Activation of CD8 T cells accelerates anti-PD-1 antibody-induced psoriasis-like dermatitis through IL-6

Use of immune checkpoint inhibitors that target programmed cell death-1 (PD-1) can lead to various autoimmune-related adverse events (irAEs) including psoriasis-like dermatitis. Our observations on human samples indicated enhanced epidermal infiltration of CD8 T cells, and the pathogenesis of which appears to be dependent on IL-6 in the PD-1 signal blockade-induced psoriasis-like dermatitis. By using a murine model of imiquimod-induced psoriasis-like dermatitis, we further demonstrated that PD-1 deficiency accelerates skin inflammation with activated cytotoxic CD8 T cells into the epidermis, which engage in pathogenic cross-talk with keratinocytes resulting in production of IL-6. Moreover, genetically modified mice lacking PD-1 expression only on CD8 T cells developed accelerated dermatitis, moreover, blockade of IL-6 signaling by anti-IL-6 receptor antibody could ameliorate the dermatitis. Collectively, PD-1 signal blockade-induced psoriasis-like dermatitis is mediated by PD-1 signaling on CD8 T cells, and furthermore, IL-6 is likely to be a therapeutic target for the dermatitis. Tanaka et al investigate the mechanism by which psoriasis-like dermatitis may occur following PD-1 antibody treatment for melanoma. They find that PD-1 loss in CD8 T-cells accelerates dermatitis in a manner depending on IL-6 signaling, suggesting IL-6 as a potential therapeutic target.

F or cancer immune therapies that regulate T cells to enhance immune responses, T cells must successfully recognize tumor antigens through their T-cell receptors (TCRs) and become activated in order to expel tumors 1,2 . In addition, a number of stimulatory and inhibitory receptor and ligand pairs expressed on T cells, antigen-presenting cells (APCs) or tumor cells, termed immune checkpoints, also play crucial roles for both T cell activation and inhibition 3 . Programmed cell death-1 (PD-1) is one of these immune checkpoint molecules, which was initially detected in activated murine T cells upon TCR engagement 4 and subsequently in exhausted T cells 5 . Its ligands, programmed cell death-ligand 1 (PD-L1) and PD-L2, are expressed on various cell types, including hematopoietic cells infiltrating tumors, including APCs, and on non-hematopoietic cells such as cancer cells 6,7 . The interaction between PD-1 and its ligands reduces T cell function by inducing exhaustion, apoptosis, anergy, and downregulation of cytokine production by T cells, leading to suppression of the antitumor immune response 8,9 . In melanoma, PD-1 expression is detected on tumorinfiltrating lymphocytes including tumor antigen-specific T cells, which are functionally impaired. Moreover, the biological activity of these cells can be partially recovered by inhibiting the PD-1 pathway [10][11][12] . Indeed, anti-PD-1 blocking antibodies such as nivolumab and pembrolizumab function as immune checkpoint inhibitors, and have proven effective for the treatment of melanoma 13,14 . However, as the PD-1 pathway also maintains peripheral T cell tolerance and regulates inflammation 15 , inhibition of this pathway may lead to autoimmune manifestations referred to as immune-related adverse events (irAEs) 16,17 . Early clinical trials and reviews have reported that anti-PD-1 antibodyrelated irAEs occur in more than 70% of patients, and cutaneous irAEs are the most frequently observed (approximately 40%). Further, most cutaneous irAEs are mild (low-grade) and manageable with topical steroids 16,[18][19][20][21] . On the other hand, it has also been recently reported that two-thirds of patients with cutaneous irAEs reportedly required systemic corticosteroids for the treatment of eruptions, and 19% of patients discontinued cancer-immunotherapy due to irAEs, even though 75% experienced antitumor responses with the therapy 22 . High-dose and/or long-term use of systemic immunosuppressive therapies are required to control such irAEs 23 , potentially resulting in prolonged interruption of cancer treatment. Moreover, these immunosuppressive therapies may also abrogate the antitumor response by counteracting lymphocyte activation 20,24 . Therefore, more efficacious, systemic therapies that resolve the symptoms of irAEs while also enabling shorter interruptions of cancer treatments and do not interfere with their antitumor effects would be ideal. In addition, a recent American Society of Clinical Oncology guideline suggests that cutaneous irAEs are increasingly recognized as a contributing factor to treatment noncompliance, discontinuation, or dose modification 24 . Plausibly, such skin manifestations cause changes in appearance along with discomfort, which reduces patient quality of life and results in loss of treatment motivation. We previously reported a case of nivolumab-induced psoriasis-like dermatitis 25 , which has been reported to develop in patients treated with anti-PD-1/PD-L1 antibody 25,26 . The latest post-marketing surveillance of nivolumab in Japan reports that 2,391 cases of cutaneous irAE occurred, of which 103 cases (4.3 %) were labeled as psoriasis. Notably, more than 18% (19 /103) of those cases were reportedly severe 27 . Importantly, the mechanism by which psoriasis-like dermatitis occurs following PD-1/PD-L1 inhibition remains unknown, and strategies to mitigate the occurrence of especially severe cases are yet to be identified. With the recent increase in use of anti-PD-1 antibody for patients with various types of cancers, clarification of the underlying mechanisms and development of more efficacious treatment for PD-1 signal blockade-induced psoriasis-like dermatitis is needed.
Application of imiquimod (IMQ), a toll-like receptor 7/8 agonist, is known to induce psoriasis-like dermatitis in both humans 28 and mice 29 . Furthermore, it has already been reported that both PD-1 genetic deficiency and blockade of PD-1 with a specific monoclonal antibody exacerbate IMQ-induced psoriasislike dermatitis in mice 30 . Therefore, it is likely that the pathophysiological mechanism of PD-1 signal blockade-induced psoriasis-like dermatitis could be elucidated using this murine model.
The present study aimed to elucidate the characteristics and mechanisms underlying psoriasis-like dermatitis induced by blocking PD-1 signaling, and to identify suitable treatments. The observations from human samples and further experiments using a preclinical murine model of IMQ-induced psoriasis-like dermatitis demonstrated that the dermatitis was accelerated by an increase of skin-infiltrating activated, cytotoxic CD8 T cells allowing pathogenic crosstalk with keratinocytes and subsequent production of IL-6. Moreover, blockade of interleukin (IL)-6 signaling by anti-IL-6 receptor blocking antibody (MR16-1) restrained the PD-1 signal blockade provoked by severe dermatitis by inhibiting both Th17 cell differentiation and cytotoxic CD8 T cell activation. Thus, this highlights the significance of IL-6 blockade therapy specifically for the regulation of PD-1 signal blockade-induced dermatitis.

Results
Increased CD8/CD4 ratio of epidermal-infiltrating lymphocytes in cases of anti-PD-1 antibody-induced psoriasis-like dermatitis compared to cases of idiopathic psoriasis. Immunohistochemical (IHC) evaluation of skin biopsy samples, as demonstrated in Fig. 1a, revealed that CD8/CD4 ratios of epidermal-infiltrating mononuclear cells were significantly increased in cases of anti-PD-1 antibody-induced psoriasis-like dermatitis (median ± standard deviation [SD], 3.48 ± 1.0) compared to that in cases of idiopathic psoriasis (1.06 ± 0.19, P = 0.008 by Mann-Whiney U test, Fig. 1b).
Elevated serum IL-6 correlates with the development of anti-PD-1 antibody-induced psoriasis-like dermatitis in humans. We reported in our preliminary study that only increased serum levels of IL-6, but not those of IL-17A, interferon (IFN)γ and IL-8, correlated with the development of anti-PD-1 antibody-induced psoriasis-like dermatitis in patients with malignant melanoma 25 . In order to validate this phenomenon, we analyzed the serum levels of IL-6 in eight cases of psoriasislike dermatitis, and 19 cases without any irAEs. Cases of psoriasis-like dermatitis exhibited significantly higher serum IL-6 levels compared to those of IL-6 in cases without any irAEs (P < 0.0001 by Mann-Whiney U test, Fig. 1c). Our additional analysis using the remaining samples showed that there was no significant difference in the serum levels of soluble IL-6 receptor alfa (sIL-6Rα) between the two groups, six cases of psoriasis-like dermatitis and 18 cases without any irAEs (Supplemental Fig. 1). Collectively, these results suggest that the pathogenesis of anti-PD-1 antibody-induced psoriasis-like dermatitis may depend on IL-6.
Moreover, we confirmed that mice treated with anti-PD-1 blocking monoclonal antibody developed clinically and histopathologically severe IMQ-induced psoriasis-like dermatitis compared to control mice treated with isotype IgG2a control (Ctrl) antibody (Supplemental Fig. 2A-E). The results corresponded to the experiments using PD-1 −/− mice.
We also conducted an experiment using the B16 melanoma murine model, in which B16F10 melanoma cells were inoculated into backs of both WT and PD-1 −/− mice, to investigate whether the presence of cancer involves PD-1 blockade-induced psoriasislike dermatitis. First, this model did not induce psoriasis-like dermatitis spontaneously nor under vehicle cream treatment (Supplemental Fig. 3A-E). Moreover, the presence of B16 melanoma did not lead to a significant difference in PASI score or ear swelling in WT or PD-1 −/− mice (Supplemental Fig. 3A-H). Fig. 1 Characteristics of anti-programmed cell death-1 (PD-1) antibody-induced psoriasis-like dermatitis. a Representative clinical images of patients with idiopathic psoriasis and anti-PD-1 antibody-induced psoriasis-like dermatitis. Both patients developed well-defined scaly plaques scattered over their trunks and extremities. b Representative hematoxylin and eosin (HE)-stained, and anti-CD8 or CD4 antibody-stained skin biopsy samples from patients with idiopathic psoriasis and anti-PD-1 antibody-induced psoriasis-like dermatitis. Scale bars = 50 μm. c CD8/CD4 ratios of epidermal-infiltrating lymphocytes (n = 6 and 7 in idiopathic psoriasis and anti-PD-1 antibody-induced psoriasis-like dermatitis, respectively). **P < 0.01 by nonparametric 2tailed Mann-Whitney U test. d Profiles of serum interleukin (IL)-6 levels in serum samples from anti-PD-1 antibody-treated cancer patients who developed psoriasis-like dermatitis as an immune-related adverse event (irAE, n = 8) and those with no irAE (n = 19). ****P < 0.0001 by nonparametric 2-tailed Mann-Whitney U test.
Taken together, these data suggest that PD-1 blockade, either by genetic knockout or antibody treatment, promotes IMQinduced psoriasis-like dermatitis, and that PD-1 blockade in the context of cancer does not increase the severity of dermatitis.
PD-1 deficiency in mice results in increased epidermal infiltration of CD8 T cells with enhanced production of IFN-γ and CXC chemokine ligand (CXCL)9. IHC analysis of murine ear skin samples revealed significantly increased numbers of CD8 T cells infiltrating into the epidermis of PD-1 −/− mice and anti-PD-1 antibody-treated mice when compared to control mice ( Fig. 3a and b, P = 0.008 by Mann-Whitney U test, and Supplemental Fig. 2F, P = 0.008 by Mann-Whitney U test), similar to what was seen in the patients with anti-PD-1 antibodyinduced psoriasis-like dermatitis. Next, qRT-PCR analysis revealed that PD-1 −/− mice have significantly higher CD8a and IFN-γ mRNA levels in CD45 + epidermal cells and CXCL9 in keratinocytes (CD45-negative epidermal cells) compared to that of WT mice (Fig. 3c, P = 0.008, P = 0.008 and P = 0.03 by Mann-Whitney U test, respectively).
In summary, these in vivo results suggest that the PD-1 deficiency enhances the numbers of infiltrating activated cytotoxic CD8 T cells, resulting in acceleration of psoriasis-like dermatitis.
Enhanced expression of cutaneous IL-6 via PD-1 deficiency in mice. qRT-PCR analysis revealed that unstimulated ear skin from PD-1 −/− and WT mice contain similar low levels of psoriasisrelated cytokines, IL-6, IL23-A, and IL-17A (P = 0.95, P = 0.57 and P = 0.21 by Mann-Whitney U test, respectively, Fig. 2g). IMQ application significantly increased mRNA expression of IL-23A and IL-17A in both WT mice and PD-1 −/− mice (P = 0.007 and P = 0.0002 in WT mice, and P = 0.0003 and P = 0.0003 in PD-1 −/− mice by Mann-Whitney U test, respectively, Fig. 2g). Notably, increased IL-6 mRNA expression induced by IMQ application was observed only in PD-1 −/− mice and not in WT mice (P = 0.0006 and P = 0.27, respectively, by Mann-Whitney U test, Fig. 2g). Expression of Ly6g, a neutrophil surface marker, mRNA was undetectable in both groups after vehicle cream application, but were increased significantly in PD-1 −/− mice compared to WT mice after IMQ application (P = 0.048 by Mann-Whitney U test, Fig. 2g). In addition, these results were also confirmed using PD-1-specific blocking antibody treatment (Supplemental Fig. 2G). Further investigations revealed a significantly higher level of IL-6 mRNA expression in the CD45positive epidermal cells, and an increased total number of CD45positive epidermal cells with specific infiltration of neutrophils in PD-1 −/− mice compared to WT mice (Supplemental Fig. 4).
Collectively, IL-6 expression related to expression of Th17 cytokines and infiltration of neutrophils correlates with PD-1 deficiency-enhanced IMQ-induced psoriasis-like dermatitis.
Taken together, blockade of IL-6 signaling with an anti-IL-6R antibody is a potential therapeutic approach to resolve psoriasislike dermatitis caused by inhibition of PD-1. Moreover, the treatment kinetics of anti-IL-6R antibody appear to be shorter than that of anti-IL-17A antibody treatment.
Importantly, there were not any differences between MR16-1treatment and IgG Ctrl-treatment in littermate Ctrl mice, highlighting the significance of IL-6 blockade therapy for the regulation of PD-1 signal blockade-activated CD8 T cells in psoriasis-like dermatitis.

Discussion
The pathogenesis of cutaneous irAEs in patients treated with anti-PD-1 antibody has yet to be elucidated. However, previous reports suggest that activated proliferative intradermal CD8 T cells evoke cutaneous irAEs such as lichen planus-like dermatitis and eczematous reaction 31,32 . The present study highlights the importance of PD-1 expression on CD8 T cells for the regulation of psoriasis-like dermatitis. We found that CD8-positive lymphocyte infiltration into the epidermis was significantly increased in patients with anti-PD-1 antibody-induced psoriasis-like dermatitis compared to that in idiopathic psoriasis. A murine model of IMQ-induced psoriasislike dermatitis clearly demonstrated that PD-1 deficiency accelerates infiltration of epidermal CD8 T cells with enhanced IFN-γ production of inflamed skin, and IFN-γ-stimulated keratinocytes produced an IFN-γ-inducible chemokine (CXCL9) for recruitment of T cells. Furthermore, the newly generated cKO mice with PD-1 deficiency specifically in CD8-positive cells demonstrated more severe IMQ-induced psoriasis-like dermatitis compared to the littermate control mice. These results suggest that PD-1 regulates skin-infiltrating CD8 T cells to engage in pathogenic crosstalk with PD-L1 expressed on various cells including keratinocytes 33 . In idiopathic psoriasis activation of conventional dendritic cells producing IL-23 lead to expansion and activation of autoreactive CD8 T cells in the dermis, which in turn acquire expression of α1β1integrin and migrate into the epidermis. The epidermis has been identified as an ideal location for CD8 T cells to engage in pathogenic crosstalk with keratinocytes 34,35 . Furthermore, intraepidermal CD8 T cells are shown to be highly pathogenic as the accumulation of epidermal T cells parallels the increase in proliferating keratinocytes in vivo 34 . Collectively, PD-1 signal blockadeinduced activation of CD8 T cells is essential to induce and accelerate anti-PD-1 antibody-induced psoriasis-like dermatitis.
We also found a significant increase in the serum levels of IL-6 in patients with anti-PD-1 antibody-induced psoriasis-like dermatitis, as we had shown in our preliminary study 25 , indicating that IL-6 could play an important role during disease development and thus, may be a suitable treatment target. As expected, a murine model of IMQ-induced psoriasis-like dermatitis enhanced via PD-1 deficiency was significantly improved by anti-IL-6R blocking antibody. These results clearly show the efficacy of IL-6-targeting therapy for PD-1 deficiency abrogated psoriasis-like dermatitis. One essential role of IL-6 is in the promotion of T helper 17 (Th17) cell production 36 . Th17 cells were recently shown to be a main pathological cell population in idiopathic psoriasis, and blockade of IL-17A and IL-23 have been established as treatments 37 , although IL-6 was not established as a potential therapeutic target. Our results also demonstrate that increased production of Th17-related cytokines, such as IL-17A and IL-23A, was accelerated in PD-1 −/− mice and was significantly suppressed by IL-6 blockade at both the tissue mRNA and serum levels to the same level as control WT mice.
IL-6 signals through the IL-6Rα and β subunit glycoprotein 130 (gp130). However, as for cells that do not express IL-6Rα on their surface, such as CD8 T cells, trans-signaling, a process whereby IL-6 signaling occurs through a complex of IL-6 and a soluble form of the IL-6Rα binding to ubiquitously expressed gp130 38 , is believed to occur. Thus, IL-6 trans-signaling likely plays an important role for the development of cytotoxic CD8 T cell function 39 . Therefore, it is likely that increased levels of soluble IL-6 in PD-1 −/− mice promotes cytotoxic CD8 T cell function via IL-6 trans-signaling. Furthermore, our analysis of human samples from anti-PD-1 antibody-treated cancer patients revealed that the serum level of sIL-6 presented correlates with that of sIL-6Rα in patients with anti-PD-1 antibody-induced psoriasis-like dermatitis, which would result in enhanced epidermal infiltration of CD8 T cells. Therefore, blocking this trans-signaling process with anti-IL-6R antibody might decrease the inflammation seen during PD-1 signal inhibition-provoked psoriasis-like dermatitis by impairing the promotion of CD8 T cells. Indeed, mice with PD-1deficiency specifically in CD8 T cells display severe psoriasis-like dermatitis, which can be restrained by blockade of IL-6 signaling.
Collectively, this treatment strategy comprised of selective blockade of IL-6 signal with anti-IL-6R blocking antibody could be effective and ideal for treating PD-1 signal blockade-induced psoriasis-like dermatitis. In fact, there have been a few case reports and a retrospective cohort study showing successful treatment of steroid-refractory irAEs with one dose of an anti-human IL-6R antibody, tocilizumab [40][41][42] , even though its use in irAE has not yet been validated. Thus, the present study for the first time demonstrates the rationale for this treatment and the pathophysiology of IL-6 signaling in PD-1 signal inhibition-provoked autoimmunity. On the other hand, a synergistic antitumor effect has been demonstrated on combined blockade of both IL-6 signaling and PD-1/PD-L1 pathways in tumor-bearing mice 43 , suggesting the efficacy of the dual signal blockade in terms of resolving the symptoms of irAEs without interfering antitumor effects. Even though psoriasis-like eruptions have been reported as a paradoxical phenomenon after use of tocilizumab 44 , our experiments and the previous clinical case report 42 demonstrate that IL-6 blockade therapy during the initial phase of PD-1 signal blockade-induced psoriasis-like dermatitis may rapidly reduce the severity of irAE and therefore, result in shorter interruptions of cancer treatments. Collectively, individuals with PD-1 signal blockade-induced psoriasis-like dermatitis can potentially benefit from IL-6-targeted therapeutic intervention, which is expected to inhibit both Th17 cell differentiation and cytotoxic CD8 T cell activation in the pathological mechanisms of irAE.
The first and foremost possible limitation of the current study is its retrospective nature in human sample collection from a limited number of institutes. Therefore, potential biases, such as selection bias and reporting bias, cannot be excluded, and functional analysis of CD8 T cells in skin and blood has yet to be completed. In addition, there are potentially some differences in the pathogenic mechanisms of psoriasis-like dermatitis between PD-1-deficient mice and anti-PD-1 antibody-treated mice/ Fig. 6 Characteristics of anti-IL-6R antibody-treated IMQ-induced psoriasis-like dermatitis in cKO mice with PD-1 deficiency specifically in CD8 T cells. Representative clinical images of IgG Ctrl-or MR16-1-treated IMQ-induced psoriasis-like dermatitis in littermate Ctrl mice or PD-1-cKO mice. b Ear swelling. c PASI score. Data are representative of two independent experiments. **P < 0.01 and ****P < 0.0001 by two-way ANOVA. d Representative histological images of HE-stained ear skin samples from these mice at day 7. Scale bars, 50 μm. e Epidermal thickness. f The number of epidermal, neutrophilic micro-abscess. g Total numbers of CD8 T cells in dLNs at day 7. h mRNA expression levels of CD8a and IFN-γ in ear skin samples at day 7. Fold changes in mRNAs levels were normalized to GAPDH mRNA levels. n = 5-6 in each group. Data are expressed as mean ± SEM. Data are representative of two independent experiments. *P < 0.05, **P < 0.01 by nonparametric 2-tailed Mann-Whitney U test.
humans. These include our results that serum levels of IL-23 were significantly elevated in anti-PD-1 antibody-treated mice with psoriasis-like dermatitis compared to control mice, while the levels were equal between PD-1-deficient mice and WT mice. Further, our study did not directly addressed if PD-1 signal blockade on CD4 T cells could have some effects, as was reported in a murine model of virus infection where the effects on CD4 T cells alter CD8 T-cell function through PD-1 signal blockade 45 . Moreover, the exact source of IL-6 is yet to be determined, even though IL-6 mRNA levels in CD45-positive epidermal cells, a potential cell population identified in the current study, were significantly elevated in PD-1-deficient mice compared to WT mice. Further prospective studies are needed to clarify those findings. Despite the limitations, data from the current study highlighted the unique characteristics of PD-1 signal blockadeinduced psoriasis-like dermatitis, most strikingly the significance of strong correlation between the enhanced IL-6 production and the dermatitis development, indicating the potential significance of IL-6-targeting for therapeutic intervention.
In summary, IL-6 plays important roles during disease development of PD-1 signal blockade-induced psoriasis-like dermatitis. Moreover, PD-1 expressed on CD8 T cells is responsible for the regulation of skin inflammation. Blockade of IL-6 signaling decreases inflammation in PD-1 signal inhibition-provoked psoriasis-like dermatitis, and specifically, causes a reduction in the levels of Th17-related cytokines in a murine model of IMQ-induced psoriasis-like dermatitis. Thus, these findings highlight the potential significance of IL-6-targeting for therapeutic intervention of PD-1 signal blockade-induced psoriasis-like dermatitis in humans.
Murine model of psoriasis-like dermatitis. In order to replicate a modified murine model of IMQ-induced psoriasis-like dermatitis 30 , 3.5% IMQ cream diluted from 5% IMQ cream (Beselna ® ; Mochida Pharmaceuticals) with vehicle control cream (Vanicream ® ; Pharmaceutical Specialties) (62.5 mg IMQ total, which was a lower dose compared to the dose used in a conventional model of IMQinduced psoriasis-like dermatitis) was applied topically on a daily basis to the shaved back and both ears for 5 or 7 consecutive days. Control mice were treated with the vehicle control cream only.
Scoring system for evaluating the severity of skin inflammation. To score the severity of inflammation of the back skin, an objective scoring system mimicking the Psoriasis Area and Severity Index (PASI) score for psoriasis patients was used as in a previous study 46 , in which independent scores of erythema, scaling, and thickening with a scale from 0 to 4 (0, none; 1, slight; 2, moderate; 3, marked; 4, very marked) were cumulated (ranged from 0 to 12). The ear thickness was measured using a micrometer (Mitutoyo).
IL-6 blockade. An anti-interleukin-6 receptor (anti-IL-6R) blocking antibody (MR16-1, Chugai Pharmaceuticals), which is a rat IgG1 monoclonal antibody against murine IL-6Rα chain, was injected intravenously at the dose of 2 mg per mouse prior to IMQ application at day 0. IgG isotype control (MP Biomedicals™) was used as a control.
Histological analysis. All FFPE human skin biopsy samples and murine ear skin samples were sectioned into 2 and 4-μm-thick slides, respectively, and subsequently undergone hematoxylin-eosin (HE) staining. Human FFPE skin samples were stained immunohistologically with anti-human CD8 and anti-human CD4 monoclonal antibodies (clone C8/144B and 4B12, respectively, Nichirei Biosciences) using an automatic slide stainer according to the manufacturer's instructions. The numbers of epidermal-infiltrating cells per sample (magnification, ×400) were counted. Murine ear FFPE samples were stained immunohistochemically with primary anti-CD3 (clone SP7, diluted 1:100, Abcam) and anti-CD8a (clone 4SM15, diluted 1:400, eBioscience) monoclonal antibodies, fluorescent-labeled secondary antibodies (Alexa Fluor ® 488-labeled goat anti-rabbit IgG, and Alexa Fluor ® 555-labeled goat anti-rat IgG, Abcam), and 4′,6-diamidino-2-phenylindole (DAPI) to detect the nucleus, by standard immunohistochemical staining techniques. A fluorescence microscope (BZ-X700, Keyence) was used for observation and to count the number of infiltrating cells per sample (magnification, ×400). Blood sample assay system. Murine blood samples were collected using the submandibular bleeding method and serum samples were subsequently isolated. Human and murine serum samples were immediately stored at ≤ −20°C for later use. In order to analyze cytokine (human and murine IL-6, murine IL-23A, and IL-17A) serum levels, the MILLIPLEX® MAP Kit (Merck Millipore) using Bio-Plex ® Luminex 200 multiplex assay system (Bio-Rad) was employed according to the manufacturer's protocol. Human serum levels of sIL-6 and sIL-6Rα were measured using Enzyme-Linked Immuno Sorbent Assay (ELISA) kit (Duoset and Quantikine; R&D systems) according to the manufacturer's protocol.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted from the murine ear samples using Trizol Reagent (Invitrogen). RNA concentrations were quantified and the OD 260/230 and the OD 260/280 ratio of the RNA samples were confirmed to be more than 1.8 and 1.6, respectively, with the NanoDrop ND-1000 (peqLab Biotechnologie GmbH). Complementary DNA (cDNA) was synthesized with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher) according to the manufacturer's instructions. Messenger RNA (mRNA) expression levels were detected by PCR amplification of cDNA using the QuantStudio™ 5 Real-Time PCR Systems (Applied Biosystems) with PrimeTime ® Gene Expression Master Mix and Prime Tim qPCR predesigned primers (Integrated DNA Technologies) listed in Supplemental Table 1. All qRT-PCR analyses were performed in triplicate. Amplification products were quantified by the comparative CT method. The mRNA level of each gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Skin separation. Murine ear skin samples were treated with 0.25% trypsin (FUJIFILM Wako Pure Chemical Corporation) solution for 40 minutes at 37°C in order to separate the epidermis and dermis. After washing two times with phosphate-buffered saline without Ca 2+ and Mg 2+ and passing through a 70 µm cell strainer, dissociated epidermal cells were then separated into CD45single cells (keratinocytes) and CD45 + single cells using MACS ® cell separation technology with CD45 MicroBeads beads (Miltenyi Biotec) according to the manufacturer's instructions. The positive selected fractions and the negative sorted fractions contained more than 95% and less than 1% of CD45-positive cells, respectively, by flow cytometry (data not shown).
Statistics and reproducibility. The differences between the groups were evaluated by Student's t test, Mann-Whiney U test or two-way ANOVA using GraphPad Prism 7.0 Software. A value of P < 0.05 was considered to be statistically significant. We repeated at least twice experiments and the exact sample size (n) for each experiment appear in the figure legend.
Study approval. All patients provided written, informed consent in compliance with the approval by the Institutional Ethics Committee at the University of Tsukuba Hospital (number: H28-045 and H30-256). All animal experiments were approved by the Animal Experiment Committee of the University of Tsukuba (Permit Number: 17-137), and performed in accordance with the Guide for the Care and Use of Laboratory Animals of the University of Tsukuba.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Raw data for graphs can be found in Supplementary Data 1. All other data are available within the manuscript files or from the corresponding author upon reasonable request.