Atsushi Tanemura

Anti-CCR4 mAb selectively depletes effector-type FoxP3+CD4+ regulatory T cells, evoking antitumor immune responses in humans

Significance Regulatory T (Treg) cells expressing the transcription factor FOXP3 play a critical role in suppressing antitumor immune responses. Here we found that, compared with peripheral blood T cells, tumor-infiltrating T cells contained a higher frequency of effector Tregs, which are defined as FOXP3hi and CD45RA−, terminally differentiated, and most suppressive. Effector Treg cells, but not FOXP3lo and CD45RA+ naïve Treg cells, predominantly expressed C-C chemokine receptor 4 (CCR4) in both cancer tissues and peripheral blood. In vivo or in vitro anti-CCR4 mAb treatment selectively depleted effector Treg cells and efficiently induced tumor-antigen-specific CD4+ and CD8+ T cells. Thus, cell-depleting anti-CCR4 mAb therapy is instrumental for evoking and enhancing tumor immunity in humans via selectively removing effector-type FOXP3+ Treg cells. CD4+ Treg cells expressing the transcription factor FOXP3 (forkhead box P3) are abundant in tumor tissues and appear to hinder the induction of effective antitumor immunity. A substantial number of T cells, including Treg cells, in tumor tissues and peripheral blood express C-C chemokine receptor 4 (CCR4). Here we show that CCR4 was specifically expressed by a subset of terminally differentiated and most suppressive CD45RA−FOXP3hiCD4+ Treg cells [designated effector Treg (eTreg) cells], but not by CD45RA+FOXP3loCD4+ naive Treg cells, in peripheral blood of healthy individuals and cancer patients. In melanoma tissues, CCR4+ eTreg cells were predominant among tumor-infiltrating FOXP3+ T cells and much higher in frequency compared with those in peripheral blood. With peripheral blood lymphocytes from healthy individuals and melanoma patients, ex vivo depletion of CCR4+ T cells and subsequent in vitro stimulation of the depleted cell population with the cancer/testis antigen NY-ESO-1 efficiently induced NY-ESO-1–specific CD4+ T cells. Nondepletion failed in the induction. The magnitude of the responses was comparable with total removal of FOXP3+ Treg cells by CD25+ T-cell depletion. CCR4+ T-cell depletion also augmented in vitro induction of NY-ESO-1–specific CD8+ T cells in melanoma patients. Furthermore, in vivo administration of anti-CCR4 mAb markedly reduced the eTreg-cell fraction and augmented NY-ESO-1–specific CD8+ T-cell responses in an adult T-cell leukemia-lymphoma patient whose leukemic cells expressed NY-ESO-1. Collectively, these findings indicate that anti-CCR4 mAb treatment is instrumental for evoking and augmenting antitumor immunity in cancer patients by selectively depleting eTreg cells.

Mesenchymal–epithelial interactions in the skin: increased expression of dickkopf1 by palmoplantar fibroblasts inhibits melanocyte growth and differentiation

We investigated whether or not the topographic regulation of melanocyte differentiation is determined by mesenchymal–epithelial interactions via fibroblast-derived factors. The melanocyte density in palmoplantar human skin (i.e., skin on the palms and the soles) is five times lower than that found in nonpalmoplantar sites. Palmoplantar fibroblasts significantly suppressed the growth and pigmentation of melanocytes compared with nonpalmoplantar fibroblasts. Using cDNA microarray analysis, fibroblasts derived from palmoplantar skin expressed high levels of dickkopf 1 (DKK1; an inhibitor of the canonical Wnt signaling pathway), whereas nonpalmoplantar fibroblasts expressed higher levels of DKK3. Transfection studies revealed that DKK1 decreased melanocyte function, probably through β-catenin–mediated regulation of microphthalmia-associated transcription factor activity, which in turn modulates the growth and differentiation of melanocytes. Thus, our results provide a basis to explain why skin on the palms and the soles is generally hypopigmented compared with other areas of the body, and might explain why melanocytes stop migrating in the palmoplantar area during human embryogenesis.

Targeted disruption of LIG-1 gene results in psoriasiform epidermal hyperplasia1

The gene encoding a transmembrane glycoprotein LIG‐1, of which the extracellular region was organized with the leucine‐rich repeats and immunoglobulin‐like domains, was disrupted in mice by gene targeting. LIG‐1‐deficient mice developed a skin change on the tail and facial area after birth. The affected skin was histologically reminiscent of the epidermis in human common skin disease ‘psoriasis’. LIG‐1 was expressed in basal cells of the epidermis and outer root sheath cells of hair follicles in mice. Interestingly, the LIG‐1 expression was apparently down‐regulated in the psoriatic lesions, suggesting that LIG‐1 inversely correlates with proliferative ability of epidermal keratinocytes.

Periostin, a matricellular protein, accelerates cutaneous wound repair by activating dermal fibroblasts

Abstract:  Cutaneous wound repair is a highly ordered and well‐coordinated process involving various cell lineages and many molecular effectors. Cell–matrix interactions through integrin molecules provide key signals important for wound repair. Periostin is a matricellular protein that may provide signals important during tissue development and remodelling by interacting with several integrin molecules, via the phosphatidylinositol 3‐kinase/Akt and MAP kinase pathways. In this study, we examined the role of periostin in the process of cutaneous wound repair using periostin‐deficient mice and by analysing the effects of periostin on dermal fibroblasts. We first determined the expression profile and localization of periostin in a well‐characterized wound repair model mice. Periostin was robustly deposited in the granulation tissues beneath the extended epidermal wound edges and at the dermal–epidermal junctions in wounded mice. Moreover, periostin‐deficient mice exhibited delayed in vivo wound repair, which could be improved by direct administration of exogenous periostin. In vitro analyses revealed that loss of periostin impaired proliferation and migration of dermal fibroblasts, but exogenous supplementation or enforced periostin expression enhanced their proliferation. Combined, these results demonstrate that periostin accelerates the process of cutaneous wound repair by activating fibroblasts.

Dysregulation of melanocyte function by Th17‐related cytokines: significance of Th17 cell infiltration in autoimmune vitiligo vulgaris

The aim of this study was to determine whether CD4+IL‐17A+Th17 cells infiltrate vitiligo skin and to investigate whether the proinflammatory cytokines related to Th17 cell influence melanocyte enzymatic activity and cell fate. An immunohistochemical analysis showed Th17 cell infiltration in 21 of 23 vitiligo skin samples in addition to CD8+ cells on the reticular dermis. An in vitro analysis showed that the expression of MITF and downstream genes was downregulated in melanocytes by treatment with interleukin (IL)‐17A, IL‐1β, IL‐6, and tumor necrosis factor (TNF)‐α. Treatment with these cytokines also induced morphological shrinking in melanocytes, resulting in decreased melanin production. In terms of local cytokine network in the skin, IL‐17A dramatically induced IL‐1β, IL‐6, and TNF‐α production in skin‐resident cells such as keratinocytes and fibroblasts. Our results provide evidence of the influence of a complex Th17 cell‐related cytokine environment in local depigmentation in addition to CD8+ cell‐mediated melanocyte destruction in autoimmune vitiligo.

Functional RET G691S Polymorphism in Cutaneous Malignant Melanoma

RET proto-oncogene encodes a receptor tyrosine kinase whose ligand is glial cell line-derived neurotrophic factor (GDNF), and its polymorphism at G691S juxtamembrane region (RETp) is a germline polymorphism. Cutaneous melanomas, particularly the desmoplastic subtype, are highly neurotropic; thus we sought to determine the frequency of RETp in cutaneous melanoma and its functional responsiveness to GDNF. RETp was assessed in 71 non-desmoplastic cutaneous melanomas (non-DMs) and 70 desmoplastic melanomas (DMs). Melanoma cell lines with RETp, RET wild type (RETwt), BRAF V600E mutation (BRAFmt) or BRAF wild type (BRAFwt) were assessed for functional activity. RETp frequency was significantly higher in DMs (61%) than in non-DMs (31%, P<0.001). BRAFmt was detected in only 11% of DMs. GDNF stimulation significantly amplified cell proliferation, migration and invasion in RETp, but not in RETwt melanoma cells. GDNF stimulation of RETp cell lines enhanced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt of the RET-RAS-RAF-ERK and RET-phosphatidylinositol 3-kinase (PI3K)-Akt pathways, respectively. GDNF response of RETp cells in signal transduction and other functional studies were not affected by BRAFmt. The study demonstrates that RETp is frequently found in cutaneous melanoma, particularly desmoplastic subtypes, and responds to GDNF inducing events favorable for tumor progression.

Nodal Lymphangiogenesis and Metastasis Role of Tumor-Induced Lymphatic Vessel Activation in Extramammary Paget's Disease

Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Pagets disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-beta1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis.

Impact of sedative and non-sedative antihistamines on the impaired productivity and quality of life in patients with pruritic skin diseases.

BACKGROUND The impairment that pruritic skin diseases have on patient productivity at work, in the classroom, and in daily activities is substantial and needs to be characterized. The objective of this study was to determine how pruritic skin diseases impact patient productivity and quality of life (QOL), in order to improve the measurement of these endpoints to allow the influence of treatment options including sedative and non-sedative antihistamines to be analyzed. METHODS The impact of pruritic skin diseases and the effect of antihistamine therapy on work, classroom, and daily productivity were evaluated using the Work Productivity Assessment Index-Allergy Specific Questionnaire. The intensity of itch and patient QOL were assessed using a visual analogue scale and Skindex-16, respectively. RESULTS Pruritic skin diseases resulted in significant impairment of work, classroom, and daily productivity. The severity of overall work impairment in atopic dermatitis (AD), urticaria, and prurigo was higher than for other diseases analyzed. However, classroom activity was more adversely affected in patients with urticaria relative to other diseases. All pruritic diseases in this study negatively impacted daily activity to a similar degree. Impaired productivity was significantly improved in patients taking non-sedative antihistamines for 1 month, and the improvements correlated with the alleviation of itch and improved QOL. CONCLUSIONS These results indicate that pruritic skin diseases reduce patient productivity at work, in the classroom, and during daily activities, and that non-sedative antihistamines may offer an advantage over sedative antihistamines for alleviating certain negative consequences of these skin diseases.

AMP kinase-related kinase NUAK2 affects tumor growth, migration, and clinical outcome of human melanoma

The identification of genes that participate in melanomagenesis should suggest strategies for developing therapeutic modalities. We used a public array comparative genomic hybridization (CGH) database and real-time quantitative PCR (qPCR) analyses to identify the AMP kinase (AMPK)-related kinase NUAK2 as a candidate gene for melanomagenesis, and we analyzed its functions in melanoma cells. Our analyses had identified a locus at 1q32 where genomic gain is strongly associated with tumor thickness, and we used real-time qPCR analyses and regression analyses to identify NUAK2 as a candidate gene at that locus. Associations of relapse-free survival and overall survival of 92 primary melanoma patients with NUAK2 expression measured using immunohistochemistry were investigated using Kaplan–Meier curves, log rank tests, and Cox regression models. Knockdown of NUAK2 induces senescence and reduces S-phase, decreases migration, and down-regulates expression of mammalian target of rapamycin (mTOR). In vivo analysis demonstrated that knockdown of NUAK2 suppresses melanoma tumor growth in mice. Survival analysis showed that the risk of relapse is greater in acral melanoma patients with high levels of NUAK2 expression than in acral melanoma patients with low levels of NUAK2 expression (hazard ratio = 3.88; 95% confidence interval = 1.44–10.50; P = 0.0075). These data demonstrate that NUAK2 expression is significantly associated with the oncogenic features of melanoma cells and with the survival of acral melanoma patients. NUAK2 may provide a drug target to suppress melanoma progression. This study further supports the importance of NUAK2 in cancer development and tumor progression, while AMPK has antioncogenic properties.

Pilomatrix carcinoma arising from pilomatricoma after 10-year senescent period : Immunohistochemical analysis

Pilomatrix carcinoma is a rare malignant counterpart of pilomatricoma. To our knowledge, only approximately 90 cases have been published in English literature. Pilomatrix carcinoma is locally aggressive and occasionally shows rapid progression infiltrating to the muscle, bone and vessels. We report a case of pilomatrix carcinoma that developed in a 38‐year‐old man and started to grow after a long stable period, relapsed for a short time and infiltrated into the muscle underneath. While the initial skin biopsy showed histopathological findings consistent with pilomatricoma, the recurrent tumor contained marked cellular atypia and an aggressive growth pattern. Although it is still controversial whether pilomatrix carcinoma arises de novo or through malignant transformation of a pilomatricoma, the present case might be caused by the latter process considering the patient’s clinical course. β‐catenin is a downstream effecter in the canonical pathway of Wnt, acting as a signal for cell differentiation and proliferation. The characteristic nuclear staining pattern of β‐catenin in the basaloid tumor cells, which is usually observed in pilomatrix carcinoma, supported the diagnosis of pilomatrix carcinoma in the present case.