Antigen-specific immune-regulation by genetically modified ES cell-derived dendritic cells

我们开发了一种从小鼠胚胎干细胞(ES)中培养树突状细胞(DC)的方法。后5天在含GM-CSF的OP9饲养层上培养ES细胞10天。将得到的ES细胞来源的漂浮细胞在含有GM-CSF的培养液中培养，不含饲养细胞。7d左右出现形状不规则、有突起的漂浮细胞，表达MHC II类、CD11c、CD80和CD86。它们具有刺激同种异体MLR和加工蛋白抗原并将其呈递给T细胞的能力。我们将其命名为ES-DC，其功能与骨髓细胞来源的DC相当。ES-DC的基因修饰可以通过将ES细胞导入并随后向DC分化来实现。ES-DC以不变链为基础的表位呈递载体在体外非常有效地在MHC II类分子的背景下呈现PCC表位。我们用…激发卵清蛋白特异性细胞毒T淋巴细胞更多注射ES-DC的小鼠引入OVA表达载体，展示了通过基因工程表达抗原的ES-DC免疫。为了提高ES-DC在体内激活T细胞的能力，我们产生了与OVA一起表达趋化因子的双转基因ES-DC。表达OVA的DC联合SLC或Mig免疫对表达OVA的肿瘤细胞的保护作用比单独使用OVA更有效。双转基因ES-DC可显著减轻髓鞘少突胶质细胞糖蛋白(MOG)多肽诱导的实验性自身免疫性脑脊髓炎(EAE)的严重程度，同时表达肿瘤坏死因子相关的凋亡诱导配体(TRAIL)或程序性死亡1配体(PD-L1)。作为一种医学技术，通过转移呈现抗原和同时表达免疫调节分子的双转基因ES-DC进行抗原特异性免疫调节是一种很有前途的技术。较少

We developed a method to generate dendritic cells (DC) from mouse embryonic stem (ES) cells. We cultured ES cells for 10 days on feeder cell layer of OP9 in the presence of GM-CSF in the latter 5 days. The resultant ES cell-derived floating cells were cultured without feeder cells in culture medium containing GM-CSF. In about 7 days, irregularly shaped floating cells with protrusions appeared and they expressed MHC class II, CD11c, CD80, and CD86. They had capacity to stimulate allogeneic MLR and to process and present protein antigen to T cells. We designated them ES-DCs, and the functions were comparable to those of DC generated from bone marrow cells. Genetic modification of ES-DC can readily be done by transfection of ES cells and subsequent their differentiation to DC. ES-DC introduced with an invariant chain-based epitope-presenting vector presented a PCC epitope in the context of MHC class II molecules very efficiently in vitro. We primed OVA-specific cytotoxic T lymphocytes by … More injecting mice with ES-DC introduced with an OVA-expression vector, demonstrating immunization with ES-DC genetically engineered to express antigen. To improve the capacity of ES-DC to prime T cells in vivo, we generated double-transfectant ES-DC expressing a chemokine along with OVA. Immunization with DC expressing OVA plus SLC or Mig provided protection from OVA-expressing tumor cells more potently than that with OVA only. Severity of myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis (EAE) was significantly reduced in mice treated with the double-transfectant ES-DC, presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TNF-related apoptosis-inducing ligand (TRAIL) or Programmed Death-1 ligand (PD-L1). Antigen-specific imnmuno-modulation by transfer of double-transfectant ES-DC presenting antigen and simultaneously expressing immune-regulatory molecules would be realized in the future as a medical technology. Less