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; 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。 目的：针对威斯康星州国家灵长类动物研究中心（National Primate Research Center）的使命，旨在通过向外部科学家提供专业知识和资源来开发人类疾病的治疗方法，产生灵长类动物生物学知识，并促进研究的进步，我们正在开发一种方法，我们将使用该方法来确定与Simian Immundicficitige virus（SIV）中的CD4 T细胞的丰度和原位定位的SIV（SIV）在SIV中。 结果：我们通过努力开发染色小鼠抗原特异性CD4 T细胞的方法开始了这些研究。 与明尼苏达大学的Marc Jenkins博士和T. Dileepan博士合作，我们正在使用感染细菌的小鼠，这些小鼠经过精心设计，以表达肽，该肽会触发肽特异性的CD4 T细胞反应，该反应可为MHC II类四聚体提供可用。 我们正在染色鼻相关淋巴组织（NALT）和脾脏组织，因为肽特异性的CD4 T细胞在这些位点积累。 我们尝试在几个不同的浓度下，在几个不同的孵育时间，在不同的温度下，并使用几种扩增技术，以几种不同的浓度染色组织。 到目前为止，我们已经成功地检测出使用NALT在NALT中弱染色的细胞，该细胞使用2-4 nm的四聚体以四度孵育，然后在室温下孵育一小时。 现在，我们正在尝试使用Tyramide信号扩增（TSA）技术通过II类四聚体对细胞的检测，并验证使用CD4，CD3和CD20抗体抗染色的染色的特异性。 此外，WNPRC的南希·威尔逊（Nancy Wilson）博士正在为开发用于SIV感染猕猴的II类试剂的进度。 到目前为止，她已经成功地生产了猕猴II类分子，现在正在努力开发一种净化这些分子的方法。 此外，已经确定了9个又有9个DP/肽三聚体，还有4个DQ夹子和10个DR Trimers。 在我们正在研究的七个四聚体的成功完成之后，工作将进展。 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，CD8- SIV特异性T细胞的局部群体在淋巴样卵泡和阴道的SIV上表皮中的SIV特异性T细胞。 Plos One，2009年； 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. 科学，2009年； 323（5922）：1726-1729。 li，Pamela J. Skinner，Lijie Duan和Ashley T. Haase，这是一种同时可视化病毒特异性CD8+ T细胞和原位病毒感染的细胞的技术，在Science和of Science of Science and of Science和of Science of of Science of of Science of可视化实验杂志上，2009年，2009年。