional methods for isolating TIL are based on enzymatic tissue digestion, most frequently with collagenase. Collagenase isolation is limited by poor cell recovery, altered expression of cell-surface molecules, and impaired TIL-functionality. To overcome these limitations, we developed and optimized conditions for a robust collagenase-free mechanical procedure for improved isolation of TIL from renal cell carcinoma samples.