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: 8173124
• 负责人: PAMELA J SKINNER
• 金额: $ 5.16万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173124
• 关键词: Acute; Antibodies; Antigens; Bacteria; Biology; CD3 Antigens; CD4 Positive T Lymphocytes; CD8B1 gene; Cells; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Detection; Engineering; Epithelium; Funding; Grant; Hour; In Situ; Incubated; Institution; Journals; Knowledge; Length; Lymphoid Follicle; Lymphoid Tissue; MHC Class II Genes; MS4A1 gene; Macaca; Macaca mulatta; Methodology; Methods; Minnesota; Mission; Mus; Nose; Outcome; Peptides; Population; Primates; Publishing; Reagent; Research; Research Personnel; Resources; SIV; Science; Scientist; Services; Signal Transduction; Site; Source; Specificity; Spleen; Staining method; Stains; T cell response; T-Lymphocyte; Techniques; Temperature; Time; Tissue Stains; Tissues; United States National Institutes of Health; Universities; Vagina; Virus; Virus Diseases; Wisconsin; Work; human disease; improved

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. Objective: To target the Wisconsin National Primate Research Center's 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 have tried staining tissues with class II tetramers at several different concentrations, at several different lengths of incubation time, at different temperatures, and using several amplification techniques. Thus far, we have been successful at detecting cells that are weakly stained with tetramers in the NALT using 2-4 nM tetramers incubated over night at four degrees followed by a one hour incubation at room temperature. We are now attempting to improve the detection of cells with the class II tetramers using tyramide signal amplification (TSA) techniques, and verifying the specificity of staining using CD4, CD3, and CD20 antibody counterstaining. In addition, Dr. Nancy Wilson at the WNPRC is making progress developing class II reagents for use in SIV-infected macaques. She has thus far successfully produced rhesus macaque class II molecules and is now working to develop a method to purify these molecules. In addition, 9 more DP/peptide trimers have been identified, as have 4 more DQ trimers and 10 more DR trimers. Work will progress on these subsequent to the successful completion of the seven tetramers we are studying. This work used WNPRC Research Services. PUBLICATIONS: *Jung Joo Hong, Teresa L. Mattila, Aaron Hage, Matthew R. Reynolds, David I. Watkins, Christopher J. Miller, and Pamela J. Skinner, Localized Populations of CD8- SIV-Specific T Cells in Lymphoid Follicles and Vaginal Epithelium of SIV infected macaques. PloS ONE, 2009; 4(1): e4131. Qingsheng Li, Pamela J. Skinner, Sang-Jun Ha, Lijie Duan, Teresa L. Mattila, Aaron Hage, Cara White, Daniel L. Barber, Leigh O'Mara, Peter J. Southern, Cavan S. Reilly, Christopher J. Miller, Rafi Ahmed and Ashley T. Haase, Visualizing antigen specific and infected cells in situ predicts outcome in early viral infection. Science, 2009; 323(5922):1726-1729. Qingsheng Li, Pamela J. Skinner, Lijie Duan, and Ashley T. Haase, A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells in situ, published in both Science and Journal of Visualized Experimentation, 2009.

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。子项目和 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。 目标：为了实现威斯康星国家灵长类研究中心的使命，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，我们将使用该方法来确定 SIV 感染期间组织中特定于猿免疫缺陷病毒 (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。 我们尝试用几种不同浓度、几种不同长度的孵育时间、不同温度并使用几种扩增技术用 II 类四聚体对组织进行染色。 到目前为止，我们已经成功地检测了 NALT 中四聚体微弱染色的细胞，使用 2-4 nM 四聚体在 4 度下孵育过夜，然后在室温下孵育一小时。 我们现在正尝试使用酪酰胺信号放大 (TSA) 技术改进对 II 类四聚体细胞的检测，并使用 CD4、CD3 和 CD20 抗体复染验证染色的特异性。 此外，WNPRC 的 Nancy Wilson 博士在开发用于感染 SIV 的猕猴的 II 类试剂方面取得了进展。 迄今为止，她已成功生产出恒河猴 II 类分子，目前正致力于开发一种纯化这些分子的方法。 此外，还鉴定出另外 9 个 DP/肽三聚体、另外 4 个 DQ 三聚体和 10 个 DR 三聚体。 在成功完成我们正在研究的七个四聚体之后，这些工作将取得进展。 这项工作使用了 WNPRC 研究服务。 出版物： *Jung Joo Hong、Teresa L. Mattila、Aaron Hage、Matthew R. Reynolds、David I. Watkins、Christopher J. Miller 和 Pamela J. Skinner，淋巴滤泡和阴道上皮中 CD8-SIV 特异性 T 细胞的局部群体感染 SIV 的猕猴。 《公共科学图书馆一号》，2009 年； 4（1）：e4131。 李庆生、Pamela J. Skinner、Sang-Jun Ha、段丽杰、Teresa L. Mattila、Aaron Hage、Cara White、Daniel L. Barber、Leigh O'Mara、Peter J. Southern、Cavan S. Reilly、Christopher J. Miller、Rafi Ahmed 和 Ashley T. Haase，原位可视化抗原特异性和感染细胞可预测早期病毒感染的结果。 科学，2009； 323（5922）：1726-1729。 Qingsheng Li、Pamela J. Skinner、Lijie Duan 和 Ashley T. Haase，《A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells in situ》，发表在 Science 和 Journal of Visualized Experimentation 上，2009 年。