Author Correction: Tissue patrol by resident memory CD8+ T cells in human skin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tissue patrol by resident memory CD8+ T cells in human skin.

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.

Inflammasome-Dependent Induction of Adaptive NK Cell Memory.

Monobenzone is a pro-hapten that is exclusively metabolized by melanocytes, thereby haptenizing melanocyte-specific antigens, which results in cytotoxic autoimmunity specifically against pigmented cells. Studying monobenzone in a setting of contact hypersensitivity (CHS), we observed that monobenzone induced a long-lasting, melanocyte-specific immune response that was dependent on NK cells, yet fully intact in the absence of T- and B cells. Consistent with the concept of "memory NK cells," monobenzone-induced NK cells resided in the liver and transfer of these cells conferred melanocyte-specific immunity to naive animals. Monobenzone-exposed skin displayed macrophage infiltration and cutaneous lymph nodes showed an inflammasome-dependent influx of macrophages with a tissue-resident phenotype, coinciding with local NK cell activation. Indeed, macrophage depletion or the absence of the NLRP3 inflammasome, the adaptor protein ASC or interleukin-18 (IL-18) abolished monobenzone CHS, thereby establishing a non-redundant role for the NLRP3 inflammasome as a critical proinflammatory checkpoint in the induction of hapten-dependent memory NK cells.

Regulatory T Cell Dysfunction in Autoimmune Diseases.

Regulatory T cells (Tregs), a suppressive subpopulation of T cells, are potent mediators of peripheral tolerance, responsible for immune homeostasis. Many autoimmune diseases exhibit disruptions in Treg function or quantity, resulting in an imbalance between protective and pathogenic immune cells. Selective expansion or manipulation of Tregs is a promising therapeutic approach for autoimmune diseases. However, the extensive diversity of Treg subpopulations and the multiple approaches used for Treg identification leads to high complexity, making it difficult to develop a successful treatment capable of modulating Tregs. In this review, we describe the suppressive mechanisms, subpopulations, classification, and identification methodology for Tregs, and their role in the pathogenesis of autoimmune diseases.

A secondary role for hypoxia and HIF1 in the regulation of (IFNγ-induced) PD-L1 expression in melanoma.

Cancer cells are able to escape immune surveillance by upregulating programmed death ligand 1 (PD-L1). A key regulator of PD-L1 expression is transcriptional stimulation by the IFNγ/JAK/STAT pathway. Recent studies suggest that hypoxia can induce PD-L1 expression. As hypoxia presents a hallmark of solid tumor development, hypoxic control of PD-L1 expression may affect the efficacy of cancer immunotherapy. This study aims to explore the hypoxic regulation of PD-L1 expression in human melanoma, and its interaction with IFNγ-induced PD-L1 expression. Analysis of the cutaneous melanoma dataset from the cancer genome atlas revealed a significant correlation of the HIF1-signaling geneset signature with PD-L1 mRNA expression. However, this correlation is less pronounced than other key pathways known to control PD-L1 expression, including the IFNγ/JAK/STAT pathway. This secondary role of HIF1 in PD-L1 regulation was confirmed by analyzing single-cell RNA-sequencing data of 33 human melanoma tissues. Interestingly, PD-L1 expression in these melanoma tissues was primarily found in macrophages. However, also in these cells STAT1, and not HIF1, displayed the most pronounced correlation with PD-L1 expression. Moreover, we observed that hypoxia differentially affects PD-L1 expression in human melanoma cell lines. Knockdown of HIF1 expression indicated a minor role for HIF1 in regulating PD-L1 expression. A more pronounced influence of hypoxia was found on IFNγ-induced PD-L1 mRNA expression, which is controlled at a 952 bp PD-L1 promoter fragment. These findings, showing the influence of hypoxia on IFNγ-induced PD-L1 expression, are relevant for immunotherapy, as both IFNγ and hypoxia are frequently present in the tumor microenvironment.

IFN-γ-induced PD-L1 expression on human melanocytes is impaired in vitiligo.

Mounting evidence shows that the PD-1/PD-L1 axis is involved in tumor immune evasion. This is demonstrated by anti-PD-1 antibodies that can reverse tumor-associated PD-L1 to functionally suppress anti-tumor T-cell responses. Since type I and II interferons are key regulators of PD-L1 expression in melanoma cells and IFN-γ-producing CD8+ T cells and IFN-α-producing dendritic cells are abundant in vitiligo skin, we aimed to study the role of PD-1/PD-L1 signalling in melanocyte destruction in vitiligo. Moreover, impaired PD-1/PD-L1 function is observed in a variety of autoimmune diseases. It is, therefore, hypothesized that manipulating PD-1/PD-L1 signalling might have therapeutic potential in vitiligo. The PD-1+ T cells were abundantly present in situ in perilesional vitiligo skin, but expression of PD-L1 was limited and confined exclusively to dermal T cells. More specifically, neither melanocytes nor other epidermal skin cells expressed PD-L1. Exposure to IFN-γ, but also type I interferons, increased PD-L1 expression in primary melanocytes and fibroblasts, derived from healthy donors. Primary human keratinocytes only showed increased PD-L1 expression upon stimulation with IFN-γ. More interestingly, melanocytes derived from non-lesional vitiligo skin showed no PD-L1 upregulation upon IFN-γ exposure, while other skin cells displayed significant PD-L1 expression after exposure. In a vitiligo skin explant model, incubation of non-lesional vitiligo skin with activated (IFN-γ-producing) T cells from vitiligo lesions was previously described to induce melanocyte apoptosis. Although PD-L1 expression was induced in epidermal cells in these explants, this induction was completely absent in melanocytes. The lack of PD-L1 upregulation by melanocytes in the presence of IFN-γ-producing T cells shows that melanocytes lack protection against T-cell attack during vitiligo pathogenesis. Manipulating PD-1/PD-L1 signalling may, therefore, be a therapeutic option for vitiligo patients.

Presence of Skin Tissue-Resident Memory T Cells in Human Nonmalignant and Premalignant Melanocytic Skin Lesions and in Melanoma.

ABSTRACT: The infiltration of tissue-resident memory (TRM) cells in melanoma correlates with improved survival, suggesting an important role for TRM cells in immunity against melanoma. However, little is known about the presence of TRM cells in nonmalignant and premalignant melanocytic lesions. This study aimed to evaluate the presence of TRM cells in human skin melanocytic lesions, representing the spectrum from healthy skin to metastatic melanoma. FFPE sections from healthy skin, sun-exposed skin, benign nevi, lentigo maligna (LM), primary LM melanoma, and primary cutaneous and metastatic melanoma were analyzed by immunohistochemistry. The number of infiltrating cells expressing TRM-associated markers, CD3, CD4, CD8, CD69, CD103, and CD49a, was quantified by digital analyses. Multiplex immunofluorescence was performed to analyze coexpression of TRM cell markers. More T cells and CD69+ cells were found in melanoma lesions, as compared with healthy skin and nevi. CD103+ and CD49a+ cell numbers did not significantly differ. More importantly, no differences were seen in expression of all markers between healthy skin and benign nevi. Similar results, except for CD69, were observed in LM melanoma, as compared with LM and sun-exposed skin. Interestingly, multiplex immunofluorescence showed that nevi tissues have comparable CD103+ T cell numbers with healthy skin but comprise more CD103+ CD8+ cells. Expression of TRM cell markers is significantly increased in melanoma, as compared with nonmalignant skin. Our data also show that TRM cells are not abundantly present already in premalignant tissues. Further studies on the specificity of TRM cells for melanocyte/melanoma antigens may reveal their significance in cancer immunosurveillance.

Does autoimmune vitiligo protect against COVID-19 disease?

The SARS-CoV-2 pandemic has evolved to a global health problem with a dramatic morbidity and mortality rate impacting our daily life and those of many patients. While there is evidence that some diseases are associated with an increased risk for development of a more severe course of COVID-19, little is known on protective conditions. Importantly, clearance of viral infection and protection against disease manifestation crucially depends on functional innate and adaptive immunity and the interferon signalling axis. Here, we hypothesize that patients with non-segmental vitiligo (NSV), an autoimmune skin (and mucosal) disorder, may clear SARS-CoV-2 infection more efficiently and have a lower risk of COVID-19 development. Conversely, in case of COVID-19 development, vitiligo autoimmunity may influence the cytokine storm-related disease burden. In addition, immune activation during SARS-CoV-2 infection or COVID-19 disease might increase vitiligo disease activity. Our hypothesis is based on the shift of the immune system in NSV towards adaptive type 1 (IFNγ and CD8 T cells) and innate immune responses. Identified susceptibility genes of NSV patients may further confer increased antiviral activity. To validate our hypothesis, we suggest an international consortium to perform a retrospective data registry and patient-reported study on a large number of NSV patients worldwide during the COVID-19 pandemic.

Differential Expression of Cancer Testis Antigens on Lentigo Maligna and Lentigo Maligna Melanoma.

The cancer/testis antigens (CTA) are a group of antigens expressed on germ cells of healthy testis and malignant tumors. We studied whether CTA are present on lentigo maligna (LM) and LM melanoma (LMM) samples. Immunohistochemical expression of a panel of CTA (MAGE-A1, A2- A3, NY-ESO-1, PRAME, SSX-2, and a MAGE-A antibody reactive with -A1, -A2, -A3, -A4, -A6, -A10, and -A12) was investigated in formalin-fixed paraffin-embedded samples from LMM (n = 20), LM (n = 8), chronically sun-exposed skin (n = 7), and healthy skin (n = 7). In 4 LMM lesions, the MAGE-A marker was positive. Another 3 LMM lesions were positive for MAGE-A1, MAGE-A2, and MAGE-A3. PRAME was positive in 18/20 LMM and 6/8 LM. We did not find expression of MAGE, NY-ESO-1, or SSX-2 in LM, thereby excluding these CTA as diagnostic markers to discern malignant melanocytes in LM from normal melanocytes. LMM did express MAGE, NY-ESO-1, and SSX-2. If a biopsy from a lesion suspect for LM shows positivity for MAGE, NY-ESO-1, and SSX-2, the lesion may actually be LMM. In contrast, PRAME expression was found in LM at low levels and in LMM at much higher levels, and absent in normal melanocytes. PRAME can potentially be used to discern normal melanocytes from malignant melanocytes.

House dust mite allergens Der f and Der p induce IL-31 production by blood-derived T cells from atopic dermatitis patients.

Aero-allergens, such as house dust mite (HDM), have been suggested to play a role in the initiation of atopic dermatitis (AD)-related skin inflammation. Here, we analysed the proliferation and the cytokine expression of blood-derived T cells from AD and healthy individuals upon HDM-allergen stimulation. The proliferating cells from healthy individuals and AD patients had a significantly different, distinct cytokine profile: in AD blood, we found increased frequencies of HDM-reactive IL-31-producing T cells, as well as a decreased Th1/Th2 and Tc1/Tc2 ratio, suggesting that allergen-specific T cells in blood of chronic AD patients are subject to pre-existent Th2-Tc2 and "Th31-Tc31" programming.

Melanoma-associated leukoderma and vitiligo cannot be differentiated based on blinded assessment by experts in the field.

BACKGROUND: Melanoma-associated leukoderma (MAL) is a depigmenting disorder that can occur spontaneously in patients with melanoma. The differences in clinical presentation between MAL and vitiligo are not well defined. This may lead to misdiagnosing MAL as vitiligo, resulting in delayed detection of melanoma. OBJECTIVE: The objective of this study was to assess whether experts in the field can distinguish between MAL and vitiligo, and to assess if discriminative features can be identified. METHODS: We designed an image comparison study in which 4 experts in the field blindly assessed photographs followed by medical history of 11 patients with MAL and 33 with vitiligo. RESULTS: The assessors misdiagnosed 72.7% of MAL cases and marked 80.0% of them as typical vitiligo. The median age at onset of the leukoderma was higher (55 years, P = .001) in MAL. No discriminative features were found. LIMITATIONS: Sampling bias because of inclusion in tertiary referral center is a limitation. CONCLUSION: The clinical presentation of leukoderma in patients with melanoma resembles that of vitiligo. We propose "melanoma-associated vitiligo" as the more appropriate term for leukoderma in patients with melanoma. Clinicians should be aware that depigmentation in vitiligo can also be caused by melanoma-associated vitiligo and a total body inspection should be performed.

Enhanced bleaching treatment: opportunities for immune-assisted melanocyte suicide in vitiligo.

Depigmentation in vitiligo occurs by progressive loss of melanocytes from the basal layer of the skin, and can be psychologically devastating to patients. T cell-mediated autoimmunity explains the progressive nature of this disease. Rather than being confronted with periods of rapid depigmentation and bouts of repigmentation, patients with long-standing, treatment-resistant vitiligo can undergo depigmentation treatment. The objective is to remove residual pigmentation to achieve a cosmetically acceptable result--that of skin with a uniform appearance. In the United States, only the use of mono-benzyl ether of hydroquinone (MBEH) is approved for this purpose. However, satisfactory results can take time to appear, and there is a risk of repigmentation. MBEH induces necrotic melanocyte death followed by a cytotoxic T-cell response to remaining, distant melanocytes. As cytotoxic T-cell responses are instrumental to depigmentation, we propose that combining MBEH with immune adjuvant therapies will accelerate immune-mediated melanocyte destruction to achieve faster, more definitive depigmentation than with MBEH alone. As Toll-like Receptor (TLR) agonists--imiquimod, CpG, and Heat Shock Protein 70 (HSP 70)--all support powerful Th1 responses, we propose that using MBEH in combination with these agents can achieve superior depigmentation results for vitiligo patients.

The Risk of Keratinocyte Cancer in Vitiligo and the Potential Mechanisms Involved.

Although light skin types are associated with increased skin cancer risk, a lower incidence of both melanoma and nonmelanoma skin cancer (NMSC) has been reported in patients with vitiligo. We performed a systematic review and meta-analysis on the NMSC risk in patients with vitiligo, indicating a reduced relative risk ratio of NMSC in vitiligo. Furthermore, we propose a series of hypotheses on the underlying mechanisms, including both immune-mediated and nonimmune-mediated pathways. This study reveals insights into the relationship between vitiligo and keratinocyte cancer and can also be used to better inform patients with vitiligo.

Inefficient exogenous loading of a tapasin-dependent peptide onto HLA-B*44:02 can be improved by acid treatment or fixation of target cells.

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by "cancer-germline" genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.

Immunity against Non-Melanoma Skin Cancer and the Effect of Immunosuppressive Medication on Non-Melanoma Skin Cancer Risk in Solid Organ Transplant Recipients.

Non-melanoma skin cancers (NMSCs) occur frequently in the Caucasian population and are considered a burden for health care. Risk factors include ultraviolet (UV) radiation, ethnicity and immunosuppression. The incidence of NMSC is significantly higher in solid organ transplant recipients (SOTRs) than in immunocompetent individuals, due to immunosuppressive medication use by SOTRs. While the immunosuppressive agents, calcineurin inhibitors and purine analogues increase the incidence of NMSC in transplant recipients, mTOR inhibitors do not. This is most likely due to the different immunological pathways that are inhibited by each class of drug. This review will focus on what is currently known about the immune response against cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC), two of the main types of NMSC. Furthermore, we will describe the different classes of immunosuppressants given to SOTRs, which part of the immune system they target and how they can contribute to NMSC development. The risk of developing NMSC in SOTRs is the result of a combination of inhibiting immunological pathways involved in immunosurveillance against NMSC and the direct (pro/anti) tumor effects of immunosuppressants.

Trained immunity in the pathogenesis of vitiligo.

Vitiligo is caused by an autoimmune reaction against melanocytes leading to melanocyte loss. The cause of vitiligo is an interaction between genetic susceptibility and environmental factors. Both the adaptive immune system-through cytotoxic CD8+ T cells and melanocyte specific antibodies-and the innate immune system are involved in these immune processes in vitiligo. While recent data stressed the importance of innate immunity in vitiligo, the question remains why vitiligo patients' immune response becomes overly activated. Could a long-term increase in innate memory function, described as trained immunity after vaccination and in other inflammatory diseases, play a role as an enhancer and continuous trigger in the pathogenesis of vitiligo? After exposure to certain stimuli, innate immune system is able to show an enhanced immunological response to a secondary trigger, indicating a memory function of the innate immune system, a concept termed trained immunity. Trained immunity is regulated by epigenetic reprogramming, including histone chemical modifications and changes in chromatin accessibility that cause sustained changes in the transcription of specific genes. In responses to an infection, trained immunity is beneficial. However, there are indications of a pathogenic role of trained immunity in inflammatory and autoimmune diseases, with monocytes presenting features of a trained phenotype, resulting in increased cytokine production, altered cell metabolism through mTOR signaling, and epigenetic modifications. This hypothesis paper focusses on vitiligo studies that have shown these indications, suggesting the involvement of trained immunity in vitiligo. Future studies focusing on metabolic and epigenetic changes in innate immune cell populations in vitiligo could help in elucidating the potential role of trained immunity in vitiligo pathogenesis.

Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy.

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to gamma-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy.

Increased frequencies of IL-31-producing T cells are found in chronic atopic dermatitis skin.

Interleukin (IL)-31 has been associated with pruritus, a characteristic feature of atopic dermatitis (AD). Local T cell responses may be responsible for the increased level of IL-31 mRNA observed in AD. We investigated the frequency of IL-31-producing T cells in AD lesions, as well as their cytokine profile. T cells were isolated from chronic AD lesions, autologous blood and healthy donor skin. Intracellular expression of IL-31, IFN-γ, IL-13, IL-17 and IL-22 was measured using flow cytometry. T cells from AD lesions contained significantly higher percentages of IL-31-producing T cells compared to autologous blood and donor skin. Many IL-31-producing T cells co-produced IL-13 and to lesser extent IL-22, but rarely IFN-γ or IL-17. A substantial part of the IL-31-producing T cells did not co-produce any of the other cytokines and could therefore not be linked to any of the known functionally different T cell subsets. The T cell infiltrates were also relatively enriched for Th2/Tc2 and Th22/Tc22 cells, while frequencies of Th1/Tc1 and Th17 cells were decreased. This is the first report describing the detection of IL-31 at protein level in skin-infiltrating T cells. We show here that T cells in chronic AD skin produce IL-31 and that AD lesions contain increased levels of these IL-31-producing T cells. This suggests that a substantial part of previously reported increased IL-31 mRNA levels in AD skin is T cell derived and that these cells may be involved in the pathogenesis of AD.

Immunophenotypic Analysis Reveals Differences in Circulating Immune Cells in the Peripheral Blood of Patients with Segmental and Nonsegmental Vitiligo.

Accumulating studies have indicated immune-based destruction of melanocytes in both segmental vitiligo (SV) and non-SV (NSV). Whereas SV often occurs unilaterally during childhood and stabilizes after an initial period of activity, the disease course of NSV is usually slowly progressive, with new lesions occurring bilaterally during life. This suggests an involvement of distinct pathophysiology pathways, specifically increased systemic immune activation in patients with NSV but not in patients with SV. This research aimed to identify the differences in immune cells in the blood of patients with SV and NSV through immunophenotyping of circulating cells. Regulatory T cells were unaffected in patients with SV compared with that in healthy controls but decreased in patients with NSV. In patients with NSV, the reduction in regulatory T cells was associated with the presence of other systemic autoimmune comorbidities, which were less present in SV. Similarly, the absence of a melanocyte-specific antibody response in patients with SV suggests less involvement of B-cell immunity in SV. These data show that in contrast to patients with NSV, no increased systemic immunity is found in patients with SV, indicating that SV pathogenesis is associated with a localized cytotoxic reaction targeting epidermal melanocytes.