Suppression of MHC expression on corneal cells to prevent immune rejection after allogeneic transplantation: Experimental investigation in a mouse model

同种异体角膜移植后，主要组织相容性复合体(MHC)、人类组织相容性白细胞抗原(HLA)和次要组织相容性抗原(MHA)的高度变异性导致免疫应答，并可能导致急性或慢性排斥反应的发生。此外，经常不可避免的第二次角膜移植总是与移植物排斥反应的风险增加有关。该项目旨在永久性地减少角膜移植后人类白细胞抗原I类和II类的表达，以降低角膜移植后移植排斥反应的风险。这种方法代表了一种新的治疗理念，可能有助于预防移植物排斥反应。将利用RNA干扰技术诱导MHC表达下调。这种方法不仅沉默了MHC分子在细胞表面的表达，而且有助于消除来自其他多态分子的次要组织相容性抗原的存在。在此之前，我们已经证明，永久沉默人类白细胞抗原的表达可以有效地消除针对同种异体细胞的有害免疫反应，并显著延长同种异体移植后细胞的存活时间。此外，我们还证明了在人角膜内皮细胞原有的三维结构中沉默人类白细胞抗原I类分子表达的可行性。在这项拟议的研究中，将在小鼠角膜移植排斥反应模型(BALB/c和C3H)中进行手术和非手术角膜的移植和排斥反应的监测。慢病毒系统将用于传递针对小鼠β2-微球蛋白或针对H2-E的阿尔法链(H2-EA)的短发夹状RNA(ShRNA)。抗原提呈细胞(APC)和排斥标志物的存在将在确定的时间点进行评估。此外，抑制MHC表达和角膜移植后的形态学变化将在显微镜下分析。此外，还将评估角膜中的细胞凋亡和坏死。这项研究提出了一种新的策略，以克服角膜移植中由于人类白细胞抗原不匹配而造成的局限性和缺陷。

The high variability of the major histocompatibility complex (MHC), in humans histocompatibility leukocyte antigen (HLA) complex, and of the minor histocompatibility antigens (mHA) significantly contributes to elicit an immune response after allogeneic cornea transplantation, and may lead to the development of acute or chronic rejection. In addition, an often inevitable second cornea transplantation is always associated with an increased risk of graft rejection. This project aims to permanently reduce HLA class I and class II expression on cornea transplants to decrease the risk of graft rejection after keratoplasty. This method represents a new therapeutic concept and may contribute to prevent graft rejection. RNA-interference technology will be used to induce the downregulation of MHC expression. This method not only silences the expression of MHC molecules on the cell surface but also contributes to abrogate the presentation of minor histocompatibility antigens derived from other polymorphic molecules. Previously, we demonstrated that silencing HLA expression in a permanent manner can efficiently abrogate a deleterious immune response against allogeneic cells and significantly prolongs the survival of cells after allogeneic transplantation. In addition we have shown the feasibility of silencing HLA class I expression in human cornea endothelium in its original 3D-structure. In the proposed study, the transplantation and monitoring of rejection of manipulated and non-manipulated corneas will be performed in a mouse keratoplasty model for graft rejection (BALB/c and C3H). A lentiviral system will be used for the delivery of short hairpin RNA (shRNA) targeting mouse beta2-microglobulin or targeting the alpha chain of H2-E (H2-Ea). The presence of antigen presenting cells (APCs) and rejection markers will be assessed at defined time points. In addition, morphological changes after silencing MHC expression and cornea transplantation will be analysed by microscopy. Also, apoptosis and necrosis in the corneas will be evaluated. This study presents a new strategy to overcome the limitations and drawbacks caused by HLA mismatches in keratoplasty.