Class II transactivator-induced expression of HLA-DO(beta) in HeLa cells

HLA-DO is an intracellular nonclassical MHC class II molecule expressed in the endocytic pathway of B lymphocytes. It shapes the repertoire of peptides bound to classical class II molecules such as HLA-DR by regulating the activity of HLA-DM. Using a peptide corresponding to the cytoplasmic tail of HLA-DO(beta), we have developed a mouse monoclonal antibody, HKC5. Immunofluorescence microscopy revealed that HKC5 recognizes HLA-DO molecules present in the endoplasmic reticulum as well as those in vesicular compartments of the endocytic pathway. In addition, the antibody detects the isolated beta chain on Western blots. Using mutants of the DO(beta) cytoplasmic tail fused to a reporter molecule and expressed in epithelial cells, we showed by flow cytometry that the antibody epitope includes one or both of the leucine residues forming the lysosomal sorting signal. Finally, we have used HKC5 to evaluate the presence of the HLA-DO(beta) chain in HeLa cells expressing the class II transactivator protein CIITA. Our flow cytometry and confocal microscopy analyses showed a marked expression of DO(beta) suggesting that HLA-DO could accumulate under the influence of CIITA in non-B cells.     

HLA-DM基因和HLA-DO基因与系统性红斑狼疮

主要组织相容性复合物（MHC）Ⅱ类分子的主要生物学功能是结合并提呈抗原肽供CD4T细胞识别，启动获得性免疫应答。MHCⅡ类分子是MHCⅡ类基因编码的产物。关于MHCⅡ类基因与系统性红斑狼疮（SLE）等自身免疫性疾病的相关性研究甚多，但过去的研究较多集中在HLA—DR基因。而对另两种位于MHCⅡ类基因区域的抗原加工提呈相关基因：HLA—DM和HLA—DO的研究相对较少。文章就近年来对这两种抗原加工提呈相关基因的研究进展及与SLE的相关性作一综述。     

MHC class II antigen presentation and immunological abnormalities due to deficiency of MHC class II and its associated genes

Antigen presentation by Major Histocompatibility Complex (MHC) class II molecules plays an important role in controlling immunity and autoimmunity. Multiple co-factors including the invariant chain (Ii), HLA-DM and HLA-DO are involved in this process. While the role for Ii and DM has been well defined, the biological function of DO remains obscure. Our data indicate that DO inhibits presentation of endogenous self-antigens and that developmentally-regulated DO expression enables antigen presenting cells to preferentially present different sources of peptide antigens at different stages of development. Disruption of this regulatory mechanism can result in not only immunodeficiency but also autoimmunity. Despite the fact that deletion of each of the three genes in experimental animals is associated with profound immunological abnormalities, no corresponding human diseases have been reported. This discrepancy suggests the possibility that primary immunodeficiencies due to a genetic defect of Ii, DM and DO in humans are under diagnosed or diagnosed as “common variable immunodeficiency”, a category of immunodeficiency of heterogeneous or undefined etiology. Clinical tests for any of these potential genetic defects are not yet available. We propose the use of multi-color flow cytometry in conjunction with intracellular staining to detect expression of Ii, DM, DO in peripheral blood B cells as a convenient reliable screening test to identify individuals with defects in antigen presentation.     

Presentation of abundant endogenous class II DR-restricted antigens by DM-negative B cell lines

Peptides derived from endogenous and exogenous antigens compete for binding and presentation via class II molecules. Studies with mutant B cell lines defective in exogenous antigen presentation suggest that HLA-DM molecules facilitate the interaction of foreign peptides and class II molecules. In contrast, presentation of self antigens is not strictly dependent upon HLA-DM, as demonstrated by the ability of these mutant cells to activate T cells specific for endogenous antigens. Two distinct classes of DM-negative cells, T2 cells generated by in vitro mutagenesis and lines derived from bare lymphocyte syndrome (BLS) patients, were able to present epitopes derived from self proteins. Transfection of DM genes into the mutant cells enhanced the presentation of some, but not all, endogenous antigens, suggesting that formation of select endogenous peptide/class II complexes is not dependent upon DM. The efficiency of endogenous antigen presentation in the absence of DM was also dependent on the mutant antigen-presenting cell studied, as the TxB hybrid T2 presented greater amounts of self peptides compared to cells from BLS patients. Thus, additional genes, aside from DM, may regulate the pathway for endogenous antigen presentation.     

HLA-DM基因多态性与子痫前期的相关性

目的 探讨HLA-DM基因多态性与子痫前期的相关性.方法 用序列特异性寡核苷酸探针杂交聚合酶链反应方法分别测定35例子痫前期患者及63例正常孕妇的HLA-DM基因型.结果 HLA-DMB*0103基因频率在子痫前期组比正常妊娠组明显升高,差异有显著性(x2=4.38,P＜0.05),其他各等位基因频率在两组间差异无显著性(P＞0.05).结论 HLA-DMB*0103频率在子痫前期患者中明显增多,提示HLA-DMB*0103可能为子痫前期的易感基因.     

尖锐湿疣患者HLA-DM基因多态性研究

目的探讨尖锐湿疣（CA）患者的HLA-DM基因的多态性，寻找可能的易感基因。方法运用PCR-RFLP方法确定98例CA患者和93例正常人群中HLA-DM的基因型。分析DM与CA的关联性。结果检测到7种DMA与DMB等位基因，HLADMA＊0101和DMB＊0101等位基因在汉族CA患者中的频率显著性增高（P〈0．05和P〈0．01），而DMA＊0102等位基因在CA患者中的频率显著性降低（P〈0．01）。DMA和DMB基因型在CA与正常对照之间无显著性差别。结论HLADMA＊0101和DMB＊0101等位基因可能是汉族人CA的易感基因或与易感基因连锁。     

中国汉族人群HLA-DM多态性与强直性脊柱炎疾病关联分析

目的比较中国汉族人群强直性脊柱炎(AS)与健康对照HLA-DMA/HLA-DMB各SNP位点及等位基因多态性,分析可能的AS疾病易感位点及基因。方法选取110名AS患者和1 000名健康无关献血者,利用Taq-Man PCR技术对7个DMA和DMB SNPs(single nucleotide polymorphisms)位点进行了基因分型,运用SPSS软件及Arlequin3.1软件统计基因频率和单体型相关参数。结果 AS组DMA*01∶02基因频率(19.55%)显著低于对照组(28.05%),AS组DMA*01∶02-DMB*01∶01单体型频率(17.51%)也显著低于对照组(26.87%);AS组与对照组DMA p496和DMB p590 SNP位点多态性、DMA*01∶02等位基因以及DMA*01∶02-DMB*01∶01单体型分布差异有统计学意义(P0.05)。结论 DMA*01∶02等位基因和DMA*01∶02-DMB*01∶01单体型可能对于AS疾病具有保护性作用,未来有必要在其他人群中进一步确认并研究其基因功能。     

The expression of HLA-DO (H2-O) in B lymphocytes

HLA-DO (H2-O in mice) is a nonpolymorphic transmembrane αβ heterodimer encoded in the class II region of the major histocompatibility complex (MHC). It is expressed selectively in B lymphocytes and thymic medullary epithelial cells. DO forms a stable complex with the peptide-loading catalyst HLA-DM in the endoplasmic reticulum (ER); in the absence of DM, DO is unstable. During intracellular transport and distribution in the endosomal compartments, the ratio of DO to DM changes. In primary B cells, only approx 50% of DM molecules are associated with DO. DO appears to regulate the peptide-loading function of DM in the MHC class II antigen-presentation pathway. Although certain di screpancies are present, results from most studies indicate that DO (as well as H2-O) inhibits DM (H2-M) function; this inhibition is pH-dependent. As a consequence, DO restrains presentation of exogenous antigens delivered through nonreceptor-mediated mechanisms, in addition, DO alters the peptide repertoire that is associated with cell-surface class II molecules. The biological function of DO remains obscure, partially because of the lack of striking phenotypes in the H2-O knockout mice. Results from recent studies indicate that DO expression in B cells is dynamic, and highly regulated during B-cell development and B-cell activation, suggesting that the physiological role of DO is to tailor the antigen presentation function of the B-line age cells to meet their primary function at each stage of B-cell development and maturation. Further investigations are needed in this direction.     

Class II Antigen Processing Compartments and the Function of HLA-DM

The DMα and DMβ genes encode a nonpolymorphic, class II-like molecule which functions by an, as yet, undefined mechanism in the assembly of Major Histocompatibility Complex class II-peptide complexes. Indeed, mutant cells which express class II molecules but fail to express DM are unable to process and present native protein antigens. A striking phenotype of the mutation is class II molecules that contain almost exclusively a nested set of invariant chain peptides, termed CLIP, for class II associated Ii peptides, instead of the normal array of endogenously and exogenously derived peptides. Thus, DM appears to be required for the correct assembly of processed antigen-class II complexes. Recently, the subcellular compartments that contain DM and in which functional processed antigen-class II complexes are first formed have been described. Here, the evidence for the function of DM in the antigen-processing compartments is reviewed.     

HLA-DM polymorphisms do not associate with multiple sclerosis: an association study with analysis of myelin basic protein T cell specificity

The MHC region on 6p harbors at least one susceptibility gene for multiple sclerosis (MS). Within this region, HLA-DM loci are of interest being involved in class II antigen processing. We investigated the association of HLA-DM polymorphisms with MS. Sixty-three patients with MS and 46 healthy controls from continental Italy were typed for HLA-DM polymorphisms and HLA-DRB1 alleles. Besides, among the donors characterized for the DM polymorphisms, we considered 6 MS patients previously studied for the fine specificity of their MBP-specific T lymphocyte lines (TLL). The frequencies of allelic variants at the DMA and DMB loci were similar between MS patients and controls, even when HLA-DRB1^*1501 positive and negative donors were analyzed separately. Patients with predominant responses to different MBP epitopes did not differ for their HLA-DM haplotype while patients with predominant responses to the same MBP epitope could present different HLA-DM haplotypes. HLA-DM polymorphisms do not associate with MS and may not affect specific patterns of T cell responses to MBP.