AIDS Animal Models: Cellular Immunology and Immunogenetics

艾滋病动物模型：细胞免疫学和免疫遗传学

• 批准号: 8555949
• 负责人: Bernard Lafont
• 金额: $ 27.84万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555949
• 关键词: AIDS vaccine development; Acidic Amino Acids; Acquired Immunodeficiency Syndrome; Activated Natural Killer Cell; Affect; Alleles; Amino Acids; Animal Model; Animals; Antiviral Agents; Arginine; Asians; Australia; Basic Amino Acids; Binding; Biological Assay; CD8B1 gene; Cell physiology; Cell surface; Cells; Cellular Immunity; Cellular Immunology; Characteristics; Charge; Cloning; Collaborations; Comparative Study; Complex; Cytomegalovirus; Development; Disease; Disease Progression; Disease susceptibility; Elements; Epitopes; Evolution; Exhibits; Family suidae; Frequencies; Genetic; Genetic Variation; Genotype; Gorilla gorilla; HIV; HIV Infections; HLA-A gene; HLA-B Antigens; Herpesviridae; Human; Immune; Immune response; Immunity; Immunogenetics; Individual; Infection; Investigation; Killer Cells; Kinetics; Lead; Light; Link; MHC Class I Genes; Macaca; Macaca mulatta; Major Histocompatibility Complex; Modeling; NK Cell Activation; NK cell receptor NKB1; Natural Immunity; Natural Killer Cells; Nature; Pan Genus; Pathogenesis; Pattern; Peptides; Phylogenetic Analysis; Pongidae; Positioning Attribute; Predisposition; Prevalence; Price; Property; Regulation; Research; Role; SIV; Screening procedure; Specificity; Structure; Subfamily lentivirinae; T cell response; T-Lymphocyte; TYROBP gene; Tail; Time; Transmembrane Domain; Universities; Vaccines; Viral; Viral Load result; Virus; Virus Replication; animal cloning; base; cohort; immunoglobulin receptor; improved; nonhuman primate; prevent; receptor; receptor binding; response; simian human immunodeficiency virus

Studies in humans and macaques have demonstrated that CD8+ T cells responses are associated with the initial control of HIV or SIV replication. Natural killer (NK) cells also influence virus control and survival. These antiviral activities are dependent on major histocompatibility complex (MHC) molecules and specific MHC genotypes have been associated with lower viral loads and slower disease progression in humans and macaques. However, the exact correlates of protection remain unidentified and the immune responses required for an effective vaccine need to be defined. Crucial information can be obtained from animal models such as the infection of Asian macaques with SIV or SHIV viruses. Three macaque species are used to mimic HIV infection in pathogenesis and vaccine studies: rhesus (Macaca mulatta), pig-tailed (M. nemestrina) and cynomolgus (M. fascicularis) macaques. Pig-tailed macaques possess unique susceptibility and disease development characteristics that make this species particularly informative for AIDS research (high level of cellular activation, rapid disease development, susceptibility to various SIV strains). Our research is focused on exploring how the host genetic background of macaques affects their innate and adaptive immune response to SIV and SHIV infection. Specifically, our research concentrated on characterizing the NK cell capacity to detect infected cells. Previously we identified a subset of macaque NK cells capable of recognizing specific macaque MHC class I alleles (Mane-A1*082/A1*084) by a specific killer cell immunoglobulin receptor (KIR) expressed at the cell surface. Engagement of specific MHC alleles by a KIR3L allele, KIR049-4, results in inhibition of NK cell functions. We have characterized the specificity of the KIR3DL allele (KIR049-4) by screening its binding properties against a panel of MHC class I tetramers generated in collaboration with Dr. David Price (Cardiff University, UK). The panel consists in different macaque and human alleles (Mane-A, Mamu-A, Mamu-B, HLA-A and HLA-B) loaded with viral peptides derived from lentiviruses (SIV, HIV) and herpes viruses (EBV, CMV). We demonstrated that the KIR3DL receptor KIR049-4 has a broad reactivity to macaque and human MHC alleles harboring Bw4, Bw6 and non-Bw4/Bw6 epitope at the end of their alpha 1 helix. This observation contrasts with the specificity of human KIR3DL1 receptors, which is limited to Bw4 bearing MHC alleles. Furthermore, we showed that the nature of the peptide loaded in the MHC class I groove affected drastically the strength of the interaction and that some viral peptides exhibited antagonist properties against KIR049-4 binding. A second receptor responsible for NK cell reactivity with tetramer was identified after cloning multiple KIR alleles from positive animals, expressing these receptors in 721.221 cells and screening with tetramer binding assays. Two KIR3DL allelic variants (KIR033-1, KIR059-5) were isolated from distinct animals harboring primary NK cells subsets binding HLA-B*44 tetramers refolded with an HIV peptide. Further characterization of their MHC specificity using multiple tetramers demonstrated a distinct and narrower reactivity compared to the initial receptor KIR049-4. The presence of a basic amino acid at position 8 in the peptide appears to be a critical requirement for this new KIR3DL receptor, whereas acidic amino acids prevent the KIR MHC interaction. A third KIR3DL receptor (KIR033-7) interacts specifically with Mamu-A1*002 molecules loaded with various peptides. This receptor is an orthologue of the KIR3DL5 receptor identified in rhesus macaques, demonstrating the common evolution of KIR receptors in different macaque species. Further characterization of chimeric receptors between KIR049-4, KIR033-1/59-5 will shed light on the structural elements contributing to the peptide selectivity in KIR/MHC interaction. We have also expanded the characterization of the macaque NK cell pool by analyzing their reactivity to a large panel of MHC class I tetramers. Studying a cohort of pig-tailed macaques with 22 tetramers from human and pig-tailed/rhesus macaque origin, we identified 10 distinct patterns of reactivity corresponding to the expression of distinct polymorphic receptors by macaque NK cells. Each animal had a least one subset of NK cell identified by our tetramer panel, some animals harboring up to five distinct specificities. The prevalence of each receptor varied between 23% and 90% among the cohort. Moreover, the frequency of each NK cell subset was highly variable between animals. Coverage of the NK cell pool by the 10 specificities differed between animals: Complete coverage was obtained for some animals exhibiting multiple reactivity patterns, while coverage was partial in other individuals. This later observation suggests that the NK cell repertoire has a broad recognition spectrum that we have not completely probed yet. Indeed macaques express a larger variety of KIR molecules than found in humans. In addition to the receptors with 3 and 2 Ig domains observed in humans, macaques also possess receptors with 0 or 1 Ig domain. We detected the presence of these new receptors in multiple animals and cloned several allelic variants, demonstrating that they are encoded by polymorphic loci. Further, we expressed representative KIR0DL and KIR1DL molecules in 721.221 cells and detected their presence at the cell surface. This new observation suggests that the macaque-specific KIR1D and KIR0D molecules are truly functional NK cell receptors and not dysfunctional remains of KIR evolution in macaques. The exact nature of their function is unclear at this time and will need further investigations. We also investigated how macaque activating KIR molecules interact with adaptor molecules to activate NK cells. Using a macaque KIR3DS allele (KIR033-5), we showed in collaboration with the group of Dr. Daniel McVicar (NCI, Frederick) that KIR3DS molecules associate with DAP12, but in contrast to human KIR3DS molecules, this association was constitutive and did not dependent of a charged amino acid in the transmembrane region. Furthermore, the macaque KIR033-5 molecules associate with the FceRI-g adaptor molecule in stimulation-dependent manner. This association was dependent on the presence of an arginine residue in KIR033-5 transmembrane domain. This observation indicates that the regulation of macaque NK cell activation is more complex than described in humans. To improve our understanding of the KIR-MHC binding properties, we performed a comparative study of human and non-human primate MHC class I alleles for KIR binding in collaboration with the teams of Profs. Jamie Rossjohn (Monach University, Australia), Andrew Brooks (University of Melbourne, Australia) and David Price. This collaboration resulted in the tridimensional structure of a human KIR3DL molecule in interaction with HLA-B*57:01 molecules. This structure identified 17 amino acids in the MHC class I molecule that contact 21 amino acids in the KIR3DL1 molecule. Phylogenetic analyses of human, chimpanzee, gorilla, rhesus and pig-tailed macaque MHC class I molecules, based on the entire extra cellular region, resulted in clustering of MHC alleles based on their locus. The analysis focused on the amino acid positions serving as point of contacts for KIR resulted in clustering of MHC alleles based on their HLA-KIR binding properties. The greater diversity of macaque MHC alleles was associated with additional MHC clusters, that were not associated with human or Apes molecules, suggesting that macaques may harbor KIR molecules with MHC binding properties not present in humans or great Apes. Our characterization of KIR receptors and the NK cell repertoire in macaques represent a major step forward in elucidating the role of NK cells in the SIV-macaque model.

对人类和猕猴的研究表明，CD8+ T 细胞反应与 HIV 或 SIV 复制的初始控制有关。自然杀伤 (NK) 细胞也会影响病毒的控制和存活。这些抗病毒活性依赖于主要组织相容性复合体 (MHC) 分子，并且特定的 MHC 基因型与人类和猕猴较低的病毒载量和较慢的疾病进展相关。然而，保护作用的确切相关性仍不清楚，并且需要确定有效疫苗所需的免疫反应。重要信息可以从动物模型中获得，例如亚洲猕猴感染 SIV 或 SHIV 病毒的情况。在发病机制和疫苗研究中，使用三种猕猴来模拟 HIV 感染：恒河猴 (Macaca mulatta)、猪尾猕猴 (M. nemestrina) 和食蟹猴 (M. fascicularis)。猪尾猕猴具有独特的易感性和疾病发展特征，使该物种对于艾滋病研究特别有用（高水平的细胞激活、快速的疾病发展、对各种 SIV 毒株的易感性）。 我们的研究重点是探索猕猴的宿主遗传背景如何影响它们对 SIV 和 SHIV 感染的先天和适应性免疫反应。具体来说，我们的研究集中于表征 NK 细胞检测受感染细胞的能力。此前，我们鉴定了一组猕猴 NK 细胞，能够通过细胞表面表达的特定杀伤细胞免疫球蛋白受体 (KIR) 识别特定猕猴 MHC I 类等位基因 (Mane-A1*082/A1*084)。 KIR3L 等位基因 KIR049-4 与特定 MHC 等位基因的结合会导致 NK 细胞功能的抑制。我们通过与 David Price 博士（英国卡迪夫大学）合作生成的一组 MHC I 类四聚体筛选其结合特性，表征了 KIR3DL 等位基因 (KIR049-4) 的特异性。该组由不同的猕猴和人类等位基因（Mane-A、Mamu-A、Mamu-B、HLA-A 和 HLA-B）组成，装载有源自慢病毒（SIV、HIV）和疱疹病毒（EBV、CMV）的病毒肽。我们证明 KIR3DL 受体 KIR049-4 对猕猴和人类 MHC 等位基因具有广泛的反应性，这些等位基因在其 α 1 螺旋末端含有 Bw4、Bw6 和非 Bw4/Bw6 表位。这一观察结果与人类 KIR3DL1 受体的特异性形成对比，后者仅限于带有 MHC 等位基因的 Bw4。此外，我们还表明，MHC I 类凹槽中加载的肽的性质极大地影响了相互作用的强度，并且一些病毒肽表现出针对 KIR049-4 结合的拮抗剂特性。在克隆来自阳性动物的多个 KIR 等位基因、在 721.221 细胞中表达这些受体并通过四聚体结合测定进行筛选后，鉴定了负责 NK 细胞与四聚体反应性的第二种受体。两个 KIR3DL 等位基因变体（KIR033-1、KIR059-5）是从不同的动物中分离出来的，这些动物含有与 HIV 肽重折叠的 HLA-B*44 四聚体结合的原代 NK 细胞亚群。使用多个四聚体对其 MHC 特异性的进一步表征表明，与初始受体 KIR049-4 相比，其具有独特且更窄的反应性。肽中第 8 位碱性氨基酸的存在似乎是这种新 KIR3DL 受体的关键要求，而酸性氨基酸则阻止 KIR MHC 相互作用。第三个 KIR3DL 受体 (KIR033-7) 与装载有各种肽的 Mamu-A1*002 分子特异性相互作用。该受体是在恒河猴中发现的 KIR3DL5 受体的直系同源物，证明了 KIR 受体在不同猕猴物种中的共同进化。 KIR049-4、KIR033-1/59-5 之间嵌合受体的进一步表征将揭示 KIR/MHC 相互作用中肽选择性的结构元件。 我们还通过分析猕猴 NK 细胞库与大量 MHC I 类四聚体的反应性，扩展了猕猴 NK 细胞库的特征。通过研究一群具有来自人类和猪尾/恒河猴起源的 22 个四聚体的猪尾猕猴，我们确定了 10 种不同的反应模式，对应于猕猴 NK 细胞不同多态性受体的表达。每只动物都有至少一个由我们的四聚体小组鉴定的 NK 细胞子集，一些动物具有多达五种不同的特异性。队列中每种受体的患病率在 23% 到 90% 之间变化。此外，每个 NK 细胞亚群的频率在动物之间存在很大差异。 10 种特异性对 NK 细胞池的覆盖率在动物之间有所不同：一些表现出多种反应模式的动物获得了完全覆盖，而其他个体的覆盖则为部分。后来的观察结果表明 NK 细胞库具有广泛的识别谱，我们尚未完全探索这一点。 事实上，猕猴表达的 KIR 分子比人类表达的 KIR 分子还要多。除了在人类中观察到的具有 3 和 2 个 Ig 结构域的受体外，猕猴还拥有具有 0 或 1 个 Ig 结构域的受体。我们在多种动物中检测到这些新受体的存在，并克隆了几种等位基因变体，证明它们是由多态性基因座编码的。此外，我们在 721.221 细胞中表达了代表性的 KIR0DL 和 KIR1DL 分子，并检测了它们在细胞表面的存在。这一新的观察表明，猕猴特异性的 KIR1D 和 KIR0D 分子是真正有功能的 NK 细胞受体，而不是猕猴 KIR 进化的功能失调的残余物。目前尚不清楚其功能的确切性质，需要进一步研究。 我们还研究了猕猴激活 KIR 分子如何与接头分子相互作用以激活 NK 细胞。使用猕猴 KIR3DS 等位基因 (KIR033-5)，我们与 Daniel McVicar 博士（NCI，Frederick）团队合作证明 KIR3DS 分子与 DAP12 相关，但与人类 KIR3DS 分子相比，这种关联是组成型的，并且没有依赖于跨膜区的带电氨基酸。此外，猕猴 KIR033-5 分子以刺激依赖性方式与 FceRI-g 接头分子结合。这种关联取决于 KIR033-5 跨膜结构域中精氨酸残基的存在。这一观察结果表明，猕猴 NK 细胞激活的调节比人类中描述的更为复杂。 为了加深我们对 KIR-MHC 结合特性的理解，我们与教授团队合作，对人类和非人类灵长类 MHC I 类等位基因的 KIR 结合进行了比较研究。 Jamie Rossjohn（澳大利亚莫纳克大学）、Andrew Brooks（澳大利亚墨尔本大学）和 David Price。此次合作产生了人类 KIR3DL 分子与 HLA-B*57:01 分子相互作用的三维结构。该结构鉴定了 MHC I 类分子中的 17 个氨基酸，这些氨基酸与 KIR3DL1 分子中的 21 个氨基酸相接触。基于整个细胞外区域，对人类、黑猩猩、大猩猩、恒河猴和猪尾猕猴 MHC I 类分子进行系统发育分析，得出基于其基因座的 MHC 等位基因聚类。分析的重点是作为 KIR 接触点的氨基酸位置，导致基于 HLA-KIR 结合特性的 MHC 等位基因聚类。猕猴 MHC 等位基因的更大多样性与其他 MHC 簇有关，而这些簇与人类或猿类分子无关，这表明猕猴可能含有具有人类或类人猿中不存在的 MHC 结合特性的 KIR 分子。 我们对猕猴中 KIR 受体和 NK 细胞库的表征代表着在阐明 NK 细胞在 SIV 猕猴模型中的作用方面向前迈出了重要一步。

项目成果

Killer cell immunoglobulin-like receptor 3DL1-mediated recognition of human leukocyte antigen B.

杀伤细胞免疫球蛋白样受体 3DL1 介导的人白细胞抗原 B 识别。

• 发表时间: 2011-10-23
• 影响因子: 64.8
• 作者: Vivian, Julian P;Duncan, Renee C;Berry, Richard;O'Connor, Geraldine M;Reid, Hugh H;Beddoe, Travis;Gras, Stephanie;Saunders, Philippa M;Olshina, Maya A;Widjaja, Jacqueline M L;Harpur, Christopher M;Lin, Jie;Maloveste, Sebastien M;Price, David
• 通讯作者: Price, David

Nomenclature report on the major histocompatibility complex genes and alleles of Great Ape, Old and New World monkey species.

关于类人猿、新旧世界猴种的主要组织相容性复合体基因和等位基因的命名法报告。

• 发表时间: 2012-08
• 影响因子: 3.2
• 作者: de Groot, Natasja G;Otting, Nel;Robinson, James;Blancher, Antoine;Lafont, Bernard A P;Marsh, Steven G E;O'Connor, David H;Shiina, Takashi;Walter, Lutz;Watkins, David I;Bontrop, Ronald E
• 通讯作者: Bontrop, Ronald E