卡马西平结合HLA-B*15:02对表皮抗原自身肽提呈的选择及在SJS-TEN发病中的作用

Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are severe cutaneous adverse drug reactions involving widespread epidermal and epithelial keratinocyte death. Many such reactions are strongly associated with specific HLA-Ⅰ alleles and driven by drug-specific activation of CD8+ cytotoxic T cells(CTL). However, the mechanism by which CTL is activated and attack epidermis/epithelia remains unclear. It was recently shown that drugs binds non-covalently within peptide-binding grooves of the genetic linked HLA-Ⅰmolecules. In this way, the drug guides the selection of new endogenous peptides, which as neoantigen drive CD8+ CTL activation. We speculate that altered HLA-peptide display also occur in epidermis and epithelia, which account for attacking epidermal cells by driving CD8+ T cells activation against epidermis and epithelia. We will search for epidermal self-peptides newly presented by HLA-B*15:02 expressed by dendritic cells under carbamazepine treatment using LC-MS/MS. Moreover, we will confirm its binding with HLA-*B15:02 by analyzing the affinity and the X-ray crystal strucutre of HLA-B*15:02+peptide+carbamzepine complex.Moreover, we will characterize its pathogenic role in stimulating specific CTL activation. Our project will reveal the mechanism by which CTL is activated and attack epidermis and epithelia, and will lay a solid foundation for development of novel therapeutic strategies for SJS-TEN.

中毒性表皮坏死松解症(TEN)和Stevens-Johson综合征(SJS) 以广泛表皮/上皮细胞死亡为主要表现。很多药物引起的SJS-TEN与HLA-Ⅰ类基因强相关，通过活化CD8+细胞毒性 T细胞(CTL)介导病理损伤。然而，CTL活化及杀伤表皮/上皮的机制仍不清楚。最新研究显示药物与HLA-Ⅰ类分子的肽结合沟槽非共价结合，选择提呈新的内源性肽段，诱导特异性CTL活化。我们推测这种自身肽的选择提呈发生于皮肤，并在介导CTL活化和杀伤表皮细胞中发挥关键作用。我们拟利用液相色谱-串联质谱技术，找到卡马西平处理树突状细胞后HLA-B*15:02提呈表皮抗原的新自身肽，用亲合力分析、晶体结构法验证其结合，并明确新自身肽、药物与HLA-B*15:02结合诱导特异性CTL的作用。本项目将从自身肽异常提呈角度揭示CTL活化及杀伤表皮/上皮机制，为探索SJS-TEN新的治疗策略奠定基础。

Stevens-Johson综合征(SJS)与中毒性表皮坏死松解症(TEN)以高热、广泛的表皮细胞坏死为表现，为以致死性的药物不良反应。中毒性表皮坏死松解症的表皮细胞坏死的机制目前仍未阐明。本研究成功完成卡马西平特异性T细胞株、T细胞克隆培养工作，完成HLA-B*15:02转染工作。由于技术的限制，未找到稳定的卡马西平处理C1R细胞后影响HLA-B*15:02自身肽提呈。我们对卡马西平代谢产物(10,11-环氧化卡马西平,CBZE)对角质形成细胞程序性坏死的作用进行了深入研究，首次发现CBZE 在SJS/TEN诱导 NLRP3炎症小体表达，NLRP3炎症小体并在SJS/TEN患者皮损高表达。CBZE可活化芳香烃受体(AhR)介导的程序性坏死，并且影响CYP1A1 表达。CBZE诱导角质形成细胞表达CXCL9与CXCL10，促进CD8+ T细胞移行，并促进其分泌多种细胞毒蛋白。从不同途径阻断AhR-RIP3-MLKL-NLRP3均显著减少上述作用。本研究首次发现AhR-MLKL- NLRP3-IL-1β轴介导CBZE刺激人角质形成细胞后NLRP3活化，其介导角质形成细胞程序性坏死、并促进CD8+ T细胞移行、活化、发挥细胞毒功能，在SJS/TEN发病中具有重要意义。更重要的是，本研究阐明CBZE刺激角质形成细胞产生的NLRP3炎症小体通过不同趋化因子、细胞因子的释放，与免疫细胞CD8+ T细胞移行至表皮、活化存在紧密联系。IL-1β并可能是SJS/TEN的治疗新的靶点。