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GW Seitz, PA Dullforce, KL Garman, SM Crespo, RJ Fleishman, SR Planck, DC Parker, JT Rosenbaum; Timelapse Imaging of T Cells Extravasating into the Anterior Segment and the Cells' Interaction With Antigen-Presenting Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1551.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: During uveitis, the in vivo extravasation of T lymphocytes into the anterior segment was monitored in real time by intravital microscopy. Further, the interaction between these infiltrating T cells and antigen-presenting cells in the tissue was investigated. Methods: Spleen cells from DO11.10 transgenic mice were either cultured with ovalbumin323-339 peptide, in vitro, to create blast cells or negatively selected to isolate CD4+ cells. Both resting and activated cells were fluorescently labeled with CFSE and transferred into syngeneic BALB/c mice. Mice were challenged in the anterior chamber one to seven days post-transfer with 150µg of soluble ovalbumin (OVA), fluorescent Alexa dye labeled OVA, or human serum albumin (HSA). Intravital microscopy was used to follow the movement of labeled cells within the iris and limbus. Mice were deeply anesthetized to minimize movement caused by eye rolling, breathing and heartbeat. An automated time-lapse machine was used to photograph at a constant rate (3 or 5 frames per minute) for at least one hour at 6, 12 and 24 hours post-challenge. Cryostat sections or whole mounts of enucleated eyes were immunohistologically stained and examined with a confocal microscope. Results: Movies of DO11.10 lymphocytes leaving the anterior segment blood vessels and infiltrating the surrounding tissue were generated. T cells exited the microvasculature at preferential locations. Confocal microscopic images taken of whole mounts and cryostat sections revealed some of these DO11.10 cells juxtaposed to cells that had taken up fluorescent OVA in vivo or expressed MHC class II. 3D confocal images ex vivo reveal dendrites of these antigen-presenting cells enwrapping T lymphocytes, implying clear interaction between the T cell and antigen-presenting cells. Conclusion: We have demonstrated the ability to observe T lymphocytes entering tissues from the blood stream during uveitis. Furthermore, some of these T cells interact closely with antigen presenting cells. With the ability to label the cells of the anterior segment with specific fluorescent antibodies and fluorescent antigen in vivo, we anticipate being able to observe the interaction of T lymphocytes with various antigen-presenting cells in vivo.
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