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E.J. Lee, D.R. Stevens, S.R. Planck, J.T. Rosenbaum; Dynamic in vivo Imaging of T Cell Migration in the Murine Cornea . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3588.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The T cell is fundamental to the immune response in both health and disease, but the movement of T cells at a site of inflammation is virtually unstudied. Here, we examined the T cell response within inflamed corneal tissue in vivo in an antigen–specific model. Methods: T cells isolated from spleens of transgenic DO11.10 mice, whose T cell receptors specifically recognize chicken ovalbumin peptide (OVA323–339), were cultured in vitro for 5 d with the peptide (2 µg/ml). Cells were labeled with the orange fluorescent dye, CMTMR, and injected into tail veins of naïve congenic mice. At 1–2 d thereafter, animals received a single combined corneal intrastromal injection (1–2 µl) of LPS (5 mg/ml) and AlexaFluor Green–labeled ovalbumin (25 mg/ml). At 6 h and 1 d, multicolor time–lapse intravital imaging (acquired at 3 frames/min for 30 min at each timepoint) was performed to track movement of T cells and of resident corneal pinocytic cells that had ingested ovalbumin. Movie files were processed with image stabilization software (MediaCybernetics), the ImageJ program (NIH) was used to track individual cell locations in each video frame, and parameters of cell movement were analyzed. Results: As expected, the number of T cells within the cornea increased from 6 h to1 d. Migratory dynamics of T cells analyzed at 1 d were heterogeneous. Approximately 13% of infiltrated T cells remained stationary during the timeframe of the recordings, while some cells registered instantaneous velocities as rapid as 38.4 µm/min. Mean cell displacement analysis revealed patterns of cell paths that could be broadly categorized as random (29% of migrating cells), directed (4%), or confined (18%). Interestingly, ∼50% of migrating cells exhibited a combination of at least two of these mobility patterns during the recording period. Although pinocytic cells exhibited no detectable migration, they were seen to move cell processes that apparently interacted with passing T cells. Conclusions: This model allows a precise characterization of T cell mobility at a site of inflammation and will prove useful in understanding T cell–mediated corneal diseases such as transplant rejection.
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