Major histocompatibility complex-peptidome in granulomatosis with polyangiitis

肉芽肿病伴多血管炎（GPA）是一种可能危及生命的慢性炎症性自身免疫性疾病。该病以全身性坏死性血管炎和血管外肉芽肿性炎症为特征。它与抗蛋白酶3（PR 3-ANCA）的高度特异性自身抗体相关。我们自己以前的研究表明，自身抗原和病原体驱动的发病机制的GPA。本授权申请中申请的研究目的是通过分析MHC-肽组（即通过外周血单核细胞和多形核细胞细胞表面上的主要组织相容性复合物（MHC）呈递给适应性免疫系统细胞的肽）来鉴定GPA的抗原触发剂。使用生物信息学算法，将来自患者的MHC-肽组的肽分配给其源蛋白，例如分配给自身抗原PR 3和其他内源性蛋白以及病原体衍生的蛋白，并且将分析患者和健康人之间的MHC-肽组的差异。此外，将测试GPA特异性MHC肽组特征的肽的免疫原性，即用这些肽刺激T细胞后触发免疫反应的能力。通过从纯化的MHC-I类和II类复合物中洗脱肽，随后通过液相色谱-串联质谱法（LC-MS/MS）分析洗脱的肽，进行MHC-肽组的分析。使用ELISpot筛选肽刺激后的T细胞应答，并使用流式细胞术对反应性T细胞进行表型和功能表征。MHC-肽组的分析将首次能够鉴定GPA中体内呈递给适应性免疫系统的肽序列。这些研究将导致未来在抗原触发的T细胞应答水平上的靶向治疗干预的发展。

Granulomatosis with polyangiitis (GPA) is a potentially life-threatening chronic inflammatory autoimmune disease. The disease is characterized by systemic necrotizing vasculitis and ex-travascular granulomatous inflammation. It is associated with highly specific autoantibodies against proteinase 3 (PR3-ANCA). Our own previous studies suggest an autoantigen- and pathogen-driven pathogenesis of GPA. The aim of the investigations applied for in this grant application is the identification of antigenic triggers of GPA by analyzing the MHC-peptidome, i.e. peptides that are presented to the cells of the adaptive immune system via the major his-tocompatibility complex (MHC) on the cell surface of mononuclear and polymorphonuclear cells from the peripheral blood. Using bioinformatic algorithms, peptides of the MHC-peptidome from patients will be assigned to their source proteins, e.g. to the autoantigen PR3 and other endogenous proteins as well as pathogen-derived proteins, and differences in the MHC-peptidome between patients and healthy persons will be analyzed. In addition, peptides of the GPA-specific MHC-peptidome signature will be tested for their immunogenicity, i.e. the ability to trigger an immune response after stimulating T-cells with these peptides. Analysis of the MHC-peptidome is performed by means of peptide elution from purified MHC-class I and II complexes and subsequent analysis of the eluted peptides by means of liquid chromatog-raphy-coupled tandem mass spectrometry (LC-MS/MS). T-cell responses following peptide stimulation are screened using ELISpot, and reactive T-cells will be phenotypically and func-tionally characterized using flow cytometry. Analysis of the MHC-peptidome will for the first time enable the identification of peptide sequences presented in vivo to the adaptive immune system in GPA. These investigations will lead to the development of future targeted therapeu-tic interventions at the level of the antigen-triggered T-cell response.