HLA基因型及HLA-E:NKG2A信号通路在EB病毒相关噬血细胞综合征发病机制的研究

Epstein-Barr virus-associated hemophagocytic lymphohistocytosis (EBV-HLH) is usually with poor prognosis. A major contributing factor for EBV-HLH is the impaired cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Still, there is a lack of mechanistic understanding of why some EBV-infected people, but not others, develop EBV-HLH. The diversity of human leukocyte antigen (HLA) class I molecules influences the capacity of antigen presentation to T cells. HLA-E is with limited polymorphisms and its cell surface expression is affected by HLA-I molecules. HLA-E inhibits the activity of NKG2A-expression NK or CTLs. We postulate that genetic diversity of HLA-I and HLA-E may associate to EBV-HLH. Results from HLH mouse model studies indicated that NK activity limited HLH-like immunopathology via reducing hyper-activated T cells and tissue-infiltration of macrophage, whereas the role of CTL cytotoxicity was on viral clearance. Therefore, we hypothesize that EBV-HLH associated HLA genotypes present elevated HLA-E which results in reduced NK activity. EBV clearance is impaired due to CTL exhaustion accompanying hyper activated T cells and macrophages. Finally, inhibited NK cytotoxicity fails to limit hyper activated T cells and HLH develops. To test our hypothesis, this proposed study will evaluate the immune status of EBV-HLH patients and healthy controls by using PBMC isolated from peripheral blood. To test the in vitro immune repose to EBV, PBMC samples will be stimulated with separated peptide pools each covering the entire length of 6 different EBV proteins. Moreover, PBMCs will be treated with antibodies against NKG2A or PD-1 to study the role of HLA-E:NKG2A signaling on EBV-HLH. To fulfill this purpose, Cytometry Time of Flight (CyTOF) will be utilized to extensively probe the phenotypic and functional parameters on a single cell. The above study is designed to investigate whether genotypes of HLA-I and HLA-E associate to EBV-HLH. It will further study whether its underlying molecular mechanism involves HLA-E:NKG2A signaling pathway and explore the possibility of blocking HLA-E/NKG2A interaction as a therapeutic target of EBV-HLH.

EB病毒相关噬血细胞综合征（EBV-HLH）病情重预后差，发病机制不明。人类白细胞抗原I类分子（HLA-I）多态性影响其抗原呈递能力。HLA-E抑制表达NKG2A的NK和CTL细胞的活性。研究结果表明NK细胞通过减少过度活化的CTL细胞等来限制HLH；而CTL细胞的功能为清除病毒。因此，我们推测HLA-E表达增高，通过HLA-E:NKG2A抑制NK及CTL细胞活性；CTL清除EBV能力下降及NK细胞活性下降等最终发展为EBV-HLH。本课题拟通过分析EBV-HLH患者外周血单个核细胞（PBMC）的免疫状态，及评估PBMC对EBV蛋白合成肽库刺激的反应来验证假说。并检验NKG2A抑制抗体能否缓解炎症反应；PD-1抑制剂可否促进病毒清除。研究旨在阐明HLA基因型与EBV-HLH的关联，验证HLA-E:NKG2A通路在EBV-HLH中的作用，探索阻滞HLA-E/NKG2A作为治疗靶点的可行性。

EB病毒相关噬血细胞综合征（EBV-HLH）病情重预后差，发病机制尚不明确。人类白细胞抗原I类分子（HLA-I）多态性影响其抗原呈递能力。HLA-E抑制表达NKG2A的NK和CTL细胞的活性，而CTL细胞的功能为清除病毒。本课题完成482例EBV感染相关疾病患者以及213例EBV阴性健康供者的HLA等位基因分型及EBV-DNA结果分析，发现存在统计学意义的有两个（B 15:01，DQB1 06:02），B 15:01组OR>1，暴露与疾病的危险度增加；DQB1 06：01组OR<1，暴露与疾病的危险度减少。发现HLA 15:01患者EBV感染主要以CD56+NK细胞（90.91%）以及CD4-CD8+T细胞（77.27%）这两组细胞为主，而其他基因型EBV则感染CD19+B细胞，差异有统计学意义（P<0.05）。通过单细胞质谱流式技术检测EBV患者治疗前后外周血中免疫细胞的变化，发现EBV感染相关疾病患者的细胞亚群与正常人存在差异，主要表现为NK细胞及B细胞的比例差异。治疗无效和治疗有效组，对比治疗前后，CD8+T和单核细胞的比例变化差异最明显；在有效治疗组，CD8+T细胞比例明显增多，单核细胞比例明显减少。EB病毒感染相关患者治疗后对比治疗前的细胞亚群，CM CD4+T细胞、EM CD8+T细胞比例在治疗后升高，提示为治疗相关作用。患者的NK细胞的具体亚群与健康人存在差异，且抑制性受体NKG2A的表达升高，并在有效治疗后出现下降。在使用PD-1阻断剂治疗后，治疗有效的患者相较于治疗无效的患者，部分细胞因子均显著升高，提示阻断PD-1后，部分患者CTL功能能够获得恢复。本研究发现HLA基因型与EBV-HLH存在相关性，探索了EBV相关疾病患者的细胞亚群特点，并为研究HLA-E/NKG2A作为治疗靶点提供相关依据。