Major histocompatibility complex-peptidome in granulomatosis with polyangiitis

肉芽肿合并多血管炎(GPA)是一种潜在威胁生命的慢性炎症性自身免疫病。本病以全身性坏死性脉管炎和血管外肉芽肿性炎症为特征。它与高度特异的抗蛋白酶3自身抗体(PR3-ANCA)有关。我们以前的研究表明GPA是由自身抗原和病原体驱动的发病机制。这项资助申请的研究目的是通过分析MHC-多肽组来识别GPA的抗原触发因素，即通过外周血单核细胞和多形核细胞表面的主要His-to相容复合体(MHC)呈现给适应性免疫系统细胞的多肽。利用生物信息学算法，患者的MHC-多肽将被分配到其来源蛋白，例如自身抗原PR3和其他内源性蛋白以及病原体来源的蛋白，并将分析患者和健康人MHC-多肽组的差异。此外，具有GPA特异性MHC-多肽组特征的多肽将被测试其免疫原性，即用这些多肽刺激T细胞后触发免疫反应的能力。通过从纯化的MHC-I和II类化合物中洗脱多肽，然后用LC-MS/MS(LC-MS/MS)对洗脱的多肽进行分析，来进行MHC-多肽的分析。使用ELISpot筛选多肽刺激后的T细胞反应，并使用流式细胞仪对反应性T细胞进行表型和功能表征。对MHC-多肽组的分析将首次能够识别体内呈现给GPA适应性免疫系统的多肽序列。这些研究将导致未来在抗原触发的T细胞反应水平上的有针对性的治疗干预的发展。

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.