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Going deeper: three‐dimensional study of γδ T cells in mouse reproductive tract using tissue clearing methods
Author(s) -
Mikolajewicz Katarzyna,
Chodaczek Grzegorz
Publication year - 2019
Publication title -
immunology and cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.999
H-Index - 104
eISSN - 1440-1711
pISSN - 0818-9641
DOI - 10.1111/imcb.12202
Subject(s) - estrous cycle , immunolabeling , epithelium , biology , stroma , laminin , microbiology and biotechnology , andrology , endocrinology , immunology , medicine , extracellular matrix , genetics , immunohistochemistry
Several tissue clearing methods have been developed for three‐dimensional imaging of thick specimens. Here, we applied CUBIC and Sca l eS approaches to whole‐mounted vaginal wall to reveal spatial distribution of γδ T lymphocytes, the key cells engaged in the epithelial homeostasis control and immune surveillance. Both methods rendered the tissue transparent and enabled detection of the green fluorescent protein ( GFP )‐expressing γδ T cells in vaginal samples of Tcrd‐H2Be GFP transgenic mice. Upon additional immunolabeling, however, only CUBIC preserved the GFP signal and allowed for cell localization assessment during the estrous cycle. Using a combination of single‐ and two‐photon microscopy, we found that during the diestrus phase the number of γδ T cells in the vaginal wall increased compared to estrus, while the proportion of cells residing in epithelium and stroma remained constant, irrespective of the cycle phase, and was close to 3:1, respectively. Moreover, the distance from epithelial γδ T cells to laminin‐positive basal membrane and collagen‐rich stroma also increased in diestrus in spite of thinning of epithelium upon shedding cornified cells. Our data indicate that γδ T cells sense sex hormone fluxes which influence their number and position them closer to the vaginal lumen in the diestrus phase.