Arakawa et al. discovered that the autoimmune response in psoriasis is directed against melanocytes. They show that the main psoriasis risk allele HLA-C*06:02 mediates melanocyte-specific autoimmunity and identify ADAMTSL5 as a melanocyte autoantigen, which stimulates IL-17 and IFN-γ production in CD8+ T cells.

Recent reports on ES cell differentiation have suggested the possibility that information on in vivo neurogenesis might be systematically linked to stem cell technology (3, 4). However, it remains to be known whether ES cell-derived neural precursors generated in vitro can produce the full dorsal-ventral range of neuroectodermal derivatives in response to embryonic positional information. To address this question, we have tested in this study whether SDIA-treated ES cells have the competence of differentiating into the dorsal-(neural crest) and ventralmost (floor plate) cells under embryologically relevant conditions. Materials and MethodsCell Culture and Treatment with Patterning Factors. Mouse ES cells (EB5), primate ES cells (cynomolgus monkey-derived; purchased from Asahi Technoglass, Funabashi, Japan), and PA6 cells were maintained and used for induction as described (1, 2, 5). Human bone morphogenetic protein (BMP)4 and mouse were purchased from R&D Systems and freshly added at each medium change. The day on which ES cells are seeded on PA6 is defined as day 0.Immunocytochemistry, Statistics, and RT-PCR. Cells were fixed with 4% paraformaldehyde, and immunostaining was performed with secondary antibodies conjugated with FITC, cy3, or cy5. For statistics, Ϸ100 colonies were observed in each experiment, and three or more experiments were performed. P values for statistical significance (t test) are described in the corresponding figure legends. The values shown in graphs represent the mean Ϯ SD. RT-PCR was performed with ES cell colonies detached from feeder cells as described (1). The primary antibodies and primers used are described in Supporting Materials and Methods, which are published as supporting information on the PNAS web site, www.pnas.org.Colony Isolation and Axon Guidance Assays. The 3D collagen gel assay for axon guidance was performed by using isolated ES cell colonies (day 8; ref. 1) and the cerebellar plate region excised from embryonic day 13 Wistar rats as a responder (6). Results Positional Identity of Neural Tissues Induced from ES Cells by SDIA.We first examined the expression of rostral-caudal CNS markers by RT-PCR (Fig. 1A). SDIA-treated mouse ES cells express the forebrain markers Otx2 and Six3, the ventral diencephalon marker Rx, the ventral forebrain marker Nkx2.1, the midbrainhindbrain border marker En2, and the hindbrain marker Gbx2, but not the spinal cord markers Hoxb4 and b9 (lane 2). These results show that a majority of neural cells induced by SDIA express rostral neural markers. This idea is consistent with our previous report that dopaminergic neurons generated by the SDIA method are those of the midbrain type (1).We next attempted to alter the rostral-caudal identity of SDIA-induced neural cells by the caudalizing factor retinoic acid (RA; ref. 7). RA treatment (0.2 M all-trans RA; Fig. 1 A, lane 3) suppressed the forebrain markers Otx2, Six3, Rx, and Nkx2.1, whereas it induced the hindbrain marker Gbx2 and the spinal cord markers Hoxb4 and b9. RA treatment did not sign...

Tumor-infiltrating lymphocytes (TILs) have been reported as a prognostic factor in various cancers and are a promising target for immunotherapy. To investigate whether TILs have any impact on the prognosis of angiosarcoma patients, 55 non-treated patients (40 patients at stage 1 with cutaneous localized tumors, 4 patients at stage 2 with lymph node metastases and 11 patients at stage 3 with distant metastases) with angiosarcoma were evaluated retrospectively by immunohistochemistry stained CD4, CD8, FOXP3 and Ki67. The Kaplan-Meier method was used to estimate overall survival with patients at stage 1. Survival differences were analyzed by the log-rank test. Patients with higher numbers of CD8 1 TILs in their primary tumors survived significantly longer compared with patients with lower values. Moreover, the number of CD8 in TILs was positively correlated with a distant metastasis-free period. The total number of primary TILs (CD4 plus CD8) and CD8 1 primary TILs of stage 3 patients with distant metastases was positively correlated with their overall survival. To evaluate whether CD8 1 effector T cells are activated or differentiated, flow cytometric analysis of peripheral blood mononuclear cells (PBMC) was performed. The percentages of CD8 1 T cells producing IFN-c in PBMC were significantly higher in patients with angiosarcoma (n 5 10) compared not only with that of healthy controls (n 5 20) but also patients with advanced melanoma (n 5 11). These results suggest that anti-tumor immunity is clinically relevant in angiosarcoma.

Recognition of cancer antigens drives the clonal expansion of cancer-reactive T cells, which is thought to contribute to restricted T-cell receptor (TCR) repertoires in tumor-infiltrating lymphocytes (TILs). To understand how tumors escape anti-tumor immunity, we investigated tumor-associated T-cell repertoires of patients with advanced melanoma and after blockade of the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1 (PD-1). TCR Vβ-gene spectratyping allowed us to quantify restrictions of T-cell repertoires and, further, diversities of T-cell clones. In this study, we show that the blood TCR repertoires were variably restricted in CD4 + and extensively restricted in CD8 + T cells of patients with advanced melanoma, and contained clones in both T-cell fractions prior to the start of immunotherapy. A greater diversification especially of CD4 + blood T-cell clones before immunotherapy showed statistically significant correlations with long-term survival upon CTLA4 or PD-1 inhibition. Analysis of TILs and corresponding blood available in one patient indicated that blood clonality may at least partially be related to the clonal expansion in the tumor microenvironment. In patients who developed severe immune-related adverse events (IrAEs), CD4 + and CD8 + TCR spectratypes became more restricted during anti-CTLA4 treatment, suggesting that newly expanded oligoclonal T-cell responses may contribute to IrAEs. This study reveals diverse T-cell clones in the blood of melanoma patients prior to immunotherapy, which may reflect the extent to which T cells are able to react against melanoma and potentially control melanoma progression. Therefore, the T-cell clonality in the circulation may have predictive value for antitumor responses from checkpoint inhibition.

Generalized pustular psoriasis (GPP) is the most severe psoriasis variant. Mutations in the IL-36 antagonist IL36RN, in CARD14 or AP1S3 provide genetic evidence for autoinflammatory etiology but cannot explain its pathogenesis completely. Here we demonstrate that unopposed IL-36 signaling promotes antigen-driven and likely pathogenic T-helper type 17 (Th17) responses in GPP. We observed that CD4 T cells in blood and skin lesions of GPP patients were characterized by intense hyperproliferation, production of the GPP key mediator, IL-17A, and highly restricted TCR repertoires with identical T-cell clones in blood and skin lesions, indicating antigen-driven T-cell expansions. The clonally expanded CD4 T cells were major producers of IL-17A. IL-36 signaling substantially enhanced TCR-mediated proliferation of CD4 T cells. Moreover, GPP patients showed preferences for HLA-DRB1∗14, HLA-DQB1∗05, and HLA-DQB1∗03. We conclude that in GPP unopposed IL-36 signaling and certain HLA-class II alleles may cooperate in promoting antigen-driven Th17 responses, which in the obvious absence of exogenous triggers may reflect autoimmune reactions. This study reveals a pathogenic pathway where innate immune dysregulation promotes T-cell-mediated inflammation in GPP.

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