Molecular genetics and population studies of the KIR and HLA gene complexes

已知HLA - i类蛋白的表达水平影响病理结果：病原体下调HLA以逃避宿主免疫反应，宿主炎症反应上调HLA，人类之间HLA稳态表达水平的差异与疾病易感性有关。然而，由于HLA具有巨大的多态性，对不同HLA位点的相对表达水平进行精确定量是困难的。除了HLAE外，大多数人还表达六种经典HLA等位基因（即HLAA、HLAB和HLAC的杂合），因此特异性检测特定位点是具有挑战性的。即使可以鉴定出每个位点的分子特异性抗体，它们的结合也不能简单地进行比较，因为它们对各自抗原的亲和力存在差异。经典HLA -i类基因座的相对表达水平在HIV感染的细胞中特别有趣，因为HIV编码Nef蛋白，其下调HLAA和HLAB。Nef具有多种功能，但具体而言，体内HLA/MHC的下调被证明是显著的。由于HLAA和HLAB被HIV降低的效率不同，而且Nef不调节HLAC，因此在HIV感染的细胞上，哪个HLA位点占主导地位尚不清楚。我们使用两种独立的方法，流式细胞术和质谱（MS），来确定HLA - i类蛋白在正常和HIV感染原代细胞上的相对表达水平。正常供体的PBLs显示，HLAA和HLAB蛋白表达水平相似，流式细胞仪检测的表达水平是HLAC的13 ~ 18倍，质谱检测的表达水平是HLAC的4 ~ 5倍；这些差异可能反映了检测到的蛋白质构象或位置的变化。由于HIV下调选择性HLA类型，我们也研究了体外感染HIV的原发性CD4 T细胞。HLAA和HLAB在HIV感染细胞上的减少程度在感染培养的细胞之间有所不同。流式细胞术对单个感染细胞进行平均，HLAA比HLAC高1 ~ 3倍，HLAB比HLAC高2 ~ 5倍。这些结果量化了来自不同HLA位点的蛋白表达水平的实质性差异，这很可能在未感染和HIV感染的细胞上具有重要的生理意义。在HLA I类位点之间观察到的表达水平的巨大差异可能在功能上具有重要意义。较高的HLA表达水平可以更有效地启动CTL反应并调节CTL分泌的细胞因子。此外，一些抑制性受体（如LILRB1/2和KIR3DL1）识别来自多个HLA位点的抗原，因此同种异体特异性表达水平可能影响先天免疫反应以及获得性免疫反应。鉴于越来越多的数据表明不同的同种异体特异性和基因座特异性表达水平对免疫反应有显著影响，确定每个HLA基因座的这种特性以确定其对人类疾病的潜在影响是很重要的。高度多态性的HLA I类和II类基因映射到人类MHC，并编码有助于适应性和先天免疫反应的分子。全基因组关联研究已经确定MHC是与几乎所有类型的复杂人类疾病相关的基因组中最丰富的4mb区域，并且对于许多这些疾病，他们强调该区域在确定全基因组范围内疾病风险方面最重要。较高的HLAC表达水平与增强的人类免疫缺陷病毒（HIV）控制有关，产生HLAC的可能性更大，限制了细胞毒性T淋巴细胞（CTL）对病毒的反应，以及通过病毒逃逸突变测量的更强的HIV免疫压力。HLA-C表达水平也与克罗恩病的风险相关，但在这种情况下，低表达似乎具有保护作用。不同疾病中不同表达水平的相互作用可能表明在HLA等位基因谱系中维持差异表达的选择压力，这一模型得到了HLA位点遗传进化分析的支持。考虑到这一点，我们最近使用实时聚合酶链反应法检测了216名欧裔美国人HLAA基因座等位基因的表达水平，并观察了与HLAA等位基因谱系相关的表达梯度。HLAA基因的DNA甲基化似乎有助于HLAA mRNA表达水平的变化，因为在未经处理的细胞中观察到HLAA mRNA表达水平与DNA甲基转移酶抑制剂处理细胞后表达增加的程度之间存在显著的负相关。此外，深度测序和免疫沉淀分析揭示了HLA-A启动子区域的等位基因谱系特异性甲基化模式，其中DNA甲基化水平的增加与HLA-A表达水平的降低显著相关。这些数据表明HLA-A等位基因在表达水平上存在特异性变异，这至少部分是由于DNA甲基化模式的差异，在健康的欧美（EA）供体中，低HLA-A表达谱系的DNA甲基化水平较高，反之亦然。这种转录调控模式不同于HLA- b和HLA-C，因为所有HLA- b和-C谱系都是完全未甲基化的，尽管它们具有与HLA- a相似的甲基化CpG靶点，这进一步区分了经典HLA I类位点的进化史。几种机制可能导致HLA-A谱系表达水平的变化，并且表征这些机制可能提供HLA-A特异性表达水平调节作为药物靶点的潜力。

The expression level of HLA class-I proteins is known to influence pathological outcomes: pathogens downregulate HLA to evade host immune responses, host inflammatory reactions upregulate HLA, and differences among people with regard to the steady-state expression levels of HLA associate with disease susceptibility. Yet precise quantification of relative expression levels of the various HLA loci is difficult because of the tremendous polymorphism of HLA. Most individuals express six classical HLA alleles (i.e., heterozygous at HLAA, HLAB, and HLAC) in addition to HLAE, so detecting a particular locus with specificity is challenging. Even if antibodies specific to molecules from each locus can be identified, their binding cannot simply be compared because of differences in affinity for their respective antigens. Relative expression levels of the classical HLA class-I loci are of particular interest on HIV-infected cells, because HIV encodes the Nef protein, which downregulates HLAA and HLAB. Nef has multiple functions but, specifically, downregulation of HLA/MHC was shown to be significant in vivo. Because HLAA and HLAB are not reduced with equal efficiency by HIV, and Nef does not modulate HLAC, it is not clear which HLA locus dominates on HIV-infected cells . We used two independent approaches, flow cytometry and mass spectrometry (MS), to determine the relative expression levels of HLA class-I proteins on normal and HIV infected primary cells. PBLs from normal donors showed that HLAA and HLAB proteins are expressed at similar levels, which are 13 to 18 times higher than HLAC by flow cytometry and 4 to 5 times higher than HLAC by mass spectrometry; these differences may reflect variation in the conformation or location of proteins detected. Primary CD4 T cells infected with HIV in vitro were also studied because HIV downregulates selective HLA types. HLAA and HLAB were reduced on HIV infected cells by a magnitude that varied between cells in an infected culture. Averaging all infected cells from an individual showed HLAA to be 1 to 3 times higher and HLAB to be 2 to 5 times higher than HLAC by flow cytometry. These results quantify substantial differences in expression levels of the proteins from different HLA loci, which are very likely physiologically significant on both uninfected and HIV infected cells. The large differences in expression levels observed between HLA class I loci are likely to be functionally significant. Higher HLA expression levels are known to more efficiently initiate CTL responses and modulate the cytokines that CTLs secrete. Further, some inhibitory receptors (such as LILRB1/2 and KIR3DL1) recognize antigens from multiple HLA loci, so allotype-specific expression levels may affect the innate-immune response, as well as the acquired-immune response. Given the accumulating data pointing to a significant impact of differential allotype-specific and locus-specific expression levels on the immune response, it is important to define this property for each HLA locus to determine its potential effect across human diseases. The highly polymorphic HLA class I and class II genes map to the human MHC and encode molecules that contribute to both the adaptive and innate immune responses. Genome-wide association studies have identified the MHC as the most rich 4 Mb region of the genome in terms of association with virtually all types of complex human disease, and for many of these diseases, they highlight this region as the most important in determining disease risk genome wide. Higher HLAC expression levels have been associated with enhanced human immunodeficiency virus (HIV) control, greater odds of generating HLAC restricted cytotoxic T lymphocyte (CTL) responses to the virus, and stronger immune pressure on HIV as measured by viral escape mutations. HLA-C expression levels also associate with risk of Crohn's disease, but in this case, low expression appears to confer protection. Opposing effects of expression levels on various diseases may indicate selection pressure to maintain differential expression across HLA allelic lineages, a model supported by genetic evolution analyses of the HLAC locus. With this in mind, we recently examined the expression levels of allelic lineages at the HLAA locus in a sample of 216 European Americans using a real-time polymerase chain reaction assay, and observed a gradient of expression that associates with HLAA allelic lineage. DNA methylation of the HLAA gene appears to contribute to the variation in HLAA mRNA expression levels, as a significant inverse correlation was observed between HLAA mRNA expression levels in untreated cells and the degree to which expression is increased after treatment of the cells with a DNA methyltransferase inhibitor. Further, deep-sequencing and immunoprecipitation assays revealed allelic lineage-specific methylation patterns within the HLA-A promoter region where increased DNA methylation levels correlated significantly with reduced HLA-A expression levels. These data demonstrate HLA-A allelic lineage-specific variation in expression levels, which is due at least in part to differential DNA methylation patterns where low HLA-A expressing lineages have higher DNA methylation levels, and vice versa, in healthy European American (EA) donors. This mode of transcriptional regulation is distinct from HLA-B and HLA-C, as all HLA-B and -C lineages are completely unmethylated in spite of having the similar CpG target sites for methylation as HLA-A, further distinguishing the evolutionary history of the classical HLA class I loci. Several mechanisms are likely to contribute to the variation in expression levels of HLA-A lineages, and characterizing these mechanisms may present the potential for HLA-A-specific regulation of expression levels as drug targets.