VERMONT COBRE (BOYSON) PROJECT 4: GENETIC DETERMINANTS OF NKT CELL FUNCTION

佛蒙特州 COBRE (Boyson) 项目 4：NKT 细胞功能的遗传决定因素

• 批准号: 8167730
• 负责人: JONATHAN E BOYSON
• 金额: $ 14.08万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167730
• 关键词: 129 Mouse; 129X1/SvJ Mouse; Address; Adoptive Transfer; Agonist; Alternative Splicing; Antigen-Presenting Cells; Apoptosis; B-Lymphocytes; Backcrossings; Bioinformatics; Bone Marrow; Bromodeoxyuridine; Candidate Disease Gene; Cell Count; Cell physiology; Cells; Centers of Research Excellence; Chimera organism; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Congenic Strain; Control Locus; DNA analysis; Data; Dendritic Cells; Event; Exhibits; Family; Funding; Galactosylceramides; Gene Family; Genes; Genetic; Genetic Determinism; Genetic Polymorphism; Glycolipids; Grant; Homeostasis; Immune response; Immune system; In Situ Nick-End Labeling; Inbred NOD Mice; Inbred Strains Mice; Institution; Interferon Type II; Interleukin-12; Interleukin-18; Interleukin-4; Label; Leukocytes; Ligands; Lipopolysaccharides; Liver; Maps; Microsatellite Repeats; Mus; Natural Killer Cells; Pathway interactions; Phenotype; Production; Protein Isoforms; Recombinants; Regulation; Research; Research Personnel; Resolution; Resources; Role; SNP genotyping; Screening procedure; Serum; Sorting - Cell Movement; Source; Spleen; Staining method; Stains; T-Lymphocyte Subsets; TLR4 gene; TNF gene; Testing; Thymus Gland; United States National Institutes of Health; Vermont; Work; arm; base; congenic; cytokine; in vivo; macrophage; member; microorganism; monocyte; receptor; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ***Please note: Dr. Boyson re-joined COBRE as a project PI as of 9/1/2009. NKT cells comprise an innate-like T cell subset that is notable for its ability to rapidly activate and/or modulate function of leukocyte subsets of the innate arm of the immune system. NKT cells can be activated through recognition of microorganism-derived glycolipids presented by CD1d (direct pathway), or through IL-12 and IL-18 produced by antigen presenting cells activated by TLR ligands (indirect pathway). Upon activation, NKT cells rapidly secrete large amounts of a wide variety of cytokines and activate other leukocyte subsets such as dendritic cells, macrophages, B cells, and natural killer cells. Therefore, NKT cells are uniquely poised to influence early events in the developing immune response. We have shown that 129X1/SvJ and 129S1/SvImJ NKT cells are severely deficient in IFN-g, IL-4, and TNF production in response to the prototypical NKT cell agonist a-galactosylceramide. Furthermore, they exhibit significantly lower numbers of liver, but not spleen or thymus, NKT cells. Since macrophage TNF production is impaired in NKT cell-deficient B6 mice, we asked whether impaired NKT cell number and function in 129 mice would result in impaired macrophage function after an in vivo LPS challenge. We found that the two 129 strains, as well as other inbred strains of mice deficient in NKT cell number and function, exhibit a severe deficiency in serum TNF production in response to an in vivo challenge with the TLR4 ligand, lipopolysaccharide. Intracellular cytokine staining demonstrated that low serum TNF levels were due to significantly impaired monocyte and macrophage TNF production in response to LPS. Strain-dependent differences in macrophage TNF production were not macrophage-intrinsic, suggesting that strain-dependent differences in macrophage TNF production after LPS challenge was due to in vivo regulation. Adoptive transfer of B6 NKT cells to the H2-matched 129 strains increased macrophage TNF production in response to an in vivo LPS challenge. Using a B6.129 congenic strain, we found that the locus controlling impaired macrophage TNF production in response to in vivo LPS challenge coincides with the Slam locus, which has previously been demonstrated to control NKT cell number and function in NOD mice. Based on these preliminary data, our central hypothesis is that genetic regulation of macrophage TNF production by Nkt1 occurs through the control of NKT cell number and function. To test this hypothesis, we propose to 1) Identify the genes in the Nkt1 locus that underlie the coordinate regulation of the in vivo response of macrophages to LPS and of NKT cell number/function by generating high-resolution congenic lines spanning the Nkt1 congenic interval, and 2) to investigate the mechanisms through which Nkt1 regulates NKT cell and monocyte/macrophage homeostasis and function. Aim 1: In the current funding period (08/09  04/10) we have constructed a high-resolution map of the B6.129-Slam congenic interval using SNP genotyping. Using these data, we have chosen appropriate microsatellite markers at the centromeric and telomeric ends of the interval for use in screening recombinants in backcross progeny. We have set up (B6.129-Slam X B6)F1 matings and we have begun to screen our first litters. Screening will be performed at the DNA Analysis facility at UVM. In addition, we have compared the transcriptional profiles of liver NKT cells from B6 and B6.129-Slam mice using microarray. This work done in collaboration with the Microarray and Bioinformatics Core, has resulted in the identification of a number of candidate genes within the congenic interval that could explain the observed phenotypes, including members of the Slam receptor and Ifi200 gene families. At present, we are confirming expression data on a limited number of candidate genes using QPCR. In the coming year, we plan on generating subcongenic lines which will be assessed for phenotypic differences in the NKT cell and macrophage compartments. We also plan on comparing transcriptional profiles of B6 and B6.129-Slam macrophages after in vivo LPS challenge. These studies will be conducted in collaboration with the Microarray and Bioinformatics Core. Aim 2: Our preliminary data indicate that liver NKT cell number and NKT cell cytokine production in response to in vivo aGalCer challenge is regulated by a gene(s) within the Slam locus. In the current funding period, we have begun to address the mechanisms underlying these phenotypes. Examination of liver NKT cell homeostasis using in vivo BrDU labeling and TUNEL staining revealed a significantly higher level of apoptosis in 129 liver NKT cells versus B6, and that a significant portion of this phenotype was regulated by the Slam locus. In the coming year, we will confirm and then extend these data by assessing liver NKT cell homeostasis in mixed B6 and B6.129-Slam bone marrow chimeras. In addition, we have begun to evaluate the role of Slam family receptors on NKT cell cytokine production. Many of the Slam family genes in the congenic interval exhibit numerous polymorphisms that define haplotypic differences between B6 and 129X1/SvJ mice. Our preliminary analysis revealed differential expression of many of these receptors on NKT cells, including the differential expression of Slamf6 (Ly108) alternative splice isoforms. Addition of anti-SLAMF6 mAbs to sorted NKT cells resulted in significantly impaired IFN-g production. In the coming year, we will confirm and extend these data using a panel of anti-SLAM receptor mAbs to assess their role in NKT cytokine production.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 ***请注意：Boyson 博士于 2009 年 9 月 1 日作为项目 PI 重新加入 COBRE。 NKT 细胞包含先天性 T 细胞亚群，其以其快速激活和/或调节免疫系统先天臂白细胞亚群功能的能力而闻名。 NKT 细胞可以通过识别 CD1d 呈递的微生物来源的糖脂（直接途径）或通过 TLR 配体激活的抗原呈递细胞产生的 IL-12 和 IL-18 来激活（间接途径）。激活后，NKT 细胞迅速分泌大量多种细胞因子，并激活其他白细胞亚群，如树突状细胞、巨噬细胞、B 细胞和自然杀伤细胞。因此，NKT 细胞具有独特的能力来影响免疫反应发展中的早期事件。 我们已经证明，129X1/SvJ 和 129S1/SvImJ NKT 细胞在响应原型 NKT 细胞激动剂 a-半乳糖神经酰胺时严重缺乏 IFN-g、IL-4 和 TNF 生成。此外，它们的肝脏 NKT 细胞数量显着减少，但脾脏或胸腺的 NKT 细胞数量却没有减少。由于 NKT 细胞缺陷的 B6 小鼠中巨噬细胞 TNF 的产生受损，我们询问 129 只小鼠中 NKT 细胞数量和功能受损是否会导致体内 LPS 攻击后巨噬细胞功能受损。我们发现，这两个 129 品系以及其他 NKT 细胞数量和功能缺陷的近交品系小鼠在响应 TLR4 配体脂多糖的体内攻击时，表现出血清 TNF 产生的严重缺陷。细胞内细胞因子染色表明，低血清 TNF 水平是由于 LPS 响应的单核细胞和巨噬细胞 TNF 产生显着受损所致。巨噬细胞 TNF 产生的菌株依赖性差异不是巨噬细胞固有的，表明 LPS 攻击后巨噬细胞 TNF 产生的菌株依赖性差异是由于体内调节所致。将 B6 NKT 细胞过继转移至 H2 匹配的 129 株中，可增加巨噬细胞 TNF 的产生，以响应体内 LPS 的挑战。使用 B6.129 同类菌株，我们发现响应体内 LPS 攻击而控制巨噬细胞 TNF 产生受损的基因座与 Slam 基因座一致，该基因座先前已被证明可以控制 NOD 小鼠中的 NKT 细胞数量和功能。 基于这些初步数据，我们的中心假设是，Nkt1 对巨噬细胞 TNF 产生的遗传调控是通过控制 NKT 细胞数量和功能而发生的。为了检验这一假设，我们建议 1) 通过生成跨越 Nkt1 同源间隔的高分辨率同源系，识别 Nkt1 基因座中巨噬细胞对 LPS 的体内反应和 NKT 细胞数量/功能协调调节的基因，2) 研究 Nkt1 调节 NKT 细胞和单核细胞/巨噬细胞稳态和功能的机制。 目标 1：在当前资助期间 (08/09 04/10)，我们使用 SNP 基因分型构建了 B6.129-Slam 同源区间的高分辨率图谱。利用这些数据，我们在着丝粒和端粒末端选择了适当的微卫星标记，用于在回交后代中筛选重组体。我们已经建立了 (B6.129-Slam X B6)F1 交配，并且我们已经开始筛选我们的第一窝幼崽。筛查将在 UVM 的 DNA 分析设施进行。此外，我们还使用微阵列比较了 B6 和 B6.129-Slam 小鼠肝脏 NKT 细胞的转录谱。这项工作是与微阵列和生物信息学核心合作完成的，在同源区间内鉴定了许多候选基因，这些基因可以解释观察到的表型，包括 Slam 受体和 Ifi200 基因家族的成员。目前，我们正在使用 QPCR 确认有限数量的候选基因的表达数据。来年，我们计划生成亚同类系，对 NKT 细胞和巨噬细胞区室的表型差异进行评估。我们还计划比较体内 LPS 攻击后 B6 和 B6.129-Slam 巨噬细胞的转录谱。这些研究将与微阵列和生物信息学核心合作进行。 目标 2：我们的初步数据表明，响应体内 aGalCer 攻击的肝脏 NKT 细胞数量和 NKT 细胞细胞因子的产生受到 Slam 基因座内的基因的调节。在当前的资助期内，我们已经开始解决这些表型背后的机制。使用体内 BrDU 标记和 TUNEL 染色检查肝脏 NKT 细胞稳态，结果显示 129 个肝脏 NKT 细胞的凋亡水平显着高于 B6，并且这种表型的很大一部分是由 Slam 基因座调节的。来年，我们将通过评估混合 B6 和 B6.129-Slam 骨髓嵌合体中的肝脏 NKT 细胞稳态来确认并扩展这些数据。此外，我们已经开始评估Slam家族受体对NKT细胞细胞因子产生的作用。许多同系区间的 Slam 家族基因表现出许多多态性，这些多态性定义了 B6 和 129X1/SvJ 小鼠之间的单倍型差异。我们的初步分析揭示了 NKT 细胞上许多受体的差异表达，包括 Slamf6 (Ly108) 选择性剪接亚型的差异表达。 向分选的 NKT 细胞中添加抗 SLAMF6 mAb 会导致 IFN-g 产生显着受损。来年，我们将使用一组抗 SLAM 受体单克隆抗体来确认和扩展这些数据，以评估它们在 NKT 细胞因子产生中的作用。