IN SITU SIV-SPECIFIC CD4 T CELL ANALYSIS DURING ACUTE SIV INFECTION

急性 SIV 感染期间 SIV 特异性 CD4 T 细胞原位分析

• 批准号: 8358220
• 负责人: PAMELA J SKINNER
• 金额: $ 6.68万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8358220
• 关键词: Acute; Affinity; Alleles; Antibodies; Antigens; Bacteria; Baculoviruses; Biology; CD4 Positive T Lymphocytes; Cells; Collaborations; DNA; Engineering; Funding; Genome; Grant; Immunology; In Situ; Infection; Knowledge; Length; Link; Lymphoid Tissue; MHC Class II Genes; Macaca; Macaca mulatta; Methodology; Methods; Minnesota; Mission; Mus; N-terminal; National Center for Research Resources; Nose; Peptides; Plasmids; Primates; Principal Investigator; Publications; Reagent; Research; Research Infrastructure; Resources; Rest; SIV; Scientist; Site; Source; Spleen; Staining method; Stains; Streptavidin; T cell response; Techniques; Temperature; Testing; Time; Tissue Stains; Tissues; Translations; United States National Institutes of Health; Universities; Virus Diseases; Wisconsin; Work; Writing; cost; human disease; meetings; monomer; reagent testing; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To target the Wisconsin National Primate Research Center's (WNPRC) mission to develop treatments for human disease, generate knowledge of primate biology, and to facilitate the progress of research by providing expertise and resources to external scientists, we are developing a methodology that we will use to determine the abundance and in situ localization of CD4 T cells specific to simian immunodeficiency virus (SIV) in tissues during SIV infection. RESULTS: We initiated these studies by working to develop methods to stain antigen specific CD4 T cells in mice. In collaboration with Dr. Marc Jenkins and Dr. T. Dileepan at the University of Minnesota we are using mice infected with bacteria that are engineered to express a peptide that triggers a peptide-specific CD4 T cell response for which MHC class II tetramers are available. We are staining nasal associated lymphoid tissues (NALT) and spleen tissues because peptide-specific CD4 T cells accumulate at these sites. We stained tissues with class II tetramers at several different concentrations, for different lengths of incubation time, at different temperatures, and using several amplification techniques. We were successful at identifying conditions that allowed us to detect antigen-specific CD4 T cells cells stained with tetramers in the NALT. These finding will be presented at the Immunology 2011 meeting and are presently being written up for publication. In addition, Dr. Nancy Wilson at the WNPRC made substantial progress developing class II reagents for use in SIV-infected macaques. She has now successfully produced, purified, and tetramerized rhesus macaque class II molecules. Lara Vojnov at the WNPRC has begun to test these reagents in tissues from SIV infected rhesus macaques. In addition, Dr. Wilson continues to expand the repertoire of class II tetramer reagents to include additional class II alleles. Details of Dr. Wilson's work is presented below. Gag102-113 (QIVQRHLVVE), along with a linker region was engineered N terminal of DRB*w606, between the leader sequence and start of translation. DRA*010401 was engineered into the other expression site for the bi-cistronic vector pBAC10. These were done using the method of Kozono, et al. This plasmid was co-transformed with linear DNA encoding the rest of the baculovirus genome and baculovirus was generated on SF9 cells, grown to high volume and titered. Hi5 cells were expanded and infected with baculovirus encoding this construct. Supernatant was collected after 6 days of infection and monomer was purified using an affinity column to which 5 mg/ml of L243 antibody had been covalently linked. The monomer was biotinylated and conjugated to Streptavidin APC. The cells used for testing the tetramer were cryopreserved CD4+ T cell clones generated in our lab (Giraldo-Vela, et al). Unfortunately the amount of tetramer produced was small, so we have only a limited amount available for testing.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 目标：为了实现威斯康星国家灵长类研究中心 (WNPRC) 的使命，开发人类疾病的治疗方法，产生灵长类生物学知识，并通过向外部科学家提供专业知识和资源来促进研究进展，我们正在开发一种方法将用于确定 SIV 感染期间组织中特定于猿猴免疫缺陷病毒 (SIV) 的 CD4 T 细胞的丰度和原位定位。 结果： 我们通过开发对小鼠抗原特异性 CD4 T 细胞进行染色的方法来启动这些研究。我们与明尼苏达大学的 Marc Jenkins 博士和 T. Dileepan 博士合作，使用感染细菌的小鼠，这些细菌经过工程改造，可表达一种肽，该肽可触发肽特异性 CD4 T 细胞反应，而 MHC II 类四聚体可用于该反应。 我们对鼻相关淋巴组织 (NALT) 和脾组织进行染色，因为肽特异性 CD4 T 细胞在这些部位积聚。我们用几种不同浓度、不同长度的孵育时间、不同温度并使用几种扩增技术对组织进行了 II 类四聚体染色。 我们成功地确定了能够检测 NALT 中用四聚体染色的抗原特异性 CD4 T 细胞的条件。这些发现将在 2011 年免疫学会议上公布，目前正在撰写出版。 此外，WNPRC 的 Nancy Wilson 博士在开发用于感染 SIV 的猕猴的 II 类试剂方面取得了实质性进展。 她现已成功生产、纯化和四聚化恒河猴 II 类分子。 WNPRC 的 Lara Vojnov 已开始在感染 SIV 的恒河猴组织中测试这些试剂。 此外，Wilson 博士继续扩展 II 类四聚体试剂的种类，以包括其他 II 类等位基因。 威尔逊博士的工作详情如下。 Gag102-113 (QIVQRHLVVE) 与接头区域一起被设计在 DRB*w606 的 N 末端，位于前导序列和翻译起始点之间。 DRA*010401 被工程化到双顺反子载体 pBAC10 的另一个表达位点。这些是使用 Kozono 等人的方法完成的。 该质粒与编码杆状病毒基因组其余部分的线性DNA共转化，并在SF9细胞上产生杆状病毒，生长至高体积并滴定。 Hi5 细胞被扩增并用编码该构建体的杆状病毒感染。 感染6天后收集上清液并使用共价连接有5mg/ml L243抗体的亲和柱纯化单体。 该单体经过生物素化并与链霉亲和素 APC 缀合。 用于测试四聚体的细胞是我们实验室（Giraldo-Vela 等人）生成的冷冻保存的 CD4+ T 细胞克隆。 不幸的是，产生的四聚体数量很少，因此我们只有有限的数量可用于测试。