Going deeper: three‐dimensional study of γδ T cells in mouse reproductive tract using tissue clearing methods

• Published in: Immunology and cell biology 2019-01, Vol.97 (1), p.104-111
• Main Authors: Mikolajewicz, Katarzyna, Chodaczek, Grzegorz
• Format: Article
• Language: English
• Subjects: Collagen; Confocal and 2‐photon microscopy; Epithelium; estrous cycle; Estrus cycle; Gamma‐delta T cells; Green fluorescent protein; Homeostasis; Immunosurveillance; Laminin; Localization; Lymphocytes; Lymphocytes T; Reproductive system; Rodents; Spatial distribution; Stroma; Transgenic mice; Vagina; vaginal epithelium
• ISSN: 0818-9641; 1440-1711
• DOI: 10.1111/imcb.12202

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 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‐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 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. Vaginal wall and tissue‐resident γδ T cells can be successfully imaged upon tissue clearing with CUBIC method irrespective of the estrous cycle phase. γδ T cells sense estrous cycle changes as their number increases during diestrus. During the estrus phase, they reside close to the basal membrane, while in diestrus after shedding cornified epithelial cells, they are localized closer to the vaginal lumen.