Keratinocytes Present Staphylococcus aureus Enterotoxins and Promote Malignant and Nonmalignant T Cell Proliferation in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma.

Vitamin D in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is characterized by the proliferation of malignant T cells in inflamed skin lesions. Mycosis fungoides (MF)-the most common variant of CTCL-often presents with skin lesions around the abdomen and buttocks ("bathing suit" distribution), i.e., in skin areas devoid of sun-induced vitamin D. For decades, sunlight and vitamin D have been connected to CTCL. Thus, vitamin D induces apoptosis and inhibits the expression of cytokines in malignant T cells. Furthermore, CTCL patients often display vitamin D deficiency, whereas phototherapy induces vitamin D and has beneficial effects in CTCL, suggesting that light and vitamin D have beneficial/protective effects in CTCL. Inversely, vitamin D promotes T helper 2 (Th2) cell specific cytokine production, regulatory T cells, tolerogenic dendritic cells, as well as the expression of immune checkpoint molecules, all of which may have disease-promoting effects by stimulating malignant T-cell proliferation and inhibiting anticancer immunity. Studies on vitamin D treatment in CTCL patients showed conflicting results. Some studies found positive effects, others negative effects, while the largest study showed no apparent clinical effect. Taken together, vitamin D may have both pro- and anticancer effects in CTCL. The balance between the opposing effects of vitamin D in CTCL is likely influenced by treatment and may change during the disease course. Therefore, it remains to be discovered whether and how the effect of vitamin D can be tilted toward an anticancer response in CTCL.

Regulation of immune response genes in the skin of allergic and clinically tolerant individuals exposed to p-phenylenediamine.

BACKGROUND: p-Phenylenediamine (PPD) is a potent contact allergen found in many hair colour products. However, not all individuals develop allergic contact dermatitis (ACD) although they are regularly exposed to PPD. It is unclear whether these asymptomatic individuals are true non-responders to PPD or whether they mount a response to PPD without showing any symptoms. METHODS: Skin biopsies were collected from 11 asymptomatic hairdressers regularly exposed to PPD and from 10 individuals with known ACD on day 4 after patch testing with 1% PPD in petrolatum and petrolatum exclusively as control. RNA sequencing and confocal microscopy were performed. RESULTS: T cell activation, inflammation and apoptosis pathways were up-regulated by PPD in both asymptomatic and allergic individuals. Compared to asymptomatic individuals with a negative patch test, individuals with a strong reaction to PPD strongly up-regulated both pro- and anti-inflammatory cytokines genes. Interestingly, PPD treatment induced significant up-regulation of several genes for chemokines, classical type 2 dendritic cell markers and regulatory T cell markers in both asymptomatic and allergic individuals. In addition, apoptosis signalling pathway was activated in both non-responders and allergic individuals. CONCLUSION: This study demonstrates that there are no true non-responders to PPD but that the immune response elicited by PPD differs between individuals and can lead to either tolerance, subclinical inflammation or allergy.

Contact allergens in African countries: A review of published patch test studies.

Only few studies on contact allergy in African countries have been published. The aim of the present study was to provide an overview of the most common contact allergens identified by the use of patch tests in African countries based on a review of the existing literature. A total of twenty-four publications from eight African countries were initially identified by search in PubMed. The abstracts and method sections were screened, and 15 studies in which patch tests were actually used to identify the allergen causing the allergic contact dermatitis (ACD) were finally selected. Nickel, cobalt, chromium, fragrance mix and p-tert-butylphenol-formaldehyde resin were the dominating contact allergens responsible for 40%-90% of the positive patch test reactions. This study indicates that a targeted effort directed towards prevention, avoidance and regulation of reliably identified contact allergens could reduce the disease burden of ACD considerable in some African countries.

Metabolic re-programming of keratinocytes in response to contact allergens.

BACKGROUND: Allergic contact dermatitis (ACD) is a common skin disease caused by the recognition of haptens by the immune system. Keratinocytes play an important role in the initiation and facilitation of inflammatory responses in ACD. Immune responses are associated with major changes in metabolism. However, metabolic re-programming is not well studied in ACD; specifically, knowledge of metabolic alterations in structural cells is lacking. METHODS: Metabolic re-programming in ACD was studied using publicly available transcriptome datasets. Primary pooled keratinocytes and a keratinocyte cell line (HaCaT) were stimulated with contact allergens, and inflammatory responses and expression of metabolic markers were measured by qPCR and flow cytometry, respectively. RESULTS: ACD is characterized by metabolic re-programming with a metabolic profile similar to atopic dermatitis. Exposure to contact allergens causes a wide array of metabolic alterations. Stimulation of keratinocytes with contact allergens induced inflammatory responses typical for ACD and was associated with an up-regulation of proteins representative for glucose uptake, fatty acid metabolism, oxidative phosphorylation and to some extent arginine biosynthesis. Changes in these metabolic pathways were also observed when comparing lesional with non-lesional contact dermatitis skin. CONCLUSIONS: ACD is, similarly to other inflammatory skin diseases, characterized by metabolic re-programming. Contact allergen exposure induces expression of a wide array of metabolic pathways, which is at least in part mediated through metabolic re-programming of keratinocytes.

CD100 boosts the inflammatory response in the challenge phase of allergic contact dermatitis in mice.

BACKGROUND: 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. METHODS: 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. RESULTS: 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-1ß and decreased recruitment of neutrophils to the epidermis. CONCLUSION: 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) is important for the inflammatory response during contact hypersensitivity.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 1ß (IL-1ß) 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. CONCLUSION: 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.

In vitro differentiated human CD4+ T cells produce hepatocyte growth factor.

Differentiation of naive CD4+ T cells into effector T cells is a dynamic process in which the cells are polarized into T helper (Th) subsets. The subsets largely consist of four fundamental categories: Th1, Th2, Th17, and regulatory T cells. We show that human memory CD4+ T cells can produce hepatocyte growth factor (HGF), a pleiotropic cytokine which can affect several tissue types through signaling by its receptor, c-Met. In vitro differentiation of T cells into Th-like subsets revealed that HGF producing T cells increase under Th1 conditions. Enrichment of HGF producing cells was possible by targeting cells with surface CD30 expression, a marker discovered through single-cell RNA-sequencing. Furthermore, pharmacological inhibition of PI3K or mTOR was found to inhibit HGF mRNA and protein, while an Akt inhibitor was found to increase these levels. The findings suggest that HGF producing T cells could play a role in disease where Th1 are present.

CD4+ T cells inhibit the generation of CD8+ epidermal-resident memory T cells directed against clinically relevant contact allergens.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Endolysin Inhibits Skin Colonization by Patient-Derived Staphylococcus Aureus and Malignant T-Cell Activation in Cutaneous T-Cell Lymphoma.

Staphylococcus aureus is suspected to fuel disease activity in cutaneous T-cell lymphomas. In this study, we investigate the effect of a recombinant, antibacterial protein, endolysin (XZ.700), on S. aureus skin colonization and malignant T-cell activation. We show that endolysin strongly inhibits the proliferation of S. aureus isolated from cutaneous T-cell lymphoma skin and significantly decreases S. aureus bacterial cell counts in a dose-dependent manner. Likewise, ex vivo colonization of both healthy and lesional skin by S. aureus is profoundly inhibited by endolysin. Moreover, endolysin inhibits the patient-derived S. aureus induction of IFNγ and the IFNγ-inducible chemokine CXCL10 in healthy skin. Whereas patient-derived S. aureus stimulates activation and proliferation of malignant T cells in vitro through an indirect mechanism involving nonmalignant T cells, endolysin strongly inhibits the effects of S. aureus on activation (reduced CD25 and signal transducer and activator of transcription 5 phosphorylation) and proliferation (reduced Ki-67) of malignant T cells and cell lines in the presence of nonmalignant T cells. Taken together, we provide evidence that endolysin XZ.700 inhibits skin colonization, chemokine expression, and proliferation of pathogenic S. aureus and blocks their potential tumor-promoting effects on malignant T cells.

Malignant T cells induce skin barrier defects through cytokine-mediated JAK/STAT signaling in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms that hamper quality of life and are entry sites for bacterial infection, a major cause of morbidity and mortality in advanced diseases. The mechanism driving the pathological processes that compromise the skin barrier remains unknown. Here, we report increased transepidermal water loss and compromised expression of the skin barrier proteins filaggrin and filaggrin-2 in areas adjacent to TOX-positive T cells in CTCL skin lesions. Malignant T cells secrete mediators (including cytokines such as interleukin 13 [IL-13], IL-22, and oncostatin M) that activate STAT3 signaling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, the repression of filaggrins can be counteracted by a cocktail of antibodies targeting these cytokines/receptors, small interfering RNA-mediated knockdown of JAK1/STAT3, and JAK1 inhibitors. Notably, we show that treatment with a clinically approved JAK inhibitor, tofacitinib, increases filaggrin expression in lesional skin from patients with mycosis fungoides. Taken together, these findings indicate that malignant T cells secrete cytokines that induce skin barrier defects via a JAK1/STAT3-dependent mechanism. As clinical grade JAK inhibitors largely abrogate the negative effect of malignant T cells on skin barrier proteins, our findings suggest that such inhibitors provide novel treatment options for patients with CTCL with advanced disease and a compromised skin barrier.

Reduced vitamin D-induced cathelicidin production and killing of Mycobacterium tuberculosis in macrophages from a patient with a non-functional vitamin D receptor: A case report.

Tuberculosis (TB) presents a serious health problem with approximately a quarter of the world's population infected with Mycobacterium tuberculosis (M. tuberculosis) in an asymptomatic latent state of which 5-10% develops active TB at some point in their lives. The antimicrobial protein cathelicidin has broad antimicrobial activity towards viruses and bacteria including M. tuberculosis. Vitamin D increases the expression of cathelicidin in many cell types including macrophages, and it has been suggested that the vitamin D-mediated antimicrobial activity against M. tuberculosis is dependent on the induction of cathelicidin. However, unraveling the immunoregulatory effects of vitamin D in humans is hampered by the lack of suitable experimental models. We have previously described a family in which members suffer from hereditary vitamin D-resistant rickets (HVDRR). The family carry a mutation in the DNA-binding domain of the vitamin D receptor (VDR). This mutation leads to a non-functional VDR, meaning that vitamin D cannot exert its effect in family members homozygous for the mutation. Studies of HVDRR patients open unique possibilities to gain insight in the immunoregulatory roles of vitamin D in humans. Here we describe the impaired ability of macrophages to produce cathelicidin in a HVDRR patient, who in her adolescence suffered from extrapulmonary TB. The present case is a rare experiment of nature, which illustrates the importance of vitamin D in the pathophysiology of combating M. tuberculosis.

The role of the different CD3γ domains in TCR expression and signaling.

The CD3 subunits of the T-cell antigen receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3γ (γ-) show reduced surface TCR expression levels and abolished phorbol ester (PMA)-induced TCR down-regulation. The response to PMA is mediated by a double leucine motif in the intracellular (IC) domain of CD3γ. However, the molecular cause of the reduced TCR surface expression in γ- lymphocytes is still not known. We used retroviral vectors carrying wild type CD3γ or CD3δ or the following chimeras (EC-extracellular, TM-transmembrane and IC): δECγTMγIC (δγγ for short), γγδ, γδδ and γγ-. Expression of γγγ, γγδ, γδδ or γγ- in the γ- T cell line JGN, which lacks surface TCR, demonstrated that cell surface TCR levels in JGN were dependent on the EC domain of CD3γ and could not be replaced by the one of CD3δ. In JGN and primary γ- patient T cells, the tested chimeras confirmed that the response to PMA maps to the IC domain of CD3γ. Since protein homology explains these results better than domain structure, we conclude that CD3γ contributes conformational cues that improve surface TCR expression, likely at the assembly or membrane transport steps. In JGN cells all chimeric TCRs were signalling competent. However, an IC domain at CD3γ was required for TCR-induced IL-2 and TNF-α production and CD69 expression, indicating that a TCR without a CD3γ IC domain has altered signalling capabilities.

Concomitant Inhibition of FASN and SREBP Provides a Promising Therapy for CTCL.

Cutaneous T cell lymphoma (CTCL) is a group of non-Hodgkin's primary cutaneous T cell lymphomas, with Mycosis Fungoides and Sézary syndrome (SS) being the two most common subtypes. Fatty acid synthase (FASN) is a crucial enzyme that catalyses the biosynthesis of fatty acids, which has been reported to play an oncogenic role in various malignancies but not in CTCL so far. Herein, we show that FASN is highly expressed in CTCL cell lines and in peripheral blood mononuclear cells (PBMCs) from CTCL patients, while it is not in PBMCs from healthy individuals. The inhibition of FASN in CTCL cell lines impairs cell viability, survival, and proliferation, but, interestingly, it also increases FASN expression. However, inhibiting sterol regulatory element binding protein (SREBP), a transcription factor that promotes the expression of FASN, partially reversed the upregulation of FASN induced by FASN inhibitors. Thus, the combination of FASN and SREBP inhibitors enhanced the effects on both CTCL cell lines and PBMCs from SS patients, where a valid inhibition on cell proliferation could be verified. Importantly, compared to non-malignant cells, primary malignant cells are more sensitive to the inhibition of FASN and SREBP, making the combination of FASN and SREBP inhibitors a promising novel therapeutic strategy in CTCL.

Induced Human Regulatory T Cells Express the Glucagon-like Peptide-1 Receptor.

The glucagon-like peptide-1 receptor (GLP-1R) plays a key role in metabolism and is an important therapeutic target in diabetes and obesity. Recent studies in experimental animals have shown that certain subsets of T cells express functional GLP-1R, indicating an immune regulatory role of GLP-1. In contrast, less is known about the expression and function of the GLP-1R in human T cells. Here, we provide evidence that activated human T cells express GLP-1R. The expressed GLP-1R was functional, as stimulation with a GLP-1R agonist triggered an increase in intracellular cAMP, which was abrogated by a GLP-1R antagonist. Analysis of CD4+ T cells activated under T helper (Th) 1, Th2, Th17 and regulatory T (Treg) cell differentiation conditions indicated that GLP-1R expression was most pronounced in induced Treg (iTreg) cells. Through multimodal single-cell CITE- and TCR-sequencing, we detected GLP-1R expression in 29-34% of the FoxP3+CD25+CD127- iTreg cells. GLP-1R+ cells showed no difference in their TCR-gene usage nor CDR3 lengths. Finally, we demonstrated the presence of GLP-1R+CD4+ T cells in skin from patients with allergic contact dermatitis. Taken together, the present data demonstrate that T cell activation triggers the expression of functional GLP-1R in human CD4+ T cells. Given the high induction of GLP-1R in human iTreg cells, we hypothesize that GLP-1R+ iTreg cells play a key role in the anti-inflammatory effects ascribed to GLP-1R agonists in humans.

CD8+ tissue-resident memory T cells recruit neutrophils that are essential for flare-ups in contact dermatitis.

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.

Normal T and B Cell Responses Against SARS-CoV-2 in a Family With a Non-Functional Vitamin D Receptor: A Case Report.

The coronavirus disease 2019 (COVID-19) pandemic has severely impacted daily life all over the world. Any measures to slow down the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to decrease disease severity are highly requested. Recent studies have reported inverse correlations between plasma levels of vitamin D and susceptibility to SARS-CoV-2 infection and COVID-19 severity. Therefore, it has been proposed to supplement the general population with vitamin D to reduce the impact of COVID-19. However, by studying the course of COVID-19 and the immune response against SARS-CoV-2 in a family with a mutated, non-functional vitamin D receptor, we here demonstrate that vitamin D signaling was dispensable for mounting an efficient adaptive immune response against SARS-CoV-2 in this family. Although these observations might not directly be transferred to the general population, they question a central role of vitamin D in the generation of adaptive immunity against SARS-CoV-2.

Macrophages Control the Bioavailability of Vitamin D and Vitamin D-Regulated T Cell Responses.

The active form of vitamin D3 (1,25(OH)2D3) has a great impact on T cell effector function. Thus, 1,25(OH)2D3 promotes T helper 2 (Th2) and regulatory T (Treg) cell function and concomitantly inhibits Th1 and Th17 cell function. Thus, it is believed that vitamin D exerts anti-inflammatory effects. However, vitamin D binding protein (DBP) strongly binds both 1,25(OH)2D3 and the precursor 25(OH)D3, leaving only a minor fraction of vitamin D in the free, bioavailable form. Accordingly, DBP in physiological concentrations would be expected to block the effect of vitamin D on T cells and dendritic cells. In the present study, we show that pro-inflammatory, monocyte-derived M1 macrophages express very high levels of the 25(OH)D-1α-hydroxylase CYP27B1 that enables them to convert 25(OH)D3 into 1,25(OH)2D3 even in the presence of physiological concentrations of DBP. Co-cultivation of M1 macrophages with T cells allows them to overcome the sequestering of 25(OH)D3 by DBP and to produce sufficient levels of 1,25(OH)2D3 to affect T cell effector function. This study suggests that in highly inflammatory conditions, M1 macrophages can produce sufficient levels of 1,25(OH)2D3 to modify T cell responses and thereby reduce T cell-mediated inflammation via a vitamin D-mediated negative feed-back loop.