Section of Transplantation Immunology, Department of Surgery, Medical University of Vienna, Vienna, Austria.
Cardiac Surgery Research Laboratory, Division of Cardiac Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria.
Front Immunol. 2020 Dec 8;11:586856. doi: 10.3389/fimmu.2020.586856. eCollection 2020.
Humoral immunity is a major barrier limiting long-term outcome after organ transplantation. Especially, the production of antibodies directed against donor HLA/MHC antigens (i.e. donor-specific antibodies (DSA)) leading to antibody-mediated rejection (ABMR) is considered to be a major factor negatively affecting allograft survival. DSAs of the IgG isotype are routinely measured in transplant patients. However, not all patients diagnosed with IgG-DSA develop ABMR events. Therefore, research in better understanding the mechanisms of ABMR is of great importance. We recently demonstrated the production of MHC-specific IgE upon allograft rejection in mice and in transplant patients. IgE is classically connected with allergy and is known to be important for the humoral defense against helminths and worms. However, its role in autoimmune diseases and cancer has been reported recently as well. The concentration of IgE in blood is extremely low compared to other antibody isotypes. Therefore, detection of MHC-specific IgE from serum requires methods of high sensitivity. Since MHC-specific IgG-typically present at much higher serum levels-develops as well, high specificity is also required of IgE detection methods. In the murine model we developed an enzyme linked immunosorbent assay (ELISA) using MHC monomers for measurement of MHC-specific IgE, allowing us to distinguish between specificities of antibodies against different class I and class II antigens. For measurement of functional activity of MHC-specific IgE , a release assay using a rat basophil cell line (RBL-2H3) was established. For functional analysis of MHC-specific IgE , a cutaneous hypersensitivity reaction assay was adapted for this purpose using MHC monomers. Humanized RBL-2H3 cells transfected with cDNA coding for the human-high affinity IgE receptor were used for functionality measurement of donor-specific IgE in sensitized transplant patients. For detection of HLA-specific IgE, a bead assay was adapted, using beads expressing single HLA antigens. The aim of this publication is to demonstrate currently established methods for the detection and characterization of MHC-specific IgE in the murine and human setting.
体液免疫是限制器官移植后长期效果的主要障碍。特别是，针对供体 HLA/MHC 抗原（即供体特异性抗体 (DSA)）产生的抗体导致抗体介导的排斥反应 (ABMR)，被认为是对移植物存活产生负面影响的主要因素。移植患者中常规测量 IgG 同种型的 DSA。然而，并非所有诊断出 IgG-DSA 的患者都会发生 ABMR 事件。因此，更好地了解 ABMR 机制的研究非常重要。我们最近证明了在小鼠和移植患者的同种异体排斥反应中产生了 MHC 特异性 IgE。IgE 经典上与过敏有关，并且已知对针对寄生虫和蠕虫的体液防御很重要。然而，最近也报道了其在自身免疫性疾病和癌症中的作用。与其他抗体同种型相比，IgE 在血液中的浓度极低。因此，需要高灵敏度的方法来检测血清中的 MHC 特异性 IgE。由于 MHC 特异性 IgG 通常也存在于更高的血清水平，因此也需要 IgE 检测方法具有高度特异性。在我们开发的小鼠模型中，我们使用 MHC 单体开发了一种酶联免疫吸附测定 (ELISA) 来测量 MHC 特异性 IgE，使我们能够区分针对不同 I 类和 II 类抗原的抗体的特异性。为了测量 MHC 特异性 IgE 的功能活性，建立了使用大鼠嗜碱性细胞系 (RBL-2H3) 的释放测定法。为了进行 MHC 特异性 IgE 的功能分析，为此目的适应了使用 MHC 单体的皮肤过敏反应测定法。用人源化 RBL-2H3 细胞转染编码人高亲和力 IgE 受体的 cDNA 用于检测致敏移植患者中的供体特异性 IgE。为了检测 HLA 特异性 IgE，适应了使用表达单个 HLA 抗原的珠粒的珠粒测定法。本出版物的目的是证明目前在小鼠和人类环境中检测和表征 MHC 特异性 IgE 的方法。
