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

已知HLA I类蛋白的表达水平会影响病理结果：病原体下调HLA以逃避宿主免疫应答，宿主炎症反应上调HLA，并且人与人之间HLA稳态表达水平的差异与疾病易感性相关。然而，由于HLA的巨大多态性，各种HLA基因座的相对表达水平的精确定量是困难的。大多数个体表达六种经典的HLA等位基因（即，在HLAA、HLAB和HLAC处杂合的基因座），因此检测具有特异性的特定基因座具有挑战性。即使可以鉴定出对来自每个基因座的分子具有特异性的抗体，也不能简单地比较它们的结合，因为它们对各自抗原的亲和力不同。经典的HLA I类基因座的相对表达水平在HIV感染的细胞上是特别感兴趣的，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，HLA/MHC的下调在体内显示显著。由于HLAA和HLAB被HIV以不同的效率降低，并且Nef不调节HLAC，因此尚不清楚哪个HLA位点在HIV感染的细胞上占主导地位。我们使用两种独立的方法，流式细胞术和质谱（MS），以确定正常和HIV感染的原代细胞的HLA I类蛋白的相对表达水平。来自正常供体的PBL显示HLAA和HLAB蛋白以相似的水平表达，通过流式细胞术比HLAC高13至18倍，通过质谱比HLAC高4至5倍;这些差异可能反映了所检测的蛋白质的构象或位置的变化。还研究了体外感染HIV的原代CD 4 T细胞，因为HIV下调选择性HLA类型。在HIV感染的细胞上，HLAA和HLAB减少的幅度在感染的培养物中的细胞之间变化。对来自个体的所有感染细胞进行平均显示，通过流式细胞术，HLAA比HLAC高1至3倍，HLAB比HLAC高2至5倍。这些结果量化了来自不同HLA基因座的蛋白质的表达水平的实质性差异，这在未感染和HIV感染的细胞上都非常可能是生理学上显著的。在HLA I类基因座之间观察到的表达水平的巨大差异可能是功能上显著的。已知较高的HLA表达水平更有效地启动CTL应答并调节CTL分泌的细胞因子。此外，一些抑制性受体（如LILRB 1/2和KIR 3DL 1）识别来自多个HLA基因座的抗原，因此同种异型特异性表达水平可能影响先天免疫应答以及获得性免疫应答。鉴于累积的数据指向差异同种异型特异性和基因座特异性表达水平对免疫应答的显著影响，重要的是定义每个HLA基因座的这种性质以确定其对人类疾病的潜在影响。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是基因组中最丰富的4 Mb区域，与几乎所有类型的复杂人类疾病相关，并且对于许多这些疾病，他们强调该区域在确定全基因组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、产生HLAC限制性细胞毒性T淋巴细胞（CTL）对病毒应答的可能性更大以及对HIV的免疫压力更强（通过病毒逃逸突变测量）相关。HLA-C表达水平也与克罗恩病的风险有关，但在这种情况下，低表达似乎提供保护。表达水平对各种疾病的相反影响可能表明选择压力，以维持HLA等位基因谱系的差异表达，HLAC基因座的遗传进化分析支持的模型。考虑到这一点，我们最近研究了等位基因谱系的表达水平在HLAA基因座的样本中的216个欧洲裔美国人使用实时聚合酶链反应测定，并观察到梯度的表达与HLAA等位基因谱系。HLAA基因的DNA甲基化似乎有助于HLAA mRNA表达水平的变化，因为在未处理细胞中的HLAA mRNA表达水平与用DNA甲基转移酶抑制剂处理细胞后表达增加的程度之间观察到显著的负相关。此外，深度测序和免疫沉淀分析揭示了HLA-A启动子区域内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。这些数据证明了HLA-A等位基因谱系特异性表达水平的变化，这至少部分是由于不同的DNA甲基化模式，其中在健康的欧洲美洲（EA）供体中，低HLA-A表达谱系具有较高的DNA甲基化水平，反之亦然。这种转录调控模式与HLA-B和HLA-C不同，因为所有HLA-B和-C谱系都是完全未甲基化的，尽管具有与HLA-A相似的甲基化CpG靶位点，进一步区分了经典HLA I类基因座的进化历史。几种机制可能有助于HLA-A谱系表达水平的变化，并且表征这些机制可能呈现作为药物靶点的HLA-A特异性表达水平调节的潜力。

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.