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Doran Spencer, Asghar Haider, Geraint John Parfitt, James V Jester; Localization and characterization of limbal T cells relative to label-retaining cells via immunofluorescence tomography. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4037.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the anatomical localization of limbal T cells relative to label-retaining cells (LRCs). Previously, we demonstrated the presence of presumed limbal regulatory (Treg) Foxp3-GFP+ T cells via in vivo epifluorescent imaging. In this study, we further characterize the localization of T cells in the limbus in conjunction with LRCs, i.e. presumed limbal stem cells, via immunofluorescence tomography.
H2B-GFP/K5tTA (HGK) mice express histone H2B-green fluorescent protein (GFP) under the control of the tetracycline and keratin 5 promoters, resulting in retention of GFP in slow-cycling LRCs after doxycycline administration. Following a 16 week pulse-chase period, the limbal region of HGK mice was fixed with 2% paraformaldehyde, sectioned and embedded in butyl-methyl-methacrylate, subjected to immunohistochemistry (IHC) with the T cell marker CD3, and imaged via immunofluorescence tomography to create high-resolution 3-D reconstructions of 200 sections. 3-D reconstructions were performed through semi-automated alignment with Amira software to allow for anatomical localization of LRCs and CD3+ T cells. DAPI was used as a nuclear stain for cellular localization and to aid image registration. Further IHC experiments with additional antibodies specific for Tregs are undergoing optimization.
In a representative experiment, 24 CD3+ and 55 GFP+ cells were localized to the limbus and were absent in the peripheral cornea, i.e. epithelium and full-thickness of the stroma. The CD3+ cells were detected within the limbal epithelium and cornea stroma in close anatomical approximation to GFP+ LRCs.
We have demonstrated the localization of CD3+ and GFP-label retaining cells of pulse-chase H2B-GFP/K5tTA mice within a narrow anatomical region corresponding to the reported niche of limbal stem cells. This limbal region corresponds qualitatively to the site of presumed regulatory Foxp3-GFP+ T cells identified previously by a separate experimental approach. Further experiments and statistical analysis are underway to allow for greater delineation of the nature of the T cells and their potential anatomical and functional association with limbal stem cells; these findings suggest possible implications for diseases involving inflammatory conditions of the ocular surface.
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