HLA-DRB1风险氨基酸变异通过改变抗原结合槽的空间构象参与氨苯砜超敏反应综合征发生的机制研究

Five amino acid variants in high disequilibrium within HLA-DRB1 were identified significantly associated with dapsone hypersensitivity syndrome by the applicant. Research findings were published in 《J Invest Dermatol》. Through Three dimensional crystal structure simulation analysis, we found HLA-DRB1 risk amino acid variants may affect the spatial conformation of antigen-binding groove. Previous research indicated that polymorphism of amino acid residues in the antigen-binding cleft influence interaction and recognition of specific drugs, peptides repertoire would be altered, evoking the immune response. Thus, we hypothesize that risk amino acid variants of HLA-DRB1 may influence the interaction between dapsone and HLA-DR, alter the peptide repertoire, activate T cells, and eventually induce dapsone hypersensitivity syndrome. In this program, we will perform molecular docking to detect the interaction between dapsone and HLA-DRB1 risk amino acid variants, LS-MS elution peptide analysis to detect whether dapsone affect the repertoire of peptides. The function and restricted pattern of HLA-DRB1 risk amino acid variants in dapsone-specific T-cells activation will be detected using lymphocyte transformation test and ELIspot, the phenotypic features and secreted cytokines of activated dapsone-specific T-cells will be analyzed by flow cytometry and ELISA. This program will reveal the function and molecular pathogenesis of HLA-DRB1 risk amino acid variants involving in DHS and provide suggestions for the precise prediction of DHS.

申请人前期发现HLA-DRB1上5个氨基酸（AA）变异与氨苯砜超敏反应综合征（DHS）发病相关，研究成果发表于《J Invest Dermatol》。三维晶体结构模拟分析发现上述AA变异影响了抗原结合槽的空间构象。既往研究已证实此类空间构象的变化可影响其与特定药物的交互作用，进而改变抗原肽谱激活免疫应答致药物超敏反应发生。因此，为证实5个风险AA变异在DHS发生中的作用机制是否遵循上述过程，申请人后续拟通过分子对接研究风险AA变异与氨苯砜间的交互作用；LC-MS洗脱肽分析研究氨苯砜与DRB1结合是否改变抗原肽谱；采用淋巴细胞活化试验、Elispot试验、流式、ELISA等分析风险AA变异在氨苯砜激活T细胞中的作用，对活化T细胞表型特征及细胞因子的影响，及对CD4+T细胞活化的限制性，以期深入探讨风险AA变异在DHS发生中的作用机制，为疾病精准预防奠定基础。

申请人前期发现HLA-DRB1上5个氨基酸（AA）变异与氨苯砜超敏反应综合征（DHS）发病相关，研究成果发表于《J Invest Dermatol》。三维晶体结构模拟分析发现上述AA变异影响了抗原结合槽的空间构象。既往研究已证实此类空间构象的变化可影响其与特定药物的交互作用，进而改变抗原肽谱激活免疫应答致药物超敏反应发生。后续课题组利用体外刺激培养成功制备多种T细胞克隆，并且通过单细胞TCR测序成功发现氨苯砜特异性T细胞为多克隆T细胞；通过全基因组关联分析和全基因组甲基化技术成功发现影响抗原递呈细胞功能的关键分子-TAP，加上课题组之前发现的氨苯砜与HLA分子的结合发生在抗原递呈细胞后，因此课题组得出TAP影响内源性抗原肽的转运，从而改变影响氨苯砜特异性T细胞应答激活的抗原肽谱；进一步通过体外TAP功能抑制实验发现，氨苯砜特异性T细胞应答的产生依赖于TAP功能的完整性；而TAP基因通过影响HLA分子的表达以及影响HLA分子与内源性抗原肽的结合发挥作用。

内容获取失败，请点击重试