Identification of novel HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from Zinc Transporter 8

Numerous evidences demonstrated that type 1 diabetes (T1D) is due to a loss of immune tolerance to islet antigens, and CD8⁺ T cells play an important role in the development of T1D. Zinc Transporter 8 (ZnT8) has emerged in recent years as a target of disease-associated autoreactive T cells in human T1D. However, ZnT8-associated CTL specific-peptides have not been identified. In this study, we predicted and identified HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from ZnT8, and utilized it to immunize HLA-A2.1/Kb transgenic (Tg) mice. The results demonstrated that peptides of ZnT8 containing residues 107–115, 115–123 and 145–153 could elicit specific CTLs in vitro, and induce diabetes in mice. The results suggest that these specific peptides are novel HLA-A*0201-restricted CTL epitopes, and could have therapeutic potential in preventing of T1D disease.     

Altered decamer and nonamer from an HLA-A0201-restricted epitope of Survivin differentially stimulate T-cell responses in different individuals

Survivin is a universal tumor antigen that is being currently targeted in vaccine approaches against cancer. Our study here examined the immunogenicity of a novel variant of an HLA-A0201-binding decamer peptide from region 95 to 104 of Survivin (ELMLGEFLKL) with a T → M modification at position 3 in the peptide. We found that this new modified 10-mer peptide had enhanced HLA-A0201 binding and induced a stronger T-cell response over its wild type counterpart peptide (ELTLGEFLKL) in select HLA-A0201⁺ normal donors. In addition, when compared to the previously characterized altered 96-104 peptide (LMLGEFLKL) from the same region of Survivin currently used in vaccine trials, we found that both peptides had similar immunogenicity, but donor T cells preferentially reacted strongly to either one or the other, but not strongly to both. These results suggest that these two closely related Survivin peptides yield distinct T-cell responses and that most individuals dominantly respond to one or the other altered peptide. We also found a novel association between positive reactivity to the new altered decamer Survivin peptide in some individuals and their expression of the HLA-C0701 allele along with HLA-A0201. Thus, vaccinating with both the 10-mer and 9-mer peptides would be required to immunize a maximum number of individuals in the HLA-A0201⁺ population and could lead to more consistent T-cell responses against this region of Survivin.     

A novel mouse model for immunogenic evaluation of human HBV vaccines

To prepare a human HBV vaccine, investigators need an animal model that can help them screen and prioritize vaccine candidates. In this study, HBV/HLA-A2.1 double transgenic mice (dTg), confirmed by analysis of genomic integration and by observation of long-term expression of HBV and HLA genes, were generated for the first time. The HBV/HLA-A2.1 dTg not only display tolerance to HBV antigens (Ags), but also have the ability to process and present HLA-A2 restricted antigenic epitopes. The animals vaccinated with HLA-A2 restricted HBc_18–27 or HBs_183–191 epitope peptide vaccine induced effective HLA-A2 restricted peptide-specific cytolytic T lymphocyte responses. This was supported by the evidence of cytotoxicity assay, ELISPOT and tetramer staining analysis. Furthermore, T cell tolerance against HBV Ags in HBV/HLA-A2.1 dTg was broken by the HBc_18–27 or HBs_183–191 peptide vaccine. In conclusion, HBV/HLA-A2.1 dTg was demonstrated to be useful model for in vivo immunogenicity testing of human HBV-based vaccines.     

新的HLA-A等位基因A~*0290的认定

目的确认1个中国人群中的HLA新等位基因。方法使用PCR-SSP和测序为基础的分型技术,分析确认HLA-A的新等位基因与A*02010101的差异。结果先证者HLA-A位点有1个等位基因的核苷酸序列与已知的HLA等位基因均不同,该基因和A*02010101的差异在第2外显子区域的292位碱基C>G,导致74编码子CAC>GAC,编码的氨基酸由组氨酸(His)变成天冬氨酸(Asp)。结论该基因为HLA新的等位基因,2005年10月被世界卫生组织HLA因子命名委员会正式命名为HLA-A*0290。     

淋巴细胞HLA-A mRNA方法学建立及在乙型肝炎病毒感染患者中的应用

目的建立外周血淋巴细胞HLA-A mRNA的检测方法,探讨其在慢性乙型肝炎、肝炎后肝硬化、肝癌患者中的应用价值。方法改良法提取人外周血淋巴细胞RNA,应用逆转录-聚合酶链反应(RT-PCR)一步法扩增HLA-A mRNA,琼脂糖凝胶电泳结果采用Bandscan软件分析相对灰度值,半定量HLA-A的基因表达。结果半定量HLA-A所得平均相对灰度值在慢性乙型肝炎组、肝硬化组、肝癌组、正常对照组分别为0．54±0．21、0．34±0．27、0．22±0．16、0．95±0．19。慢性乙型肝炎组、肝硬化组、肝癌组均明显低于正常对照组(P<0．05),慢性乙型肝炎组与肝癌组,肝硬化组与肝癌组相比差异有统计学意义(P<0．05),慢性乙型肝炎组与肝硬化组有下降趋势。结论成功建立检测HLA-A基因的RT-PCR一步法,从基因转录水平上证实慢性乙型肝炎、肝炎后肝硬化、肝癌患者外周血淋巴细胞HLA-A下调,且与相应患者细胞免疫功能减弱密切相关。     

聚合酶链反应方法检测外周血淋巴细胞HLA-A mRNA方法学的建立及在肾移植中的初步应用

目的通过建立检测宿主外周血淋巴细胞(PBL)表面HLA-AmRNA表达程度的实时荧光定量逆转录聚合酶链反应(RT-FQ-PCR)方法,检测肾移植患者移植后外周血淋巴细胞表面HLA-AmRNA的表达程度,探讨其在诊断移植后早期排斥反应的发生的价值及机体免疫状态的关系。方法以6-磷酸葡萄糖脱氢酶(G6PDH)作内参照,用Taqman探针荧光定量技术,建立检测PBLHLA-AmRNA的RT-FQ-PCR方法;并检测35例随访移植患者的不同时间的60个标本和60名正常人的阈循环数(Ct),然后用REST软件分析PBLHLA-AmRNA的表达水平。结果正常人和51个移植标本的PBLHLA-AmRNA对G6PDH的相对表达量分别为0·036±0·012和0·026±0·015(双侧t=1·981,P<0·05),提示服用免疫抑制药(目前临床用药剂量均偏大)后患者PBLHLA-AmRNA表达减弱,机体免疫状态低,无排斥反应发生但易导致感染、肿瘤等并发症的产生;其中9个移植标本的PBLHLA-AmRNA对G6PDH的相对表达量为0·042±0·017(双侧t=2·002,P<0·05),提示较其他51例移植标本患者PBLHLA-AmRNA表达增强的患者,经病例回访查询发现该5例移植患者在这期间有排斥反应(根据临床表现、肾功能检查、影像学及核医学检查的临床排斥反应期)的发生,其中1例在移植后6个不同时间的标本显示2次有增高,每次经治疗后均降低。结论建立的宿主PBLHLA-AmRNART-FQ-PCR检测方法简便、特异、重复性好、可信度高,可用于肾移植后PBLHLA-AmRNA表达水平的检测,评估机体的免疫状态,预测排斥反应的发生。     

中国汉族个体HLA全长序列新等位基因A＊74：02：01：02的鉴定

目的对中国汉族个体人类白细胞抗原（HLA）-A基因全长序列进行测定,发现和鉴定突变位于常规检测区域以外的全长序列新等位基因。方法应用已对常规检测区域第2～4外显子进行测序分型、结果已知的中国汉族个体的样本进行HLA—A基因全长4．3kb序列的高保真扩增、克隆及测序;利用序列比对、进化树等方法分析突变所在位置及其产生的原因。结果发现1个HLA—A全长序列新等位基因,序列与国际IMGT／HLA数据库中最接近的等位基因A＊74：02：01：01的全长序列相比共有5个位于内含子的变异,其中第3内含子1个：NT1427C〉T,第7内含子5个,分别为NT2842G〉A,NT2850C〉T,NT2862T〉C,NT2863G〉A以及NT2868T〉C。该HLA—A新等位基因于2010—07—31被世界卫生组织人类白细胞抗原因子命名委员会正式命名为HLA—A＊74：02：01：02。结论新等位基因A＊74：02：01：02的变异产生的机制可能是随机突变和等位基因之间的交换重组。