VTCN1 regulation of MHC in early human placental development

植入围产期胎盘发育不当可能导致母胎疾病(早产、宫内 生长迟缓、先兆子痫)和高妊娠率。由于这些事件发生在临床发现怀孕之前，对人类胎盘早期发育的研究在伦理和后勤方面受到限制。该项目使用创新的人类胚胎干细胞(HESC)来源的模型在体外研究原始滋养层细胞。胎盘类似于癌症，因为植入中的胎儿和正在发展的癌症都涉及宿主组织入侵、大量细胞增殖、新生血管和即时免疫微环境的改变。植入胎儿的胎盘是仅有的几个主要组织相容性复合体(MHC)I类(MHC-I)移植抗原、人类白细胞抗原(HLA)-A和-B出现生理性下调的非癌症组织之一。虽然MHC II类调控的正调控元件已被描述，但对包括胎盘在内的非癌组织中MHC I类表达的调控知之甚少。人类胚胎干细胞(HESCs)已被用于一种新的人类胎盘发育阶段的体外模型，该模型发生在临床检测怀孕之前。通过使用该模型，已经识别了几个在怀孕早期选择性高水平表达的mRNAs和蛋白质。在这些被研究的人中，似乎都在入侵过程中发挥了作用。值得注意的是，在我们的早期滋养层细胞模型中，其中一种蛋白-V-SET结构域包含T细胞激活抑制物1(VTCN1)的siRNA沉默强烈上调了MHC-I类人类白细胞抗原(HLAA)-A和-B的表达，而这两种抗原通常在人类滋养层细胞中不表达，并略微上调了正常驻留的HLAC，但对胎盘特异性HLAG的表达没有影响。VTCN1被认为是调节T细胞受体与MHC分子相互作用的B7超家族中的一员。据推测，VTCN1neo的表达影响癌细胞逃避宿主免疫检测的能力。这些结果突出了我们的体外模型在填补几个重要的知识空白方面的有效性。体外模拟围植入期胎盘发育的独特能力将使我们能够剖析人类滋养层细胞(TB)上MHC-I表达的调控，特别是验证Tb缺乏HLA-A和-B表达是由VTCN1通过主要间接机制控制的假设。该提案有三个目的：1)通过VTCN1的下调和过表达，在包括hESC来源的原始滋养层细胞在内的各种TB模型中展示VTCN1对MHC-I类的经典抑制；2)确定与VTCN1相关的TB MHC-I相关变化所涉及的转录途径和转录后调控 3)在早期结核的体外细胞模型中寻找VTCN1的蛋白伙伴，以发现和证实VTCN1调节MHC-I类分子表达的途径。这些研究将应用于常见胎盘异常疾病的起源以及对癌症免疫学的理解。

Improper peri-implantation placental development may contribute to feto-maternal diseases (preterm birth, intrauterine growth retardation, preeclampsia) and high rates of pregnancy loss. Since these events occur prior to clinical detection of pregnancy, studies of very early human placental development are ethically and logistically constrained. The proposed project uses an innovative human embryonic stem cell (hESC)-derived model to study primitive trophoblast in vitro. The placenta resembles a cancer in that an implanting fetus and a developing cancer both involve host tissue invasion, massive cellular proliferation, neo-vascularization, and alterations in the immediate immune microenvironment. The placenta of the implanting fetus is one of the only non-cancerous tissues that displays physiologic downregulation of the classical major histocompatibility complex (MHC) class I (MHC-I) transplantation antigens, human leukocyte antigens (HLA)-A and -B. While positive regulatory elements for MHC class II regulation are described, little is known about such regulation of MHC class I expression in noncancerous tissues, including the placenta. Human embryonic stem cells (hESCs) have been used in a novel in vitro model of the stages of human placental development that occur prior to clinical detection of pregnancy. Several mRNAs and proteins that are selectively expressed at high levels in early pregnancy have been identified by using the model. Of those studied, all appear to play roles in invasion. Remarkably, siRNA silencing of one of these proteins, V-Set Domain Containing T Cell Activation Inhibitor 1 (VTCN1), in our model for early trophoblast strongly up-regulated the expression of the MHC class I human leukocyte antigens (HLA)-A and -B, which are normally not expressed in human trophoblast, slightly up-regulated the normally resident HLA-C, but had no effect on expression of the placenta-specific HLA-G. VTCN1 is recognized as a member of the B7 superfamily of cell-surface co-receptors that regulate T cell receptor interactions with MHC molecules. It has been postulated that VTCN1 neo-expression affects cancer cells’ ability to evade host immune detection. These results highlight the efficacy of our in vitro models for filling several important knowledge gaps. The unique ability to model peri-implantation placental development in vitro will enable dissection of the control of MHC-I expression on human trophoblast (TB), particularly testing the hypothesis that the lack of expression of HLA-A and -B by TB is controlled by VTCN1 through largely indirect mechanisms. There are three aims to the proposal: 1) Demonstrate classical MHC class I suppression by VTCN1 in various TB models, including hESC-derived primitive trophoblast, by using VTCN1 knock-down and overexpression; 2) Define the transcriptional pathways and transcriptional and immediate post-transcriptional regulation involved in VTCN1-related alterations in TB MHC-I expression and 3) Identify protein partners of VTCN1 in in vitro cell models of early TB to discover and confirm pathways involved in VTCN1 regulation of MHC class I expression. These studies will have application to the origins of common disorders of abnormal placentation and to the understanding of cancer immunology.