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。     

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     

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。     

人肝癌TIL体外抗瘤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MTTI地检测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，ASMMC－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。     

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。     

缓激肽体外选择性开放血肿瘤屏障的机制

目的原代培养大鼠脑微血管内皮细胞及星形胶质细胞，在体外探讨不同剂量缓激肽的作用靶细胞。方法细胞原代培养成功后，运用免疫荧光测定脑血管内皮细胞、星形胶质细胞及C6胶质瘤细胞在不同剂量缓激肽作用前后的细胞内钙离子变化，根据给药前后的荧光改变来确定大、小剂量缓激肽的作用靶点细胞。结果小剂量缓激肽可以引起肿瘤细胞内的钙离子水平升高。而只有大剂量缓激肽才能触发星形胶质细胞内的钙离子水平变化，脑微血管内皮细胞对大、小剂量缓激肽均无任何反应。结论缓激肽的直接作用靶点是胶质细胞及肿瘤细胞，缓激肽调节脑血管内皮细胞通透性的作用可能需要某些细胞间信使的参与。     

乳腺癌体外药敏实验结果分析及价值

目的 探讨肿瘤体外药敏实验对乳腺癌个体化化疗的应用价值.方法 采用组织块培养-终点染色-计算机图像分析(TECIA)法检测38例乳腺癌对6种常用化疗药物的体外肿瘤药物敏感性.结果 38例乳腺癌组织对化疗药物的敏感性由高到低依次为: 多柔比星、紫杉醇、长春瑞滨、环磷酰胺、顺铂及氟尿嘧啶.结论 TECIA法的体外肿瘤药敏实验对指导乳腺癌的临床用药及个体化化疗具有重要价值.     

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。     

TNF-α与放线菌酮协同作用促体外培养黑素细胞凋亡的实验研究

目的 观察肿瘤坏死因子-α和放线菌酮对体外培养的黑素细胞的影响。方法 取包皮环切术的包皮标本，在体外进行黑素细胞的分离和培养，肿瘤坏死因子-α和放线菌酮分别处理体外培养黑素细胞24h，以及它们共同作用后，利用MTT法测定MC活力、流式细胞术测定MC凋亡率、电镜观察MC凋亡时形态学改变。结果 黑素细胞活力明显降低，放线菌酮和共同作用后出现典型的凋亡特征。电镜下，染色质边集，核固缩，凋亡小体出现，流式细胞仪检测可见典型的凋亡峰，且共同作用强于单独用药。结论 肿瘤坏死因子-α和放线菌酮共同作用可以明显的抑制黑素细胞增殖和促黑素细胞凋亡，具有协同作用。     

新城疫病毒对人肺腺癌A549细胞系的作用

目的 观察新城疫病毒对人肺腺癌A549细胞的作用.方法 应用新城疫病毒(NDV)强毒株D90对体内、体外培养人肺腺癌A549细胞进行抗癌实验,通过体外培养A549细胞观察D90对肿瘤细胞的杀伤作用,进而建立14例A549肿瘤细胞裸鼠模型,对实验后裸鼠肿瘤组织进行病理学检查,通过光镜、电镜等方法观察肿瘤细胞形态学改变从而探讨新城疫病毒D90对人肺腺癌A549细胞的杀伤作用,为临床应用奠定基础.结果 NDV强毒株D90可在体外培养条件下溶解、杀伤A549细胞.实验组与对照组比较肿瘤组织体积明显缩小(t'=4.753,P<0.01),光镜和电镜下可见肿瘤组织内血管分布减少,肿瘤细胞坏死与凋亡.结论 新城疫病毒强毒株D90能够抑制体外培养的人肺腺癌A549细胞的增殖并溶解、杀伤肿瘤细胞,能够抑制裸鼠体内肿瘤组织的生长和转移,对正常组织无影响.     

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.     

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.

     

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.     

Regulatory T Cell Infiltration Predicts Outcome Following Resection of Colorectal Cancer Liver Metastases

Background

Tumor-infiltrating lymphocyte (TIL) counts in colorectal cancer liver metastases (CRCLM) predict survival following resection. While CD4 and CD8 T cells have been correlated with outcome following CRCLM resection, the role of regulatory T cells (Treg) is not well defined.

Methods

TIL in 188 patients who underwent CRCLM resection between 1998 and 2000 were analyzed by immunohistochemistry using tissue microarrays. Correlation between TIL composition and outcome was determined while controlling for established prognostic factors. Total T cells (CD3), helper T cells (CD4), cytotoxic T cells (CD8), and Treg (FoxP3) were analyzed.

Results

Median follow-up time was 40 months for all patients and 95 months for survivors. Overall survival (OS) at 5 and 10 years was 40 and 25 %, respectively. The CD4 T cell count correlated with OS (p = .02) and recurrence-free survival (p = .04). A high number of CD8 T cells relative to total T cells (CD8:CD3 ratio) predicted longer OS times (p = .05). Analysis of Treg revealed that high FoxP3:CD4 (p = .03) and FoxP3:CD8 (p = .05) ratios were independent predictors of shorter OS. Patients with a high clinical risk score (CRS) were more likely to have a high number of intratumoral Treg, and patients ≥65 years old had a less robust CRCLM T cell infiltration.

Conclusions

A high number of Treg relative to CD4 or CD8 T cells predicted poor outcome, suggesting an immunosuppressive role for FoxP3 + TIL. The intratumoral immune response was an independent predictor of outcome in patients with colorectal liver metastases.     

Tumor infiltrating T lymphocytes in colorectal cancer: Tumor-selective activation and cytotoxic activity in situ

OBJECTIVE: To examine whether tumor-selective infiltration, activation, and cytotoxic activity of tumor infiltrating T lymphocytes (TIL) can be demonstrated in situ in colorectal cancer samples. SUMMARY BACKGROUND DATA: Recent studies indicated a correlation between the presence of TIL and an improved prognosis in colorectal cancer. However, tumor-selective activation and cytotoxic activity of CD8 TIL in situ in colorectal cancer patients have not yet been examined. METHODS: Tumor samples from 49 patients and corresponding normal mucosa samples from 23 patients with colorectal cancer (UICC stages II-IV) were examined for TIL. Two-color fluorescence immunohistochemistry and multicolor flowcytometric (FACS) analysis were used for quantification of CD8 T cells and measurement of their activation status (CD69-expression) and cytotoxic activity (CD107a-expression) in situ. Presence of tumor antigen-reactive T cells in tumor, blood, and bone marrow was evaluated by IFN-gamma Elispot analysis. RESULTS: While absolute numbers of CD8 T cells were similar, CD4 T helper cells were significantly increased in tumor tissue compared with normal mucosa. There was a significantly higher proportion of activated and cytotoxically active CD8 TIL in colorectal cancer compared with normal mucosa. Increased activation, cytotoxic activity, and functional reactivity of TIL were correlated with the presence of functional tumor antigen-reactive T cells in the blood and bone marrow. The proportion of activated TIL decreased significantly with higher tumor stage. CONCLUSIONS: Tumor-selective activation and cytotoxic activity of CD8 TIL and tumor-selective migration of CD4 T helper cells were demonstrated in colorectal cancer for the first time. Our data support the immunogenicity of colorectal cancer and suggest clinical significance of tumor-specific immune responses.     

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。     

Detection and Functional Analysis of Tumor Infiltrating T-Lymphocytes (TIL) in Liver Metastases from Colorectal Cancer

Background  Tumor-infiltrating T lymphocytes (TIL) play an important role in primary colorectal cancer, but their activity in liver metastases has not yet been investigated. The aim of this study was to examine whether tumor-selective infiltration, activation, and cytotoxic activity of TIL can be demonstrated in situ in colorectal liver metastases. Methods  TIL were obtained from liver metastases and corresponding normal liver tissue of 16 patients with colorectal liver metastases. Characterization of TIL in situ was performed by multicolor flowcytometric analysis. Presence of tumor antigen-reactive T cells was evaluated by interferon gamma Elispot analysis. Results  TIL in colorectal liver metastases responding against tumor antigens were present in most patients. Although the proportions of CD3⁺ T cells were comparable in liver metastasis and normal liver tissue, metastases contained significantly enhanced proportions of CD4⁺ cells (49% vs. 22%, P < .001). Among all CD4⁺ T helper cells, the proportion of activated (CD4⁺CD25⁺) effector cells was significantly increased in liver metastases (15.0% vs. 7.8%, P = .003). Metastases showed significantly higher proportions of activated (CD69⁺ [70.1% vs. 49.8%, P = .02] and CD25⁺ [4.1% vs. .6%, P = .06]) and cytotoxically active (CD107a⁺) CD8⁺ TIL (3.2% vs. 1.3%, P = .03). Importantly, the presence of activated T helper cells correlated with the frequencies of cytotoxic T lymphocytes that exerted cytotoxic activity in situ (P = .02). Conclusion  CD4⁺ and CD8⁺ TIL are selectively activated in liver metastases, and cytotoxic T lymphocytes exert tumor-selective cytotoxic activity in situ in the presence of activated T helper cells, suggesting the requirement of in-situ-activated T helper cells for efficient cytotoxic T lymphocytes effector function. P. Wagner and M. Koch contributed equally to this study. An erratum to this article can be found at     

Improved in vivo efficacy of tumor-infiltrating lymphocytes after restimulation with irradiated tumor cells in vitro

Background: We investigated different culture conditions for tumor-infiltrating lymphocytes (TILs) with regard to proliferation, phenotypic changes, in vitro cytotoxicity, and in vivo therapeutic efficacy. Methods: After enzymatic digestion of the murine fibrosarcoma, MCA-105, TIL cultures were initiated as pure lymphocyte (groups 1 and 2) or mixed lymphocyte/tumor suspensions (groups 3 and 4). Group 1 TILs were grown in culture medium containing 100 IU/ml recombinant interleukin-2 (rIL-2). Group 2 TILs were stimulated with solid-phase anti-CD3 monoclonal antibody (mAb) for 48 h and cultured in rIL-2 (100 IU/ml)-containing medium. Group 3, which consisted initially of a surplus of tumor cells, received the same treatment as group 2. Group 4 was also activated with anti-CD3 mAb and rIL-2 but was additionally restimulated weekly with irradiated tumor cells (TILs to tumor, 20:1). Results: Groups 1 and 2 showed up to twofold higher increases in TIL numbers compared with groups 3 and 4 by the end of culture week 5. Although the original lymphocyte/tumor cell suspension consisted of 12.0 ± 3.8% CD4⁺ T cells and 5.3 ± 3.3% CD8⁺ T cells, all four TIL cultures showed 80% CD8⁺ TILs and no CD4⁺ TILs by the end of culture week 4. In vitro cytotoxicity did not correlate with in vivo efficacy of the examined TIL cultures. By using the MCA-105 pulmonary metastases model in C57BL/6 mice, only suboptimal doses of TILs (2 × 10⁶) from group 4, which had been restimulated weekly with irradiated tumor, showed significant tumor eradication compared with all other treatment groups (p<0.01). Conclusions: We conclude that in vitro tumor restimulation of TILs improves in vivo efficacy, most likely through the education of tumor-reactive T cells.Presented at the 48th Annual Meeting of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.