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

项目总结 这项建议的目的是探索多肽结合域中的体细胞突变的功能意义。 获得性红斑狼疮患者存活血细胞中人类白细胞抗原I类等位基因的研究 再生障碍性贫血(AA)。再生障碍性贫血是一种自身免疫性骨髓再生障碍性疾病，发生于既往健康的个体。 导致危及生命的细胞减少症。多方面的证据，最引人注目的是T细胞定向免疫的疗效- 再生障碍性贫血的非抑制治疗，涉及T淋巴细胞对造血干细胞的自身免疫破坏和 再生障碍性贫血的祖细胞(HSPC)。尽管取得了一些进展，再生障碍性贫血患者的预后仍然很差，而且 迫切需要为再生障碍性贫血开发更有效的治疗方法。进步的主要障碍一直是以下的穷人- 再生障碍性贫血自身免疫特殊机制的研究现状。无论是抗原靶向还是自体免疫的原因- 社区已经被确定了。了解再生障碍性贫血的免疫发病机制对于开发治疗方法至关重要。 这可以更有效地治疗再生障碍性贫血，预防其并发症。在先前对AA获得性突变的研究中， 我们在再生障碍性贫血患者存活的造血细胞中发现了人类白细胞抗原I类等位基因的反复突变。恩-- HLAI类突变细胞存活率的提高突显了HLAI类在介导自身免疫反应中的重要作用。 再生障碍性贫血中HSPC的MUN识别。被体细胞突变破坏的HL A等位基因被认为是导致 受影响患者的自身抗原呈递(作为“危险等位基因”)。它们的躯体失活使得 突变的HSPC以逃避自身免疫攻击。而80%的已鉴定突变导致突变的丢失 人类白细胞抗原(HL A)等位基因的表达，预测有20%的突变可形成功能性的人类白细胞抗原蛋白。值得注意的是，八个MUTA- TIONS是位于人类白细胞抗原多肽结合袋的α(α)-1和α-2区域的氨基酸变化， 这可能是关于AA自身抗原结合的关键残基的特别信息。这些数据支持我们的 中心假说：再生障碍性贫血的自身免疫攻击是针对再生障碍性贫血自身抗原(S)提出的 人类白细胞抗原I类等位基因和体细胞性人类白细胞抗原突变可通过取消人类白细胞抗原来干扰再生障碍性贫血患者的自身抗原提呈 危险等位基因表达或其自身抗原结合。为了解决我们的假设，我们将评估功能连接- 两个综合目标中再生障碍性贫血患者人类白细胞抗原等位基因多肽结合区的突变序列。目标1 将使用细胞分析来确定人类白细胞抗原错义突变对人类白细胞抗原加工和表面的影响 稳定性。Aim 2将使用蛋白质组学来确定表达野生型的细胞的HLAI类免疫多肽 和突变的人类白细胞抗原等位基因。我们的结果将确定自身抗病所必需的AA-HLA风险等位基因的结构特征。 基因结合，为将来研究候选AA自身抗原奠定了基础。再生障碍性贫血是一种毁灭性的疾病， 然而，其潜在的免疫学机制仍不清楚。我们的学习将满足这一迫切需求，开启 为今后深入研究门机和今后的发展提供更有效、合理的设计 再生障碍性贫血的治疗方法。

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.