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Cancer immunotherapy is now the first-line treatment for many unresectable cancers. However, it remains far from a complete cure for all patients. Therefore, it is necessary to develop innovative methods for cancer immunotherapy, and immune cell therapy could be an option. Currently, several institutions are attempting to generate immune cells from induced pluripotent stem cells (iPSCs) for use in cancer immunotherapy. A method for generating dendritic cells (DCs) and macrophages (MPs) from iPSC has been established. iPSC-derived DCs (iPS-DCs) can activate T cells via antigen presentation, and iPSC-derived macrophages (iPS-MPs) attack cancer. Since iPSCs are used as the source, genetic modification is easy, and various immune functions, such as the production of anti-tumour cytokines, can be added. Furthermore, when iPS-DCs and iPS-MPs are immortalized, cost reduction through mass production is theoretically possible. In this review, the achievements of cancer research using iPS-DCs and iPS-MPs are summarized, and the prospects for the future are discussed.

We observed malignant tumor development from human iPS-ML-IFN cells in SCID mice for up to 12 weeks. During the observation period, the mice were healthy, and no significant difference was observed in the body weight of human iPS-ML-IFN-injected mice and control mice. (2A). Twelve weeks after the injection of human iPS-ML-IFN cells, the mice were sacrificed, and examined under a stereomicroscope. We found no differences in the organs including lung, spleen, liver and greater omentum between human iPS-ML-IFN-injected mice and control mice. (2B)

In recent years, immunotherapy for advanced melanoma has been gaining increased attention. The efficacy of anti-cytotoxic T-lymphocyte antigen 4 antibodies, anti-programmed cell death 1 antibodies, and the BRAFV600E kinase inhibitor has been proven in metastatic melanoma. At the same time, adoptive cell transfer has significant effects against metastatic melanoma; however, it is difficult to apply on a broad scale because of the problems related to cell preparation. To overcome these problems, we developed immune cell therapy using induced pluripotent stem (iPS) cells. The benefit of our method is that a large number of cells can be readily obtained. We focused on macrophages for immune cell therapy because macrophage infiltration is frequently observed in solid cancers. In this study, the efficacy of human iPS cell–derived myeloid cell lines (iPS-ML) genetically modified to express type I IFNs against human melanoma cells was examined. The morphology, phagocytic ability, and surface markers of iPS-ML were similar to those of macrophages. The iPS-ML that express type I IFNs (iPS-ML-IFN) showed significant effects in inhibiting the growth of disseminated human melanoma cells in SCID mice. The infiltration of iPS-ML into the tumor nests was confirmed immunohistologically. The iPS-ML-IFNs increased the expression of CD169, a marker of M1 macrophages that can activate antitumor immunity. The iPS-ML-IFNs could infiltrate into tumor tissue and exert anticancer effects in the local tumor tissue. In conclusion, this method will provide a new therapeutic modality for metastatic melanoma. Cancer Immunol Res; 4(3); 248–58. ©2015 AACR.

Recently emerging cancer immunotherapies combine the applications of therapeutics to disrupt the immunosuppressive conditions in tumor-bearing hosts. In this study, we found that targeting the proinflammatory cytokine IL6 enhances tumor-specific Th1 responses and subsequent antitumor effects in tumor-bearing mice. IL6 blockade upregulated expression of the immune checkpoint molecule programmed death-ligand 1 (PD-L1) on melanoma cells. This PD-L1 induction was canceled in IFNγ-deficient mice or CD4+ T cell–depleted mice, suggesting that CD4+ T cell–derived IFNγ is important for PD-L1 induction in tumor-bearing hosts. In some patients with melanoma, however, treatment with the anti–PD-1 antibody nivolumab increased systemic levels of IL6, which was associated with poor clinical responses. This PD-L1 blockade-evoked induction of IL6 was reproducible in melanoma-bearing mice. We found that PD-1/PD-L1 blockade prompted PD-1+ macrophages to produce IL6 in the tumor microenvironment. Depletion of macrophages in melanoma-bearing mice reduced the levels of IL6 during PD-L1 blockade, suggesting macrophages are responsible for the IL6-mediated defective CD4+ Th1 response. Combined blockade of the mutually regulated immunosuppressive activities of IL6 and PD-1/PD-L1 signals enhanced expression of T cell–attracting chemokines and promoted infiltration of IFNγ-producing CD4+ T cells in tumor tissues, exerting a synergistic antitumor effect, whereas PD-L1 blockade alone did not promote Th1 response. Collectively, these findings suggest that IL6 is a rational immunosuppressive target for overcoming the narrow therapeutic window of anti–PD-1/PD-L1 therapy.Significance: These findings advance our understanding of IL6-PD1/PD-L1 cross-talk in the tumor microenvironment and provide clues for targeted interventional therapy that may prove more effective against cancer. Cancer Res; 78(17); 5011–22. ©2018 AACR.

Immune checkpoint inhibitors improved the survival rate of patients with unresectable melanoma. However, some patients do not respond, and variable immune-related adverse events have been reported. Therefore, more effective and antigen-specific immune therapies are urgently needed. We previously reported the efficacy of an immune cell therapy with immortalized myeloid cells derived from induced pluripotent stem cells (iPS-ML). In this study, we generated OX40L-overexpressing iPS-ML (iPS-ML-Zsgreen-OX40L) and investigated their characteristics and in vivo efficacy against mouse melanoma. We found that iPS-ML-Zsgreen-OX40L suppressed the progression of B16-BL6 melanoma, and prolonged survival of mice with ovalbumin (OVA)-expressing B16 melanoma (MO4). The number of antigen-specific CD8+ T cells was higher in spleen cells treated with OVA peptide-pulsed iPS-ML-Zsgreen-OX40L than in those without OX40L. The OVA peptide-pulsed iPS-ML-Zsgreen-OX40L significantly increased the number of tumor-infiltrating T lymphocytes (TILs) in MO4 tumor. Flow cytometry showed decreased regulatory T cells but increased effector and effector memory T cells among the TILs. Although we plan to use allogeneic iPS-ML in the clinical applications, iPS-ML showed the tumorgenicity in the syngeneic mice model. Incorporating the suicide gene is necessary to ensure the safety in the future study. Collectively, these results indicate that iPS-ML-Zsgreen-OX40L therapy might be a new method for antigen-specific cancer immunotherapy.

A 66-year-old woman with diabetes who was treated with prednisolone (15 mg/day) for autoimmune hepatitis developed multiple erythematous nodules with retention of purulent fluid on her lower right limb. Candida albicans was cultured from the nodules. She was started on oral fluconazole, and the lesions subsided. However, multiple dark-red abscesses and indurations newly appeared on the left crus. Histopathological examination showed numerous branched hyphae, and tissue culture yielded a Rhizopus microsporus-related fungus. She was treated with liposomal amphotericin B combined with drainage and debridement. However, she died because of poor control of the infection and hepatic disorder.

A 66-year-old woman with diabetes who was treated with prednisolone (15 mg/day) for autoimmune hepatitis developed multiple erythematous nodules with retention of purulent fluid on her lower right limb. Candida albicans was cultured from the nodules. She was started on oral fluconazole, and the lesions subsided. However, multiple dark-red abscesses and indurations newly appeared on the left crus. Histopathological examination showed numerous branched hyphae, and tissue culture yielded a Rhizopus microsporus-related fungus. She was treated with liposomal amphotericin B combined with drainage and debridement. However, she died because of poor control of the infection and hepatic disorder.

Type I interferons (IFNs) play important roles in antitumor immunity. We generated IFN-α-producing cells by genetically engineering induced pluripotent stem cell (iPSC)-derived proliferating myeloid cells (iPSC-pMCs). Local administration of IFN-α-producing iPSC-pMCs (IFN-α-iPSC-pMCs) alters the tumor microenvironment and propagates the molecular signature associated with type I IFN. The gene-modified cell actively influences host XCR1+ dendritic cells to enhance CD8+ T cell priming, resulting in CXCR3-dependent and STING-IRF3 pathway-independent systemic tumor control. Administration of IFN-α-iPSC-pMCs in combination with immune checkpoint blockade overcomes resistance to single-treatment modalities and generates long-lasting antitumor immunity. These preclinical data suggest that IFN-α-iPSC-pMCs might constitute effective immune-stimulating agents for cancer that are refractory to checkpoint blockade.

Background Anti-programmed cell death protein (PD)-1 antibody treatment is associated with a notable improvement in only 30%–40% of patients. Thus, a predictive and easily measured marker of the clinical benefit of anti-PD-1 antibody treatment is necessary; therefore, in this study, we focused on the serum concentration of hepatocyte growth factor (HGF). Objectives To evaluate whether the serum concentration of HGF can be used as a biomarker for the clinical response to anti-PD-1 antibody therapy. Methods This study included 29 metastatic melanoma patients receiving nivolumab or pembrolizumab. Nine patients responded to anti-PD-1 antibody treatment, whereas the other 20 patients did not. The serum concentrations of HGF were analyzed by using ELISA. In 28 patients, immunohistochemical analysis of the HGF protein in patients’ cancer tissues was also performed. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured with an anti-CD3 antibody in the presence or absence of HGF and c-MET inhibitor. The expression of perforin in CD8+ T cells were evaluated by using flow cytometry. Results Among the 29 recruited patients, the non-responders displayed higher serum concentrations of HGF than the responders (P = 0.00124). Patients with low serum concentrations of HGF showed longer overall survival (N = 28, P = 0.039; HR 0.3125, 95% CI 0.1036–0.9427) and progression-free survival (N = 24, P = 0.0068; HR 0.2087, 95% CI 0.06525–0.6676) than those with high concentrations of HGF. We observed a significant correlation between the serum concentration of HGF and immunohistochemical-positive staining (P = 0.000663). In a flow cytometry analysis of PBMCs from healthy donors, HGF was found to downregulate perforin secretion. Furthermore, the addition of capmatinib, a specific inhibitor of c-MET, increased the expression of perforin in CD8+ T cells. Conclusions HGF concentration represents a valid biomarker that can be further developed for the evaluation of anti-PD-1 therapy. Our results suggested that c-MET inhibition promotes perforin expression in CD8+ T cells. Therefore, c-MET inhibitors can activate the immune system and may play an important role in combined immunotherapy.

Recently emerging cancer immunotherapies combine the applications of therapeutics to disrupt the immunosuppressive conditions in tumor-bearing hosts. In this study, we found that targeting the proinflammatory cytokine IL6 enhances tumor-specific Th1 responses and subsequent antitumor effects in tumor-bearing mice. IL6 blockade upregulated expression of the immune checkpoint molecule programmed death-ligand 1 (PD-L1) on melanoma cells. This PD-L1 induction was canceled in IFNγ-deficient mice or CD4+ T cell–depleted mice, suggesting that CD4+ T cell–derived IFNγ is important for PD-L1 induction in tumor-bearing hosts. In some patients with melanoma, however, treatment with the anti–PD-1 antibody nivolumab increased systemic levels of IL6, which was associated with poor clinical responses. This PD-L1 blockade-evoked induction of IL6 was reproducible in melanoma-bearing mice. We found that PD-1/PD-L1 blockade prompted PD-1+ macrophages to produce IL6 in the tumor microenvironment. Depletion of macrophages in melanoma-bearing mice reduced the levels of IL6 during PD-L1 blockade, suggesting macrophages are responsible for the IL6-mediated defective CD4+ Th1 response. Combined blockade of the mutually regulated immunosuppressive activities of IL6 and PD-1/PD-L1 signals enhanced expression of T cell–attracting chemokines and promoted infiltration of IFNγ-producing CD4+ T cells in tumor tissues, exerting a synergistic antitumor effect, whereas PD-L1 blockade alone did not promote Th1 response. Collectively, these findings suggest that IL6 is a rational immunosuppressive target for overcoming the narrow therapeutic window of anti–PD-1/PD-L1 therapy. Significance: These findings advance our understanding of IL6-PD1/PD-L1 cross-talk in the tumor microenvironment and provide clues for targeted interventional therapy that may prove more effective against cancer. Cancer Res; 78(17); 5011–22. ©2018 AACR.

We previously demonstrated that Th1 differentiation of tumor-specific CD4+ T cells was attenuated in tumor-bearing or aged mice in an IL-6-dependent manner (Nat. Comm. 6: e6702, 2015, Cancer Immunol. Res. 1: 64, 2013). Furthermore we found that accompanied by the systemic increase of soluble IL-6 receptor (sIL-6R) in cancer patients, inhibition of IL-6 trans-signaling mediated through the sIL-6R restored the Th1 responses, their helper activity toward CD8+ T cells, and anti-tumor activity in tumor-bearing mice (Cancer Res. 77: 2279, 2017). The systemically increased sIL-6Rs were mainly produced by myeloid cells. Moreover, cMaf-deficient CD4+ T cells were resistant to Th1 suppression and impairment of T cell-mediated anti-tumor immunity induced by IL-6/sIL-6R indicating that c-Maf activity was responsible for Th1 suppression. Myeloid cell-derived sIL-6R was also possibly associated with Th1 suppression and c-Maf expression in head and neck cancer patients. These results suggest that targeting a pro-inflammatory cytokine, IL-6 enhanced the tumor-specific Th1 responses and subsequent anti-tumor effects. However, IL-6 blockade in turn up-regulated the expression of immune-checkpoint molecule, PD-L1 on melanoma cells. This PD-L1 induction was canceled in IFN-γ-deficient mice or mice depleted of CD4+ T cells, suggesting an important role of CD4+ T cell-derived IFN-γ in the PD-L1 induction in tumor-bearing hosts. On the other hand, in some patients with melanoma, anti-PD-1 antibody, Nivolumab treatment increased the systemic level of IL-6, which were associated with their poor clinical responses. This PD-L1 blockade-evoked IL-6 induction was also observed in melanoma-bearing mice (Cancer Res. 78: 5011, 2018). Considering the mechanistic linkage between IL-6 and PD-1/PD-L1 signals, we found that PD-1/PD-L1 blockade prompted PD-1+macrophages to produce IL-6 in tumor microenvironment. Depletion of macrophages in melanoma-bearing mice revealed that macrophages functioned as a source of IL-6 during PD-L1 blockade, which was responsible for the defective Th1 response. Furthermore, combined blockade of the mutually regulated-immunosuppressive activities mediated by IL-6 and PD-1/PD-L1 signals enhanced the infiltration of IFN-γ-producing CD4+ T cells in tumor tissues, and exerted a synergistic anti-tumor effect, whereas PD-L1 blockade alone did not promote Th1 response. Collectively, these findings suggest that IL-6 is considered to be a rational immunosuppressive target to overcome a narrow therapeutic window of anti-PD-1/PD-L1 therapy. [ This research was financially supported by the JSPS KAKENHI grant nos. 26430165 and 18K07325 to HT, nos. 15H04311 and 16H06498 to YN, and the P-CREATE from the AMED, Japan to YN and HT. ] Citation Format: Yasuharu Nishimura, Koji Fujieda, Azusa Miyashita, Satoshi Fukushima, Tokunori Ikeda, Yosuke Kubo, Satoru Senju, Hironobu Ihn, Hiroyuki Oshiumi, Hirotake Tsukamoto. Combination of anti-IL-6 and anti-PD-L1 antibodies synergistically reinvigorates cancer immunity by activating both CTL and Th1 cells in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3213.

Immune checkpoint inhibitors have increased the median survival of melanoma patients. To improve their effects, antigen-specific therapies utilizing melanoma-associated antigens should be developed. Cell division cycle-associated protein 1 (CDCA1), which has a specific function at the kinetochores for stabilizing microtubule attachment, is overexpressed in various cancers. CDCA1, which is a member of cancer-testis antigens, does not show detectable expression levels in normal tissues. Quantitative reverse transcription polymerase chain reaction and immunoblotting analyses revealed that CDCA1 was expressed in all of the tested melanoma cell lines, 74% of primary melanomas, 64% of metastatic melanomas and 25% of nevi. An immunohistochemical analysis and a Cox proportional hazards model showed that CDCA1 could be a prognostic marker in malignant melanoma (MM) patients. CDCA1-specific siRNA inhibited the cell proliferation of SKMEL2 and WM115 cells, but did not reduce the migration or invasion activity. These results suggest that CDCA1 may be a new therapeutic target of melanoma.

In recent years, immunotherapy for advanced melanoma has been gaining increased attention. The efficacy of anti-cytotoxic T-lymphocyte antigen 4 antibodies, anti-programmed cell death 1 antibodies, and the BRAFV600E kinase inhibitor has been proven in metastatic melanoma. At the same time, adoptive cell transfer has significant effects against metastatic melanoma; however, it is difficult to apply on a broad scale because of the problems related to cell preparation. To overcome these problems, we developed immune cell therapy using induced pluripotent stem (iPS) cells. The benefit of our method is that a large number of cells can be readily obtained. We focused on macrophages for immune cell therapy because macrophage infiltration is frequently observed in solid cancers. In this study, the efficacy of human iPS cell–derived myeloid cell lines (iPS-ML) genetically modified to express type I IFNs against human melanoma cells was examined. The morphology, phagocytic ability, and surface markers of iPS-ML were similar to those of macrophages. The iPS-ML that express type I IFNs (iPS-ML-IFN) showed significant effects in inhibiting the growth of disseminated human melanoma cells in SCID mice. The infiltration of iPS-ML into the tumor nests was confirmed immunohistologically. The iPS-ML-IFNs increased the expression of CD169, a marker of M1 macrophages that can activate antitumor immunity. The iPS-ML-IFNs could infiltrate into tumor tissue and exert anticancer effects in the local tumor tissue. In conclusion, this method will provide a new therapeutic modality for metastatic melanoma. Cancer Immunol Res; 4(3); 248–58. ©2015 AACR.

Anaplastic large cell lymphoma (ALCL) is a high grade non-Hodgkin lymphoma (NHL) that is comprised of the malignant proliferation of large lymphoid cells, which express CD30. Primary ALCL of the skeletal muscle is extremely uncommon. A 51-year-old Japanese female presented at our hospital with a 2-month history of severe pain and swelling of the right leg. A gallium-67 SPECT/CT scan showed a large mass involving the skeletal muscles from the gluteus to femoris. A biopsy of the mass demonstrated diffuse infiltration of medium and large neoplastic cells with round or lobulated hyperchromatic pleomorphic nuclei. A subset of Reed-Sternberg-like cells was also identified. Immunohistochemically, the neoplastic cells were strongly positive for CD4 and CD30, but negative for CD3, CD8, anaplastic lymphoma kinase (ALK), CD20, CD79α, CD21 and CD23. Based on the histological examination, this patient was diagnosed to have ALK-negative ALCL of the skeletal muscle. Further studies are needed to clarify the biological behavior of primary skeletal muscle ALCL.

Background Adiponectin is protein hormone produced by visceral and subcutaneous fat cells. In many past studies, adiponectin plays some roles in inflammation, angiogenesis and tissue remodeling, and induces the production of the anti-inflammatory mediators. Recently, adiponectin has also attracted attention for its involvement in the pathogenesis of collagen diseases. De Sanctis et al. Objectives In this study, we determined the adiponectin expression in the sera and lesional skin of patients with systemic sclerosis (SSc). Methods Serum adiponectin concentrations were measured in 32 patients with SSc, 10 patients with SLE, 12 patients with dermatomyositis and 13 healthy subjects with specific enzyme-linked immunosorbent assays. Adiponectin mRNA was determined in skin tissues of 5 patients with diffuse cutaneous SSc (dcSSc), 7 patients with limited cutaneous SSc (lcSSc) and 7 healthy subjects with real-time polymerase chain reaction. Results There was significant reduction in serum adiponectin levels in patients with dcSSc compared with controls (11.2±8.0 vs 139.7±54.2pg/ml, P Conclusions Serum adiponectin levels may be a useful biomarker for fibrotic condition in patients with SSc. Clarifying the role of adiponectin in collagen diseases may lead to further understanding of the pathogenesis and new therapeutic approach. References Motoshima H, Wu X, Sinha MK, et al. Differential regulation of adiponectin secretion from cultured human omental and subcutaneous adipocytes: effects of insulin and rosiglitazone. J Clin Endocrinol Metab 2002;87(12):5662-7. De Sanctis JB, Zabaleta M, Bianco NE, Garmendia JV, Rivas L. Serum adipokine levels in patients with systemic lupus erythematosus. Autoimmunity 2009;42(4):272-4. Disclosure of Interest None Declared

MoO3 is one of the efficient hole-transporting materials for organic photovoltaic cells (OPVs). Here, a facile method of preparing the MoO3 buffer layer will be introduced. MoO3 powder was added into methanol and ultrasonication of the dispersion solution was carried out. Then the solution was centrifuged, and the supernatant was spin-cast on an indium–tin-oxide anode. On it, a photoconversion layer was prepared by spin-casting a poly(3-hexylthiophene):[6,6]-phenyl-C61 butyric acid methyl ester (P3HT:PCBM) composite solution. The fabricated OPVs revealed an efficiency as high as 3.05%, which is better than that of P3HT:PCBM OPV with a poly(3,4-ethylene dioxythiophene):poly(styrene sulfonate) hole-transporting layer.

We introduce a new method to fabricate a MoO3 hole-transporting layer for organic photovoltaic cells (OPVs). We fabricated a MoS2 film from its solution and converted it to MoO3. MoS2 has a lamellar crystal structure similar to graphite, and it can be exfoliated into monolayer MoS2 dispersible in water. Li atoms were first intercalated into van der Waals gaps of MoS2, and the compound was immersed in water to generate H2 bubbles, which broke the van der Waals bond between adjacent MoS2 layers. The produced solution of MoS2 was spin-casted on an indium tin oxide substrate, and the film was oxidized by ozone. On the converted MoO3 hole-transporting layer, an organic photoconversion layer was fabricated by spin-casting a poly(3-hexylthiophene):[6,6]-phenyl-C61 butyric acid methyl ester (P3HT:PCBM) composite solution. Fabricated OPVs revealed a power conversion efficiency as large as 3.14%, which was superior to that of P3HT/PCBM OPV with a poly(3,4-ethylene dioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) hole-transporting layer.