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PDF file - 1482K, Proposed model for MDSC-mediated inhibition of anti-tumor CD4+ T cell response.

Protection against subcutaneously inoculated OVA-expressing melanoma by OVA-peptide loaded iPS-pMC-DCs.

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow–derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2Kb-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs. Cancer Immunol Res; 3(6); 668–77. ©2015 AACR.

PDF file - 830K, Representative plots showing IFN-γproduction in tumor-specific CD4+ T cells.

PDF file - 1746K, The effect of MDSC-derived IL-6 and sIL-6R on tumor-specific CD4+ T cells.

Supplementary Figure Legends

PDF file - 790K, IL-6 production related to MDSC, and phenotypic characterization of MDSC.

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)

Collaborative action between tumor cells and host-derived suppressor cells leads to peripheral tolerance of T cells to tumor antigens. Here, we showed that in tumor-bearing mice, generation of tumor antigen-specific effector T-helper cells (TH1) was significantly attenuated, and impaired TH1 differentiation was restored by the temporal blockade of interleukin (IL)-6 activity at the T-cell priming phase. Furthermore, we found that Gr-1+ myeloid-derived suppressor cells (MDSC) served as a source of IL-6 in tumor-bearing mice. Adoptive transfer of effector CD4+ T cells revealed that MDSC-sensitized effector CD4+ T cells were less potent in mounting antitumor immune responses, although effector T cells generated together with Gr-1+ cells from tumor-free mice eradicated established tumors. CD8+ T cells, IFN-γ, and MHC-class II expression in host mice were indispensable for the antitumor activity initiated by effector CD4+ T cells. Despite comparable suppressive activity of IL-6+/+ and IL-6−/− MDSC on primary T-cell activation, transfer of IL-6+/+ MDSC, but not IL-6−/− MDSC, dampened the efficient induction of effector TH1 cells and counteracted CD4+ T cell–mediated antitumor immunity including cognate help for CD8+ T cells in vivo. These findings suggest that, apart from the inhibitory effects on primary T-cell activation, MDSC promote tumor progression by attenuating functional differentiation of tumor-specific CD4+ T cells into effector TH1 cells through IL-6 production to promote tumor progression. This novel mode of MDSC-induced tolerance of effector CD4+ T cells should be considered as the basis for the rational design of effective T cell–mediated antitumor therapies. Cancer Immunol Res; 1(1); 64–76. ©2013 AACR.

Supplementary Materials and Methods

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.

PDF file - 920K, The effect of MDSC-derived IL-6 on Th1 differentiation.

Supplementary Figure 4 - PDF file 105K, Induction of KIF20A-A2466-75 SP-specific CTLs in mice immunized with KIF20A60-84-LP

IL6 produced by tumor cells promotes their survival, conferring a poor prognosis in patients with cancer. IL6 also contributes to immunosuppression of CD4+ T cell–mediated antitumor effects. In this study, we focused on the impact of IL6 trans-signaling mediated by soluble IL6 receptors (sIL6R) expressed in tumor-bearing hosts. Higher levels of sIL6R circulating in blood were observed in tumor-bearing mice, whereas the systemic increase of sIL6R was not prominent in tumor-bearing mice with myeloid cell–specific conditional deletion of IL6R even when tumor cells produced sIL6R. Abundant sIL6R was released by CD11b+ cells from tumor-bearing mice but not tumor-free mice. Notably, IL6-mediated defects in Th1 differentiation, T-cell helper activity for tumor-specific CD8+ T cells, and downstream antitumor effects were rescued by myeloid-specific deletion of sIL6R. Expression of the T-cell transcription factor c-Maf was upregulated in CD4+ T cells primed in tumor-bearing mice in an IL6-dependent manner. Investigations with c-Maf loss-of-function T cells revealed that c-Maf activity was responsible for IL6/sIL6R-induced Th1 suppression and defective T-cell–mediated antitumor responses. In patients with cancer, myeloid cell–derived sIL6R was also possibly associated with Th1 suppression and c-Maf expression. Our results argued that increased expression of sIL6R from myeloid cells and subsequent c-Maf induction were adverse events for counteracting tumor-specific Th1 generation. Overall, this work provides a mechanistic rationale for sIL6R targeting to improve the efficacy of T-cell–mediated cancer immunotherapy. Cancer Res; 77(9); 2279–91. ©2017 AACR.

Supplementary Text - PDF file 195K, Supplementary Methods and Figure legends

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.

CCR Translation for This Article from Identification of Promiscuous KIF20A Long Peptides Bearing Both CD4+ and CD8+ T-cell Epitopes: KIF20A-Specific CD4+ T-cell Immunity in Patients with Malignant Tumor

Purpose: To identify long peptides (LP) derived from a novel tumor-associated antigen (TAA), kinesin family member 20A (KIF20A), which induce tumor-specific T-helper type 1 (TH1) cells and CTLs.Experimental Design: We combined information from a recently developed computer algorithm predicting HLA class II–binding peptides with KIF20A-derived CTL-epitope sequences presented by HLA-A2 (A*02:01) or HLA-A24 (A*24:02) to select candidate promiscuous TH1-cell epitopes containing CTL epitopes. Peripheral blood mononuclear cells (PBMC) derived from healthy donors or patients with head-and-neck malignant tumor (HNMT) were used to study the immunogenicity of KIF20A-LPs, and the in vitro cross-priming potential of KIF20A-LPs bearing CTL epitopes. We used HLA-A24 transgenic mice to address whether vaccination with KIF20A-LP induces efficient cross-priming of CTLs in vivo. The TH1-cell response to KIF20A-LPs in HNMT patients receiving immunotherapy with TAA-derived CTL-epitope peptides was analyzed using IFN-γ enzyme-linked immunospot assays.Results: We identified promiscuous KIF20A-LPs bearing naturally processed epitopes recognized by CD4+ T cells and CTLs. KIF20A-specific CTLs were induced by vaccination with a KIF20A-LP in vivo. KIF20A expression was detected in 55% of HNMT by immunohistochemistry, and significant frequencies of KIF20A-specific TH1 cell responses were detected after short-term in vitro stimulation of PBMCs with KIF20A-LPs in 50% of HNMT patients, but not in healthy donors. Furthermore, these responses were associated with KIF20A expression in HNMT tissues.Conclusions: These are the first results showing the presence of KIF20A-specific TH1 cell responses in HNMT patients and underline the possible utility of KIF20A-LPs for propagation of TH1 cells and CTLs. Clin Cancer Res; 19(16); 4508–20. ©2013 AACR.

Supplementary Figure 2 - PDF file 39K, Induction of KIF20A-LPs-specific Th cells from healthy donors

Supplementary Figure 3 - PDF file 157K, Enhanced induction of KIF20A-SP-specific CTLs by KIF20A-LP-specific CD4+ T-cells

Supplementary Data from Identification of a Novel Tumor-Associated Antigen, Cadherin 3/P-Cadherin, as a Possible Target for Immunotherapy of Pancreatic, Gastric, and Colorectal Cancers

Myeloid cell-derived sIL-6R in tumor-bearing mice modulates CD4+ T-cell property.

Purpose: To establish cancer immunotherapy, it is important to identify the tumor-associated antigens (TAA) that are strongly expressed in the tumor cells but not in the normal cells. In this study, to establish an effective anticancer immunotherapy, we tried to identify the useful TAA of pancreatic cancer.Experimental Design: Based on a previous genome-wide cDNA microarray analysis of pancreatic cancer, we focused on cadherin 3 (CDH3)/P-cadherin as a novel candidate TAA for anticancer immunotherapy. To identify the HLA-A2 (A*0201)–restricted CTL epitopes of CDH3, we used HLA-A2.1 (HHD) transgenic mice (Tgm). Furthermore, we examined the cytotoxicity against the tumor cells in vitro and in vivo of CTLs specific to CDH3 induced from HLA-A2–positive healthy donors and cancer patients.Results:CDH3 was overexpressed in the majority of pancreatic cancer and various other malignancies, including gastric and colorectal cancers, but not in their noncancerous counterparts or in many normal adult tissues. In the experiment using HLA-A2.1 Tgm, we found that the CDH3-4655-663 (FILPVLGAV) and CDH3-7757-765 (FIIENLKAA) peptides could induce HLA-A2–restricted CTLs in Tgm. In addition, peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A2–positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both CDH3 and HLA-A2. Furthermore, the adoptive transfer of the CDH3-specific CTLs could inhibit the tumor growth of human cancer cells engrafted into nonobese diabetic/severe combined immunodeficiency mice.Conclusions: These results suggest that CDH3 is a novel TAA useful for immunotherapy against a broad spectrum of cancers, including pancreatic cancer.

Supplementary Figure 1 - PDF file 188K, KIF20A-derived and promiscuous HLA class II-binding peptides predicted by a recently developed computer algorithm

Supplementary Table 3 - PDF file 33K, Results of the immunohistochemical analysis of KIF20A in head and neck tumor

Macrophages and MDSC produce sIL-6R in tumor-bearing mice.

Supplementary Table 1 Demographics and disease characteristics of HNT patients. Supplementary Table 2 Healthy donor information. Supplementary Table 3 The comparison of pathway parameters without covariates between HD and HNT. Supplementary Table 4 Result of multilevel linear models with covariates.

This file includes "Supplementary Materials and Methods" and figure legends for "Supplementary Figures 1 to 4"

Supplementary Table 1 - PDF file 19K, Binding algorithm scores of KIF20A-derived peptides for HLA-class II molecules

Comparative analysis in immunological parameters from cancer patients and healthy donors.

Despite effective antiretroviral therapy, HIV-1 persists in cells, including macrophages, which is an obstacle to cure. However, the precise role of macrophages in HIV-1 infection remains unclear because they reside in tissues that are not easily accessible. Monocyte-derived macrophages are widely used as a model in which peripheral blood monocytes are cultured and differentiated into macrophages. However, another model is needed because recent studies revealed that most macrophages in adult tissues originate from the yolk sac and fetal liver precursors rather than monocytes, and the embryonic macrophages possess a self-renewal (proliferating) capacity that monocyte-derived macrophages lack. Here, we show that human induced pluripotent stem cell–derived immortalized macrophage-like cells are a useful self-renewing macrophage model. They proliferate in a cytokine-dependent manner, retain macrophage functions, support HIV-1 replication, and exhibit infected monocyte-derived macrophage–like phenotypes, such as enhanced tunneling nanotube formation and cell motility, as well as resistance to a viral cytopathic effect. However, several differences are also observed between monocyte-derived macrophages and induced pluripotent stem cell–derived immortalized macrophage-like cells, most of which can be explained by the proliferation of induced pluripotent stem cell–derived immortalized macrophage-like cells. For instance, proviruses with large internal deletions, which increased over time in individuals receiving antiretroviral therapy, are enriched more rapidly in induced pluripotent stem cell–derived immortalized macrophage-like cells. Interestingly, inhibition of viral transcription by HIV-1–suppressing agents is more obvious in induced pluripotent stem cell–derived immortalized macrophage-like cells. Collectively, our present study proposes that the model of induced pluripotent stem cell–derived immortalized macrophage-like cells is suitable for mimicking the interplay between HIV-1 and self-renewing tissue macrophages, the newly recognized major population in most tissues that cannot be fully modeled by monocyte-derived macrophages alone.

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.

Although several in vitro assays that predict the sensitizing potential of chemicals have been developed, none can distinguish between chemical respiratory and skin sensitizers. Recently, we established a new three-dimensional dendritic cell (DC) coculture system consisting of a human airway epithelial cell line, immature DCs derived from human peripheral monocytes, and a human lung fibroblast cell line. In this coculture system, compared to typical skin sensitizers, typical respiratory sensitizers showed enhanced mRNA expression in DCs of the key costimulatory molecule OX40 ligand (OX40L), which is important for T helper 2 (Th2) cell differentiation. Herein, we established a new two-step DC/T cell coculture system by adding peripheral allogeneic naive CD4+ T cells to the DCs stimulated in the DC coculture system. In this DC/T cell coculture system, typical respiratory sensitizers but not skin sensitizers enhanced mRNA expression of the predominant Th2 marker interleukin-4 (IL-4) and its transcription factor GATA-binding protein 3. To improve the versatility, in place of peripheral monocytes, monocyte-derived proliferating cells called CD14-ML were also used in the DC coculture system. Similar to peripheral monocytes, enhanced mRNA expression of OX40L was observed by typical respiratory sensitizers compared to skin sensitizers. When these cell lines were applied to the DC/T cell coculture system with peripheral allogeneic naive CD4+ T cells, typical respiratory sensitizers but not skin sensitizers enhanced the mRNA expression of IL-4. Thus, this DC/T cell coculture system might be useful for discriminating between respiratory and skin sensitizers by differential mRNA upregulation of IL-4 in T cells.

We previously established a method to generate myeloid cells with a proliferative capability from pluripotent stem cells and designated them iPS‐ML. Human iPS‐ML cells share features with physiological macrophages including the capability to infiltrate into cancer tissues. We observed therapeutic effects of human iPS‐ML cells expressing interferon β (iPS‐ML/interferon (IFN)‐β) in xenograft cancer models. However, assessment of host immune system‐mediated therapeutic and adverse effects of this therapy is impossible by xenograft models. We currently evaluated the therapeutic effects of a mouse equivalent of human iPS‐ML/IFN, a mouse embryonic stem (ES) cell‐derived myeloid cell line producing IFN (ES‐ML/IFN). The ES‐MLs producing IFN‐β (β‐ML) and IFN‐γ (γ‐ML) and originating from E14 ES cells derived from the 129 mouse strain (H‐2b) were generated, and the MHC (H‐2K b , D b , and I‐A b) genes of the ES‐ML/IFN were disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 method. We used the ES‐ML/IFN to treat allogeneic BALB/c mice (H‐2d) transplanted with Colon26 cancer cells. Treatment with β‐ML but not with γ‐ML cells repressed the growth of colon cancer in the peritoneal cavity and liver. The transferred ES‐ML/IFN infiltrated into cancer tissues and enhanced infiltration of T cells into cancer tissues. ES‐ML/IFN therapy increased the number of immune cells in the lymphoid organs. Sensitization of both cancer antigen‐specific CD8+ T cells and natural killer (NK) cells were enhanced by the therapy, and CD8+ T cells were essential for the therapeutic effect, implying that donor MHC‐deficient β‐ML exhibited a therapeutic effect through the activation of host immune cells derived from allogeneic recipient mice. The results suggested the usefulness of HLA‐deficient human iPS‐ML/IFN‐β cells for therapy of HLA‐mismatched allogeneic cancer patients.

Immunotherapy using dendritic cells (DCs) is a promising treatment modality for cancer. However, the limited number of functional DCs from peripheral blood has been linked to the unsatisfactory clinical efficacies of current DC-based cancer immunotherapies. We previously generated proliferating antigen-presenting cells (APCs) by genetically engineering myeloid cells derived from induced pluripotent stem cells (iPSC-pMCs), which offer infinite functional APCs for broad applications in cancer therapy. Herein, we aimed to further enhance the antitumor effect of these cells by genetic modification. GM-CSF gene transfer did not affect the morphology, or surface phenotype of the original iPSC-pMCs, however, it did impart good viability to iPSC-pMCs. The resultant cells induced GM-CSF-dependent CD8+ T cell homeostatic proliferation, thereby enhancing antigen-specific T cell priming in vitro. Administration of the tumor antigen-loaded GM-CSF-producing iPSC-pMCs (GM-pMCs) efficiently stimulated antigen-specific T cells and promoted effector cell infiltration of the tumor tissues, leading to an augmented antitumor effect. To address the potential tumorigenicity of iPSC-derived products, irradiation was applied and found to restrict the proliferation of GM-pMCs, while retaining their T cell-stimulatory capacity. Furthermore, the irradiated cells exerted an antitumor effect equivalent to that of bone marrow-derived DCs obtained from immunocompetent mice. Additionally, combination with immune checkpoint inhibitors increased the infiltration of CD8+ or NK1.1+ effector cells and decreased CD11b+/Gr-1+ cells without causing adverse effects. Hence, although GM-pMCs have certain characteristics that differ from endogenous DCs, our findings suggest the applicability of these cells for broad clinical use and will provide an unlimited source of APCs with uniform quality.