Contribution of HLA class II genes to susceptibility in achalasia

Abstract: Achalasia is a motor disorder of the esophagus resulting in functional obstruction. The cause of the lesion is unknown although genetic and immunologic factors have been suggested. An association with serological HLA epitopes has been previously reported. In this study, we have further examined this HLA class II association with susceptibility to achalasia by DNA based methods. Achalasia patients ( n =40) and healthy controls ( n = 275), all Caucasians and unrelated, were included in the analysis. The strongest associations were with HLA-DQAl*0101 and two HLA-DQαβ heterodimers having their a chain encoded by this allele. Moreover, relative risk was significantly higher in DQAl*0101 homozygotes as compared to heterozygotes and results suggested that DQB1*02 may have a protective role.     

可溶性HLA-G1与NK-92细胞表面ILT2及KIR2DL4受体相互作用的研究

 目的 探讨可溶性 HLA-G1（sHLA-G1）对人 NK-92 细胞杀伤活性的抑制与细胞表面免疫球蛋白样转录分子 2（ILT2）和杀伤细胞免疫球蛋白样受体 2DL4（KIR2DL4）受体的关系。 方法 ①通过原核表达技术获得 sHLA-G1 重组蛋白（重组蛋白），并采用蛋白质印迹法进行鉴定。②取 NK-92 细胞，加入终浓度 20 μg/ml 的重组蛋白分别培养 10、30 min，再分别加入抗 HLA-G1/G5、抗ILT2 和抗 KIR2DL4 抗体，采用流式细胞术检测各组 NK-92 细胞表面 sHLA-G1 和 ILT2、KIR2DL4 受体表达阳性率；以 NK-92 细胞单独培养作为对照组。③以人白血病 K562 细胞为靶细胞，以经不同方式处理的 NK-92 细胞为效应细胞，效靶比为5:1，共同培养 2 h，采用流式细胞术检测 NK-92 细胞对 K562 细胞的杀伤率。NK-92 细胞处理方式为单纯重组蛋白处理（分别加入终浓度为 0、10、20 μg/ml 的重组蛋白培养 30 min）和表面受体封闭 + 重组蛋白处理（分别加入抗 ILT2、抗 KIR2DL4、抗 LT2 + 抗 KIR2DL4 抗体培养 30 min，再分别加入终浓度为 0、10、20 μg/ml 的重组蛋白培养 30 min）。 结果 ①蛋白质印迹分析表明所获重组蛋白为带有组氨酸标签的特异蛋白。②NK-92 细胞与 20 μg/ml 重组蛋白共培养 30 min 后，sHLA-G1 表达阳性率明显高于而 ILT2、KIR2DL4 受体表达阳性率均明显低于对照组（均 P < 0.05）。③以终浓度 0、10、20 μg/ml 的重组蛋白处理的 NK-92 细胞对 K562 细胞的杀伤率分别为 39.79% ± 2.00%、27.79% ± 0.75%、21.36% ± 0.67%（两两比较，均 P < 0.01）；单独封闭 ILT2 受体，杀伤率分别为 23.09% ± 1.63%、21.13% ± 0.38%、18.42% ± 0.47%（两两比较，均 P < 0.01）；单独封闭 KIR2DL4 受体，杀伤率分别为 30.74% ± 0.44%、26.03% ± 0.38%、21.15% ± 0.35%（两两比较，均 P < 0.01）。 结论 sHLA-G1 通过与 NK-92 细胞表面 ILT2 和 KIR2DL4 受体直接结合而抑制 NK-92 细胞的杀伤活性。     

Use of class II tetramers for identification of CD4+ T cells

Multivalent MHC class II molecules containing peptide antigens are useful tools for the detection of antigen specific human CD4+ T cells. Tetramers produced by exogenous peptide loading onto empty class II molecules are comparable to tetramers with peptide tethered to the class II chain covalently, but have many practical advantages. Conditions for optimal peptide loading to generate tetramers are discussed and optimal conditions of using tetramers for staining T cells are examined. As the frequency of antigen specific CD4+ T cells in peripheral blood is low, we demonstrate that an in vitro expansion step is effective in detecting low frequency T cells. Two new applications with tetramers, their uses for mapping T cell epitopes and for the detection of low affinity T cells are described. In a clinical setting, potential applications include using these reagents for monitoring disease progression during clinical intervention.     

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.     

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.     

Analysis of children with type 1 diabetes in Korea: high prevalence of specific anti-islet autoantibodies, immunogenetic similarities to Western populations with "unique" haplotypes, and lack of discrimination by aspartic acid at position 57 of DQB

This study analyzed the expression of anti-islet autoantibodies and HLA-DR and -DQ genotypes in Korean children with type 1 diabetes mellitus (T1DM). The positivity of the anti-ICA512, anti-GAD65, and anti-insulin autoantibodies in the newly onset T1DM patients (n = 15) was 66.7%, 86.7%, and 46.7%, respectively, and all of them had one or more of the autoantibodies. HLA analysis showed higher frequencies of HLA-DRB1*0301, *0405, *09012 and -DQB1*0201, *0401, *03032 alleles in T1DM patients compared to controls (P(c) < 0.05). Because HLA-DQB1*0401, *03032 alleles carry aspartic acid at position 57 of DQB, susceptibility to T1DM in Korean children was not related to the presence of aspartic acid at position 57 of DQB1 locus. We suggest this unique HLA-DR, -DQ allele distribution might be an important factor for the low incidence of T1DM in Korea, and the combined anti-islet autoantibody assays could be valuable screening markers for the early detection of T1DM in Korea.     

Are HLA-DR or TAP Genes Genetic Markers of Severity in Ulcerative Colitis?

The pathogeny of ulcerative colitis (UC) is not yet elucidated, but some arguments suggest the implication of genetic factors. Among the candidate genes, those encoding for HLA class II genotypes have been extensively studied in UC; however, discordant data may be imputable to heterogeneity, characterized by immunological markers such as atypical ANCA (p-ANCA), or to inclusion of more or less intractable UC. The aim of our study is to evaluate the interest of HLA class II and TAP genetic markers to identify different clinical forms of UC, according to p-ANCA status. Unrelated patients with a history of UC (n=91) and healthy control subjects with no personal or family history of inflammatory bowel diseases (IBD) (n=200) were included. HLA-DRB103 was less frequent in UC patients than in healthy controls (8% vs 28%,PC<0.03). No association was found with any TAP genotypes. Moreover, there was no association with the HLA-DR2 specificity, either in the entire group of UC patients (38% vs 28%) or in the p-ANCA-positive subgroup of patients (30%). The most consistent finding in the present study is that some genetic markers may characterize intractability in UC patients. HLA-DR2 was associated with poor prognosis, regardless of p-ANCA status. In HLA-DR2 and non-HLA-DR2 groups, colectomy was done in 55% and 27% of patients, respectively (PC<0.05). Furthermore, in non-HLA-DR2 patients, p-ANCA could be of interest to characterize those with more severe prognosis. Our results confirm the interest of genetic studies to define UC genetic susceptibility, taking into account intractability of the disease. They do not support the hypothesis that p-ANCA is a subclinical marker of genetic susceptibility to UC.     

Differences between T-cell reactivities to major myelin protein-derived peptides in opticospinal and conventional forms of multiple sclerosis and healthy controls

In Japanese, susceptibility to the conventional form of multiple sclerosis (C-MS) is associated with the HLA-DRB1*1501-DRB5*0101 haplotype while susceptibility to the opticospinal form of MS (OS-MS) is associated with HLA-DPA1*0202-DPB1*0501. To clarify the characteristics of T cells autoreactive to myelin proteins in each MS subtype, we established T-cell lines reactive to such myelin antigens as myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) from 5 of 10 OS-MS patients, 6 of 11 C-MS patients and 7 of 13 healthy controls (HCs), and T-cell epitopes and their restriction molecules were determined. We found that (a) intermolecular epitope spreading was found to be significantly more frequent in MS patients than in HCs (P=0.0128), (b) intramolecular epitope spreading also tended to occur more frequently in MS patients than in HCs (P=0.0584), (c) in OS-MS, HLA-DR-restricted and MOG-autoreactive T cells were more frequently established as compared with those reactive to MBP or PLP epitopes and (d) in C-MS, HLA-DQ-restricted and PLP-autoreactive T cells dominated those autoreactive to MBP or MOG epitopes. A DPB1*0501-restricted MBP-reactive T-cell clone from a patient with OS-MS provided evidence that the first HLA class II anchor amino acid of peptide bound to disease-susceptible DP5 molecule was distinct from that for the DR2 molecule. Taken together, these differences in specificities of myelin-autoreactive T cells between C-MS and OS-MS as well as the difference in the anchor motif of the binding peptides between each MS subtype-susceptible HLA class II molecule may contribute to the development of distinct clinical phenotypes.     

Use of HLA marker associations and HLA haplotype linkage to estimate disease risks in families with gluten-sensitive enteropathy

Based on a two-locus, double recessive model, we derive formulas for the risks that relatives of individuals with gluten-sensitive enteropathy (GSE) will also develop the disease. The calculations take advantage of: the linkage between the HLA locus and one of the two proposed GSE loci, and the preferential association of the HLA-DR3 and DR7 alleles with the GSE disease allele that occupies the HLA-linked locus. We use Bayes' rule to quantitate the strength of the association between the GSE disease allele and the HLA marker allele. This method predicts that siblings of the proband have an overall 10% risk for GSE, which is consistent with observed family data. This predicted risk rises to 30% when siblings are HLA-identical to the proband (also consistent with observed data) or when the sibling has the DR3 allele in the HLA haplotypes not shared with the proband. In those populations where DR7 also is associated with GSE, siblings of probands have a 10% predicted risk for GSE when only one HLA haplotype is shared with the proband and DR7 is included in the unshared haplotype. Other DR alleles are associated with much lower disease risks. By separating individuals into high and low risk groups, HLA typing identifies those individuals who would benefit from further diagnostic procedures. This general strategy should be applicable to other multilocus, marker-associated diseases.