Tissue-resident memory T (TRM) cells are important in allergic contact dermatitis (ACD). In this review we investigate how TRM cells differ from central and effector memory T (TCM and TEM) cells in their contributions to disease recurrence, severity, and chronicity in ACD. We further discuss how classic experimental models may not fully capture TRM cells in ACD research and how this can be avoided in future research. TRM cells persist locally in previously sensitized skin and can initiate rapid immune responses upon allergen re-exposure. Allergen-naïve skin requires recruitment of TEM cells from the circulation, leading to a delayed response compared to allergen-experienced skin. Due to their resident phenotype, TRM cells might be activated independently of specific allergen recognition through bystander mechanisms, further amplifying inflammation and chronicity. TRM cells are key drivers of both flare-ups and sustained inflammation in ACD. Their local presence, rapid responsiveness, and potential for non-specific activation make them important therapeutic targets. Understanding the differences between TRM cells compared to TCM/EM cells is very important for improving both experimental models and clinical strategies for long-term management of ACD.

CD8+ epidermal-resident memory T (TRM) cells play a significant role in fighting off pathogens. However, CD8+ TRM cells are also central in the pathogenesis of a variety of inflammatory skin diseases. It is unclear whether the generation and persistence of CD8+ TRM cells are dependent on the presence of cognate antigen and TCR signaling. The purpose of this study was to determine whether TCR signaling is required for the generation and persistence of epidermal CD8+ TRM cells in a mouse model for allergic contact dermatitis. We examined the responses to 4 different contact allergens in combination with adoptive transfer and prime-pull experiments. We determined the presence of contact allergen in the skin by western blot analysis. We found that epidermal CD8+ TRM cells can develop in the absence of the cognate antigen and TCR signaling as determined by Nur77 induction, whereas persistence of epidermal CD8+ TRM cells requires the presence of the cognate antigen and correlates with Nur77 expression. In the presence of contact allergen, a selective expansion of specific TCR clonotypes was seen. In conclusion, this study supports that cognate antigen and TCR signaling are required for the persistence of allergen-specific CD8+ TRM cells in the skin.

BACKGROUND:Dysfunction of the skin barrier is regarded as a key event in the initiation and progression of inflammatory skin diseases. In many cases of allergic contact dermatitis (ACD), epidermal-resident memory CD8+ T (TRM) cells play a central role in the immune response to contact allergens. However, if and how allergen-specific CD8+ TRM cells affect the expression of skin barrier molecules is not known. METHODS:The expression level of skin barrier molecules was determined by RT-qPCR and immunofluorescence in a mouse model of ACD. The role of CD8+ T cells on the expression of skin barrier molecules was investigated by depletion of CD8+ cells. Human primary keratinocytes were used to assess the direct effect of IFN-γ and contact allergen on their expression of skin barrier molecules. RESULTS:Sensitization with the contact allergen 1-fluoro-2,4-dinitrobenzene (DNFB) resulted in epidermal accumulation of CD8+ TRM cells and prolonged upregulation of Ifng and downregulation of keratin 5 (Krt5) and Krt14 even after complete macroscopic remission of the inflammatory response. Challenge with DNFB lead to an additionally rapid downregulation of Krt5 and Krt14 and the downregulation of several other skin barrier molecules. Depletion of CD8+ cells abolished both the prolonged and rapid downregulation of skin barrier molecules. In keratinocytes, IFN-γ and contact allergen synergistically down-regulated the expression of KRT5 and KRT14. CONCLUSION:CD8+ TRM cells contribute to a prolonged reduction in the expression of skin barrier molecules, which might exacerbate allergen permeation and the inflammatory response during succeeding exposures of the skin to allergens and antigens.

It is still a major question whether patients with atopic dermatitis (AD) have an altered response to contact allergens compared to controls without AD.1, 2 In an effort to address this question, we compared the response to the contact allergen 1-fluoro-2,4-dinitrobenzene (DNFB) in wild-type (WT), Flgft/ft and Tmem79ma/ma mice in a well-established model for allergic contact dermatitis (ACD).3 Flgft/ft and Tmem79ma/ma mice have defects in the skin barrier, but in contrast to Flgft/ft mice, Tmem79ma/ma mice spontaneously develop skin inflammation dominated by IL17-producing T cells4, 5 (Figures S1 and S2). Thus, in the present study WT mice represent healthy skin, Flgft/ft mice represent skin with an impaired skin barrier without skin inflammation, and Tmem79ma/ma mice represent skin with impaired skin barrier and pre-existing skin inflammation. Sensitization with DNFB generated similar numbers of pathogenic epidermis-resident CD8+ memory T (TRM) cells in Flgft/ft and Tmem79ma/ma mice as in WT mice as determined by flow cytometry at Day 21 after sensitization (Figure 1A,B), whereas it did not affect the number of epidermal CD4+ TRM cells that was constitutively increased in Tmem79ma/ma mice (Figures 1C and S1J). The mice were challenged with DNFB at Day 21 and in accordance with previous studies,3 the inflammatory response peaked 24 h after the challenge. However, the peak response in Tmem79ma/ma mice was significantly reduced compared to WT and Flgft/ft mice (Figure 1D). The inflammatory response is dependent on CD8+ TRM cells and recruitment of neutrophils.3 We found reduced neutrophil recruitment to the epidermis of Tmem79ma/ma mice compared to WT mice (Figure 1E), although the numbers of CD8+ TRM cells after DNFB challenge were similar in the groups (Figure 1F). In parallel with the impaired inflammatory response and recruitment of neutrophils, the expression of il1b, cxcl5 and ifng was reduced in the skin of Tmem79ma/ma mice compared to WT mice (Figure 1G). Furthermore, exposure to DNFB slightly down-regulated il17a expression in Tmem79ma/ma (Figure 1G). The observation of similar numbers of CD8+ TRM cells but a 5–10 fold reduction in IFNγ suggested that active suppression of the CD8+ TRM cells took place in the skin of the Tmem79ma/ma mice resulting in impaired chemokine production and neutrophil recruitment. Accordingly, Tmem79ma/ma mice had higher numbers of CD4+ T cells, including FoxP3+CD25+CD4+ Treg cells, in the epidermis compared to WT mice (Figure 1H,I). The number of FoxP3+CD25+CD4+ Treg cells generally correlated with the expression levels of il10 (Figure 1G,I). To distinguish the effect of circulating from epidermal-resident Treg cells, we took advantages of the observation that infusion of T-cell depletion antibodies effectively eliminates circulating T cells while sparing tissue-resident T cells6 (Figure 2A). Depletion of circulating CD4+ T cells resulted in an enhanced challenge response in the mouse strains. However, the response in the Tmem79ma/ma mice was still reduced compared to the responses in WT and Flgft/ft mice (Figure 2B) despite higher numbers of CD8+ TRM cells in Tmem79ma/ma mice (Figure 2C). The numbers of CD4+ TRM cells and FoxP3+CD25+CD4+ Treg cells were significantly higher in the epidermis of Tmem79ma/ma mice than in WT mice (Figure 2D,E), supporting that pre-existing Treg cells in the skin suppressed the immune response to DNFB in Tmem79ma/ma mice. In conclusion, our study demonstrates that pre-existing skin inflammation, as seen in AD patients, can result in anti-inflammatory mechanisms that suppress immune responses to new allergens/antigens. Further studies should determine whether this is general for all allergens/antigens. We believe that our observations are relevant for future research in the role of pro- and anti-inflammatory mechanisms in contact allergy and in the development of dermal vaccines. Conceptualization of this study was led by MHJ and CMB and supported by JDJ, CG, NØ and AW. Formal analysis was led by MHJ and supported by CMB, CG, ABF, JFW and KY. Funding acquisition was done by CMB, CG and JDJ. Investigation was led by MHJ and supported by ABF, JFW, KY, TS, VM and AG. Methodology was led by MHJ and CMB and supported by CG. Project administration was done by MHJ. Resources were obtained by CMB and CG. Supervision was done by CMB and supported by JDJ. Validation was led by MHJ and supported by ABF, JFW, KY and TS. Visualization was done by MHJ. Writing of the original draft was done by MHJ, CMB and CG. Review and editing were done by all authors. We would like to thank Rebecca Kitt Davidson Lohmann for her excellent technical support. This study was supported by The Danish Environmental Protection Agency, The LEO Foundation, The A.P. Møller Foundation for the Advancement of Medical Science and The Aase and Ejnar Danielsen Foundation. The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND:Rubber accelerators are used in the vulcanization of rubber. However, rubber accelerators for example tetraethylthiuram disulfide (TETD) and zinc diethyldithiocarbamate (ZDEC) may cause contact allergy. Concomitant reactions between ZDEC and TETD have been observed in patients which could be explained by co- or cross-reactivity. OBJECTIVES:To investigate cross-reactivity between TETD and ZDEC and vice versa. METHODS:Groups of mice were sensitized with TETD or ZDEC based on reported EC3-values. Proliferation of lymphocytes were measured on day 5. To test cross-reactivity, mice were sensitized and challenged 3 weeks later with TETD or ZDEC. The inflammatory response was measured by changes in ear thickness and the proliferative response in CD4+ and CD8+ T cells in the submandibular and cervical draining lymph nodes. RESULTS:Sensitization of mice with doses of ZDEC 3%, TETD 5.6% or TETD 16.2% induced significant increased ear thickness and proliferation of CD4+ and CD8+ T cells. Challenge with ZDEC or TETD in these groups induced significant increased ear thickness. Challenge with ZDEC in mice sensitized to TETD 5.6% or TETD 16.2% induced significant increased proliferation of CD4+ and CD8+ T cells. CONCLUSIONS:We show cross-reactivity between TETD and ZDEC. Patients sensitized to TETD or ZDEC should avoid exposure to both ZDEC and TETD.

CD8+ epidermal-resident memory T (TRM ) cells play central roles in local flare-up responses to experimental contact allergens by inducing massive influx of neutrophils to the epidermis upon allergen challenge. Whether similar immunopathogenic mechanisms are involved in the responses to clinically relevant contact allergens is unknown.The immune response to cinnamal, ρ-phenylenediamine (PPD) and methylisothiazolinone (MI) was studied in a well-established mouse model for allergic contact dermatitis that includes formation of TRM cells by ELISA, flow cytometry, fluorescence microscopy analyses and cell depletion protocols.We show that the formation of CD4+ and CD8+ epidermal TRM cells and the inflammatory response are highly allergen-dependent. However, the magnitude of the flare-up responses correlated with the number of epidermal CD8+ TRM cells, CXCL1/CXCL2 release and recruitment of neutrophils to the epidermis. Finally, depletion of CD4+ T cells strongly enhanced the number of epidermal CD8+ TRM cells, the flare-up response and the epidermal infiltration of neutrophils for all allergens.As the first, this study demonstrates that clinically relevant contact allergens have the ability to generate pathogenic, epidermal CD8+ TRM cells that recruit neutrophils following re-exposure to the allergen, but that this normally is counteracted by the simultaneous induction of anti-inflammatory CD4+ T cells.

Allergic contact dermatitis (ACD) is an inflammatory disease with a complex pathophysiology in which epidermal-resident memory CD8+ T (TRM ) cells play a key role. The mechanisms involved in the activation of CD8+ TRM cells during allergic flare-up responses are not understood.The expression of CD100 and its ligand Plexin B2 on CD8+ TRM cells and keratinocytes before and after allergen exposure was determined by flow cytometry and RT-qPCR. The role of CD100 in the inflammatory response during the challenge phase of ACD was determined in a model of ACD in CD100 knockout and wild-type mice.We show that CD8+ TRM cells express CD100 during homeostatic conditions and up-regulate it following re-exposure of allergen-experienced skin to the experimental contact allergen 1-fluoro-2,4-dinitrobenzene (DNFB). Furthermore, Plexin B2 is up-regulated on keratinocytes following exposure to some contact allergens. We show that loss of CD100 results in a reduced inflammatory response to DNFB with impaired production of IFNγ, IL-17A, CXCL1, CXCL2, CXCL5, and IL-1β and decreased recruitment of neutrophils to the epidermis.Our study demonstrates that CD100 is expressed on CD8+ TRM cells and is required for full activation of CD8+ TRM cells and the flare-up response of ACD.

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.

BACKGROUND:Allergic contact dermatitis (ACD) is classically described as a delayed-type hypersensitivity reaction. However, patients often experience flare-ups characterized by itching erythema, edema, and often vesicles occurring within hours after re-exposure of previously sensitized skin to the specific contact allergen. Recent studies have indicated that skin-resident memory T (TRM ) cells play a central role in ACD. However, the pathogenic role of TRM cells in allergen-induced flare-ups is not known.METHODS:By the use of various mouse models and cell depletion protocols, we investigated the role of epidermal TRM cells in flare-up reactions to the experimental contact allergen 1-fluoro-2,4-dinitrobenzene. The inflammatory response was measured by changes in ear thickness, and the cellular composition in epidermis was determined by flow cytometry and confocal microscopy. Finally, adaptive transfer and inhibitors were used to determine the role of TRM cells, neutrophils, and CXCL1/CXCL2 in the response.RESULTS:We show that CD8+ TRM cells initiate massive infiltration of neutrophils in the epidermis within 12 h after re-exposure to the contact allergen. Depletion of neutrophils before re-exposure to the allergen abrogated the flare-up reactions. Furthermore, we demonstrate that CD8+ TRM cells mediate neutrophil recruitment by inducing CXCL1 and CXCL2 production in the skin, and that blockage of the C-X-C chemokine receptor type 1 and 2 inhibits flare-up reactions and neutrophil infiltration.CONCLUSION:As the first, we show that epidermal CD8+ TRM cells cause ACD flare-ups by rapid recruitment of neutrophils to the epidermis.

BACKGROUND:Epidermal T cells play a central role in immune surveillance and in inflammatory skin diseases. Major differences in the epidermal T cell composition are found between adult humans and antigen-inexperienced laboratory mice. Whether this is due to inborn species differences, to different environmental exposures, or a combination of the two is a matter of debate.OBJECTIVES:To investigate the role of age and exposure to antigens on epidermal T cell subsets in human and mouse skin.METHODS:We isolated T cells from the epidermis from 19 infants and 26 adults, and determined the frequency of CD4+ and CD8+ αβ T cells and γδ T cells by flow cytometry. In addition, we determined the epidermal T cell composition in antigen-inexperienced and antigen-experienced mice.RESULTS:We found that humans are born with very few epidermal T cells. The number increases and the composition changes with age. In antigen-inexperienced mice, the epidermal T cell composition is unaffected by age, but it is dramatically affected by antigen exposure.CONCLUSION:Taken together, we show that antigen exposure, as opposed to age, is the major factor determining the composition of epidermal T cells, suggesting that the skin of antigen-experienced mice better reflects the immunological conditions in human skin.

Approximately 25% of the population suffers from skin diseases. The most common forms of skin diseases are the inflammatory skin diseases such as allergic contact dermatitis, psoriasis, and atopic dermatitis. These diseases are described as T cell-mediated diseases induced by either allergens or autoantigens. Classically, the focus has been on the role of alpha beta T cells, but it is becoming increasingly clear that gamma delta T cells play a central role in inflammatory skin diseases. In particular, an important role of IL-17A-producing gamma delta T cells in these inflammatory skin diseases has been shown in various disease models in mice. Interestingly, various epidermal proteins, which appear to be linked to inflammatory conditions in the skin by yet undescribed mechanisms, are expressed by specific subsets of thymic epithelial cells and mutations in these proteins seem to affect gamma delta T cell development. The focus of this review is how mutations in epidermal proteins affect gamma delta T cell development and how gamma delta T cells, and in particular of IL-17A-producing gamma delta T cells, contribute to inflammatory skin diseases such as allergic contact dermatitis, psoriasis, and atopic dermatitis.

Approximately 25% of the population suffers from skin diseases. The most common forms of skin diseases are the inflammatory skin diseases such as allergic contact dermatitis, psoriasis, and atopic dermatitis. These diseases are described as T cell-mediated diseases induced by either allergens or autoantigens. Classically, the focus has been on the role of αβ T cells, but it is becoming increasingly clear that γδ T cells play a central role in inflammatory skin diseases. In particular, an important role of IL-17A-producing γδ T cells in these inflammatory skin diseases has been shown in various disease models in mice. Interestingly, various epidermal proteins, which appear to be linked to inflammatory conditions in the skin by yet undescribed mechanisms, are expressed by specific subsets of thymic epithelial cells and mutations in these proteins seem to affect γδ T cell development. The focus of this review is how mutations in epidermal proteins affect γδ T cell development and how γδ T cells, and in particular of IL-17A-producing γδ T cells, contribute to inflammatory skin diseases such as allergic contact dermatitis, psoriasis, and atopic dermatitis.

Approximately 25% of the population suffers from skin diseases. The most common forms of skin diseases are the inflammatory skin diseases such as allergic contact dermatitis, psoriasis, and atopic dermatitis. These diseases are described as T cell-mediated diseases induced by either allergens or autoantigens. Classically, the focus has been on the role of αβ T cells, but it is becoming increasingly clear that γδ T cells play a central role in inflammatory skin diseases. In particular, an important role of IL-17A-producing γδ T cells in these inflammatory skin diseases has been shown in various disease models in mice. Interestingly, various epidermal proteins, which appear to be linked to inflammatory conditions in the skin by yet undescribed mechanisms, are expressed by specific subsets of thymic epithelial cells and mutations in these proteins seem to affect γδ T cell development. The focus of this review is how mutations in epidermal proteins affect γδ T cell development and how γδ T cells, and in particular of IL-17A-producing γδ T cells, contribute to inflammatory skin diseases such as allergic contact dermatitis, psoriasis, and atopic dermatitis.

This panel was designed to quantify the distribution of developing γδ T cells within seven development stages related to the programming of distinct effector phenotypes in the murine thymus. Furthermore, the panel was designed to assess the expression of additional surface markers at each development stage within two of the major γδ T-cell subsets identified by the usage of different V-segments in their T-cell receptor (TCR): TCRVγ1.1 and TCRVγ2. The panel was developed using thymus from adult C57Bl/6 mice magnetically depleted of CD4 and CD8 expressing cells in order to enrich for the γδ T-cell population.

The skin is our interface with the outside world, and consequently it is exposed to a wide range of microbes and allergens. Recent studies have indicated that allergen-specific skin-resident memory T (TRM) cells play a role in allergic contact dermatitis (ACD). However, the composition and dynamics of the epidermal T-cell subsets during ACD are not known. Here we show that exposure of the skin to the experimental contact allergen DNFB results in a displacement of the normally occurring dendritic epidermal T cells (DETC) concomitant with an accumulation of epidermal CD8+CD69+CD103+ TRM cells in mice. By studying knockout mice, we provide evidence that CD8+ T cells are required for the displacement of the DETC and that DETC are not required for recruitment of CD8+ TRM cells to the epidermis following allergen exposure. We demonstrate that the magnitude of the allergic reaction correlates with the number of CD8+ epidermal TRM cells, which again correlates with allergen dose and number of allergen exposures. Finally, in an attempt to elucidate why CD8+ epidermal TRM cells persist in the epidermis, we show that CD8+ epidermal TRM cells have a higher proliferative capability and are bioenergetically more stable, displaying a higher spare respiratory capacity than DETC.

Mutations in the filaggrin gene (Flg) are associated with increased systemic levels of Th17 cells and increased IL-17A production following antigen exposure in both humans and mice. In addition to Th17 cells, γδ T cells can produce IL-17A. The differentiation of γδ T cells to either IFNγ or IL-17A-producing (γδT17) cells is mainly determined in the thymus. Interestingly, it has been reported that filaggrin is expressed in the Hassall bodies in the human thymic medulla. However, whether filaggrin affects γδ T cell development is not known. Here, we show that filaggrin-deficient flaky tail (ft/ft) mice have an increased number of γδT17 cells in the spleen, epidermis, and thymus compared to wild-type (WT) mice. We demonstrate that filaggrin is expressed in the mouse thymic medulla and that blocking the egress of cells from the thymus results in accumulation of Vγ2+ γδT17 cells in the thymus of adult ft/ft mice. Finally, we find increased T cell receptor expression levels on γδ T cells and increased levels of IL-6 and IL-23 in the thymus of ft/ft mice. These findings demonstrate that filaggrin is expressed in the mouse thymic medulla and that production of Vγ2+ γδT17 cells is dysregulated in filaggrin-deficient ft/ft mice.

Background: Nickel allergy and dermatitis have been associated with filaggrin gene mutations in epidemiological studies, but the mechanisms mediating these associations are unknown. Objectives: To investigate whether filaggrin-deficient flaky tail (ft/ft) mice show increased immune reactivity to nickel and elucidate the mechanisms mediating this. Methods: The immune responses to nickel, 2,4-dinitrofluorobenzene (DNFB), cinnamal and p-phenylenediamine were assessed in ft/ft and wild-type (WT) mice. The amounts of nickel in the skin of ft/ft and WT mice were determined 20 hours after nickel exposure. The effect of blocking either the interleukin (IL)-17A pathway or the IL-1 pathway on the response to nickel in ft/ft mice was evaluated. Results: Increased responsiveness to nickel, DNFB and cinnamal was observed in ft/ft mice as compared with controls. A reduced amount of nickel was found in the skin of ft/ft mice as compared with WT mice, suggesting increased nickel absorption by the skin of ft/ft mice. Blocking either the IL-17A pathway or the IL-1 pathway reduced nickel responsiveness in ft/ft mice. Conclusions: These findings suggest that the increased nickel responsiveness associated with epidermal filaggrin deficiency is mediated by a combination of increased nickel penetration and the steady-state inflammation found in the skin of filaggrin-deficient mice.