Li Shengbin, Mwakalundwa Gwantwa, Skinner Pamela J
Department of Veterinary and Biomedical Sciences, University of Minnesota.
Department of Veterinary and Biomedical Sciences, University of Minnesota;
J Vis Exp. 2017 Sep 22(127):56130. doi: 10.3791/56130.
T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled "Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8 Cells In Vivo." The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.
T细胞对许多免疫过程至关重要，包括检测和清除病毒感染细胞、预防自身免疫、协助B细胞和浆细胞产生抗体以及检测和清除癌细胞。通过流式细胞术分析抗原特异性T细胞的MHC四聚体染色技术的发展，彻底改变了我们研究和理解T细胞免疫生物学的能力。虽然流式细胞术对于确定抗原特异性T细胞的数量和表型极为有用，但它无法确定抗原特异性T细胞在组织中与其他细胞和结构的空间定位，而且目前用于提取流式细胞术所需T细胞的解离技术在非淋巴组织中的效果有限。原位MHC四聚体染色（IST）是一种在组织中可视化针对感兴趣抗原的T细胞的技术。与免疫组织化学（IHC）相结合，IST可以确定组织中抗原特异性CD8和CD4 T细胞的丰度、位置和表型。在这里，我们描述了一种对位于特定组织隔室内具有特定表型的抗原特异性CD8 T细胞进行染色和计数的方案。这些程序与我们最近由Li等人发表的题为“卵泡中产生猿猴免疫缺陷病毒的细胞在体内被CD8细胞部分抑制”的文章中所使用的相同。所描述的方法具有广泛的适用性，因为它们可用于在任何组织中定位、鉴定表型并量化基本上任何有可用MHC四聚体的抗原特异性CD8 T细胞。
