Epidermal Γδ Research Articles

Determining the Effects of Obesity and Type 2 Diabetes on CCR6+ Epidermal GD T Cell Number and Function in Inflammatory Disease and Wound Healing

Abstract A maladaptive healing cascade causes chronic wounds. γδ T cells are affected, the dysregulation of which causes a reduction of cytokines and growth hormones. We are studying the Chemokine Receptor 6 (CCR6)/Chemokine Ligand 20 (CCL20) axis and how it functions in the wounds of obese and diabetic mice. When keratinocytes are stressed by wounding or psoriasis, CCL20 is released and interacts with CCR6 on Dermal T cells to recruit them to the epidermis. Epidermal T cells and their role in this axis are not as well understood. Upon wounding, it was found that a subset of epidermal γδ T cells express CCR6 within 24 hours, and it is downregulated by 72 hours. The role of the CCR6 epidermal γδ T cells express was also investigated. Epidermal γδ T cells, when co stimulated with CCL20 and anti-CD3, generate IL-17A, suggesting that CCR6 plays a role in TH17 responses during inflammation. Animals and humans with obesity and diabetes both exhibit elevated IL-17, so obese and diabetic mice were examined during wound healing and psoriasis. During keratinocyte stress, epidermal gd T cells experienced significantly elevated levels of CCR6 and IL-17 in obese and diabetic mice. This shows that in chronic diseases like type 2 diabetes, resident T cell IL-17 responses are amplified. In better understanding the mechanisms used by resident T cells during their inflammatory responses to keratinocyte stress, a targeted therapy can be developed to help treat chronic wounds and psoriasis.

TCR signaling and cellular metabolism regulate the capacity of murine epidermal γδ T cells to rapidly produce IL-13 but not IFN-γ.

Resident epidermal T cells of murine skin, called dendritic epidermal T cells (DETCs), express an invariant γδ TCR that recognizes an unidentified self-ligand expressed on epidermal keratinocytes. Although their fetal thymic precursors are preprogrammed to produce IFN-γ, DETCs in the adult epidermis rapidly produce IL-13 but not IFN-γ early after activation. Here, we show that preprogrammed IFN-γ-producing DETC precursors differentiate into rapid IL-13 producers in the perinatal epidermis. The addition of various inhibitors of signaling pathways downstream of TCR to the in vitro differentiation model of neonatal DETCs revealed that TCR signaling through the p38 MAPK pathway is essential for the functional differentiation of neonatal DETCs. Constitutive TCR signaling at steady state was also shown to be needed for the maintenance of the rapid IL-13-producing capacity of adult DETCs because in vivo treatment with the p38 MAPK inhibitor decreased adult DETCs with the rapid IL-13-producing capacity. Adult DETCs under steady-state conditions had lower glycolytic capacity than proliferating neonatal DETCs. TCR stimulation of adult DETCs induced high glycolytic capacity and IFN-γ production during the late phase of activation. Inhibition of glycolysis decreased IFN-γ but not IL-13 production by adult DETCs during the late phase of activation. These results demonstrate that TCR signaling promotes the differentiation of IL-13-producing DETCs in the perinatal epidermis and is needed for maintaining the rapid IL-13-producing capacity of adult DETCs. The low glycolytic capacity of adult DETCs at steady state also regulates the rapid IL-13 response and delayed IFN-γ production after activation.

Multimodal profiling reveals site-specific adaptation and tissue residency hallmarks of γδ T cells across organs in mice

γδ T cells perform heterogeneous functions in homeostasis and disease across tissues. However, it is unclear whether these roles correspond to distinct γδ subsets or to a homogeneous population of cells exerting context-dependent functions. Here, by cross-organ multimodal single-cell profiling, we reveal that various mouse tissues harbor unique site-adapted γδ subsets. Epidermal and intestinal intraepithelial γδ T cells are transcriptionally homogeneous and exhibit epigenetic hallmarks of functional diversity. Through parabiosis experiments, we uncovered cellular states associated with cytotoxicity, innate-like rapid interferon-γ production and tissue repair functions displaying tissue residency hallmarks. Notably, our observations add nuance to the link between interleukin-17-producing γδ T cells and tissue residency. Moreover, transcriptional programs associated with tissue-resident γδ T cells are analogous to those of CD8+ tissue-resident memory T cells. Altogether, this study provides a multimodal landscape of tissue-adapted γδ T cells, revealing heterogeneity, lineage relationships and their tissue residency program.

The junctional adhesion molecule-like protein (JAML) is important for the inflammatory response during contact hypersensitivity.

The junctional adhesion molecule-like protein (JAML) plays important roles in wound healing and activation of epidermal γδ T cells in mice. Whether JAML plays a role in contact hypersensitivity (CHS), the animal model of allergic contact dermatitis (ACD), is not known. To examine the role of JAML in CHS, we used various mouse models of CHS in JAML knockout (KO) and wild-type (WT) mice. Furthermore, the expression of the JAML ligand coxsackievirus and adenovirus receptor (CXADR) on keratinocytes was accessed in vitro and in vivo. JAML KO mice had a diminished inflammatory response during both the sensitization and elicitation phase of CHS and had reduced numbers of CD8+ and CD4+ T cells in the epidermis. Furthermore, interferon γ (IFNγ), interleukin 1β (IL-1β) and CXCL10 production were significantly reduced in JAML KO mice during the elicitation phase. We found that CD8+ T cells express JAML and that JAML is essential for rapid flare-up responses to contact allergens. Finally, we show that keratinocytes up-regulate the JAML ligand CXADR following exposure to contact allergens. Our study is the first to show a central role of JAML in CHS and reveals a potential new target for the treatment of ACD in humans.

Regulation of CCR6 Expression on Epidermal γδ T Cells in Response to Damage or Stress in the Skin

Abstract Chronic conditions, including type 2 diabetes and psoriasis, interfere with the timing and location of necessary inflammatory cellular functions. Inflammation in the skin is at least partly mediated by epidermal γδ T cells in humans and mice, which secrete growth factors, cytokines and chemokines. However, less is known about which chemokines regulate epidermal γδ T cells and whether functional epidermal T cell subsets exist. One signaling pathway of interest is the C-C Motif Chemokine Receptor 6 (CCR6)/C-C Motif Chemokine ligand 20 (CCL20) axis, which recruits T cells to the epidermis for wound repair. To investigate how this signaling pathway initiates and regulates epidermal γδ T cell functions, we utilized flow cytometry, publicly available scRNA-seq data. Luminex and immunofluorescence microscopy were utilized to evaluate the location and timing of CCR6 expression by epidermal γδ T cells. Initial findings indicate that CCR6 expression is upregulated in a subset of activated epidermal γδ T cells and that CCL20 induces pro-inflammatory IL-17-biased gene expression. Analysis of publicly available single cell RNA sequencing data from mice with psoriasis show that predominantly proinflammatory genes are upregulated in the CCR6+ epidermal γδ T cell subset. Some proinflammatory genes that are expressed include IL-22, IL-17F and IL-17A. Further, upregulation of CCR6 expression by a subset of epidermal γδ T cells begins within the first hours post wounding and remains elevated until a decline is observed 72 hours post wounding.Further understanding of the role of CCR6 in inflammation can lead to the development of novel treatments for conditions such as chronic non healing wounds or psoriasis.

Identification of BST2 as a biomarker for alopecia areata in both mice and humans

Abstract Alopecia areata is an autoimmune disease characterized by aberrant non-scarring hair loss. 147 million people worldwide are currently diagnosed with alopecia areata with a lifetime incidence of 2.1%. The hair follicle is a site of immune privilege however in alopecia areata, CD4+ helper T cells and CD8+ cytotoxic T cells infiltrate and attack the hair follicle. To better understand the initiating mechanisms surrounding alopecia areata, we examined the function of epidermal γδ T cells and dermal macrophages in the initiation of or protection from the disease. We show that epidermal γδ T cells and keratinocytes are elevated in the skin of C3H/HeJ mice with alopecia areata. Analysis of publicly available single cell RNA sequencing data delineated the top differentially expressed genes of epidermal γδ T cells and dermal macrophages. Bone marrow stromal antigen 2 (BST2) is significantly upregulated among both cell types and is a novel gene associated with alopecia areata. Using immunofluorescence microscopy we determined that a small population of hair follicle-associated epidermal γδ T cells express BST2 in C3H/HeJ mice and C57Bl/6J mice without alopecia areata. We have also identified a significant increase in BST2 expression in skin sections from mice affected by alopecia areata. BST2+CD11b+ macrophages upregulate genes associated with antiviral activity indicating an association between the pathogenesis of alopecia areata and infectious viral particles. BST2+ epidermal γδ T cells upregulate genes associated with FAK signaling in alopecia areata, which indicates a role for FAK in the increased number of epidermal T cells. These studies identify a role for BST2 in alopecia areata that is mediated by epidermal γδ T cells and macrophages.

Diminished γδ T Cells during allergic skin inflammation is mediated by IL-4 signaling in keratinocytes

Abstract Atopic dermatitis results in profound changes in the function of the skin that include diminished barrier function and altered production of antimicrobial peptides. Dendritic epidermal γδ T cells (DETC) are a subset of γδ T cells that play an important role in the wound repair and inflammation process. Our previous work identified a loss of DETC in Stat6VT mice and in MC903 induced skin inflammation mouse model that was dependent on IL-4. However, the mechanisms through which IL-4 mediates this loss of DETCs are still not clear. In this study, we show that IL-4Rα germline knockout mice (Il4ra−/−) have an increased DETC population in skin that correlates with a higher rate of wound closure and increased EDC gene and fibronectin 1 expression. In Il4ra−/− mice, MC903 induced loss of DETCs was alleviated. Bone marrow chimeras of Il4ra−/− and WT bone marrow into Il4ra−/− mice retained the increased DETCs in the skin of Il4ra−/− host mice. Importantly, IL-15 secretion from skin keratinocytes was decreased dramatically in the MC903 induced skin inflammation model and injection of IL-15 rescued the loss of DETCs. Furthermore, conditional knock out of IL-4Rα from Keratinocytes of Il4ra fl/fl K14cre mice also showed an increase of this DETC populations in skin, increased IL-15 expression and less skin inflammation. Together, these results identify an IL-4-dependent circuit that regulates DETCs in the skin by repressing IL-15 expression in keratinocytes. This provides further insights into the ability of IL-4 to promote the development of atopic lesions and impair wound healing. Supported by grants from NIH (R01AI095282)

908 T-cell receptor signaling and cellular metabolism regulate IL-13-producing capacity of murine epidermal γδ T cells

908 T-cell receptor signaling and cellular metabolism regulate IL-13-producing capacity of murine epidermal γδ T cells

Examining the relationship between epidermal gamma delta T cells and neuropeptide Y

Abstract Neuropeptide Y (NPY) is one of the most abundant peptides present in the central nervous system and is responsible for regulating food intake, energy metabolism, and emotional expression. Emerging evidence indicates that NPY could potentially play a role in serving as a transmitter between the nervous system and immune system and even within the immune system itself. Epidermal gamma delta (γδ) T cells play a role in governing wound repair and homeostasis within the skin by producing growth factors, cytokines, and performing cytolysis. Wound repair involves the transmission of pain signals and immune functions simultaneously, suggesting interaction and collaboration between the nervous and immune system. We isolated and cultured epidermal γδ T cells from wild type mice and used flow cytometry to determine the expression of NPY1R on epidermal γδ T cells prior to and after activation. The data indicates that NPY1R is present on epidermal γδ T cells and that expression increases when upon activation. In addition, we performed immunofluorescence microscopy to acquire images of skin sections and epidermal sheets to determine expression of NPY1R on epidermal γδ T cells during skin homeostasis. This data shows that NPY1R is present on select populations of epidermal γδ T cells in the skin and future directions will examine how this impacts the role of epidermal γδ T cells in wound repair and homeostasis in the skin. Supported by the Arnold & Mabel Beckman Foundation (Beckman Scholars Program)

Increased proliferation of epidermal gamma delta T cells and expression of the transmembrane protein, BST2, in Alopecia areata

Abstract Alopecia areata is an autoimmune disease characterized by patchy non-scarring hair loss in affected individuals. Typically, research has focused on hair follicle infiltration and disruption via CD8+ and CD4+ T cells. Our research attempts to elucidate the role of resident epidermal γδ T cells in disease. Analysis of publicly available bioinformatics data shows a statistically significant downregulation in a number of immune-related genes after patients were treated with a JAK inhibitor, Tofacitinib. We have focused on one of these proteins, bone marrow stromal antigen 2 (BST2), in vitro and in vivo using the C3H mouse model of alopecia areata. We have identified populations of epidermal γδ T cells expressing BST2 in the follicle using immunofluorescence microscopy. In addition, BST2 expression is upregulated in unaffected skin from mice with active alopecia areata suggesting BST2 expression may precede loss of hair follicle immune privilege. Furthermore, epidermal γδ T cell number is increased in skin affected by alopecia areata. Together our findings suggest that BST2 is expressed by epidermal T cells prior to hair loss and is followed by an increase in epidermal γδ T cell number. Our lab is focused on how these changes in epidermal γδ T cell number and BST2 expression impacts disease. Supported by grants from NIH Supported by grants from NSF

Skin γδ T Cells and Their Function in Wound Healing.

For the skin immune system, γδ T cells are important components, which help in defensing against damage and infection of skin. Compared to the conventional αβ T cells, γδ T cells have their own differentiation, development and activation characteristics. In adult mice, dendritic epidermal T cells (DETCs), Vγ4 and Vγ6 γδ T cells are the main subsets of skin, the coordination and interaction among them play a crucial role in wound repair. To get a clear overview of γδ T cells, this review synopsizes their derivation, development, colonization and activation, and focuses their function in acute and chronic wound healing, as well as the underlining mechanism. The aim of this paper is to provide cues for the study of human epidermal γδ T cells and the potential treatment for skin rehabilitation.

Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8+ T cells

SummarySuccessful immune responses rely on a regulated delivery of the right signals to the right cells at the right time. Here we show that natural killer (NK) and dendritic epidermal γδ T cells (DETCs) use similar mechanisms to spatiotemporally orchestrate conventional type 1 dendritic cell (cDC1) functions in the spleen, skin, and its draining lymph nodes (dLNs). Upon MCMV infection in the spleen, cDC1 clusterize with activated NK cells in marginal zones. This XCR1-dependent repositioning of cDC1 toward NK cells allows contact delivery of IL-12 and IL-15/IL-15Rα by cDC1, which is critical for NK cell responses. NK cells deliver granulocyte-macrophage colony-stimulating factor (GM-CSF) to cDC1, guiding their CCR7-dependent relocalization into the T cell zone. In MCMV-infected skin, XCL1-secreting DETCs promote cDC1 migration from the skin to the dLNs. This XCR1-dependent licensing of cDC1 both in the spleen and skin accelerates antiviral CD8+ T cell responses, revealing an additional mechanism through which cDC1 bridge innate and adaptive immunity.

Hspa8 and ICAM-1 as damage-induced mediators of γδ T cell activation.

Tissue-resident γδ T cells form the first line of defense at barrier surfaces where they survey host tissue for signs of stress or damage. Following recognition of injury, γδ T cells play a crucial role in the wound-healing response through the production of growth factors and cytokines that promote proliferation in surrounding epithelial cells. To initiate this response, γδ T cells require interactions with a variety of epithelial-expressed costimulatory molecules in addition to primary signaling through their TCR. In the epidermis these signals include the coxsackie and adenovirus receptor (CAR), histocompatibility antigen 60c (H60c), and plexin B2, which interact with γδ T cell-expressed junctional adhesion molecule-like protein (JAML), NKG2D, and CD100, respectively. Here we identify heat shock protein family A member 8 (Hspa8) and ICAM-1 as two additional keratinocyte-expressed costimulatory molecules for epidermal resident γδ T cells (termed DETC). These molecules were rapidly up-regulated in the epidermis following wounding in both mouse and human tissue. Both Hspa8 and ICAM-1 had a costimulatory effect on DETC, inducing proliferation, CD25 up-regulation, and IL-2 production. We also provide evidence that DETC can be activated through the potential ICAM-1 and Hspa8 receptors LFA-1 and CD316. Finally, knockdown of Hspa8 in keratinocytes reduced their ability to activate DETC in culture and ICAM-1-/- mice exhibited impaired rates of healing in skin-organ culture suggesting a role for these proteins in the DETC-mediated damage response. Together with previous work on CAR, H60c, and plexin B2, these results add to a picture of a complex keratinocyte wound signature that is required for efficient DETC activation.

Skin Resident γδ T Cell Function and Regulation in Wound Repair.

The skin is a critical barrier that protects against damage and infection. Within the epidermis and dermis reside γδ T cells that play a variety of key roles in wound healing and tissue homeostasis. Skin-resident γδ T cells require T cell receptor (TCR) ligation, costimulation, and cytokine reception to mediate keratinocyte activity and inflammatory responses at the wound site for proper wound repair. While both epidermal and dermal γδ T cells regulate inflammatory responses in wound healing, the timing and factors produced are distinct. In the absence of growth factors, cytokines, and chemokines produced by γδ T cells, wound repair is negatively impacted. This disruption in γδ T cell function is apparent in metabolic diseases such as obesity and type 2 diabetes. This review provides the current state of knowledge on skin γδ T cell activation, regulation, and function in skin homeostasis and repair in mice and humans. As we uncover more about the complex roles played by γδ T cells in wound healing, novel targets can be discovered for future clinical therapies.

Epidermal resident γδ T cell development and function in skin.

Epidermal resident γδ T cells, or dendritic epidermal T cells (DETCs) in mice, are a unique and conserved population of γδ T cells enriched in the epidermis, where they serve as the regulators of immune responses and sense skin injury. Despite the great advances in the understanding of the development, homeostasis, and function of DETCs in the past decades, the origin and the underlying molecular mechanisms remain elusive. Here, we reviewed the recent research progress on DETCs, including their origin and homeostasis in the skin, especially at transcriptional and epigenetic levels, and discuss the involvement of DETCs in skin diseases.

Modulation of Epidermal γδ T Cell Functionality by Obesity due to Acute versus Chronic TNF-α Reception

Abstract Epidermal γδ T cells exhibit functional plasticity with roles varying from wound healing to tumor cell lysis. Activation of epidermal γδ T cells occurs via the γδ TCR along with costimulation, suggesting that costimulatory ligand expression regulates functional outcome. Data from our lab indicates that the cytokine milieu adds an additional pressure on epidermal γδ T cells to mediate or inhibit a particular functionality. Previous work from our lab shows that obesity impairs epidermal γδ T cell wound repair function in a TNF-α dependent manner. Here we identify TNF-α as a modulator of epidermal γδ T cell function in wound repair or cellular lysis depending on reception of acute or chronic signals. TNF-α signals through two receptors, tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2), thus the signaling dynamics are complex and induce varied responses including apoptosis, survival and inflammation. We show that epidermal γδ T cells exhibit low TNFR1 and TNFR2 expression prior to TCR activation. Even with low TNFR expression epidermal γδ T cells produce cytokines and chemokines involved in inflammation and macrophage recruitment in response to acute TNF-α signals without TCR activation. During TCR activation or IL-2 stimulation epidermal γδ T cells to upregulate TNFR2 indicating a reliance on TNFR2 signaling during activation. Alternatively, chronic TNF-α stimulation, similar to levels in obese patients, induces a reduction in epidermal γδ T cell cytotoxicity, proliferation and viability. Normal function is restored with IL-2 addition suggesting the inhibition is not permanent, but instead can be modulated. These studies provide potential targets for regulating epidermal γδ T cell functions such as wound repair and tumor lysis.

Role of C-C Motif Chemokine Ligand 20 in the Regulation of Epidermal γδ T Cells

Abstract 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 effects of α-MSH on epidermal γδ T cell activation and function

Abstract The hair follicle is a site of immune privilege that, when disrupted, results in the autoimmune disorder alopecia areata. Although there are a variety of cell types that reside near the hair follicle and may be involved in the initiation of alopecia areata, few studies have focused on γδ T cells. γδ T cells are resident in epithelial tissues where they normally play roles in inflammation and wound healing. Alpha-melanocyte-stimulating(α-MSH) is an immunoregulator locally produced within the hair follicle. α-MSH ensures the immune system is suppressed during anagen phase (growth phase) and restored during catagen phase (resting phase). The effects of α-MSH on epidermal γδ T cells via MC1R, one of five melanocortin receptors, is unknown. We therefore purified primary epidermal γδ T cells to investigate the immunomodulatory effects of α-MSH. Flow cytometry and the Luminex platform were used to compare changes in MC1R expression levels and cytokine production in response to α-MSH. Here we show that resting epidermal γδ T cells express MC1R, and TCR stimulation increases MC1R expression. Upregulation of MC1R expression is augmented further by stimulation with α-MSH. Epidermal γδ T cells significantly upregulate immunostimulatory cytokines when stimulated with anti-CD3, but cytokine production did not significantly change with the addition of α-MSH. Additional findings suggest that α-MSH instead plays a role in MC1R regulated pathways such as DNA repair and cell survival. This study begins to delineate the mechanisms by which α-MSH impacts γδ T cell function and leads to the disruption of immune privilege that occurs in alopecia areata.

Isolation and Analysis of Mouse and Human Skin γδ T Cells.

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.

Keratinocyte-derived CCL20 orchestrates epidermal localization of IL-17-producing γδ T cells and ILCs to mediate psoriasis-like skin inflammation

Abstract CCR6 is important for imiquimod (IMQ)-induced psoriatic dermatitis in mice, which depends on motile CCR6+ γδ T cells that produce IL-17 and IL-22. CCR6 ligands include CCL20 and β-defensins. We used new strains of Ccl20 tdTomato knock-in mice, Ccl20fl/flmice, and Ccr6fl/fl GFP knock-in mice to determine sites of production and action of CCL20 and its role in the localization of γδ T cells and ILCs during imiquimod (IMQ)-induced inflammation. IMQ treatment of Ccl20+/tdT mice led to expression of Ccl20 in keratinocytes (KCs), including in the infundibulum and isthmus of the hair follicles. Ccl20−/− mice showed reduced inflammation and expression of Il17 and Il22 in the treated skin. Deletion of Ccl20 specifically in the KCs by treating Ccl20fl/flK14-CreERT mice with tamoxifen before applying IMQ significantly decreased the accumulation of IL-17-producing gd T cells and ILCs in the epidermis. Depleting KC-derived CCL20 at day 4 when inflammation was well established also reduced the number of epidermal γδ T cells and ILCs. In both cases the percent of IL-17-producing γδ T cells and ILCs did not change, demonstrating that migration to the epidermis and the ability to produce IL-17 are regulated independently. Multi-photon microscopy of IMQ-treated Ccr6+/gfp and Ccr6gfp/gfp(CCR6-deficient) micerevealed a CCR6-independent step whereby dermal CCR6+ cells accumulate at the hair follicles, followed by a CCR6-dependent step whereby the cells move into the superficial epidermis.