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）生成树突状细胞（DC）的方法。后5天在GM-CSF存在下，在OP9的饲养细胞层上培养ES细胞10天。在含GM-CSF的培养基中培养无饲养细胞的ES细胞衍生的漂浮细胞。约7 d时，出现形状不规则且有突出物的漂浮细胞，表达MHCⅱ类、CD11c、CD80和CD86。它们有能力刺激同种异体MLR，加工并向T细胞呈递蛋白抗原。我们将其命名为es -DC，其功能与骨髓细胞产生的DC相当。ES-DC的遗传修饰可以很容易地通过转染胚胎干细胞并随后将其分化为DC来完成。引入基于不变链的表位提呈载体的ES-DC在体外非常有效地表达了MHC II类分子背景下的PCC表位。我们通过向小鼠注射含有ova表达载体的ES-DC，引发ova特异性细胞毒性T淋巴细胞，证明了ES-DC基因工程表达抗原的免疫作用。为了提高ES-DC在体内诱导T细胞的能力，我们制备了双转染ES-DC，表达一种趋化因子和OVA。表达OVA的DC与SLC或Mig联合免疫对表达OVA的肿瘤细胞的保护作用比仅用OVA更强。双转染ES-DC治疗小鼠髓鞘少突胶质细胞糖蛋白（MOG）肽诱导的实验性自身免疫性脑脊髓炎（EAE）的严重程度显著降低，在MHC II类分子背景下呈现髓鞘少突胶质细胞糖蛋白（MOG）肽，同时表达tnf相关凋亡诱导配体（TRAIL）或程序性死亡-1配体（PD-L1）。通过传递双转染ES-DC递呈抗原并同时表达免疫调节分子来实现抗原特异性免疫调节将在未来成为一种医学技术。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