H7N9禽流感患者恢复期T细胞受体库特征研究

Features of T Cell Receptor Repertoires of Influenza H7N9 Virus Infected Patients in Convalescence

目的建立H7N9禽流感患者恢复期T细胞受体库,寻找H7N9特异性T细胞受体变化。方法收集2013年3月至5月确诊的8例人感染禽流感H7N9患者恢复期血液以及10例健康人血液,分离基因组DNA,采用Ⅲumina He Seq2000测序平台对T细胞受体库β链(TRB)抗原互补决定区3进行高通量测序,分析TRB库的多样性等特征。结果与正常组比较,H7N9患者某些TRBV基因使用频率如TRBV30、TRBV27存在显著差异。使用主成分分析降维后,发现恢复期患者TRBV-TRBJ基因配对模式与正常人有显著差别,可用于区分H7N9患者和正常人群。患者—患者、正常人—正常人之间的共享序列数量和频率明显大于患者-正常人,且患者间TRB库具有较高相似度。多样性指数在患者组明显低于正常人群,显示在感染恢复期其免疫功能仍处于相对低下状态。同时H7N9患者中超高表达克隆明显增多,且序列间存在较高相似性。结论 H7N9恢复期患者T细胞受体库有H7N9特征性变化,这些特征将为疾病的诊断和治疗性T细胞的研究提供重要信息。

Objective To investigate specific changes of T cell repertoire in convalescent patients infected by influenza A （H7N9 virus. Methods Peripheral blood samples from 8 convalescent patients infected by H7N9 virus and 10 healthy donors were collected. After extracting whole DNA from these samples,arm-PCR were performed and the products were submitted to Illumina HiSeq2000 platform to produce deep sequencing data of the nucleotide sequences of complementary determining region 3 of T cell receptor 13 chain （TRB）. Differences were compared in TRB diversity and V-D-J gene usage and similarities of sequences between the patients and the healthy donors. Results Frequency of V-D-J gene usage was different between the H7N9 patient group and the healthy group, such as TRBV30,TRBV27, and TRBV18 （ Student＇ s t test, P 〈 0. 05 ）. Main component analysis showed V-J pairing pattern was significantly different between two groups, which may have potential in identifying patients from healthy people. A considerable number of shared CDR3s were found in patient-patient pairs and normal-normal pairs,while seldom were found in patient-normal pairs. The similarity between patients was also confirmed by overlap distance analysis. Indexes for assessing diversity of immune repertoires, Shannon-Weiner index and Simpson index, were both lower in the patients （ Student＇ s t test, P 〈 0. 05 ）, suggesting that the immune system of the patients had not recovered 6 months after H7N9 infection. Compared with the healthy donors ,the number of hyper-expression clones increased in the patient group, and some of them showed similarity among patients. Conclusions TRB repertoires are less diverse in patients with increased hyper-expressed clones and identifiable V-J usage pattern,which is identifiable from normal population. These results suggest that thereare HTN9-specific changes in TRB repertoires of H7N9 infected patients in convalescent phase, which have potential implication in diagnosis and therapeutic T cell development.