Skin‐resident and infiltrating γδ T lymphocytes are components of the cutaneous immune system that provide the first line of defense against pathogens and the environment. Research that employs the isolation and culture of T cells from murine and human skin can help delineate the molecular and cellular mechanisms utilized by T lymphocytes in skin‐specific immunity. However, obtaining high numbers of T cells from epithelial tissue without resorting to long‐term culture or transformation can be difficult. Here, specific approaches are described for the isolation and culture of γδ T lymphocytes from murine skin and human skin explant cultures. In addition, a protocol to assess the morphology and activation of epidermal γδ T cells in situ using immunofluorescent microscopy is detailed. These techniques can be used to analyze resident and infiltrating γδ T lymphocytes in the skin via flow cytometry, RNA‐seq, or proteomics to further study inflammatory diseases, cancer, or autoimmunity. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Isolation, culture, and analysis of γδ T cells from murine epidermis Basic Protocol 2: Examination of γδ T cells in epidermal sheets to assess activation and morphology Basic Protocol 3: Preparation of human skin explant cultures for analysis of skin T cells Support Protocol 1: Counting live cells with hemocytometer Support Protocol 2: Preparing a Matrigel

The epidermis provides a barrier that protects against environmental damage and infection by pathogens. Epidermal T cells and keratinocytes work together to maintain skin homeostasis upon damage. During tissue repair, epidermal γδ T cells become activated to secrete cytokines, chemokines and growth factors to initiate inflammatory responses. Previous work in our lab has shown that during obesity and type 2 diabetes, Chemokine Receptor 6 (CCR6) is downregulated by murine epidermal T cells. The ligand for CCR6, C-C Motif Chemokine Ligand 20 (CCL20), is produced by keratinocytes and is responsible for cellular migration. Little is known about how CCR6 and CCL20 ligation affects epidermal γδ T cells. In this study, murine epidermal γδ T cells were harvested and stimulated in the absence or presence of CCL20 to discover how CCL20 affects activation and function of the cells. Epidermal γδ T cells that have been stimulated with anti-CD3ɛ and CCL20 downregulate their T cell receptor and upregulate CD25 showing cell activation. Interestingly, CCL20 costimulates T cell receptor stimulation to activate epidermal γδ T cells. Immunofluorescent microscopy was used to visualize the impact of CCL20 on activated cells verses non-activated cells in situ. Epidermal γδ T cells treated with CCL20 did not migrate within or out of the epidermis unlike previous studies on dermal γδ T cells. Flow cytometric analysis and Luminex assays were performed to determine which cytokines and chemokines are produced in response to CCL20. We show that CCL20 skews epidermal γδ T cells to produce pro-inflammatory cytokines and chemokines. Future studies will determine how CCL20 and CCR6 ligation affects murine models with obesity and type 2 diabetes.

The skin is the largest organ on the body and is responsible for providing protection from foreign pathogens. When skin tissue damage occurs, immune cells aid in wound repair. Epidermal T cells play a key role in maintaining skin homeostasis by their secretion of cytokines and chemokines to facilitate inflammatory responses following tissue damage. During obesity and type 2 diabetes, epidermal T cells exhibit reduced production of select cytokine and chemokine receptors one of which is C-C Chemokine receptor 6 (CCR6). The principle ligand for CCR6 is C-C Chemokine Ligand 20 (CCL20). Upon CCL20 binding, CCR6 induces lymphocyte migration however, little is known about the regulatory role CCR6 plays in cellular functions such as cytokine production and cell surface receptor expression. For this study, murine epidermal T cells were harvested and stimulated in the presence or absence of CCL20. Epidermal T cell production of cytokines and chemokines in response to CCL20 were characterized via Luminex assay. Consequently to being stimulated with CCL20 epidermal T cells upregulate IL-2R and downregulate their T cell receptor both of which signify cell activation. Additionally, CCL20 costimulates TCR stimulation to activate epidermal T cells. Using immunofluorescence microscopy, we show that CCR6 has a pivotal role in activation causing morphological changes to skin resident T cells. Future studies will investigate CCR6 activation and receptor expression of αβ and γδ in human skin.