4-1BB信号拮抗肿瘤细胞培养上清诱导的树突状细胞凋亡

4-1BB signal inhibits the dendritic cell apoptosis induced by supernatants of the tumor cell cultures

为研究4-1BB信号拮抗肿瘤细胞培养上清诱导的树突状细胞(DC)凋亡的作用,采用rmGM-CSF联合rmIL-4诱导小鼠骨髓细胞分化为未成熟树突状细胞(imDC),分别经rmTNF-α、4-1BB激发型单克隆抗体(2A)或TNF-α联合2A激发DC成熟。未成熟或成熟DC分别与不同浓度的肿瘤培养上清混合培养。流式细胞仪测定DC表面CD80、CD86的表达,3H-TdR掺入法测定DC激发T细胞活化增殖能力;JAM法测定未成熟或成熟DC与肿瘤上清混合培养后DNA片断形成率。结果显示:经2A或TNF-α刺激后,DC激发T细胞活化的能力显著提高;5%、10%、20%、50%小鼠肿瘤细胞株A20或B16培养上清与未成熟DC共育24h后,DNA片断形成率分别为16%、29%、41%、51%和20%、27%、37%、58%,且该效应具有时间依赖性;DC经2A或TNF-α激发48h后,不同浓度肿瘤培养上清诱导DNA片断形成率显著降低。因此,肿瘤细胞可以通过分泌可溶性因子诱导未成熟DC凋亡;4-1BBmAb能促进DC的发育成熟,并能有效地抑制肿瘤细胞培养上清诱导的DC凋亡。

The role of 4-1BB signal to inhibit the dendritic cell（DC） apoptosis was investigated by observation of the inhibition of DC apoptosis by supernatants of the tumor cell culture in the present study. Mouse DC were generated in vitro from mouse bone marrow ceils with recombinant mouse GM-CSF （rmGM-CSF） and recombinant mouse IL-4（rmIL-4）, and the maturation of DC was induced by TNF-α and 4-1BB mAb（2A） separately or in combination. Immature or mature DC were co-cultivated with supernatants of the tumor cell cultures, and the expressions of CD80 and CD86 on DC were measured by FCM. The ability of DC to prime T cell proliferation was analyzed by ^3H-TdR incorporation, and JAM assay was used to determine the DNA fragmentation of DC which was co-cuhivated with supernatants of tumor cell cultures. The experimental results showed that the ability of DC to prime T cell proliferation was strikingly improved after stimulation with TNF-α or 2A（5μg）, however, the expressions of CD80 and CD86 on DC were not significantly up-regulated. Meanwhile, the DNA fragmentation of immature DC co-cultivated with 5%, 10%, 20%, 50% of supernatants of mouse tumor cells A20 or B16 cultures were 16%, 29%, 41%, 51% as well as 20%, 27%, 37%, 58% respectively with significant time-dependence correlation. When pre treated with TNF-α or 2A for 48 hours, the DNA fragmentation induced by the supernatants of tumor cell A20 or B16 cultures was significantly reduced. Our resuhs demonstrate that tumor cells can induce apoptosis of DC by secretion of soluble factors, and the corss-linking of 4-1BB signal induces maturation of DC and resistance to tumor cell-induced apoptosis as well.