Preparation and Identification of HLA-A＊1101 Tetramer Loading with Human Cytomegalovirus pp65 Antigen Peptide

MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8^＋ T cells in humans. The tetramer molecule is composed of HLA heavy chain, β2-microglobulin （β2m）, an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A＊1101-restricted CD8^＋ T cell responses against human cytomegalovirus （HCMV）, we established an approach to prepare HLA-A＊1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A＊1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A＊1101 fused with a BirA substrate peptide （HLA-A＊1101-BSP） at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichla coli. Moreover, HLA-A＊1101-BSP protein was refolded in the presence of β2m and an HCMV peptide pp6516.24 （GPISGHVLK, GPI）. Soluble HLA-A＊1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of 〉 80%. The HLA-A＊1101-GPI tetramers could bind to virus-specific CD8^＋ T cells, suggesting soluble HLA-A＊1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A＊1101-restricted CD8^＋ T cell responses against HCMV infection.     

小儿急性淋巴细胞白血病HLA-A,B等位基因多态性研究

 目的 研究北方汉族人群儿童急性淋巴细胞性白血病（ALL）患者与HLA-A,B等位基因多态性的遗传关联。方法 采用聚合酶链反应-序列特异性寡核苷酸探针（PCR-SSO）方法,对197例北方汉族儿童ALL患者和5 841例健康脐带血样本HLA-A,B等位基因多态性进行研究。结果 在HLA-A,B等位基因中,儿童ALL患者的A11,A24,B40,B15,B56,B67,B27等基因的基因频率都显著高于正常人群,而HLA-B48基因的基因频率显著下降（P＜0.05）。结论 HLA-A11,A24,B15,B58,B67,B27等基因对儿童ALL患者有遗传易感作用,尤其是B40与ALL具有强相关性;而HLA-B48基因对儿童ALL患者有遗传拮抗作用。     

Molecular mimicry of human cytochrome P450 by hepatitis C virus at the level of cytotoxic T cell recognition.

Hepatitis C virus (HCV) is thought to be involved in the pathogenesis of autoimmune hepatitis (AIH) type 2, which is defined by the presence of type I antiliver kidney microsome autoantibodies directed mainly against cytochrome P450 (CYP)2D6 and by autoreactive liver infiltrating T cells. Virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) that recognize infected cells and contribute to viral clearance and tissue injury during HCV infection could be involved in the induction of AIH. To explore whether the antiviral cellular immunity may turn against self-antigens, we characterized the primary CTL response against an HLA-A*0201-restricted HCV-derived epitope, i.e., HCV core 178-187, which shows sequence homology with human CYP2A6 and CYP2A7 8-17. To determine the relevance of these homologies for the pathogenesis of HCV-associated AIH, we used synthetic peptides to induce primary CTL responses in peripheral blood mononuclear cells of healthy blood donors and patients with chronic HCV infection. We found that the naive CTL repertoire of both groups contains cross-reactive CTLs inducible by the HCV peptide recognizing both CYP2A6 and CYP2A7 peptides as well as endogenously processed CYP2A6 protein. Importantly, we failed to induce CTLs with the CYP-derived peptides that showed a lower capacity to form stable complexes with the HLA-A2 molecule. These findings demonstrate the potential of HCV to induce autoreactive CD8(+) CTLs by molecular mimicry, possibly contributing to virus-associated autoimmunity.     

Effect of interferon α, γ, and tumor necrosis factor α on the HLA-A,B, C expression of cell lines derived from human liver

ABSTRACT— Our study was undertaken to determine whether human recombinant interferon α(rIFNα), γ(rIFNγ), and tumor necrosis factor α(rTNFα) exert an effect on the HLA-A, B, C expression of human liver cell lines. The HLA-A, B, C expression was assayed by immunoperoxidase staining and enzyme-linked immunosorbent assay. rIFNα and γ enhanced the HLA-A, B, C expression of the three cell lines tested, Chang cells, SK-Hep-1, and PLC/PRF/5. The activity of rIFNγ proved more than 8000 times more potent than that of rIFNα in Chang cells, 30 times in SK-Hep-1, and 20 times in PLC/PRF/5, respectively. rTNFα also enhanced the HLA-A, B, C expression of the three cell lines. The enhancement of HLA-A, B, C expression by rIFNα and γ reached a peak on day 3, and that by rTNFα on day 5. These findings suggest that IFNα, IFNγ, and TNFα may play similar roles in enhancement of HLA-A, B, C expression of hepatocytes in hepatitis and hepatoma cells.     

Increase of HLA haplotype A9-Bw16 in familial insulin-dependent diabetes mellitus in Northern Finland

Summary Twenty-one families, each with two diabetic children, from North Finland were HLA typed. The grade of HLA identity between diabetic siblings was evaluated, and frequencies of various HLA antigens in familial cases were compared to those of non-familial cases from the same area. Ten pairs of diabetic children were HLA-identical and eleven haploidentical; two of the latter were identical for the HLA-D locus. These figures emphasize the significance of HLA-region associated genetic factors in the susceptibility to the disease, but do not support a simple dominant/recessive gene theory. There was a significant difference between familial and non-familial IDDM cases in the frequencies of B15, Bw16, B40 and Cw3 antigens. Bw16 was greatly increased and B15, B40 and Cw3 decreased among familial cases. The haplotype A9, Bw16 was common in familial cases, but, compared to healthy controls, the frequencies of the two antigens were also slightly increased among non-familial cases. Neither Dw3 nor Dw4 was associated with Bw16 antigen. The differences between familial and non-familial IDDM cases and the significance of the A9, Bw16 combination in the patients emphasize the heterogeneity of IDDM.     

Mouse Homologue of the Human SART3 Gene Encoding Tumor-rejection Antigen

We recently isolated a human SART3 ( hSART3 ) gene encoding a tumor-rejection antigen recognized by HLA-A2402-restricted cytotoxic T lymphocytes (CTLs). The hSART3 was also found to exist as an RNA-binding nuclear protein of unknown biological function. In this study, we cloned and analyzed the homologous mouse SART3 ( mSART3 ) gene in order to understand better the function of hSART3, and to aid in establishing animal models of specific immunotherapy. The cloned 3586-bp cDNA encoded a 962-amino acid polypeptide with high homology to hSART3 (80% or 86% identity at the nucleotide or protein level, respectively). Nonapeptides recognized by the HLA-A2402-restricted CTLs and all of the RNA-binding motifs were conserved between hSART3 and mSART3. The mSART3 mRNA was ubiquitously expressed in normal tissues, with low level expression in the liver, heart, and skeletal muscle. It was widely expressed in various organs from as early as day 7 of gestation. mSART3 was mapped to chromosome 5, a syntenic region for human chromosome 12q23–24, and its genomic DNA extended over 28-kb and consisted of 19 exons. This information should be important for studies of the biological functions of the SART3 protein and for the establishment of animal models of specific cancer immunotherapy.     

肿瘤增殖细胞核抗原的人白细胞抗原-A2限制性细胞毒T淋巴细胞表位鉴定

目的 鉴定肿瘤增殖细胞核抗原(PCNA)的人白细胞抗原(HLA)-A2限制性细胞毒T淋巴细胞(CTL)表位,为肿瘤疫苗制备提供依据.方法 根据PCNACTL表位评分结果,合成8条候选肽;采用经典T2-肽结合实验检测候选肽与HLA-A2分子亲和力;采用酶联免疫斑点法(ELISPOT)检测候选肽刺激的HLA-A2阳性CTL分泌干扰素(IFN)-γ能力.结果 T2-肽结合实验结果显示PCNA 14-22、110-118位置候选肽与HLA-A2分子结合力较强,其荧光系数FI值最高,分别为0.30和0.34,显著高于其他候选肽.ELISPOT检测结果显示14-22和39-47位置候选肽形成的IFN-γ斑点数目(477.25±52.08、640.50±145.74)明显高于阴性对照(0.75±0.50)及其余候选肽.结论 PCNA抗原14-22位置候选肽KVLEALKDL可作为HLA-A2限制性CTL表位用于制备肿瘤疫苗.     

Association study of HLA-A gene and schizophrenia in Han Chinese from Taiwan

Dysregulation of the immune response has been proposed as a precipitating factor of schizophrenia, and human leukocyte antigens (HLA) play a critical role in regulating the cascade of immunological reaction. Hence, many studies have investigated the relationship between the HLA system and schizophrenia. HLA is a complex gene family that contains several highly polymorphic genes, while the HLA-A gene is the most often studied gene to be associated with schizophrenia in the literature. A recent study reported that the interaction of the HLA-A10 allele and Chlamydial infection was highly associated with schizophrenia in a German population, which prompted us to investigate whether the HLA-A gene was also associated with schizophrenia in our population. Using a sequencing-based HLA typing method, we determined the HLA-A genotypes in 377 Han Chinese patients with schizophrenia (214 males, 163 females) and 321 non-psychotic Han Chinese control subjects (164 males, 157 females) from Taiwan. In total, 26 DNA-defined HLA-A alleles were identified in this sample. However, no significant differences of these allelic frequencies were found between the patients and the control subjects, suggesting that the HLA-A gene was unlikely a major risk factor of schizophrenia in this sample. As different populations have different HLA polymorphisms, an examination of the relationship of other HLA genes and schizophrenia in our population, with a larger sample size, is warranted in the future.     

海南地区汉族人群HLA及其单倍型频率分析

目的了解海南地区汉族人群HLA抗原及单倍型的频率。方法采用微量淋巴细胞毒试验，对海南地区209例非血缘关系的汉族正常人进行HLA-A、B抗原分型。结果在209例中检出前9位HLA-A、HIA-B高频率抗原为A11，A2，A24、A33；B40，B46，B57，B13，B62。通过家系抗原遗传分析获得418条海南地区HLA-A／B单倍型，查出6条海南地区高频率出现的单倍型为A33／B57、A2／B46、A11／B60、A11／B13、A24／B60、A11／B62。结论海南地区汉族人群的HLA-A、B抗原频率与广东地区相同，而单倍型频率有所不同。不同种族、不同地区的人群有着不同单倍型频率分布。     

Identification of an HLA-A*24:02-restricted α-fetoprotein signal peptide-derived antigen and its specific T-cell receptor for T-cell immunotherapy

Hepatocellular carcinoma (HCC) is the most common type of liver cancer with limited treatments. Asia has the highest HCC incidence rates; China accounts for over 50% of all HCC cases worldwide. T-cell receptor (TCR) -engineered T-cell immunotherapies specific for human leukocyte antigen (HLA) -A*02:01-restricted α-fetoprotein (AFP) peptide have shown encouraging results in clinics. HLA-A*24:02 is more common than HLA-A*02:01 in Asian countries, including China. Here we identified a novel HLA-A*24:02-restricted peptide KWVESIFLIF (AFP_2–11) located in AFP signal peptide domain by mass spectrometric analysis of HLA-bound peptides from HepG2 cells. A TCR (KWV3.1) specific for AFP_2–11-HLA-A*24:02 was isolated from peripheral blood mononuclear cells of a healthy donor. The binding affinity of soluble KWV3.1 to its antigen was determined to be ~55 μm , within the affinity range of native TCRs for self-antigens. KWV3.1-transfected T cells could specifically activate and kill AFP_2–11 pulsed T2-A24 cells and AFP⁺ HLA-A*24:02⁺ tumor cell lines, demonstrating that AFP_2–11 can be naturally presented on the surface of AFP⁺ tumor cell lines. The newly identified antigenic peptide can provide a novel target for immunotherapeutic strategies for patients with AFP⁺ HLA-A*24:02⁺ HCC.