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M.B. Ronick, J.T. Rosenbaum, S.R. Planck; Quantification of T Cell–Antigen Presenting Cell Interaction in the Iris in a Murine Uveitis Model . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4552.
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T lymphocytes are educated by antigen–presenting cells (APCs) in secondary lymphoid organs, but the interaction of T cells with APCs at a site of inflammation is less well characterized. Here we report quantification of T cell–APC physical interaction in the inflamed iris in a murine uveitis model.
OVA–specific T cells were isolated from spleens of DO11.10 transgenic mice, cultured with OVA323–329 peptide, stained with orange CMTMR and injected IV into three naïve BALB/c mice. After 2–3 days, these mice were challenged with a 4–µl intravitreal injection of 100 µg Alexa Fluor 350 (blue)–conjugated chicken ovalbumin (OVA) and ∼0.5 µg E. coli strain 055:B5 endotoxin. After 24 hours, mice were euthanized immediately following IV injection of FITC–dextran to label blood vessels. Anterior segments were dissected and 3–dimensional image reconstructions were made from Z–stacks of iris regions acquired by 3–color deconvolution fluorescence microscopy. T cell–APC interactions were quantified by determining the percent of infiltrated CMTMR–labeled T cells that were physically contacting probable APCs labeled by uptake of blue OVA. T cells were categorized as one of the following three types: A) T cells in clear, close physical contact with an APC; B) T cells within one T–cell diameter of an APC that may be in contact, though clear cell–cell contact is not visible; and C) T cells not obviously in contact with an APC and greater than one T–cell diameter from the nearest APC. Although the precise number of APCs was difficult to count, the percent of antigen–containing APCs in close contact with at least one T cell was estimated.
The injection of OVA with endotoxin into the vitreous of BALB/c mice that have received DO11.10 T cells results in reproducible T cell infiltration in the iris in an antigen–specific manner. The replacement of OVA with a control protein results in little or no T cell infiltration in the iris. We found that 22±11% (Mean±SD; n=3 eyes) of infiltrated T cells are in close physical contact with an antigen–specific APC, 36±16% are clearly not in direct contact with an APC, and in 44±6% a definitive conclusion was not clear. Conversely, a substantial number of OVA–labeled APCs appeared to be in contact with an OVA–specific T cell (range: 48%–70%).
The interaction of T cells with APCs is a fundamental component of the immune response and the visualization of such interactions at a site of inflammation is a novel observation. At the time point tested, a substantial number of T cells in the inflamed irises are affiliated with a probable APC containing the specific antigen recognized by the T cell.
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