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。     

肿瘤浸润淋巴细胞研究的进展

自从1986年Rosenberg首次报道直接从小鼠肿瘤制备的肿瘤浸润淋巴细胞(TIL)，清除转移灶效率比LAK细胞高50～100倍以来。TIL作为继LAK细胞之后的第二代抗瘤细胞，杀瘤率高及依赖IL-2程度低、副作用小日益受到重视。近几年来TIL分离培养技术、杀瘤机制及基因治疗等方面发展迅速。本文就这些方面的进展作一简要综述。     

膀胱癌肿瘤浸润淋巴细胞表型分析

应用免疫组织化学技术对从16例膀胱移行细胞癌分离出的肿瘤浸润淋巴细胞(TIL)进行培养前后细胞表型分析。结果显示:培养前TIL中CD_3~ 细胞和CD_4~ 细胞随肿瘤分级、分期升高而减少;培养前后相比,培养后TIL中CD_3~ 细胞和CD_4~ 细胞较培养前显著增多。提示膀胱移行细胞癌TIL细胞表型变化与肿瘤局部免疫之间存在因果关系。     

肝癌肿瘤浸润淋巴细胞的培养及抗CD3单抗对其增殖及细胞毒的 …

目的 研究人原发性肝癌肿瘤浸润淋巴细胞（ＴＩＬ）的分离，培养方法和增殖及体外细胞毒变化特点以及抗ＣＤ３单克隆抗体（Ａｎｔｉ－ＣＤ３ＭｃＡｂ）对ＴＩＬ增殖和细胞毒的影响。方法 采用Ｉ型和Ⅳ型胶原酶，Ｖ型透明质酸酶和Ｉ型ＤＮＡ酶四联酶消化，应用不连续密度梯度的离心分离，ＴＩＬ，采用ＭＴＴ比色法检测ＴＩＬ对自体癌细胞及肝癌细胞株的细胞毒。结果 ＴＩＬ对自体肝癌细胞的杀伤具有特异性（Ｐ〈０．０１）。Ａｎｔ     

葡萄球菌肠毒素A对肝癌肿瘤浸润淋巴细胞抗瘤活性的作用

目的探讨葡萄球菌肠毒素A(SEA)对肝癌肿瘤浸润淋巴细胞(TIL)抗瘤活性的诱导作用。方法取5例手术切除肝癌标本,分离TIL,在SEA作用下进行培养。定时记数,了解其增殖情况。流式细胞仪检测其CD3、CD4、CD8表达情况,噻唑蓝(MTT)比色法测定其对HepG-2肝癌细胞株的细胞毒活性,酶联免疫吸附试验(ELISA)测定培养上清液中肿瘤坏死因子(TNF)-α和γ-干扰素(IFN-γ)浓度。结果在SEA刺激下,2周TIL扩增100倍。1周后CD3+细胞占95%以上,CD8+细胞较CD4+细胞增殖更迅速。TIL细胞毒活性随培养逐渐增强。在培养的前10d内,TIL产生大量的TNF-α峰值达(453.70±9.26)ng/L和IFN-γ,其峰值达(2013.22±20.41)ng/L。结论SEA可高效、迅速诱导肝癌TIL的抗瘤活性。     

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。     

肿瘤浸润淋巴细胞在肿瘤免疫研究中的进展

ＴＩＬｓ的应用进入了新阶段，也存在许多亟待解决的问题。本文综述原位ＴＩＬｓ与临床预后的相关性、体外扩增方法及临床应用效果和前景等方面的主要进展。     

人体肿瘤浸润淋巴细胞杀伤胃癌细胞的形态学观察

在光镜和电镜下观察人肿瘤浸润淋巴细胞（ＴＩＬ）杀伤ＭＫＮ４５传代人胃癌细胞的形态学变化。发现ＴＩＬ细胞与细胞共同孵育１小时后，前者向癌细胞靠近，并伸出伪足接触之３小时后，胃细胞结构模糊，胞浆内出现空泡，胞膜绒毛脱落。６小时后，癌细胞呈空泡状，膜裂解，细胞呈“凋零状”坏死，结果表明，ＴＩＬ细胞的主要作用方式是通过与癌细胞接触进行杀伤。     

Enhanced radioinduced cytotoxicity of cultured human bladder cancer cells using 43°C hyperthermia or anticancer drugs

Summary Using a colony-forming technique and 2 human bladder cancer cell lines, T24 and KK-47, the enhanced radioinduced cytotoxicity in combination with 43°C hyperthermia (HPT) or 4 anticancer drugs; bleomycin (BLM), cis-dichlorodiammineplatinum (II) (CDDP), mitomycin C (MMC), carbazilquinone (CQ), has been studied. In the series of both cell lines, the combination of 43 °C hyperthermia and irradiation resulted in exceedingly enhanced cytotoxicity. This was characterized by a marked decline of the slope of the radiation dose-survival curve as compared with slope in the combination of each of the anticancer drugs and irradiation. Among the 4 anticancer drugs, BLM was thought to be the most promising agent as a potentiator of the radiotherapy, on the basis of a remarkable decrease in the shoulder portion of the radiation dose-survival curve. The other 3 anticancer drugs showed a certain degree of potentiation of radiosensitivity.     

Effector cells in natural cytotoxicity against human bladder cancer cell lines

Summary Human peripheral blood mononuclear cells obtained by ficoll-hypaque sedimentation were depleted of Fc-receptor-bearing (FcR+) cells. Cytotoxicity (direct killing of target cells by effector cells), tested in a 40 h assay, was significantly decreased against a variety of target cells. Tests in which no FcR+ cells could be detected were also positive for natural killing (NK) against a spectrum of target cells from normal donors. NK in this system was mediated by more than one subpopulation of lymphocytes. Monocytes probably did not play a significant role.Decreasing the FcR+ cells in peripheral blood mononuclear cells in patients with bladder cancer and in controls did not reveal specific antitumour activity.This work was supported by NTH grant CA16880 through the National Bladder Cancer Project, and grant CA12800 from the National Cancer Institute     

红细胞生成素对血透患者自然杀伤细胞活性的影响

目的 探讨血透患者自然杀伤细胞(NK)活性与贫血之间的关系。方法 采用LDH释放法,对12例血透接受重组人红细胞生成素(rHuEPO)治疗前后血红蛋白(Hb)水平和NK活性进行观察。结果 经rHuEPO治疗后,患者Hb和NK活性均有明显提高,但rHuEPO对NK细胞并无刺激作用,而红细胞对NK细胞活性有明显的增强作用。结论 血透患者的NK细胞活性低下与贫血有关,rHuEPO提高NK细胞活性的作用是通过改善贫血而非rHuEPO的直接作用。     

Aromatase inhibitors increase the sensitivity of human tumor cells to monocyte-mediated, antibody-dependent cellular cytotoxicity

OBJECTIVE: A randomized, placebo-controlled phase III trial of the breast cancer vaccine Theratope (Biomira Corporation, Edmonton, Alberta, Canada), which expresses the underglycosylated, mucin-associated peptide STn showed that patients treated concomitantly with hormone therapy plus vaccine survived significantly longer than patients treated with hormone therapy plus a control vaccine. The objective of this study was to elucidate a mechanism to explain this effect. METHODS: Tumor cells characterized for expression of estrogen receptor (ER), STn, and Mucin-1 (Muc1) were pretreated (24 hours) with the aromatase inhibitor (AI) formestane, followed by assessment of sensitivity to monocyte-mediated killing in the presence and absence of STn or Muc1 antibodies (Abs) using the (51)Cr-release assay. RESULTS: ER+/STn+/Muc1+ tumor cells cultured in medium were equally sensitive to killing by monocytes in the absence or presence of STn and Muc1 Abs (mean = 54% and 55% cytolysis, respectively, P = not significant). Formestane-pretreated cells showed decreased sensitivity to killing by monocytes in the absence of Abs (mean = 45% cytolysis, P = .07) but significantly increased sensitivity to monocyte-mediated, antibody-dependent cellular cytotoxicity (MM-ADCC) (mean = 65%, P = .003). These effects were not seen with either ER+/STn-/Muc1+ cells or ER-/STn+/Muc1+ cells, indicating the need for both ER and STn positivity of the target tumor cells. CONCLUSIONS: Tumor cells treated with an AI exhibit increased sensitivity to MM-ADCC. The capacity of an AI to "sensitize" tumor cells to this form of antitumor immunity represents a heretofore, undescribed mechanism whereby a hormone-based treatment may collaborate with antigen-specific tumor immunity to produce improved tumor control in vivo in metastatic breast cancer patients.     

Cellular cytotoxicity in transitional cell carcinoma of the human urinary bladder — A summary

Summary The cytotoxicity in vitro of peripheral blood lymphocytes from patients with carcinoma of the urinary bladder (TCC-bladder) against allogeneic target cells from established cell lines was studied by the ⁵¹Cr-release assay. Lymphocytes from both untreated and treated TCC-bladder patients have a significantly elevated mean cytotoxicity to TCC-bladder target cells. Tumour cell destruction by lymphocytes from TCC-bladder patients shows a clear disease related specificity. In TCC-bladder patients a superimposed cytotoxicity exists, probably reflecting reactions against one or several tumour-associated antigens. In treated patients this cytotoxicity may be masked by higher incidence of cross reaction.     

Dialysis fluid cytotoxicity and inhibition of host defence in cultured human mesothelial cells are neutralized rapidly with incubation in the peritoneum

BACKGROUND.: A recent survey puts the global dialysis population at 535 100;of those on peritoneal dialysis, 85% are on continuous ambulatoryperitoneal dialysis. Current hyperosmolar dialysis fluids aretoxic to peritoneal cells and inhibit certain host-defence functions.An alternative preparation, glucose polymer, has recently beendeveloped. METHODS.: Mesothelial cell viability, interleukin-6 and prostacyclin synthesis,after exposure to 7.5% glucose polymer, 1.36% glucose or 3.86%glucose peritoneal dialysis effluent solution was assessed. RESULTS.: In its neat form, at an original pH of 5.4, glucose polymerwas as toxic as hyperosmolar solutions (P <0.01). Synthesisof interleukin-6 and prostacyclin were significantly inhibitedby neat dialysis fluid, (P <0.01). However, after an in vivointraperitoneal incubation of only 15 min the toxicity of allsolutions tested in vitro was lost. CONCLUSFONS.: Despite rapid in situ neutralization of dialysis fluid toxicity,mesothelial injury and inhibition of host-defence function,early in the dialysis cycle, may affect peritoneal physiologygiven the complex network of pathways to which these cells contribute.Although recent trials indicate improved ultrafiltration isachievable with glucose polymer, it is not a biocompatible dialysisfluid in its current manufactured form.     

Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity

BACKGROUND: The susceptibility of porcine endothelial cells (pEC) to human natural killer (NK) cells is related to the failure of human major histocompatibility complex (MHC)-specific killer inhibitory receptors to recognize porcine MHC class I molecules. The aims of this study were (i) to assess the protection of pEC against xenogeneic NK-mediated cytotoxicity afforded by the stable expression of HLA-E single chain trimers (SCT) composed of a canonical HLA-E binding peptide antigen, VMAPRTLIL, the mature human beta2-microglobulin, and the mature HLA-E heavy chain, and (ii) to test whether HLA-E expression on pEC and porcine lymphoblastoid cells affects the adhesion of human NK cells. METHODS: Porcine EC lines expressing different levels of HLA-E SCT were generated by Ca(2)PO(4)-transfection followed by limiting dilution cloning. Surface expression of HLA-E was measured by flow cytometry. Susceptibility of transfected pEC lines against human NK cells was tested in (51)Cr-release cytotoxicity assays. Interactions between human NK cells and HLA-E positive pEC or porcine lymphoblastoid cells were further addressed in adhesion and conjugation assays. RESULTS: The level of protection of pEC from human NK-mediated cytotoxicity correlated with the intensity of surface HLA-E expression. Furthermore, the HLA-E SCT-mediated protection was specifically reversed by blocking the HLA-E specific NK inhibitory receptor CD94/NKG2A. HLA-E expression does neither affect the adhesion of human NK cells to pEC nor the heteroconjugate formation between human NK and porcine 13271.10 cells. CONCLUSIONS: Stable surface expression of HLA-E on pEC was achieved in the absence of extrinsic peptide pulsing and provided partial protection from human NK cytotoxicity. Though insufficient to inhibit xenogeneic NK cell reactivity completely, transgenic HLA-E expression on pig organs might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.     

Patterns of human tumor-infiltrating lymphocytes in 120 human cancers.

Tumor-infiltrating lymphocytes from 120 samples of human cancers, including melanoma, renal cell carcinoma, breast cancer, sarcoma, and colon cancer, were examined. The percentage of lymphocytes recovered from the cancer varied widely; that of renal cell carcinoma was higher than that of breast or colon cancer (65% vs 45%), which was higher than that of melanomas or sarcomas (30% to 35%). The types of lymphocytes before and after interleukin 2 activation showed specific patterns. CD4+ helper T cells predominated in all tumors except melanomas, which had more CD8+ cytotoxic T cells. CD16+ natural killer cells were recovered in renal cell carcinoma and sarcomas. Three different cytotoxic lymphocytes were identified among interleukin 2-activated tumor-infiltrating lymphocytes: (1) CD3+ CD16- cytotoxic T lymphocytes with cytotoxicity restricted to autologous tumor cells in melanomas, (2) CD3-CD16+ natural killer cells with vigorous major histocompatibility complex-nonrestricted cytotoxicity in renal cell carcinoma, and (3) CD3+ CD16- T cells with modest levels of major histocompatibility complex-nonstricted cytotoxicity in all cancers except melanomas. Thus, there was considerable diversity of tumor-infiltrating lymphocytes among these histologically distinct tumors with respect to magnitude of lymphocyte infiltration, phenotypic expression, and functional capacity.     

人肝癌裸鼠NKG2D受体的表达及对NK细胞毒活性的影响

目的 通过研究原发性肝癌中NK细胞的细胞毒活性变化及其活化性受体NKG2D的表达,观察肝脏和脾脏组织病理变化,探讨NK细胞免疫抑制机制,为原发性肝癌的免疫治疗提供理论依据。方法 ①建立人肝癌裸鼠皮下-肝原位移植瘤模型：先用人肝癌细胞株Hep3B接种于裸鼠皮下,形成皮下移植瘤,然后用此移植瘤组织再接种于裸鼠肝内,建立肝原位移植瘤模型（间接肝原位移植瘤模型）;②检测裸鼠原发性肝癌对NK细胞免疫活性的影响：分离裸鼠外周血、肝脏及脾脏组织NK细胞,LDH方法检测NK细胞的细胞毒活性,流式细胞技术检测不同组织NK细胞NKG2D表达百分率,H-E染色观察肝脏移植瘤对肝脏和脾脏淋巴细胞的影响。结果 ①外周血、肝脏及脾脏的NK细胞毒活性及NKG2D受体表达随着肿瘤生长逐渐下降,其中肝脏NK细胞毒活性及NKG2D表达下降明显;②荷瘤裸鼠肝脏癌组织与皮下瘤相同,癌组织异型性与时间呈正相关,脾脏淋巴小结在第4周增生明显,第8周小梁结构增多。结论 原发性肝癌通过下调NKG2D的表达,对NK细胞有免疫抑制作用,这种作用主要发生在肝脏,但对外周血和脾脏也有影响。