Going Deeper: Three-Dimensional Study Of Gamma Delta T Cells In Mouse Reproductive Tract Using Tissue Clearing Methods

Several tissue clearing methods have been developed for three-dimensional imaging of thick specimens. Here, we applied CUBIC and ScaleS approaches to whole-mounted vaginal wall to reveal spatial distribution of gamma delta 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 gamma delta T cells in vaginal samples of Tcrd-H2BeGFP 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 gamma delta 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 gamma delta 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 gamma delta T cells sense sex hormone fluxes which influence their number and position them closer to the vaginal lumen in the diestrus phase.