The effects of somatic HLA class I allele mutations on antigen presentation in acquired aplastic anemia

项目摘要 本研究的目的是探讨肽结合结构域体细胞突变的功能意义 获得性骨髓增生异常综合征患者存活造血细胞中人类白细胞抗原（HLA）I类等位基因的检测 再生障碍性贫血（AA）。AA是一种自身免疫性骨髓发育不全，发生在以前健康的个体中 导致危及生命的血细胞减少多种证据，最明显的是T细胞定向免疫的功效， AA的非抑制性治疗，涉及T淋巴细胞在造血干细胞的自身免疫性破坏中， 祖细胞（HSPCs）在AA中。尽管取得了一些进展，AA患者的结果仍然很差， 迫切需要开发更有效的AA疗法。进步的主要障碍是贫困人口- AA自身免疫机制的研究进展。无论是抗原靶还是自身免疫的原因， 社区已被确认。了解AA的免疫发病机制对于开发治疗方法至关重要 可以更有效地治疗AA并预防其并发症。在先前对AA获得性突变的研究中， 我们在AA患者存活的造血细胞中鉴定了HLA I类等位基因的复发性突变。恩- HLA I类突变细胞的存活率提高强调了HLA I类在介导自体免疫中的关键作用。 AA中HSPCs的免疫识别。被体细胞突变破坏的HLA等位基因被认为是导致 在受影响的患者中的自身抗原呈递（作为“风险等位基因”起作用）。它们的体细胞失活 突变的HSPC以逃避自身免疫攻击。而80%的突变导致了突变体的丢失， 在HLA等位基因的表达中，预测20%的突变形成功能性HLA蛋白。值得注意的是，八个穆塔- 结果显示，HLA肽结合口袋的α（α）-1和α-2结构域发生了氨基酸变化， 这对于AA自身抗原结合至关重要的残基可能特别有用。这些数据支持我们 中心假设，AA中的自身免疫攻击是针对由特异性抗原呈递的AA自身抗原， HLA I类等位基因和体细胞HLA突变可通过废除HLA破坏AA中的自身抗原呈递 风险等位基因表达或其自身抗原结合。为了解决我们的假设，我们将评估功能性条件- 序列突变的肽结合结构域的HLA等位基因在AA患者在两个综合目标。要求1 将使用细胞分析来确定HLA错义突变对HLA加工和表面的影响。 稳定目的2：利用蛋白质组学技术，确定表达野生型HLA-I类抗原的细胞的免疫肽组 和突变的HLA等位基因。我们的研究结果将明确AA HLA风险等位基因的结构特征， 基因结合，形成了候选AA自身抗原的未来研究的基础。AA是一种毁灭性的疾病， 但其潜在的免疫学机制仍不清楚。我们的研究将填补这一关键需求，打开 为今后深入开展机理研究和今后开发更有效、设计合理的 治疗AA。

PROJECT SUMMARY The aim of this proposal is to explore the functional significance of somatic mutations in peptide-binding domains of Human Leukocyte Antigen (HLA) class I alleles in surviving hematopoietic cells of patients with acquired aplastic anemia (AA). AA is an autoimmune bone marrow aplasia that occurs in previously healthy individuals causing life-threatening cytopenias. Multiple lines of evidence, most notably the efficacy of T-cell directed immu- nosuppressive therapy in AA, implicate T lymphocytes in autoimmune destruction of hematopoietic stem and progenitor cells (HSPCs) in AA. Despite some progress, AA patients’ outcomes remain poor, and there is a critical need to develop more effective therapies for AA. The main barrier to progress has been the poor under- standing of specific mechanisms of autoimmunity in AA. Neither the antigenic target nor the causes of autoim- munity have been identified. Understanding the immune pathogenesis of AA is essential for developing therapies that can more effectively treat AA and prevent its complications. In previous studies of acquired mutations in AA, we identified recurrent mutations in HLA class I alleles in surviving hematopoietic cells of AA patients. The en- hanced survival of HLA class I-mutant cells underscored the critical role of HLA class I in mediating the autoim- mune recognition of HSPCs in AA. HLA alleles disrupted by somatic mutations are thought to be responsible for autoantigen presentation in the affected patients (function as “risk alleles”). Their somatic inactivation allows mutant HSPCs to evade the autoimmune attack. While 80% of the identified mutations led to the loss of mutant HLA alleles’ expression, 20% of mutations were predicted to form a functional HLA protein. Notably, eight muta- tions were amino acid changes located in the alpha (α)-1 and α-2 domains of the HLA peptide-binding pocket, which could be especially informative about residues critical for AA autoantigen binding. These data support our central hypothesis that autoimmune attack in AA is directed against AA autoantigen(s) presented by specific HLA class I alleles, and somatic HLA mutations can disrupt autoantigen presentation in AA by abrogating HLA risk allele expression or its autoantigen binding. To address our hypothesis, we will evaluate the functional con- sequences of mutations in peptide-binding domains of HLA alleles in AA patients in two integrated aims. Aim 1 will use cellular assays to determine the effects of HLA missense mutations on HLA processing and surface stability. Aim 2 will use proteomics to define the HLA class I immunopeptidome of cells expressing wild-type and mutant HLA alleles. Our results will define structural features of AA HLA risk alleles necessary for autoanti- gen binding, forming the foundation for future studies of candidate AA autoantigens. AA is a devastating disease, yet its underlying immunological mechanisms remain unknown. Our studies will fill this critical need, opening the door to future in-depth mechanistic studies and the future development of more effective, rationally-designed therapies for AA.