Melanocyte lysis by cytotoxic T lymphocytes recognizing the MART-1 melanoma antigen in HLA-A2 patients with Vogt Koyanagi Harada disease

The MART-1/Melan-A melanoma antigen recognized by the majorityof HLA-A2-restricted tumorinfiltrating lymphocytes is a selfantigen expressed on melanocytes and the retina. We have investigatedwhether Vogt–Koyanagi–Harada (VKH) disease and sympatheticophthalmia (SO), systemic inflammatory disorders affecting variousorgans containing melanocytes, are autoimmune diseases directedtoward the MART-1 antigen. In two of three patients with VKHdisease and one patient with SO, CD8⁺ T cell clones (TCC) fromintraocular fluid of HLA-A2⁺ patients lysed T2 cells when pulsedwith a HLA-A2-binding MART-1 peptide, but not a HLA-A2-bindingpMel-17 or tyrosinase peptide, in a HLA-A2-restricted manner.These CD8⁺ TCC lysed both melanocytes and melanoma cells ina HLA-A2-restricted manner. In addition, CD8⁺ TCC recognizinga HLA-A2-binding MART-1 peptide were also established from peripheralblood mononuclear cells of a patient with VKH disease. In contrast,either CD4⁺ TCC from these patients or CD8⁺ TCC from the intraocularfluid of HLA-A2⁺ patients with uveitis associated with Behcet'sdisease or HTLV-I uveitis did not show this cytotoxicity. Theresults demonstrate that the MART-1 peptide-specific cytotoxicT lymphocytes lyse melanocytes in the eye of patients with VKHdisease or SO, suggesting that these diseases are autoimmunediseases directed toward the MART-1 antigen in HLA-A2⁺ patients.     

HLA-A*0201与EGFP融合蛋白表达载体的构建及其在K562细胞的表达和定位

目的:构建增强型绿色荧光蛋白(EGFP)与HLA-A^*0201(A^*0201)融合蛋白哺乳动物细胞表达载体,分析其在K562细胞中的表达和亚细胞定位。方法:以RT-PCR方法克隆A^*0201cDNA,构建A^*0201-EGFP融合蛋白表达载体,转染K562细胞,以流式细胞仪和激光共聚焦显微镜分析融合蛋白的表达及其亚细胞定位。结果:从两个HLA-A2阳性供者外周血细胞中克隆到A^*0201cDNA编码区全长序列。通过PCR方法在起始位点前加入Kozak序列并删除终止密码,成功构建A^*0201-EGFP融合蛋白表达载体。以该质粒转染K562细胞,5h后A^*0201和EGFP的表达百分率分别为25.12±2.26、27.37±3.59,24h后表达水平无明显提高。表达的融合蛋白主要分布于细胞膜上,胞内分布较少。相反,转染空载体的细胞不表达A^*0201分子,仅表达EGFP,且其在细胞内呈弥散样分布。结论:成功构建A^*0201-EGFP融合蛋白表达载体,并在K562细胞中得到表达,表达产物主要分布于细胞膜表面,提示表达该融合蛋白的K562细胞是潜在的人工抗原提呈细胞。     

Identification of a novel HLA-DQA1 null allele, DQA1*0403N, from an East African woman

We report a novel DQA1 allele (DQA1*0403N) identified during sequence-based HLA-DQA1 typing of a Kenyan population. The new allele is identical to DQA1*0401 at exon 2 except for a single-nucleotide substitution at codon 53, changing it from lysine to a stop codon (CAA-->TAA). The substitution at codon 53 was confirmed by sequencing two separate polymerase chain reaction products and by sequencing multiple clones obtained following TOPO-TA cloning. The resulting stop codon at position of codon 53 in exon 2 is predicted to produce a non-functional DQA1 alpha-chain. The new allele has been named by the WHO nomenclature committee as DQA1*0403N. This is the first report of a null allele detected in the DQA1 gene.     

Identification of a new HLA-B*56 variant, B*5614

In this report, the novel allele B*5614 is presented. The allele was identified in a Chinese individual by sequence-based typing. HLA-B*5614 differs from B*5608 by a single nucleotide at position 277G-->C in exon 2. This results in an amino acid change from Gly to Arg at codon 93.     

An HLA-binding-motif-aided peptide epitope library: A novel library design for the screening of HLA-DR4-restricted antigenic peptides recognized by CD4+T cells

Susceptibility to a series of autoimmune diseases is strongly associated with particular HLA class II alleles. Identification of T cell clones and antigenic epitopes bound by HLA class II molecules involved in autoimmune diseases is critical to understanding the etiology of these HLA class II-associated diseases. However, establishment of T cell clones in autoimmune diseases is difficult because the antigenic peptides are unknown. Peptide library methods which include all possible peptide sequences offer a potentially powerful tool for the detection of cross-reactive antigenic peptides recognized by T cells. Here, we reduced the number of peptides per mixture by utilizing the known binding motifs of peptides for the HLA-DRB1*0405 molecule and evaluated the effectiveness of this library design. Each library mixture evoked a strong proliferative response in the unprimed peripheral blood lymphocytes (PBL) from HLA-DRB1*0405-positive donors but little or no response in the PBL from HLA-DRB1*0405-negative donors. The library also detected antigenic peptides that activated three antigen-specific T cell lines restricted by HLA-DRB1*0405, with different specificities. The motif-based approach thus presents a powerful method for monitoring T cells in large, heterogeneous T cell populations and is useful for the identification of the mimic peptide epitopes of T cell lines and clones. Received: October 3, 1997 / Accepted: October 23, 1997     

Sequence of a novel HLA-A*0301 intronic variant (A*03010103)

We report here the full-length sequence of a novel HLA-A*0301 allele, A*03010103, which differs from A*03010101 by a single nucleotide substitution (G>T) at position 492 within intron 2. The variant was originally identified by Reference Strand-mediated Conformational Analysis (RSCA) and was confirmed by cloning and sequencing. The difference in RSCA mobility between A*03010101 and A*03010103 demonstrates the sensitivity of RSCA to detect single nucleotide polymorphisms.     

Twelve novel HLA-B*15 alleles carrying previously observed sequence motifs are placed into B*15 subgroups

Twelve new B*15 alleles are described. All of the known B*15 alleles are divided into subgroups based on serologic assignments and/or nucleotide sequence polymorphisms. These groups might be used as a reference for DNA-based testing at an intermediate (i.e. "serologic") level of resolution.     

5-HT 102T/C多态性与Tourette综合征的病例对照及核心家系关联分析

目的 研究5－羟色胺受体102T／C多态性是否与Tourette综合征（TS）相关联,方法对157个核心家系样本采用病例－对照关联分析,传递不平衡检验方法,聚合酶链反应及RFLP等技术,根据TS与强迫症（obsessive compulsive disorder,OCD)的同病现象,将TS划分亚组进行与5－羟色胺受体102T／C多态性的关联分析。结果 合并OCD的TS与该位点的基因型102C／C（X2＝8．38,P＝0．004）及等位基因102C／（X2＝4．84,P＝0．028）存在关联,进一步采用传递不平衡分析,发现合并（美国精神疾病诊断和统计手册IV》论断标准的OCD的TS与该位点存在关联或连锁不平衡（X2＝5．12,,P＝0．02）,而在TS总体样本及单纯TS样本中未发现与该位点的关联,结论 5－羟色胺受体102T／C多态性与中国人群合并OCD的TS存在关联,合并OCD的TS可能是TS中相对独立的一个亚型。     

Novel HLA-A*11 allele, A*1120, identified by sequence-based typing

In this report, we describe the identification of a human leucocyte antigen-A*11 (HLA-A*11) nucleotide sequence variant, a new HLA-A*1120 by using sequence-based typing (SBT). The new allele was detected during routine HLA typing by high-resolution SBT. Allele A*1120 showed one nucleotide difference with A*110101 at codon 152 (GCG-->GAG) resulting in an amino acid change from alanine to glutamate. Residue 152 is located on alpha(2)-helix of HLA class I molecule and involved in peptide binding by constructing E pocket of peptide-binding groove, implying that the change of the residue 152 would affect the binding affinity of peptides to A*1120 allele.     

HLA-A*0203-BSP的表达和复性及其四聚体的鉴定

目的：优化诱导条件大批量表达生物素化酶BirA底物肽（BSP）与HLA-A＊0203重链胞外域的融合蛋白（HLA—A＊0203、BSP），并制备负载HLA-A＊0203限制性EB病毒抗原肽EBNA3 596-604的四聚体（HIA—A}0203／SVR）。方法：以HLA—A＊0203-BSP原核表达载体转化E．coli BL21（DE3）菌株，优化诱导条件进行大批量重组蛋白的表达。通过稀释法复性可溶性HLA-A＊0203／SVR单体，然后以BirA对其进行生物素化，并以阴离子交换树脂纯化。将纯化的HLA-A＊0203／SVR单体与荧光素标记的链亲和素按4：1的比例混合形成四聚体，通过对特异性CTL进行染色验证其结合活性。结果：当IPTG的浓度为0．4mmol／L，于37℃诱导过夜后，融合蛋白的表达最多。该重组蛋白相对分子质量（Mr）为34003，与HLA—A＊0203-BSP的理论Mr相一致。该重组蛋白以包涵体形式存在于沉淀部分，约占菌体总蛋白的30％。负载抗原肽的可溶性HLA-A＊0203／SVR单体是在同时存在HLA-A＊0203，BSP、β2微球蛋白及HLA-A＊0203限制性抗原肽SVR的情况下通过稀释法复性而获得。该单体生物素化并纯化后与荧光素标记的链亲和素按4：1的比例混合后即形成四聚体。流式细胞术（FCM）分析证实，该四聚体具有与HLA—A2^＋供者特异性CTL结合的活性。结论：HLA—A＊0203-BSP融合蛋白在优化条件下获得高效表达。以此蛋白制备的HLA-A＊0203／SVR四聚体具有与HLA-A2^＋供者特异性CTL结合的活性，为研究HLA—A＊0203个体EB病毒特异性CTL的免疫应答打下了基础。