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

主要组织相容性复合体（MHC）、人类组织相容性白细胞抗原（HLA）复合体和次要组织相容性抗原（mHA）的高度可变性显著有助于引起同种异体角膜移植后的免疫应答，并且可能导致急性或慢性排斥反应的发展。此外，通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降低角膜移植物上的HLA I类和II类表达，以降低角膜移植术后移植物排斥反应的风险。这种方法代表了一种新的治疗概念，可能有助于防止移植排斥反应。RNA干扰技术将用于诱导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，而且还有助于消除来自其他多态性分子的次要组织相容性抗原的呈递。以前，我们证明了以永久的方式沉默HLA表达可以有效地消除针对同种异体细胞的有害免疫应答，并显着降低同种异体移植后细胞的存活率。此外，我们已经显示了在人角膜内皮的原始3D结构中沉默HLA I类表达的可行性。在拟定研究中，将在小鼠角膜移植术移植排斥模型（BALB/c和C3 H）中进行移植和监测操作和非操作角膜的排斥反应。慢病毒系统将用于递送靶向小鼠β 2-微球蛋白或靶向H2-E α链（H2-Ea）的短发夹RNA（shRNA）。将在规定的时间点评估抗原呈递细胞（APC）和排斥标志物的存在。此外，将通过显微镜分析沉默MHC表达和角膜移植后的形态学变化。此外，还将评价角膜中的细胞凋亡和坏死。本研究提出了一种新的策略，以克服HLA错配所造成的局限性和缺点，在角膜移植。

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.