Tumor cell-induced macrophage senescence plays a pivotal role in tumor initiation followed by stable growth in immunocompetent condition.

BACKGROUND: The cancer stem cell theory proposes that tumor formation in vivo is driven only by specific tumor-initiating cells having stemness; however, clinical trials conducted to test drugs that target the tumor stemness provided unsatisfactory results thus far. Recent studies showed clear involvement of immunity in tumors; however, the requirements of tumor-initiation followed by stable growth in immunocompetent individuals remain largely unknown. METHODS: To clarify this, we used two similarly induced glioblastoma lines, 8B and 9G. They were both established by overexpression of an oncogenic H-RasL61 in p53-deficient neural stem cells. In immunocompromised animals in an orthotopic transplantation model using 1000 cells, both show tumor-forming potential. On the other hand, although in immunocompetent animals, 8B shows similar tumor-forming potential but that of 9G's are very poor. This suggests that 8B cells are tumor-initiating cells in immunocompetent animals. Therefore, we hypothesized that the differences in the interaction properties of 8B and 9G with immune cells could be used to identify the factors responsible for its tumor forming potential in immunocompetent animals and performed analysis. RESULTS: Different from 9G, 8B cells induced senescence-like state of macrophages around tumors. We investigated the senescence-inducing factor of macrophages by 8B cells and found that it was interleukin 6. Such senescence-like macrophages produced Arginase-1, an immunosuppressive molecule known to contribute to T-cell hyporesponsiveness. The senescence-like macrophages highly expressed CD38, a nicotinamide adenine dinucleotide (NAD) glycohydrolase associated with NAD shortage in senescent cells. The addition of nicotinamide mononucleotide (NMN), an NAD precursor, in vitro inhibited to the induction of macrophage senescence-like phenotype and inhibited Arginase-1 expression resulting in retaining T-cell function. Moreover, exogenous in vivo administration of NMN after tumor inoculation inhibited tumor-initiation followed by stable growth in the immunocompetent mouse tumor model. CONCLUSIONS: We identified one of the requirements for tumor-initiating cells in immunocompetent animals. In addition, we have shown that tumor growth can be inhibited by externally administered NMN against macrophage senescence-like state that occurs in the very early stages of tumor-initiating cell development. This therapy targeting the immunosuppressive environment formed by macrophage senescence-like state is expected to be a novel promising cancer therapeutic strategy.

Induced pluripotent stem cell-derived hematopoietic stem and progenitor cells induce mixed chimerism and donor-specific allograft tolerance.

In transplantation using allogeneic induced pluripotent stem cells (iPSCs), strategies focused on major histocompatibility complexes were adopted to avoid immune rejection. We showed that minor antigen mismatches are a risk factor for graft rejection, indicating that immune regulation remains one of the most important issues. In organ transplantation, it has been known that mixed chimerism using donor-derived hematopoietic stem/progenitor cells (HSPCs) can induce donor-specific tolerance. However, it is unclear whether iPSC-derived HSPCs (iHSPCs) can induce allograft tolerance. We showed that 2 hematopoietic transcription factors, Hoxb4 and Lhx2, can efficiently expand iHSPCs with a c-Kit+Sca-1+Lineage- phenotype, which possesses long-term hematopoietic repopulating potential. We also demonstrated that these iHSPCs can form hematopoietic chimeras in allogeneic recipients and induce allograft tolerance in murine skin and iPSC transplantation. With mechanistic analyses, both central and peripheral mechanisms were suggested. We demonstrated the basic concept of tolerance induction using iHSPCs in allogeneic iPSC-based transplantation.

Induction of allograft tolerance by adoptive transfer of donor B cells: an immune regulatory strategy for transplantation using MHC-matched iPS cells.

For cellular or tissue transplantation using induced pluripotent stem cells (iPSCs), from the viewpoint of time and economic cost, the use of allogeneic ones is being considered. Immune regulation is one of the key issues in successful allogeneic transplantation. To reduce the risk of rejection, several attempts have been reported to eliminate effects of the major histocompatibility complex (MHC) on the iPSC-derived grafts. On the other hand, we have shown that minor antigen-induced rejection is not negligible even when the MHC's impact is mitigated. In organ transplantation, it is known that donor-specific transfusion (DST) can specifically control immune responses to the donor. However, whether DST could control the immune response in iPSC-based transplantation was not clarified. In this study, using a mouse skin transplantation model, we demonstrate that infusion of donor splenocytes can promote allograft tolerance in the MHC-matched but minor antigen-mismatched conditions. When narrowing down the cell types, we found that infusion of isolated splenic B cells was sufficient to control rejection. As a mechanism, the administration of donor B cells induced unresponsiveness but not deletion in recipient T cells, suggesting that the tolerance was induced in the periphery. The donor B cell transfusion induced allogeneic iPSC engraftment. These results suggest for the first time a possibility that DST using donor B cells could induce tolerance against allogeneic iPSC-derived grafts.

Macrophage-like iPS-derived Suppressor Cells Reduce Th1-mediated Immune Response to a Retinal Antigen.

PURPOSE: To investigate the immunotherapeutic effects of macrophage-like induced pluripotent stem (iPS) cell-derived suppressor cells (SCs) in ocular immune response and experimental autoimmune uveoretinitis (EAU). METHODS: The genes of Oct3/4, Sox2, Klf4, and c-Myc were transferred to B cells enriched from the spleen cells of C57BL/6 mice by using retrovirus vectors. Transferred B cells were cultured for 17 days to obtain colonies of iPS cells. Through additional steps, iPS-SCs were induced. An antigen-specific T cell proliferation assay was performed with CD4+ T cells collected from draining lymph nodes of the mice immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide and co-cultured with iPS-SCs. Cytokine concentrations in the culture supernatant were examined. Mice were immunized with hIRBP peptide to induce EAU. The iPS-SCs were administered into the mice one day before the induction of EAU. RESULTS: The iPS-SCs decreased hIRBP-specific T cell proliferation depending on the number of cells. Productions of tumor necrosis factor-α and interferon-γ were significantly decreased; however, transforming growth factor-ß1, nitric oxide, interleukin (IL)-13, IL-17A, and IL-17 F levels were elevated in the supernatant when the collected T cells were co-cultured with iPS-SCs. The iPS-SCs had immunosuppressant effects even without cell-to-cell contact, and their effects were non-specific to the antigen preloaded on iPS-SCs. EAU was significantly milder in the mice administered iPS-SCs prior to immunization. CONCLUSIONS: Macrophage-like iPS-SCs reduced Th1 immune response to a retinal antigen and Th1-mediated EAU in mice. These results showed the possibility of the application of iPS technology to the treatment of noninfectious ocular inflammation, endogenous uveitis, in the future.

Establishment of Human Leukocyte Antigen-Mismatched Immune Responses after Transplantation of Human Liver Bud in Humanized Mouse Models.

Humanized mouse models have contributed significantly to human immunology research. In transplant immunity, human immune cell responses to donor grafts have not been reproduced in a humanized animal model. To elicit human T-cell immune responses, we generated immune-compromised nonobese diabetic/Shi-scid, IL-2RγKO Jic (NOG) with a homozygous expression of human leukocyte antigen (HLA) class I heavy chain (NOG-HLA-A2Tg) mice. After the transplantation of HLA-A2 human hematopoietic stem cells into NOG-HLA-A2Tg, we succeeded in achieving alloimmune responses after the HLA-mismatched human-induced pluripotent stem cell (hiPSC)-derived liver-like tissue transplantation. This immune response was inhibited by administering tacrolimus. In this model, we reproduced allograft rejection after the human iPSC-derived liver-like tissue transplantation. Human tissue transplantation on the humanized mouse liver surface is a good model that can predict T-cell-mediated cellular rejection that may occur when organ transplantation is performed.

Establishment of an experimental model for MHC homo-to-hetero transplantation.

Preventing rejection is a major challenge in transplantation medicine, even when using pluripotent stem cell-derived grafts. In iPS cell (iPSC)-based transplantation, to reduce the risk of rejection, it is thought to be optimal that preparing the cells from donors whose human leukocyte antigen-haplotype are homozygous. Generally, this approach is referred to as major histocompatibility complex (MHC) homo-to-hetero transplantation, which is MHC-matched but minor antigen-mismatched. To investigate the immune response in the MHC homo-to-hetero transplantation, we established a murine experimental system in which MHC-matched but minor antigen-mismatched tissue (skin) grafts were transplanted into MHC-heterozygous recipients. Unexpectedly, only minor antigen-mismatched grafts were rejected at the same time points as rejection of fully allogeneic grafts. A vigorous anti-donor type T cell response was detected in vitro and conventional immunosuppressants targeting T cell activation had limited effects on controlling rejection. However, anti-donor antibodies were not detected only in the minor antigen-mismatched transplantation. This murine transplantation model can be used to further analyze immunological subjects for MHC homo-to-hetero iPSC-based transplantation.

Induction of macrophage-like immunosuppressive cells from common marmoset ES cells by stepwise differentiation with DZNep.

Recent progress in regenerative medicine has enabled the utilization of pluripotent stem cells (PSCs) as the resource of therapeutic cells/tissue. However, immune suppression is still needed when the donor-recipient combination is allogeneic. We have reported previously that mouse PSCs-derived immunosuppressive cells contribute to prolonged survival of grafts derived from the same mouse PSCs in allogeneic recipients. For its clinical application, a preclinical study using non-human primates such as common marmoset must be performed. In this study, we established the induction protocol of immunosuppressive cells from common marmoset ES cells. Although similar immunosuppressive macrophages could not be induced by same protocol as that for mouse PSCs, we employed an inhibitor for histone methyltransferase, DZNep, and succeeded to induce them. The DZNep-treated macrophage-like cells expressed several immunosuppressive molecules and significantly inhibited allogeneic mixed lymphocyte reaction. The immunosuppressive cells from non-human primate ESCs will help to establish an immunoregulating strategy in regenerative medicine using PSCs.

Immune reaction and regulation in transplantation based on pluripotent stem cell technology.

The development of pluripotent stem cell (PSC)-based technologies provides us a new therapeutic approach that generates grafts for transplantation. In order to minimize the risk of immune reaction, the banking of induced pluripotent stem cells (iPSCs) from donors with homozygous human leukocyte antigen (HLA) haplotype is planned in Japan. Even though pre-stocked and safety validated HLA-homozygous iPSCs are selected, immunological rejection may potentially occur because the causes of rejection are not always due to HLA mismatches. A couple of studies concerning such immunological issues have reported that genetic ablation of HLA molecules from PSC combined with gene transduction of several immunoregulatory molecules may be effective in avoiding immunological rejection. Also, our research group has recently proposed a concept that attempts to regulate recipient immune system by PSC-derived immunoregulatory cells, which results in prolonged survival of the same PSC-derived allografts. PSC-based technologies enable us to choose a new therapeutic option; however, considering its safety from an immunological point of view should be of great importance for safe clinical translation of this technology.

Efficient generation of thymic epithelium from induced pluripotent stem cells that prolongs allograft survival.

The thymus plays a significant role in establishing immunological self-tolerance. Previous studies have revealed that host immune reaction to allogeneic transplants could be regulated by thymus transplantation. However, physiological thymus involution hinders the clinical application of these insights. Here, we report an efficient generation of thymic epithelial-like tissue derived from induced pluripotent stem cells (iPSCs) and its potential to regulate immune reaction in allogeneic transplantation. We established an iPSC line which constitutively expresses mouse Foxn1 gene and examined the effect of its expression during in vitro differentiation of thymic epithelial cells (TECs). We found that Foxn1 expression enhances the differentiation induction of cells expressing TEC-related cell surface molecules along with upregulation of endogenous Foxn1. iPSC-derived TECs (iPSC-TECs) generated T cells in nude recipient mice after renal subcapsular transplantation. Moreover, iPSC-TEC transplantation to immuno-competent recipients significantly prolonged the survival of allogeneic skin. Our study provides a novel concept for allogeneic transplantation in the setting of regenerative medicine.

Prognostic value of IL-34 in colorectal cancer patients.

The mortality of colorectal cancer is expected to increase in some countries including the United States, which necessitates the identification of new molecules that help in prognosis assessment and survival improvement. In this brief report, we evaluated the potential of interleukin-34 (IL-34) as a prognostic factor in colorectal cancer. IL-34 was reported for the first time in 2008 as a novel cytokine that controls the biology of the myeloid cell lineage. Accumulating evidence suggests important roles for IL-34 in modifying the tumor microenvironment and enhancing therapeutic resistance of cancer. In this study, we found that IL-34 expression was detectable in various colorectal cancer cell lines in addition to primary cancer tissues from a cohort of Japanese colorectal cancer patients, ranging from high to absent. A Kaplan-Meier analysis showed that high expression of IL-34 correlated with poor survival of colorectal cancer patients. Importantly, in both univariate and multivariate analysis, high IL-34 expression correlated with unfavorable prognosis. A similar relationship between IL-34 expression and the poorer prognosis was also observed in a cohort of colorectal cancer patients registered at The Cancer Genome Atlas. Together, these findings indicate a potential role for IL-34 as a prognostic factor in colorectal cancer.

A role for IL-34 in osteolytic disease of multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy that grows in multiple sites of the axial skeleton and causes debilitating osteolytic disease. Interleukin-34 (IL-34) is a newly discovered cytokine that acts as a ligand of colony-stimulating factor-1 (CSF-1) receptor and can replace CSF-1 for osteoclast differentiation. In this study, we identify IL-34 as an osteoclastogenic cytokine that accelerates osteolytic disease in MM. IL-34 was found to be expressed in the murine MM cell line MOPC315.BM, and the expression of IL-34 was enhanced by stimulation with proinflammatory cytokines or by bone marrow (BM) stromal cells. MM-cell-derived IL-34 promoted osteoclast formation from mouse BM cells in vitro. Targeting Il34 by specific small interfering RNA impaired osteoclast formation in vitro and attenuated osteolytic disease in vivo. In BM aspirates from MM patients, the expression levels of IL-34 in CD138+ populations vary among patients from high to weak to absent. MM cell-derived IL-34 promoted osteoclast formation from human CD14+ monocytes, which was reduced by a neutralizing antibody against IL-34. Taken together, this study describes for the first time the expression of IL-34 in MM cells, indicating that it may enhance osteolysis and suggesting IL-34 as a potential therapeutic target to control pathological osteoclastogenesis in MM patients.