Increased Regulatory T Cells and Decreased Myeloid-Derived

International Journal of Molecular Sciences

Article Increased Regulatory T Cells and Decreased Myeloid-Derived Suppressor Cells Induced by High CCL17 Levels May Account for Normal Incidence of Cancers among Patients with Atopic Dermatitis

Sohshi Morimura 1, Makoto Sugaya 1,* , Tomonori Oka 2 , Hiraku Suga 2, Tomomitsu Miyagaki 3 , Yuichiro Tsunemi 4 , Yoshihide Asano 2 and Shinichi Sato 2

1 Department of Dermatology, International University of Health and Welfare, Chiba 286-8520, Japan; [email protected] 2 Department of Dermatology, The University of Tokyo Graduate School of Medicine, Tokyo 113-8655, Japan; [email protected] (T.O.); [email protected] (H.S.); [email protected] (Y.A.); [email protected] (S.S.) 3 Department of Dermatology, St. Marianna University School of Medicine, Kanagawa 216-8511, Japan; [email protected] 4 Department of Dermatology, Saitama Medical University, Saitama 350-0495, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-476-35-5600; Fax: +81-476-35-5586

Abstract: The incidence of cancers in atopic dermatitis (AD) is not increased, although the Th2-

Citation: Morimura, S.; Sugaya, M.; dominant environment is known to downregulate tumor immunity. To gain mechanistic insights Oka, T.; Suga, H.; Miyagaki, T.; regarding tumor immunity in AD, we utilized CCL17 transgenic (TG) mice overexpressing CCL17, Tsunemi, Y.; Asano, Y.; Sato, S. which is a key chemokine in AD. Tumor formation and lung metastasis were accelerated in CCL17 TG Increased Regulatory T Cells and mice when melanoma cells were injected subcutaneously or intravenously. Flow cytometric analysis Decreased Myeloid-Derived showed increases in regulatory T cells (Tregs) in lymph nodes in CCL17 TG mice with high mRNA Suppressor Cells Induced by High levels of IL-10 and Foxp3 in tumors, suggesting that Tregs attenuated tumor immunity. The frequency CCL17 Levels May Account for of myeloid-derived suppressor cells (MDSCs), however, was signiﬁcantly decreased in tumors of Normal Incidence of Cancers among CCL17 TG mice, suggesting that decreased MDSCs might promote tumor immunity. Expression of Patients with Atopic Dermatitis. Int. J. CXCL17, a chemoattractant of MDSCs, was decreased in tumors of CCL17 TG mice. Depletion of Mol. Sci. 2021, 22, 2025. https:// Tregs by the anti-CD25 antibody markedly reduced tumor volumes in CCL17 TG mice, suggesting doi.org/10.3390/ijms22042025 that tumor immunity was accelerated by the decrease in MDSCs in the absence of Tregs. Thus, CCL17

Academic Editor: Genji Imokawa attenuates tumor immunity by increasing Tregs and Th2 cells, while it decreases MDSCs through reductions in CXCL17, which may work as a “safety-net” to reduce the risk of malignant tumors in Received: 15 January 2021 the Th2-dominant environment. Accepted: 16 February 2021 Published: 18 February 2021 Keywords: atopic dermatitis; tumor immunity; CCL17; myeloid-derived suppressor cells

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Atopic dermatitis (AD) is a common skin disorder characterized by an itchy skin rash. The disease is now considered as a complex inflammatory process resulting from activated Th2 cells and disorders of barrier function [1]. Patients with AD have a high risk of allergic comorbidities such as asthma, hay fever, allergic rhinitis, and allergic conjunctivitis [2]. Copyright: © 2021 by the authors. Three clinical patterns (the persistent form, the relapsing form, and the adult-onset form) Licensee MDPI, Basel, Switzerland. and various clinical phenotypes have been reported in adult AD [3]. This article is an open access article There is increasing epidemiological evidence that AD is generally negatively associ- distributed under the terms and ated with cancer development. AD has a significantly reduced risk of acute lymphoblastic conditions of the Creative Commons leukemia and acute myeloid leukemia [4,5]. Most research has shown that there is an Attribution (CC BY) license (https:// inverse association between brain tumors and AD [6,7]. With regard to non-Hodgkin’s creativecommons.org/licenses/by/ lymphoma, one report showed a significantly lower risk in patients with AD [8], while two 4.0/).

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papers reported a positive association between non-Hodgkin’s lymphoma and AD [9,10]. There was an increased risk of basal cell carcinoma in AD, while no significantly increased risk of malignant melanoma and squamous cell carcinoma [11]. Thus, AD may prevent the development of malignant tumors in some ways, although the Th2-dominant environment is known to downregulate tumor immunity. The underlying mechanism of protecting against malignant tumors in patients with AD is still unclear. Chemokines are small proteins that stimulate the recruitment of leukocytes. CCL17, known as thymus and activation-regulated chemokine, plays a role in inducing chemotaxis of Th2 cells [12,13]. CCL17 is produced by dendritic cells, T cells, endothelial cells, and fibroblasts. Serum CCL17 levels were found to be significantly higher in patients with AD than in healthy controls [14]. CCL17 is considered as a key chemokine in the development of AD and serves as one of the most reliable biomarkers that can reflect the disease activity of AD. CCL17 has been reported to be related to the development of several other diseases such as cutaneous T-cell lymphoma (CTCL) [15], asthma [16], and acute eosinophilic pneumonia [17]. It has been reported that CCL17 positively regulates tumor development in hepatocellular and gastric carcinomas [18,19]. As CCL17 is overexpressed in AD, it is interesting to focus on the role of CCL17 in tumor immunity. The tumor microenvironment is an emerging concept that specific types of cells contribute to promoting tumor development. Regulatory T cells (Tregs) have an important function in maintaining the tumor microenvironment [20]. Naïve Tregs develop into effector Tregs after activation by antigens when moving from the thymus to the peripheral blood. Effector Tregs express cytotoxic T-lymphocyte antigen-4 (CTLA-4) to suppress dendritic cell maturation. They also produce cytotoxic substances, such as perforin and granzyme B and cytokines such as TGF-β and IL-10 to suppress cytotoxic T cells, attenuating anti-tumor immunity in the tumor microenvironment. Myeloid derived suppressor cells (MDSCs) are myeloid immature cells that have the ability to suppress T cell responses [21]. MDSCs expand rapidly after activation during infection, cancer, and inflammation. Tumor cells stimulate MDSCs to promote tumor expansion in the tumor microenvironment. We hypothesized that Tregs and/or MDSCs are involved in the development of tumor formation in AD. Herein, we utilized CCL17 transgenic (TG) mice that were generated in our department [22], to elucidate key functions of CCL17 in tumor immunity in vivo models. The mice overexpressing the CCL17 protein in keratinocytes showed enhanced Th2 responses after antigen challenge, implicating a new AD mouse model. In the present study, to elucidate the role of CCL17 in tumor immunity, skin tumors and lung metastasis were evaluated. As expected, skin tumor formation by melanoma cells and lymphoma cells was significantly enhanced in CCL17 TG mice compared with wild-type (WT) mice. In addition, lung metastasis, made by intravenous injection of melanoma cells, was significantly augmented in CCL17 TG mice. Interestingly, while Tregs were increased in the skin, lymph nodes, and spleen of CCL17 TG mice, MDSCs in the skin tumor were significantly decreased. After Tregs depletion, tumor formation was attenuated in CCL17 TG mice compared with that in WT mice depleted of Tregs, suggesting that the decrease in MDSCs in a high level of CCL17 may positively contribute to tumor immunity in the absence of Tregs. These results implicate that tumor formation may largely depend on the balance of increased Tregs and decreased MDSCs, thereby inducing a normal incidence of tumors in AD.

2. Results 2.1. Enhanced Skin Tumor Formation in CCL17 TG Mice We ﬁrst assessed skin tumor formation of B16 melanoma cells via subcutaneous injection in CCL17 TG mice. Signiﬁcantly larger tumors were formed in CCL17 TG mice than in WT mice (Figure1A,B). We next evaluated tumor formation by EL4 mouse lymphoma cells. Tumor formation by EL4 cells in CCL17 TG mice was also enhanced compared with WT mice (Figure1C). Thus, tumor growth was enhanced where CCL17 was highly expressed. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 14

lymphoma cells. Tumor formation by EL4 cells in CCL17 TG mice was also enhanced Int. J. Mol. Sci. 2021, 22, 2025 compared with WT mice (Figure 1C). Thus, tumor growth was enhanced where3 of 14 CCL17 was highly expressed.

Figure 1. Tumor formation of back skin was enhanced in CCL17 TG mice. Shaved back skin of wild-type (WT) and CCL17 transgenic (TG) mice was treated with injection of B16F1 cells or EL4 cells. The tumor size was calculated using the equation: FigureL1 1.× TumorL2, where formation L1 = longest of back diameter skin was (mm) enhanced and L2 = in shortest CCL17 diameter TG mice (mm). Shaved and evaluatedback skin onof dayswild-type 4, 7, 10, (WT) and and 14. CCL17 transgenic(A) Phenotypical (TG) mice manifestation was treated ofwith back injection skin from of WT B16F1 and CCL17 cells or TG EL4 mice cells. 14 days The after tumor B16F1 size cells was injection calculated (yellow using circle). the equation: L1(B) x Tumor L2, where sizes were L1 = significantly longest diameter increased (mm) in CCL17 and TGL2 mice= shor comparedtest diameter with those (mm) in WTand mice evaluated on days on 7, 10, days and 4, 14 7, after 10, and 14. (A) Phenotypicalsubcutaneous manifestation injection of B16F1 of cells. back (C skin) Tumor from formation WT and was CCL17 also significantly TG mice promoted14 days after in CCL17 B16F1 TG cells mice comparedinjection (yellow circle).with (B) that Tumor in WT sizes mice were on days significantly 10 and 14 after increased injection ofin EL4 CCL17 cells. TG Data mi arece presented compared as meanwith± thoseSEM ofin threeWT independentmice on days 7, 10, and 14experiments after subcutaneous (n = 6 for each injection group). of * p B16F1< 0.05, **cells.p < 0.01,(C) versusTumor WT formation mice. was also significantly promoted in CCL17 TG mice compared with that in WT mice on days 10 and 14 after injection of EL4 cells. Data are presented as mean ± SEM of three independent experiments2.2. (n Promotion= 6 for each of Lunggroup). Metastasis * p < 0.05, in CCL17** p < 0.01, TG Miceversus WT mice. We next evaluated lung metastasis by injecting B16 melanoma cells intravenously 2.2.because Promotion the pathophysiology of Lung Metastasis of primary in CCL17 tumor TG formation Mice and metastasis formation differs. MoreWe tumors next evaluated were formed lung in CCL17 metastasis TG mice by thaninjecting in WT B16 mice melanoma after 21 days cells (Figure intravenously2A becauseupper panel).the pathophysiology Histologically, the of occupiedprimary colonytumor areaformation of tumor and cells metastasis was significantly formation dif- larger in CCL17 TG mice than in WT mice (Figure2A lower panel). We histologically fers. More tumors were formed in CCL17 TG mice than in WT mice after 21 days (Figure evaluated the number of colonies in each section of the three lobes of the right lung and 2Acalculated upper panel). as the Histologically, following method the that occupied when >60% colony of thearea section of tumor was cells occupied was significantly with largertumor, in theCCL17 colony TG number mice wasthan defined in WT as mice 1000 (Figure [23]. Lung 2A metastasis lower panel). was significantly We histologically evaluatedenhanced the at the number background of colonies of high in CCL17. each section of the three lobes of the right lung and calculated as the following method that when >60% of the section was occupied with tumor, the colony number was defined as 1000 [23]. Lung metastasis was significantly enhanced at the background of high CCL17.

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FigureFigure 2. 2.Metastatic Metastatic lunglung tumor was was promoted promoted in in CCL17 CCL17 TG TG mice. mice. Wild-type Wild-type (WT) (WT) and and CCL17 CCL17 transgenic transgenic (TG) (TG) mice mice were weretreated treated with with B16F1 B16F1 melanoma melanoma cells cells inject injectionion intravenously. intravenously. Metastatic Metastatic lung lung area area waswas evaluated evaluated 21 days 21 days after after B16F1 B16F1 cells cellswere were injected injected intravenously. intravenously. (A) Phenotypical (A) Phenotypical manifestation manifestation of lungs of lungsfrom WT from and WT CCL17 and CCL17 TG mice TG 21 mice days 21 after days applica- after applicationtion of B16F1 of B16F1 cells injection cells injection (upper (upper panel). panel). Representative Representative path pathologicalological images images of lung of lung metast metastaticatic clones clones in WT in WT mice mice and andCCL17 CCL17 TG TGmice mice are are shown shown (lower (lower panel). panel). Original Original magnification magnification ×100;×100; scale scale bar bar = 200 = 200μm.µ (m.B) (MetastaticB) Metastatic lung lung area area was significantly larger in CCL17 TG mice compared with that in WT mice. Metastatic lung nodules were evaluated by calcu- was significantly larger in CCL17 TG mice compared with that in WT mice. Metastatic lung nodules were evaluated by lating colony areas as described following. When >60% of the section was occupied with tumor, the colony number was calculating colony areas as described following. When >60% of the section was occupied with tumor, the colony number defined as 1000. Each section of the three lobes of the right lung was investigated. Data are presented as mean ± SEM of ± wasthree defined independent as 1000. experiments Each section ( ofn = the 6 for three each lobes group). of the * p right < 0.05 lung WT was versus investigated. CCL17 TG Data mice. are presented as mean SEM of three independent experiments (n = 6 for each group). * p < 0.05 WT versus CCL17 TG mice. 2.3. Increased Tregs in CCL17 TG Mice 2.3. IncreasedSkin tumors Tregs inand CCL17 draining TG Mice lymph nodes were harvested for mRNA expression 14 daysSkin after tumors subcutaneous and draining injection lymph of nodesB16 cells. were Lung harvested tissues for were mRNA harvested expression 21 days 14 days after afterthe intravenous subcutaneous injection. injection Expression of B16 cells. levels Lung of Foxp3 tissues and were IL-10 harvested, both of 21which days are after related the intravenouswith Tregs, injection. were evaluated. Expression Foxp3 levels mRNA of Foxp3 levelsand wereIL-10 significantly, both of which elevated are related in skin with tu- Tregs,mors, werewhile evaluated. IL-10 mRNAFoxp3 levelsmRNA were levels significantly were significantly elevated elevatedin skin tumors, in skin tumors,draining whilelymphIL-10 nodes,mRNA and lung levels metastatic were significantly nodules in elevated CCL17 TG in skinmice tumors, (Figure draining3A). Focusing lymph on nodes,Tregs, andflowcytometric lung metastatic analysis nodules of drain in CCL17 lymph TGnodes mice and (Figure spleen3 A).was Focusing performed. on The Tregs, fre- flowcytometricquencies of Foxp3 analysis+ CD4 of+ draincells and lymph Foxp3 nodes+ CD25 and+ spleencells were was increased performed. in TheCCL17 frequencies TG mice of14 Foxp3 days+ afterCD4 +inoculationcells and Foxp3 (Figure+ CD25 3B,C).+ cellsThus, were Tregs increased were increased in CCL17 in TG draining mice 14 lymph days afternodes inoculation and the spleen (Figure in3 CCL17B,C). Thus, TG mice Tregs 14 were days increased after inoculation, in draining which lymph may nodes be respon- and thesible spleen for enhanced in CCL17 tumor TG mice formation. 14 days after inoculation, which may be responsible for enhanced tumor formation.

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FigureFigure 3. Regulatory3. Regulatory T T cells cells (Tregs) (Tregs) were were increased increased inin skin tumors, draining draining lymph lymph nodes, nodes, and and lung lung metastasis metastasis of CCL17 of CCL17 transgenictransgenic (TG) (TG) mice. mice. Skin Skin tumors, tumors, draining draining lymph lymph nodes, nodes, and and spleen spleen werewere harvestedharvested 1414 days after subcutaneous subcutaneous injection injection of B16F1of B16F1 melanoma melanoma cells, cells, while while metastatic metastatic lung lung tissues tissues were were harvested harvested 21 days21 days after after intravenous intravenous injection injection of of B16F1 B16F1 melanoma melanoma cells. Expression levels of markers of Tregs were evaluated by quantitative reverse transcription-PCR in skin tumors, cells. Expression levels of markers of Tregs were evaluated by quantitative reverse transcription-PCR in skin tumors, draining lymph nodes, and metastatic lung tissues from wild-type (WT) mice and CCL17 TG mice. The frequency of Tregs drainingwas analyzed lymph nodes,by flowcytometric and metastatic approach lung tissues in lymph from nodes wild-type and spleen (WT) micefrom andWT CCL17mice and TG CCL17 mice. TheTG mice. frequency (A) Foxp3 of Tregs wasmRNA analyzed levels by were flowcytometric significantly higher approach in skin in lymphtumors nodesof CCL17 and TG spleen mice than from those WT of mice WT andmice. CCL17 IL-10 mRNA TG mice. levels (A were) Foxp3 mRNAsignificantly levels were upregulated significantly in skin higher tumors, in skin lymph tumors nodes, of CCL17and metastatic TG mice lung than tissues those ofof WTCCL17 mice. TGIL-10 micemRNA compared levels with were significantlythose of WT upregulated mice. (B) Representative in skin tumors, flowcytometric lymph nodes, imag andes metastatic showing lungthe frequency tissues of of CCL17 Foxp3 TG+ CD4 mice+ cells compared in the draining with those lymph nodes from WT mice and CCL17 mice on days 0 and 14 after tumor injection. (C) The frequency of Foxp3+ CD4+ of WT mice. (B) Representative flowcytometric images showing the frequency of Foxp3+ CD4+ cells in the draining lymph cells was significantly increased in lymph nodes and spleen on day 14. The frequency of Foxp3+ CD25+ cells was signifi- nodes from WT mice and CCL17 mice on days 0 and 14 after tumor injection. (C) The frequency of Foxp3+ CD4+ cells cantly upregulated in lymph nodes on day 14. Data are presented as mean ± SEM of three independent experiments (n = + + was6 for significantly each group). increased * p < 0.05 in WT lymph versus nodes CCL17 and TG spleen mice.on day 14. The frequency of Foxp3 CD25 cells was significantly upregulated in lymph nodes on day 14. Data are presented as mean ± SEM of three independent experiments (n = 6 for each group). * p < 0.05 WT versus2.4. Decreased CCL17 TG Myeloid-Derived mice. Suppressor Cells in CCL17 TG Mice Next, we performed flowcytometric analysis of skin tumors. MDSCs have the capac- 2.4.ity Decreasedto suppress Myeloid-Derived the adaptive immune Suppressor response Cells inmediated CCL17TG by MiceT cells, promoting a tumor- friendly microenvironment. MDSCs are usually defined in mouse models as myeloid cells expressingNext, we high performed levels of flowcytometricCD11b and Gr-1. analysis Therefore, of skin we tumors.focused on MDSCs CCR4 have+, which the is capacity ex- topressed suppress by Tregs the adaptive and Th2 cells, immune and CD11b response+Gr-1 mediated+ population. by As T cells,expected, promoting the frequency a tumor- friendlyof CCR4 microenvironment.+ cells was increased MDSCs in the skin are tumors usually of defined CCL17 in TG mouse mice (Figure models 4A,B). as myeloid On the cells + expressingother hand, high the frequency levels of CD11bof CD11b and+Gr-1 Gr-1.+ cells Therefore, was significantly we focused decreased on CCR4 in CCL17, which TG is expressed by Tregs and Th2 cells, and CD11b+Gr-1+ population. As expected, the frequency of CCR4+ cells was increased in the skin tumors of CCL17 TG mice (Figure4A,B). On the other hand, the frequency of CD11b+Gr-1+ cells was significantly decreased in CCL17 TG

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mice.mice. ThereThere waswas aa negativenegative correlationcorrelation betweenbetween thethe frequencyfrequency ofof CCR4 CCR4++ cellscells andand thatthat ofof CD11bCD11b++Gr-1+ cellscells (Figure (Figure 44C).C).

Figure 4. Decreased Myeloid-derived suppressor cells (MDSCs) in CCL17 transgenic (TG) mice. MDSCs are defined as CD11b+Gr-1+ in mice. CCR4 is expressed by Tregs and Th2 cells. Expression levels of CCR2, CCL5, and CXCL17, which are allFigure related 4. toDecreased MDSCs, andMyeloid-derivedIL-4, which is suppressor produced by cells Th2 (MDSCs) cells, were in evaluated CCL17 transgenic by quantitative (TG) mice. reverse MDSCs transcription-PCR are defined inas skinCD11b tumors+Gr-1 from+ in mice. wild-type CCR4 (WT) is expressed and CCL17 by Tregs TG mice. and (Th2A) Representative cells. Expression flowcytometric levels of CCR2 images, CCL5 showing, and CXCL17 the frequency, which are of CCR4all related+ cells, to Gr-1MDSCs,+ cells and−, andIL-4 CD11b, which+ iscells. produced (B) The by frequency Th2 cells, of were CCR4 evalua+ cellsted was by significantlyquantitative higherreverse in transcription-PCR CCL17 TG mice, whilein skin the tumors frequency from of wild-type CD11b+Gr-1 (WT)+ cells and was CCL17 significantly TG mice. lower (A) comparedRepresentative with thatflowcytometr of WT mice,ic respectively.images showing (C) There the fre- is quency of CCR4+ cells, Gr-1+ cells-, and CD11b+ cells. (B) The frequency of CCR4+ cells was significantly higher in CCL17 a negative correlation between the frequency of CCR4+ cells and that of CD11b+Gr-1+ cells. (D) Expression levels of CCR2, TG mice, while the frequency of CD11b+Gr-1+ cells was significantly lower compared with that of WT mice, respectively. CCL5, and CXCL17 mRNA were significantly downregulated in CCL17 TG mice, while IL-4 mRNA levels were significantly (C) There is a negative correlation between the frequency of CCR4+ cells and that of CD11b+Gr-1+ cells. (D) Expression upregulatedlevels of CCR2 in, CCL17 CCL5, TGand mice CXCL17 compared mRNA with were those significantly in WT mice. downregulated * p < 0.05 WT in versus CCL17 CCL17 TG mice, TG while mice. IL-4 mRNA levels were significantly upregulated in CCL17 TG mice compared with those in WT mice. * p < 0.05 WT versus CCL17 TG mice. Messenger RNA expression levels of MDSC-related markers in skin tumors were eval- CXCL17 CCR2 CCL5 uated.Messenger, RNA, andexpressionmRNA levels levelsof MDSC were-related significantly markers decreased in skin in tumors skin tumors were of CCL17 TG mice compared with WT mice (Figure4D). IL-4 mRNA was increased in evaluated. CXCL17, CCR2, and CCL5 mRNA levels were significantly decreased in skin CCL17 TG mice, as had been expected. Thus, MDSCs were decreased in the tumor microen- tumors of CCL17 TG mice compared with WT mice (Figure 4D). IL-4 mRNA was in- vironment in CCL17 TG mice, which might function as a negative regulator preventing creased in CCL17 TG mice, as had been expected. Thus, MDSCs were decreased in the severe tumor immune dysfunction. tumor microenvironment in CCL17 TG mice, which might function as a negative regulator 2.5.preventing Enhanced severe Anti-Tumor tumor Immunityimmune dysfunction. after Depletion of Regulatory T Cells in CCL17 TG Mice Tregs promote tumor cell proliferation in the tumor microenvironment. We next 2.5. Enhanced Anti-Tumor Immunity after Depletion of Regulatory T Cells in CCL17 TG Mice determined if depletion of Tregs could change tumor formation in CCL17 TG mice. After peritonealTregs injectionpromote oftumor the anti-CD25cell proliferation antibody in the or tumor phosphate-buffered microenvironment. saline We (PBS), next wede- termined if depletion of Tregs could change tumor formation in CCL17 TG mice. After

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Int. J. Mol. Sci. 2021, 22, 2025 7 of 14 peritoneal injection of the anti-CD25 antibody or phosphate-buffered saline (PBS), we evaluated tumor formation 14 days after subcutaneous injection of B16 melanoma cells (Figureevaluated 5A,B). tumor As expected, formation CCL17 14 days TG after mice subcutaneous injected with injection PBS showed of B16 melanoma larger tumors cells than WT(Figure mice 5injectedA,B). As with expected, PBS. CCL17Depletion TG mice of Treg injecteds significantly with PBS showed decreased larger tumor tumors size than both in CCL17WT miceTG mice injected and with WT PBS. mice Depletion (Figure of 5C). Tregs Interestingly, signiﬁcantly decreasedtumor formation tumor size in both CCL17 in TG miceCCL17 was markedly TG mice and reduced WT mice compared (Figure5 C).with Interestingly, WT mice after tumor depletion formation of inTregs CCL17 (Figure TG 5C). mice was markedly reduced compared with WT mice after depletion of Tregs (Figure5C). This is probably because decreases in MDSCs in CCL17 TG mice leads to enhanced tumor This is probably because decreases in MDSCs in CCL17 TG mice leads to enhanced tumor immunityimmunity in inthe the absence absence of of Tregs. Tregs.

Figure 5. Tumor immunity was markedly enhanced by depletion of regulatory T cells (Tregs) in CCL17 transgenic (TG) mice. (A) Phenotypical manifestation of back skin from wild-type (WT) and CCL17 TG mice treated with anti-CD25 Ab or Figurephosphate-buffered 5. Tumor immunity saline was (PBS) markedly peritoneally enhanced 14 days by after depletion injection of of regulatory B16F1 cells subcutaneously.T cells (Tregs) in (B )CCL17 Schema transgenic describing (TG) mice. strategy(A) Phenotypical for Tregs depletion manifestation with anti-CD25 of back skin Ab. from Anti-CD25 wild-typ Abe was (WT) injected and CCL17 peritoneally TG mice two daystreated before with subcutaneous anti-CD25 Ab or phosphate-bufferedinoculation of B16F1 saline cells (PBS) and peritoneally tumor sizes 14 were days evaluated after injection on days of 3, B16F1 7, 10, cells and 14.subcutaneously. (C) Tumor size (B was) Schema dramatically describing strategydecreased for Tregs in CCL17 depletion TG mice with treated anti-CD25 with anti-CD25 Ab. Anti-CD25 Ab compared Ab was with injected that in WTperitoneally treated with two anti-CD25 days before Ab and subcutaneous that in inoculationCCL17 of TG B16F1 micetreated cells and with tumor PBS. * sizesp < 0.05, we **rep evaluated< 0.01. on days 3, 7, 10, and 14. (C) Tumor size was dramatically decreased in CCL17 TG mice treated with anti-CD25 Ab compared with that in WT treated with anti-CD25 Ab and that in CCL17 TG mice treated with PBS.2.6. * Decrease p < 0.05, in** CXCL17,p < 0.01. a Chemoattractant of MDSCs, in Th2-Dominant Situation To identify the mechanism by which MDSCs were decreased in CCL17 TG mice, we 2.6. Decrease in CXCL17, a Chemoattractant of MDSCs, in Th2-Dominant Situation focused on CXCL17, which induces chemotaxis of MDSCs. IL-4, which was increased in CCL17To identify TG mice, the signiﬁcantly mechanism decreased by whichCXCL17 MDSCsmRNA were decreased expression in by CCL17 normal TG human mice, we focusedepidermal on CXCL17, keratinocytes which (NHEK; induces Figure chemotaxis6A). Serum of levels MDSCs. of CXCL17 IL-4, inwhich AD patients was increased were in CCL17signiﬁcantly TG mice, decreased significantly compared decreased with normal CXCL17 controls mRNA (Figure expression6B). Thus, by CXCL17 normal was human epidermaldecreased keratinocytes in Th2-dominant (NHEK; situation, Figure which 6A). may Serum account levels for of decreased CXCL17 MDSCs in AD inpatients CCL17 were significantlyTG mice. decreased compared with normal controls (Figure 6B). Thus, CXCL17 was decreased in Th2-dominant situation, which may account for decreased MDSCs in CCL17 TG mice.

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FigureFigure 6. 6.Downregulation Downregulation of of CXCL17 CXCL17 production production in in IL-4-stimulated IL-4-stimulated normal normal human human epidermal epidermal keratinocytes keratinocytes (NHEK) (NHEK) and and decreaseddecreased CXCL17 CXCL17 expression expression in patientsin patients with with atopic atopic dermatitis dermatitis (AD). (AD). Expression Expression of CXCL17 of CXCL17mRNA mRNA was assessed was assessed in NHEK in cellsNHEK treated cells with treated IL-4 with stimulation. IL-4 stimulation. CXCL17 expressionCXCL17 expression levels were levels evaluated were evaluated in sera of in patients sera of with patients AD. (withA) CXCL17 AD. (A) mRNACXCL17 expression mRNA expression by NHEK wasby NHEK signiﬁcantly was significantly attenuated by attenuated IL-4 stimulation. by IL-4 (Bstimulation.) Serum levels (B) ofSerum CXCL17 levels in patientsof CXCL17 with in ADpatients were signiﬁcantlywith AD were decreased significantly compared decreased with compared normal controls. with normal * p < 0.05. controls. *p < 0.05.

3. Discussion 3. Discussion In the present study, we utilized CCL17 TG mice as a relevant in vivo model for AD to exploreIn the the present mechanism study, weof tumor utilized immunity. CCL17 TGCCL17 mice TG as mice a relevant recapitulatedin vivo keymodel features for ADof AD, to explore demonstrating the mechanism increased of production tumor immunity. and secretion CCL17 of TG CCL17 mice [22]. recapitulated CCL17 overex- key featurespression of enhanced AD, demonstrating skin tumor increased formation production and lung metastasis and secretion with of concomitant CCL17 [22]. increases CCL17 overexpression enhanced skin tumor formation and lung metastasis with concomitant in Tregs in the skin, lymph nodes, and spleen. The pathophysiology of primary tumor increases in Tregs in the skin, lymph nodes, and spleen. The pathophysiology of primary formation and metastasis formation differs. The latter requires multiples steps, such as tumor formation and metastasis formation differs. The latter requires multiples steps, such survival in the blood or lymphatic stream, arrest at distant organ sites, extravasation into as survival in the blood or lymphatic stream, arrest at distant organ sites, extravasation into the tissues, and proliferation in the foreign microenvironment. Although direct injection the tissues, and proliferation in the foreign microenvironment. Although direct injection of the tumor cell line may not reflect true situations in metastasis, it is still interesting to of the tumor cell line may not reflect true situations in metastasis, it is still interesting to find that tumor formation is enhanced even in the lung in CCL17 TG mice. The effects of find that tumor formation is enhanced even in the lung in CCL17 TG mice. The effects of CCL17 should be systemic, not only in the skin, because serum CCL17 levels were ex- CCL17 should be systemic, not only in the skin, because serum CCL17 levels were extremely tremely high [22]. MDSCs were significantly decreased in CCL17 TG mice and tremen- high [22]. MDSCs were significantly decreased in CCL17 TG mice and tremendous recovery dous recovery of tumor immunity was upregulated by deletion of Tregs in CCL17 TG of tumor immunity was upregulated by deletion of Tregs in CCL17 TG mice, indicating thatmice, tumor indicating immunity that wastumor accelerated immunity by was the a decreaseccelerated in by MDSCs the decrease in the absence in MDSCs of Tregs. in the CCL17absence has of atTregs. least CCL17 two distinct has at mechanisms least two distinct to regulate mechanisms tumor development.to regulate tumor In one develop- way, CCL17ment. increasesIn one way, both CCL17 Tregs and increases Th2 cells, both upregulating Tregs and tumorTh2 cells, formation. upregulating At the sametumor time, for- CCL17mation. attenuates At the same MDSCs time, via CCL17 a pathway attenuates including MDSCs IL-4 via and a pathway CXCL17, including downregulating IL-4 and tumorCXCL17, formation downregulating (Figure7). tumor formation (Figure 7).

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Figure 7. Figure 7. SchemaSchema describing describing relationship relationship among among regulatory regulatory T T ce cellslls (Tregs),(Tregs), Th2 Th2 cells, cells, and and myeloid-derived myeloid-derived suppressor suppressor cells + cells (MDSCs)(MDSCs) in inatopic atopic dermatitis dermatitis (AD).(AD). CCL17 CCL17 is overexpressedis overexpressed in AD in and AD recruits and recruits CCR4 cellsCCR4 such+ cells as Tregs such and as Th2Tregs cells and into Th2 cells intothe the tumor tumor microenvironment. microenvironment. Tregs, Tr producingegs, producing IL-10 and IL-10 Foxp3, and attenuateFoxp3, attenuate tumor immunity tumor immunity and increase and tumor increase formation. tumor formation.IL-4, IL-4, which which is produced is produced by Th2 by cells, Th2 downregulatescells, downregula CXCL17tes CXCL17 expression. expression. Decreases Decreases in CXCL17 in lead CXCL17 to reduced lead frequency to reduced frequencyof MDSCs,of MDSCs, which which results results in enhanced in enhanced tumor tumor immunity. immunity. Tumor Tumor formation formation is determined is determined by the balance by the between balance positive between positive regulationregulation of of Tregs Tregs and and negative negative regulation regulation of MDSCs. of MDSCs. We demonstrated that Th2-type contact hypersensitivity was enhanced and Th1 cells Wewere demonstrated suppressed in that CCL17 Th2-type TG mice contact [22]. The hypersensitivity frequencies of CCR4was enhanced+ cells and and mast Th1 cells cells were weresuppressed high in in CCL17 CCL17 TG TG mice mice with [22]. increased The frequenciesIL-4 expression of CCR4 levels.+ cells CCL17 and modified mast cells were contacthigh in hypersensitivityCCL17 TG mice by with attracting increased CCR4 IL-4+ cells expression into the skinlevels. lesions CCL17 and modified generating contact hypersensitivitya Th2-dominant environment.by attracting InCCR4 the present+ cells into study, the the skin frequency lesions ofand CCR4 generating+ cells in a the Th2- dominantskin tumorenvironment. was increased In the inpresent CCL17 study, TG mice the as frequency had been of expected. CCR4+ cells Interestingly, in the skin the tu- + mor wasfrequency increased of MDSCs in CCL17 was decreasedTG mice andas had there been was expected. a negative Interestingly, correlation between the frequency CCR4 cells and MDSCs. Since CCR4 is expressed by Tregs and Th2 cells, we hypothesized that of MDSCs was decreased and there was a negative correlation between CCR4+ cells and Tregs and/or Th2 cells could negatively regulate MDSCs directly or indirectly. To gain MDSCs. Since CCR4 is expressed by Tregs and Th2 cells, we hypothesized that Tregs mechanistic insights, we next focus on CXCL17, a chemoattractant of MDSCs. and/or Th2CXCL17 cells could is a chemokine negatively known regulate to chemoattract MDSCs directly MDSCs, or macrophages, indirectly. To and gain dendritic mecha- nisticcells. insights, CXCL17 we isnext strongly focus expressed on CXCL17, by epithelial a chemoattractant cells and vascular of MDSCs. endothelial cells. There CXCL17has been increasingis a chemokine evidence known that CXCL17 to chemoattract enhances tumorMDSCs, formation macrophages, in several and carcinomas dendritic cells. suchCXCL17 as lung is strongly metastasis expressed [24], hepatocellular by epithelial carcinoma cells and [ 25vascular], and colon endothelial cancer [cells.26]. Our There has beenstudy increasing showed that evidenceCXCL17 thatexpression CXCL17 was enhances decreased tumor in the formation skin tumor in several of CCL17 carcino- TG mas suchmice, as while lungIL-4 metastasisexpression [24], was increased.hepatocellular We next carcinoma investigated [25], human and colon samples cancer to gain [26]. Our studyfurther showed insights. that In CXCL17 sera of AD expression patients, was CXCL17 decreased levels werein the significantly skin tumor lowerof CCL17 com- TG pared with healthy controls. CXCL17 expression levels were significantly attenuated in mice, while IL-4 expression was increased. We next investigated human samples to gain NHEK stimulated with IL-4. These results suggest that low levels of CXCL17 results in furtherdecreased insights. chemotaxis In sera of ofAD MDSCs, patients, eventually CXCL17 leading levels to were promotion significantly of tumor lower immunity. compared In with healthy controls. CXCL17 expression levels were significantly attenuated in NHEK stimulated with IL-4. These results suggest that low levels of CXCL17 results in decreased chemotaxis of MDSCs, eventually leading to promotion of tumor immunity. In Th2-dominant environment, IL-4 production was upregulated by Th2 cells and it may act as a key cytokine to reduce CXCL17 expression, as mentioned previously. In physiological conditions, MDSCs induce chemotaxis of Tregs. In this study, CCL17, a chemoattractant of

Int. J. Mol. Sci. 2021, 22, 2025 10 of 14

Th2-dominant environment, IL-4 production was upregulated by Th2 cells and it may act as a key cytokine to reduce CXCL17 expression, as mentioned previously. In physiological conditions, MDSCs induce chemotaxis of Tregs. In this study, CCL17, a chemoattractant of Tregs, was genetically overexpressed, making it difficult to document direct association between Tregs and MDSCs. Another shortcoming of our study was that we provided only data using primary keratinocytes in vitro. There are other multiple players in the immuno- logical state of the skin, such as dendritic cells, fibroblasts, endothelial cells, resident T cells, and macrophages. Further studies using other cell types would be necessary to elucidate the role of CXCL17 in tumor immunity in the skin. Several additional publications have suggested an inverse relationship between tumors and AD. The risk of gastric cancer was reduced in patients with AD, especially in males [27]. AD was shown to be associated with a reduced risk of pancreatic cancer in a meta-analysis study [28]. However, none of the articles demonstrated the mechanism by which AD reduced incidence of cancer. Our study indicates that decreases in MD- SCs, induced by reductions in CXCL17 as a consequence of CCL17 levels, led to tumor suppression. In the present study, tumor formation was enhanced in CCL17 TG mice. This is probably because the positive impact on tumor formation induced by increased Tregs was much larger than the negative impact by decreased MDSCs in our mouse model. In AD patients, the balance between the positive and negative impacts of tumor formation may be changeable in various circumstances, which may lead to a normal, higher, or lower incidence of malignant tumors, dependent on different types of cancers. IL-4, a representative Th2 cytokine, is also important for maturation of dendritic cells. Mice inoculated with IL-4-producing tumor cells showed rejection and a long-lasting anti-tumor immunity [29]. Therefore, IL-4 itself may have a negative impact on tumor development. Moreover, not only Th2 cytokines, Th17 cytokines and Th1 cytokines are expressed in AD lesional skin [30]. These cytokines may also prevent tumor development. The treatment of AD has dramatically changed in recent years. Dupilumab, a human monoclonal antibody against IL-4 receptor alpha, blocks signaling of IL-4 and IL-13, improving severe dermatitis in AD patients [31]. This drug is one of the most effective treatments for AD. In the present experiments, we showed that increased production of IL-4 induced a decrease in CXCL17, leading to suppression of tumor formation by blocking the recruitment of MDSCs into the tumor microenvironment. This is probably a protective immune reaction to prevent excessive tumor development. From this aspect, it is important for us to pay attention to the incidence of malignant tumors in AD patients treated with dupilumab. CTCL shares many clinical characteristics with AD, such as a skin rash with severe pruritus and increased serum CCL17, LDH, and IgE levels, making it difficult to distinguish between these diseases [32]. Moreover, some CTCL cases have a history of AD. There have been several papers reporting progression of CTCL after treatment with dupilumab [33,34]. Together with our results, it should be remembered that blocking IL-4/13 signaling may be disadvantageous in some types of malignancy. In conclusion, we have shown that CCL17 attenuates tumor immunity by increasing the levels of Tregs and Th2 cells, while it decreases MDSCs through CXCL17 reductions. This may be one of the reasons why the incidence of cancers in AD is not increased, working as a “safety-net” to reduce the risk of malignant tumors in the Th2-dominant environment.

4. Materials and Methods 4.1. Mice CCL17 TG mice were previously generated in our department [19]. Brieﬂy, CCL17 TG mice show increased production and secretion of the CCL17 protein with biological and functional activity in keratinocytes. All mice were healthy, fertile, and free of signs of infection or disease. Mice used for experiments were 9–12 weeks old. Age-matched wild-type littermates and C57/BL/6 mice were used as controls for CCL17 TG mice. All mice were housed in a pathogen-free barrier facility and they were healthy, fertile, and did Int. J. Mol. Sci. 2021, 22, 2025 11 of 14

not display evidence of infection or disease. All studies and procedures were approved by the Committee an Animal Experimentation of the University of Tokyo.

4.2. B16 Melanoma Cells and EL4 Lymphoma Cells B16 F1 murine melanoma cells and EL4 lymphoma cells were kindly donated by Dr. Sam T Hwang (University of California Davis School of Medicine, Sacramento, CA, USA). They were cultured in MEM medium (Invitrogen, Carsbad, CA, USA) supplemented with 10% heat-inactivated FCS (Harlan)/2 mM (Gemini Bio Products, West Sacramento, CA, USA), L-glutamine /100 units/mL (Wako, Osaka, Japan), penicillin/100 g/mL and streptomycin/0.05 M (Sigma-Aldrich, St Louis, MO, USA), 2-mercaptoethanol (Invitrogen, ◦ Carsbad, CA, USA) at 37 C in 5% CO2. Cells were passaged twice a week with trypsin (Sigma-Aldrich, St Louis, MO, USA).

4.3. Primary Cutaneous Tumor Growth B16 F1 cells (5 × 106) in 100 µL of PBS were injected subcutaneously into the shaved lateral ﬂank of anaesthetized mice. The tumor size was calculated using the equation: L1 × L2, where L1 = longest diameter (mm) and L2 = shortest diameter (mm). The size of primary tumors was measured on days 3 or 4, 7, 10, and 14.

4.4. Lung Metastasis B16 F1 cells (5 × 106) in 100 µL of PBS were injected intravenously into the tail vein. The mice were sacriﬁced on day 21 after injection, and lungs were removed. At the time-point it was not possible to count accurately the number of surface metastatic colonies using a stereomicroscope (Nikon, Tokyo, Japan). because of the small size of the colonies. Therefore, to evaluate lung metastasis, we counted histologically the number of colonies in each section of the three lobes of the right lung. The sections were stained using hematoxylin and eosin (H&E), as described below. When >60% of the section was occupied with tumors, the colony number was deﬁned as 1000 [20]. Each section was examined independently by three investigators in a blinded fashion, and the mean of the results was used for analysis.

4.5. RNA Isolation and Quantitative Reverse Transcription-PCR Skin tumor tissues and lymph nodes were harvested on day 14 after subcutaneous injection of B16F1 melanoma cells. Lung tissues were harvested on day 21 after intravenous injection of B16F1 melanoma cells. RNA was obtained from skin tumor tissues, lymph nodes, and lung tissues with RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA, USA). Complementary DNA was synthesized according to the Taqman Gene Expression Protocol (Applied Biosystems, Foster City, CA, USA). Expression mRNA levels of IL- 10, Foxp3, CCR2, CCL5, CXCL17, and IL-4 were analyzed using quantitative reverse transcription-PCR method with THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) on an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, CA, USA). The mRNA levels were normalized to those of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The relative change in the levels of genes of interest was determined by the 2−∆∆CT. Primers used for GAPDH, IL-10, Foxp3, CCR2, CCL5, CXCL17, and IL-4 were as follows: GAPDH forward, 50-CGT GTT CCT ACC CCC AAT GT-30 and reverse, 50-TGT CAT ACT TGG CAG GTT TCT-30; IL-10 forward, 50-TTT GAA TTC CCT GGG TGA GAA-30 and reverse, 50-ACA GGG GAG AAA TCG ATG ACA -30; Foxp3 forward, 50-TAC TTC AAG TTC CAC AAC ATG CGA CC -30 and reverse, 50-CGC ACA AAG CAC TTG TGC AGA CTC AG -30; CCR2 forward, 50-GTT ACC TCA GTT CAT CCA-30 and reverse, 50-CAA GGC TCA CCA TCA TCG TAG TC-30; CCL5 forward, 50-CAT ATG GCT CGG ACA CCA-30 and reverse, 50-ACA CAC TTG GCG GTT CCT-30; CXCL17 forward, 50-TGT TGC TTC CAG TGA TGC TC -30 and reverse, 50-GCT GTG GCT TTT CTC TTT GG -30; human CXCL17 forward, 50-ACC GAG GCC AGG CTT CTA-30 and reverse, Int. J. Mol. Sci. 2021, 22, 2025 12 of 14

50-GGC TCT CAG GAA CCA ATC TTT-30, and IL-4 forward, 50-ACG GAG ATG GAT GTG CCA AAC GTC-30 and reverse, 50-CGA GTA ATC CAT TTG CAT GAT GC-30.

4.6. Histologic Examination Skin tumor tissues and lymph nodes were harvested on day 14 after subcutaneous injection of B16F1 melanoma cells. Lung tissues were harvested on day 21 after intravenous injection of B16F1 melanoma cells. They were formalin (Sigma-Aldrich, St Louis, MO, USA)-ﬁxed and stained with H&E (Sigma-Aldrich, St Louis, MO, USA).

4.7. Flow Cytometric Analysis of Lymph Nodes Draining lymph nodes of the lesional skin (axillary and inguinal lymph nodes) and spleen from WT mice and CCL17 TG mice were harvested on day 14 after subcutaneous injection of B16F1 melanoma cells. Lymph nodes were stained with anti-mouse/rat Foxp3 staining set (71-5775; eBioscience, San Diego, CA, USA), PerCP-Cy TM 5.5-conjugated CD4 (550954; BD Pharmingen, San Diego, CA, USA), PerCP-Cy TM 5.5-conjugated CD25 (551071; BD Pharmingen), PE-conjugated CCR4 (131204; BioLegend, San Diego, CA, USA), FITC- conjugated Gr-1 (108405; BioLegend), and anti-CD11b (101211; BioLegend). Background staining was assessed using non-reactive isotype-matched control monoclonal antibodies (Biolegend). Cells were analyzed on a FACS Verse ﬂow cytometer (BD Biosciences, San Jose, CA, USA).

4.8. CXCL17 Expression Analysis of ELISA and In Vitro Cell Culture Immunoreactive CXCL17 in the serum of patients with AD and healthy controls was quantified by human ELISA kits (MyBioSource, Inc., San Diego, CA, USA). Optical den- sities were measured at 450 nm, with the correction wavelength set at 570 nm, using a Bio-Rad Model 550 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The con- centrations were calculated from the standard curve generated by a curve-fitting program according to the manufactures’ instructions. NHEK were cultured in 75 cm2 cell culture flasks (CORNING, Corning, NY, USA) at ◦ 37 C, 5% CO2 in Humedia-KB2 (Kurabo Industries, Osaka, Japan) supplemented with Human Keratinocyte Growth Supplement sets Kurabo Industries, Osaka, Japan). When confluence was achieved, the cells were trypsinized, washed, and resuspended in the medium at 5 × 105 cells/mL, and 2 mL was added to each well of the 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA). When the cells reached semi-confluence, the medium was completely removed and 2 mL of growth supplement-free medium was added to each well. Simultaneously, recombinant human IL-4 (R&D Systems, Minneapolis, ◦ MN, USA) was added, and the cells were incubated at 37 C and 5% CO2. The concentration of IL-4 was 0.1, 1, 10 ng/mL. After 24 h, cells were processed by TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) for isolation of total RNA, according to the manufacturers’ instructions.

4.9. Statistical Analysis Data obtained are presented as mean ± SEM. Statistical analysis was carried out with one-way ANOVA with Bonferroni post hoc tests for multiple group comparisons and the two-tailed unpaired t-test for two group comparisons, using Prism Version 7 software [35] (GraphPad, San Diego, CA, USA). For comparing two group values that did not follow Gaussian distribution, the two-tailed Mann–Whitney u test was used. Values of p < 0.05 were considered to represent a signiﬁcant difference.

Author Contributions: Conceptualization, S.M., Y.T. and M.S.; data curation, S.M., and T.O.; formal analysis, S.M.; funding acquisition, M.S.,Y.T., and S.S.; investigation, S.M.; methodology, S.M., T.O., and Y.T.; project administration, M.S.; resources, S.M., M.S., T.O., H.S., T.M., and Y.T.; supervision, T.M., M.S., and S.S.; validation, S.M.; visualization, S.M.; writing—original draft preparation, S.M.; writing—review and editing, M.S., T.O., H.S., T.M., Y.T., Y.A., and S.S. All authors have read and agreed to the published version of the manuscript. Int. J. Mol. Sci. 2021, 22, 2025 13 of 14

Funding: This research was partially funded by JSPS KAKENHI Grant (M.S. and Y.T.). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Committee on Animal Experimentation of the University of Tokyo (H07-116) and the medical ethical committee of the University of Tokyo (0695-18). Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to involvement of clinical samples. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

AD atopic dermatitis CTCL cutaneous T-cell lymphoma Tregs regulatory T cells TG transgenic MDSC myeloid-derived suppressor cell WT wild-type PBS phosphate-buffered saline NHEK normal human epidermal keratinocytes GAPDH glyceraldehyde 3-phosphate dehydrogenase

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