过敏原家族特征肽调节树突状细胞共刺激分子差异表达的免疫生物信息学解析

前期我们创建过敏原数据集，自编程序汰选得到531条过敏原家族特征肽AFFP，藉此构建过敏原判别软件SORTALLER获国际公认优势，但未区分AFFP之过敏原性强弱；脱敏治愈后过敏病例体外激发试验仍呈阳性；鼻/咽遇激多优先发病。推测：过敏原刺激树突状细胞DC产生共刺激分子与否决定免疫耐受的形成，其效能差异是区别AFFP强弱的关键。本研究以软件初步估分531条AFFP与HLA结合力，用不同类AFFP刺激扁桃体及外周血DC和人DC细胞模型，检测CD86/CD83等共刺激分子、I/II类HLA分子、TLRs及细胞因子IL-12等的表达，分析控制IFNs及其应答基因表达的上游信号分子MyD88、NF-κB、IRFs、MAPKs、JNK等的激活时序，洞悉过敏原-DC应答-免疫耐受的三角关系；明确AFFP过敏原性及激活性能并创建过敏原性评估软件1套，为变态反应疾病诊治和蛋白过敏原性评估改造提供先导平台。

We've created an allergen data set containing 2290 real allergen sequences, from which 531 allergen family featured peptides (AFFPs) were panned by a self-programmed procedure. An online allergen-classifier SORTALLER was molded based on AFFPs, which outperforms significantly other existing software and has been utilized by the researchers from more than 20 countries worldwide. Nevertheless, the allergenicity of each AFFP is due to be deciphered. Furthermore, we recently demonstrated that in vitro challenge assay for desensitized allergic patients still produced positive results. And lots of patients would firstly develop allergic disease in tonsil or nasal cavity. We hence speculate that the absence of costimulatory molecules from upstream antigen-presenting dendritic cells (DC) confers immune tolerance; and the stimulatory capacity to produce costimulatory molecules can be applied to discriminate the allergenicity of each AFFP. This project, including the following experiments, aims to construct an allergenicity evaluation software and to define the triangle relationship among the stimulatory capacity and allergenicity of each AFFP, DC response and the formation of immune tolerance, hence to decipher the mechanism underlying DC activation and the secretion of costimulatory molecules on dendritic cells after allergen stimulation. The results would provide a prelude for the diagnosis and immunotherapy of allergic diseases and protein engineering. In this study, HLA binding affinity of 531 AFFPs will be initially estimated and hence all AFFPs would be classified into different groups. Dendritic cells (DC) enriched from peripheral blood and the surgically removed tonsils, and the DCs generated from MUTZ-3 will be stimulated by all types of AFFPs. The DC maturation markers CD86, CD83, CD11c, and HLA class I and class II molecules will be investigated using flow cytometry, laser scanning confocal microscope, Western Blotting, and real-time PCR. Differential expression of the cytokines IL-1b, IL-6, IL-8, IL-12, NF-κB, etc will be monitored by ELISA. The expression of Toll-like receptors TLR1~ TLR9 will be quantitatively screened by real-time PCR and will be further evidenced by Western Blotting. Semi-quantitative Western Blotting and the corresponding inhibitors/antagonists will be applied to analyze whether signaling through TLRs, MyD88, NF-κB, IRFs, or MAPKs, etc can orchestrate the differential expression of the costimulatory molecules and whether the activation sequential relationship of signaling molecules (MyD88, PI3K, BTK, JNK, ERK, IRFs, TRAFs, IRAKs, p38, etc.), would regulate the expression of interferon-α/β and their responsive genes and subsequently modulate the formation of immune tolerance. The allergenicity and the stimulatory capacity of different AFFPs will be paraphrased by mast cell challenge assay and will hence be evidenced reciprocally with the definite pathways and an allergenicity evaluation software will hence be constructed.