Fas配体的过表达并未赋予胰腺β肿瘤细胞系（βTC - 3）免疫豁免权。 - Suppr

Okamoto S, Takamizawa S, Bishop W, Wen J, Kimura K, Sandler A

Department of Surgery, University of Iowa, Iowa City, Iowa, USA.

J Surg Res. 1999 Jun 1;84(1):77-81. doi: 10.1006/jsre.1999.5613.

BACKGROUND

Fas ligand (Fas-L) is thought to provide immune privilege to specific tissues and tumors by inducing an apoptotic signal of cytotoxic T cells expressing its Fas receptor. Purpose. The purpose of this work was to evaluate whether an immortalized insulin-secreting cell line (betaTC-3) gains immune privilege by inducing overexpression of Fas-L.

METHODS

A lipofection technique was used to transfect a betaTC-3 tumor cell line with a plasmid (pcDNA3.1/Zeo) carrying the Fas-L gene and a zeocin resistance gene. Insertion of Fas-L into betaTC was characterized by reverse transcription polymerase chain reaction (RT-PCR) and the ability of transfectants (betaTC-3/Fas-L) to induce apoptosis of Fas-sensitive T cells. Transfectants and control cells were tested for insulin secretion following which 1 x 10(6) insulin-secreting betaTC-3 and betaTC-3/Fas-L cells were subcutaneously implanted into syngeneic, allogeneic, and Fas mutant (lpr) syngeneic mice. Survival of the insulin-secreting cells was then determined by monitoring serum glucose levels in recipients.

RESULTS

Successful transfection of vector resistance gene was achieved in the transfected betaTC-3 cells, which was confirmed by zeocin resistance. RT-PCR in resistant Fas-L clones confirmed the transcription of Fas-L, which was absent in controls. Fas-L transfectants induced 20 +/- 4.2% apoptosis of Fas-sensitive T cells, while controls induced 3.47 +/- 2.3% by flow cytometry (P = 0.04, n = 3). Insulin secretion was equivalent in both betaTC-3 and betaTC-3/Fas-L cells. Syngeneic mice implanted with control betaTC-3 cells died within 3 weeks from hypoglycemia due to overgrowth of betaTC-3 tumor. Implanted Fas-L transfected betaTC-3 cells were killed and had no effect on glycemic status except in Fas mutant hosts, where tumors formed in two of three mice.

CONCLUSIONS

Despite the ability of transfected betaTC-3 cells to induce apoptosis of T cells in vitro, expression of Fas-L provided no immune privilege to these cells in vivo, but paradoxically induced killing of betaTC-3 cells even in syngeneic hosts.

背景

Fas配体（Fas-L）被认为通过诱导表达其Fas受体的细胞毒性T细胞产生凋亡信号，从而赋予特定组织和肿瘤免疫豁免权。目的：本研究旨在评估永生化胰岛素分泌细胞系（betaTC-3）是否通过诱导Fas-L的过表达而获得免疫豁免权。

方法

采用脂质体转染技术，用携带Fas-L基因和博来霉素抗性基因的质粒（pcDNA3.1/Zeo）转染betaTC-3肿瘤细胞系。通过逆转录聚合酶链反应（RT-PCR）和转染细胞（betaTC-3/Fas-L）诱导Fas敏感T细胞凋亡的能力来鉴定Fas-L在betaTC中的插入情况。检测转染细胞和对照细胞的胰岛素分泌，然后将1×10⁶个分泌胰岛素的betaTC-3和betaTC-3/Fas-L细胞皮下植入同基因、异基因和Fas突变（lpr）同基因小鼠体内。通过监测受体的血糖水平来确定胰岛素分泌细胞的存活情况。

结果

在转染的betaTC-3细胞中成功实现了载体抗性基因的转染，这通过博来霉素抗性得到证实。抗性Fas-L克隆中的RT-PCR证实了Fas-L的转录，而对照中不存在这种转录。通过流式细胞术检测，Fas-L转染细胞诱导Fas敏感T细胞凋亡20±4.2%，而对照诱导凋亡3.47±2.3%（P=0.04，n=3）。betaTC-3和betaTC-3/Fas-L细胞的胰岛素分泌相当。植入对照betaTC-3细胞的同基因小鼠在3周内死于低血糖，原因是betaTC-3肿瘤过度生长。植入Fas-L转染的betaTC-3细胞被杀死，除了在Fas突变宿主中，三只小鼠中有两只形成了肿瘤外，对血糖状态没有影响。