Tetramers were added at a concentration of 0.5 μg/ml with 2% normal goat serum (NGS) 3 and 0.5 μg/ml rat anti-CD8a Abs clone 53-6.7 (PharMingen, San Diego, CA) or CTCD8a (Caltag, South San Francisco, CA) and incubated overnight. Materials and Methodsįresh sections were stained free floating in 1 ml solution with four sections per well in 24-well tissue culture plates, and incubations were conducted at 4☌ on a rocking platform. In this report, we describe such a technique that should be generally applicable to visualizing Ag-specific T cells in tissues. To that end, we set out to find conditions in an optimal well-characterized transgenic model for directly staining Ag-specific T cells in tissues. With the eventual goal of investigating the spatial and temporal relationships between viral replication and the specific CTL response in tissue, we sought to adapt the tetramer staining technology to tissue analysis. Thus far, however, these studies have been directed to cells isolated from blood, semen, or tissues that were stained with tetramer-peptide complexes and then analyzed by flow cytometry. Enumerating virus-specific CD8 + T lymphocytes in cross-sectional or longitudinal studies has made it possible to track expansion and contraction of the CTL response in these infections and, in HIV-1 infection, to document the inverse correlation between the CTL response and viral load and progression to disease ( 4). of a method to identify and phenotypically characterize Ag-specific T lymphocytes has quickly advanced understanding of the immune response in general and particularly the virus-specific CTL response to a number of acute and chronic infections that include influenza virus, lymphocytic choriomeningitis virus, EBV, human T cell leukemia virus-1, hepatitis C, Listeria monocytogenes, and HIV-1 and SIV infections ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11).
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