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 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 目的： 针对威斯康星州国家灵长类动物研究中心的使命，开发人类疾病的治疗方法，产生灵长类动物生物学的知识，并通过提供专业知识和资源，以促进研究的进展，外部科学家，我们正在开发一种方法，我们将用于确定的丰度和原位定位的CD 4 T细胞特异性猴免疫缺陷病毒（SIV）在组织中SIV感染。 结果：我们开始这些研究的工作，以开发方法来染色抗原特异性的CD 4 T细胞在小鼠中。 与Marc Jenkins博士和T.明尼苏达大学的Dileepan博士说，我们正在使用感染了细菌的小鼠，这些细菌被改造成表达一种肽，这种肽可以触发肽特异性CD 4 T细胞反应，而MHC II类四聚体是可用的。 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CD 4 T细胞在这些部位聚集。 我们已经尝试用几种不同浓度的II类四聚体、几种不同的孵育时间长度、不同的温度和几种扩增技术对组织进行染色。 到目前为止，我们已经成功地检测了在NALT中用四聚体弱染色的细胞，使用2-4 nM四聚体在4度下孵育过夜，然后在室温下孵育1小时。 我们现在正在尝试使用酪胺信号放大（TSA）技术来改善对具有II类四聚体的细胞的检测，并使用CD 4、CD 3和CD 20抗体复染来验证染色的特异性。 此外，WNPRC的Nancy Wilson博士正在开发用于SIV感染猕猴的II类试剂。 到目前为止，她已经成功地产生了恒河猴II类分子，现在正在努力开发一种纯化这些分子的方法。 此外，还鉴定了另外9种DP/肽三聚体，另外4种DQ三聚体和另外10种DR三聚体。 在我们正在研究的七个四聚体成功完成之后，这些工作将取得进展。 这项工作使用WNPRC研究服务。 出版物： *Jung Joo Hong，Teresa L.放大图片创作者：John R. Reynolds，大卫Watkins，Christopher J.米勒和Pamela J. Skinner，SIV感染猕猴的毛囊和阴道上皮中CD 8-SIV特异性T细胞的局部群体。 PLoS ONE，2009; 4（1）：e4131. 李庆生，Pamela J. Skinner，Sang-Jun Ha，Lijie Duan，Teresa L.放大图片作者：Aaron Hage，Cara白色，丹尼尔L.放大图片作者：Peter J.作者：Christopher J.米勒，Rafi Ahmed和阿什利T. Haase，可视化抗原特异性和感染细胞原位预测早期病毒感染的结果。 Science，2009; 323（5922）：1726-1729. Qingsheng Li，Pamela J. Skinner，Lijie Duan，and阿什利T. Haase，一种同时原位可视化病毒特异性CD 8 + T细胞和病毒感染细胞的技术，发表在《科学》和《可视化实验杂志》，2009年。