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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 ***请注意：截至2009年9月1日，Boyton博士将Cobre重新加入了PI。 NKT细胞包含一个先天的T细胞子集，该子群的能力迅速激活和/或调节免疫系统先天组的白细胞子集的功能。 NKT细胞可以通过识别CD1D（直接途径）呈现的微生物衍生的糖脂，或通过TLR配体激活的抗原表现出的细胞（间接途径）产生的IL-12和IL-18。激活后，NKT细胞迅速分泌大量的各种细胞因子，并激活其他白细胞子集，例如树突状细胞，巨噬细胞，B细胞和天然杀伤细胞。因此，NKT细胞被唯一地准备影响发展免疫反应中的早期事件。 我们已经表明，响应于原型的NKT细胞激动剂A-半乳糖酰胺，129x1/SVJ和129S1/SVIMJ NKT细胞严重缺乏IFN-G，IL-4和TNF产生。此外，它们表现出明显较低的肝脏数量，但脾脏或胸腺NKT细胞的数量明显较低。由于NKT细胞缺陷型B6小鼠中巨噬细胞TNF的产生受损，因此我们询问129只小鼠的NKT细胞数和功能受损是否会导致体内LPS挑战后的巨噬细胞功能受损。我们发现，缺乏NKT细胞数量和功能的两种129种菌株以及其他近交菌株，表现出血清TNF产生的严重缺陷，响应于TLR4配体脂多糖的体内攻击。细胞内细胞因子染色表明，低血清TNF水平是由于响应LP的单核细胞和巨噬细胞TNF产生严重受损所致。巨噬细胞TNF产生的菌株依赖性差异不是巨噬细胞的内在，表明LPS挑战后巨噬细胞TNF产生的菌株依赖性差异是由于体内调节引起的。响应体内LPS挑战，将B6 NKT细胞传递给H2匹配的129株增加了巨噬细胞TNF的产生。使用B6.129的先天性菌株，我们发现控制巨噬细胞TNF产生受损的基因座响应于体内LPS挑战与SLAM基因座相吻合，该基因座以前已被证明可以控制NOD小鼠中NKT细胞的数量和功能。 基于这些初步数据，我们的中心假设是通过控制NKT细胞的数量和功能，通过NKT1产生巨噬细胞TNF的遗传调节。为了检验这一假设，我们提出1）确定NKT1基因座中的基因，这些基因是基于对巨噬细胞对LPS和NKT细胞数量/功能的体内响应的坐标调节，通过生成跨越NKT1的高分辨率的高分辨率相似线，并通过NKT1相关间隔和2）来调查NKT1的机构，并在NKT1上进行NKT1的机械性，并将其定向NKT1。和功能。 AIM 1：在当前的资金期（08/09 04/10），我们使用SNP基因分型构建了B6.129-SLAM持续间隔的高分辨率图。使用这些数据，我们选择了间隔的丝粒和端粒端的合适的微卫星标记，以用于筛选后代后代的重组分子。我们已经设置了（B6.129-Slam X B6）F1垫片，我们已经开始筛选第一个垃圾。筛选将在UVM的DNA分析设施上进行。此外，我们使用微阵列比较了B6和B6.129-SLAM小鼠的肝NKT细胞的转录谱。这项与微阵列和生物信息学核心合作完成的工作，导致在先天间隔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。在来年，我们计划生成亚综合品系，该线将对NKT细胞和巨噬细胞室中的表型差异进行评估。我们还计划比较体内LPS挑战后B6和B6.129-Slam巨噬细胞的转录曲线。这些研究将与微阵列和生物信息学核心合作进行。 AIM 2：我们的初步数据表明，肝脏NKT细胞数和NKT细胞细胞因子的产生，响应于体内Agalcer挑战，受SLAM基因座中的基因调节。在当前的资金期间，我们已经开始解决这些表型的基础机制。使用体内BRDU标记和TUNEL染色检查肝NKT细胞稳态的检查表明，在129个肝NKT细胞中，与B6相比，该表型的很大一部分受SLAM基因座调节。在来年，我们将通过评估混合B6和B6.129-Slam骨髓嵌合体中的肝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家族受体在NKT细胞细胞因子产生中的作用。先天性间隔中的许多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。我们的初步分析表明，其中许多受体在NKT细胞上的差异表达，包括SlAMF6（LY108）替代剪接同工型的差异表达。 在分类的NKT细胞中添加抗SlamF6 mAB会导致IFN-G产生严重受损。在来年，我们将使用一组抗Slam受体mAB来确认并扩展这些数据，以评估其在NKT细胞因子生产中的作用。