Fas-L (CD95-Ligand) 屬於腫瘤壞死因子家族的一員，Fas-L能作用在帶有Fas的細胞上並經由Fas/Fas-L作用導致細胞程式化凋亡。Fas/Fas-L的系統在抑制免疫細胞的反應中扮演一個重要的角色，Fas-L不僅表現在活化的T細胞以及自然殺手細胞上，也表現在不同來源的腫瘤細胞上。腫瘤細胞可能借由表現Fas-L來躲避免疫細胞的攻擊。本實驗使用兩株人類腦神經膠質瘤細胞細胞株為U373 MG及U118MG，並且利用Fas-L ribozyme抑制腦神經膠質瘤細胞株表現Fas-L。在免疫細胞包括：Jurkat、Molt-4、U937、HL-60、neutrophils-like以及人體周邊血液neutrophils等細胞與U373MG及U118MG衍生細胞株混合培養的實驗中。結果顯示腫瘤表現Fas-L可以影響不同免疫細胞產生不同量的IL-10、IFN-g及TNF-a mRNA。此外，Jurkat 細胞與Fas-L表現量較高的腦神經膠質瘤細胞培養或是以anti-Fas 抗體CH-11刺激後，IL-10的mRNA亦會增加。反之，以ZB4阻斷Jurkat細胞的Fas後，Jurkat細胞IL-10的表現便不受混合培養以及CH-11的影響。另外，caspase抑制劑Z-VAD-fmk及Z-IETD-fmk可以抑制Fas訊息所誘發的IL-10的表現。當neutrophils與腦神經膠質瘤衍生細胞混合培養時，neutrophils會經由Fas/Fas-L的作用使腦神經膠質瘤衍生細胞產生IL-6以及IL-8細胞激素，也因此維持了neutrophils的存活。但是其他免疫細胞的存活率並不因為與腦神經膠質瘤衍生細胞混合培養而有太大的變化。綜合以上的結果，我們認為Fas/Fas-L的作用不只是造成細胞死亡，也可能透過這樣的機制調節免疫細胞產生細胞激素。

Fas-L (CD95-Ligand) is a member of the TNF family. Engagement of Fas-L with Fas induces apoptosis in Fas-bearing cells. Fas/Fas-L system plays a crucial role in down-regulation of immune response. Fas-L is not only expressed on activated T and NK cells but also on tumors of diverse cellular origins. Recent reports suggested that tumor cells could evade immune attack through the Fas-L on the tumor cell surface (tumor Fas-L). To investigate how tumor Fas-L affects the immune response, we used in vitro co-culture system. Two human cell lines, U373MG and U118MG, were stably transfected with Fas-L- ribozyme plasmid or pEGFP-N1 control plasmid. Fas-Lribozyme was able to inhibit the expression of Fas-L in glioblastoma cell lines. To explore the effect of Fas-L, U373MG- or U118MG-derived cells were co-cultured with different immune cells including Jurkat cells, Molt-4 cells, U937 Cells, HL-60 cells, neutrophil-like cells and human peripheral circulation neutrophils. Transcripts of cytokines in immune cells were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Viability of target cells was determined by PI or MC540 staining.Tumor Fas-L on tumors affected the expressions of IFN-γ, IL-10 and TNF-a in immune cells to various levers. In addition, the IL-10 production was induced in Jurkat cells co-cultured with glioma cells having high level of Fas-L or treated with agonistic antibody CH-11 recognizing Fas. In contract, after neutrolizing the Fas on Jurket cells with antagonistic antibody ZB4, the induction of IL-10 production by CH-11 or tumor Fas-L upon co-culture glioma cells was blocked. Caspase inhibitor, Z-VAD-fmk and Z-IETD-fmk were also potent to inhibit the induction of IL-10 in Jurkat cells. Besides, we also found that the IL-6 and IL-8 production glioma cells would be enhanced via the interaction between Fas-L on neutrophils and Fas on glioma cells in this co-culture system. The coculture-induced IL-6/IL-8 of glioma cells could improve the viability of neutrophils. Other immune cells used in this study showed no significant change in cell viability, when they were in co-culture with glioma cells, carrying Fas-L –ribozyme or not. Taken together, our results suggest that the Fas/Fas-L interaction would not only induce cell death but also modulate the cytokine production on immune cells.

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