Yasushi Uemura

Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation

Adoptive immunotherapy with functional Txa0cells is potentially an effective therapeutic strategy for combating many types of cancer and viral infection. However, exhaustion of antigen-specific Txa0cells represents a major challenge to this type of approach. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8(+) Txa0cells from an HIV-1-infected patient to pluripotency. The Txa0cell-derived induced pluripotent stem cells were then redifferentiated into CD8(+) Txa0cells that had a high proliferative capacity and elongated telomeres. These rejuvenated cells possessed antigen-specific killing activity and exhibited Txa0cell receptor gene-rearrangement patterns identical to those of the original Txa0cell clone from the patient. We also found that this method can be effective for generating specific Txa0cells for other pathology-associated antigens. Thus, this type of approach may have broad applications in the field of adoptive immunotherapy.

Prevention of Experimental Autoimmune Encephalomyelitis by Transfer of Embryonic Stem Cell-Derived Dendritic Cells Expressing Myelin Oligodendrocyte Glycoprotein Peptide along with TRAIL or Programmed Death-1 Ligand

Experimental autoimmune encephalomyelitis (EAE) is caused by activation of myelin Ag-reactive CD4+ T cells. In the current study, we tested a strategy to prevent EAE by pretreatment of mice with genetically modified dendritic cells (DC) presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TRAIL or Programmed Death-1 ligand (PD-L1). For genetic modification of DC, we used a recently established method to generate DC from mouse embryonic stem cells (ES cells) in vitro (ES-DC). ES cells were sequentially transfected with an expression vector for TRAIL or PD-L1 and an MHC class II-associated invariant chain-based MOG epitope-presenting vector. Subsequently, double-transfectant ES cell clones were induced to differentiate to ES-DC, which expressed the products of introduced genes. Treatment of mice with either of the double-transfectant ES-DC significantly reduced T cell response to MOG, cell infiltration into spinal cord, and the severity of MOG peptide-induced EAE. In contrast, treatment with ES-DC expressing MOG alone, irrelevant Ag (OVA) plus TRAIL, or OVA plus PD-L1, or coinjection with ES-DC expressing MOG plus ES-DC-expressing TRAIL or PD-L1 had no effect in reducing the disease severity. In contrast, immune response to irrelevant exogenous Ag (keyhole limpet hemocyanin) was not impaired by treatment with any of the genetically modified ES-DC. The double-transfectant ES-DC presenting Ag and simultaneously expressing immune-suppressive molecules may well prove to be an effective therapy for autoimmune diseases without inhibition of the immune response to irrelevant Ag.

Enhanced Priming of Antigen-Specific CTLs In Vivo by Embryonic Stem Cell-Derived Dendritic Cells Expressing Chemokine Along with Antigenic Protein: Application to Antitumor Vaccination

Dendritic cell (DC)-based immunotherapy is regarded as a promising means for anti-cancer therapy. The efficiency of T cell-priming in vivo by transferred DCs should depend on their encounter with T cells. In the present study, we attempted to improve the capacity of DCs to prime T cells in vivo by genetic modification to express chemokine with a T cell-attracting property. For genetic modification of DCs, we used a recently established method to generate DCs from mouse embryonic stem cells. We generated double-transfectant DCs expressing a chemokine along with a model Ag (OVA) by sequential transfection of embryonic stem cells, and then induced differentiation to DCs. We comparatively evaluated the effect of three kinds of chemokines; secondary lymphoid tissue chemokine (SLC), monokine induced by IFN-γ (Mig), and lymphotactin (Lptn). All three types of double transfectant DCs primed OVA-specific CTLs in vivo more efficiently than did DCs expressing only OVA, and the coexpression of SLC or Lptn was more effective than that of Mig. Immunization with DCs expressing OVA plus SLC or Mig provided protection from OVA-expressing tumor cells more potently than did immunization with OVA alone, and SLC was more effective than Mig. In contrast, coexpression of Lptn gave no additive effect on protection from the tumor. Collectively, among the three chemokines, expression of SLC was the most effective in enhancing antitumor immunity by transferred DCs in vivo. The findings provide useful information for the development of a potent DC-based cellular immunotherapy.

Genetically Manipulated Human Embryonic Stem Cell‐Derived Dendritic Cells with Immune Regulatory Function

Genetically manipulated dendritic cells (DC) are considered to be a promising means for antigen‐specific immune therapy. This study reports the generation, characterization, and genetic modification of DC derived from human embryonic stem (ES) cells. The human ES cell‐derived DC (ES‐DC) expressed surface molecules typically expressed by DC and had the capacities to stimulate allogeneic T lymphocytes and to process and present protein antigen in the context of histocompatibility leukocyte antigen (HLA) class II molecule. Genetic modification of human ES‐DC can be accomplished without the use of viral vectors, by the introduction of expression vector plasmids into undifferentiated ES cells by electroporation and subsequent induction of differentiation of the transfectant ES cell clones to ES‐DC. ES‐DC introduced with invariant chain‐based antigen‐presenting vectors by this procedure stimulated HLA‐DR‐restricted antigen‐specific T cells in the absence of exogenous antigen. Forced expression of programmed death‐1‐ligand‐1 in ES‐DC resulted in the reduction of the proliferative response of allogeneic T cells cocultured with the ES‐DC. Generation and genetic modification of ES‐DC from nonhuman primate (cynomolgus monkey) ES cells was also achieved by the currently established method. ES‐DC technology is therefore considered to be a novel means for immune therapy.

Biomarkers for the early diagnosis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer-related deaths worldwide. Although the prognosis of patients with HCC is generally poor, the 5-year survival rate is > 70% if patients are diagnosed at an early stage. However, early diagnosis of HCC is complicated by the coexistence of inflammation and cirrhosis. Thus, novel biomarkers for the early diagnosis of HCC are required. Currently, the diagnosis of HCC without pathological correlation is achieved by analyzing serum α-fetoprotein levels combined with imaging techniques. Advances in genomics and proteomics platforms and biomarker assay techniques over the last decade have resulted in the identification of numerous novel biomarkers and have improved the diagnosis of HCC. The most promising biomarkers, such as glypican-3, osteopontin, Golgi protein-73 and nucleic acids including microRNAs, are most likely to become clinically validated in the near future. These biomarkers are not only useful for early diagnosis of HCC, but also provide insight into the mechanisms driving oncogenesis. In addition, such molecular insight creates the basis for the development of potentially more effective treatment strategies. In this article, we provide an overview of the biomarkers that are currently used for the early diagnosis of HCC.

Involvement of regulatory T cells in the experimental autoimmune encephalomyelitis-preventive effect of dendritic cells expressing myelin oligodendrocyte glycoprotein plus TRAIL.

We previously reported the protection from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by the adoptive transfer of genetically modified embryonic stem cell-derived dendritic cells (ES-DC) presenting MOG peptide in the context of MHC class II molecules and simultaneously expressing TRAIL (ES-DC-TRAIL/MOG). In the present study, we found the severity of EAE induced by another myelin autoantigen, myelin basic protein, was also decreased after treatment with ES-DC-TRAIL/MOG. This preventive effect diminished, if the function of CD4+CD25+ regulatory T cells (Treg) was abrogated by the injection of anti-CD25 mAb into mice before treatment with ES-DC-TRAIL/MOG. The adoptive transfer of CD4+CD25+ T cells from ES-DC-TRAIL/MOG-treated mice protected the recipient mice from MOG- or myelin basic protein-induced EAE. The number of Foxp3+ cells increased in the spinal cords of mice treated with ES-DC-TRAIL/MOG. In vitro experiments showed that TRAIL expressed in genetically modified ES-DC and also in LPS-stimulated splenic macrophages had a capacity to augment the proliferation of CD4+CD25+ T cells. These results suggest that the prevention of EAE by treatment with ES-DC-TRAIL/MOG is mediated, at least in part, by MOG-reactive CD4+CD25+ Treg propagated by ES-DC-TRAIL/MOG. For the treatment of organ-specific autoimmune diseases, induction of Treg reactive to the organ-specific autoantigens by the transfer of DC-presenting Ags and simultaneously overexpressing TRAIL therefore appears to be a promising strategy.

Systematic Analysis of the Combinatorial Nature of Epitopes Recognized by TCR Leads to Identification of Mimicry Epitopes for Glutamic Acid Decarboxylase 65-Specific TCRs

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4+ T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.

Zoledronate sensitizes neuroblastoma-derived tumor-initiating cells to cytolysis mediated by human γδ T cells.

Neuroblastoma is the most common extracranial solid tumor in children that is refractory to intensive multimodal therapy. In particular, tumor-initiating cells (TICs) derived from neuroblastoma are believed responsible for tumor formation and resistance to the conventional therapy; an optimal strategy therefore should target this population. Technically, TICs can be enriched from neuroblastoma-derived spheres when the tumor cells are cultured in a serum-free medium supplemented with certain growth factors. Recently, a line of evidence has suggested antitumor potential of V&ggr;9V&dgr;2 T cells (&ggr;&dgr; T cells), a T-cell population that recognizes and kills target cells independent of surface HLA expressions. Furthermore, a mevalonate pathway inhibitor, zoledronate, has been reported to enhance cytolytic activity of &ggr;&dgr; T cells. On the basis of these findings, we hypothesized that zoledronate would sensitize neuroblastoma TICs to &ggr;&dgr; T-cell–mediated cytolysis and promote therapeutic efficacy against neuroblastoma. In the current study, we show that zoledronate efficiently sensitizes both neuroblastoma-derived adherent cells and sphere-forming cells to &ggr;&dgr; T-cell–mediated cytolysis. Subsequently, in vitro colony formation inhibition assay and in vivo animal studies reveal that the presence of &ggr;&dgr; T cells decelerates outgrowth of neuroblastoma TICs. We finally show that addition of interleukin-15 and/or interleukin-18 in culture enhances the cytolytic activity of &ggr;&dgr; T cells. On the basis of these data, we conclude that ex vivo expanded &ggr;&dgr; T cells are a promising tool for antineuroblastoma immunotherapy with options for further improvement.

Prospectively Isolated Human Bone Marrow Cell‐Derived MSCs Support Primitive Human CD34‐Negative Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM‐derived lineage‐ and CD45‐negative cells, according to their cell surface expression of CD271 and stage‐specific embryonic antigen (SSEA)−4. Among them, the MSCs established from the Lineage−CD45−CD271+SSEA‐4+ fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well‐characterized HSC‐supportive genes, including IGF‐2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo‐chondrogenic DP MSCs possessed the ability to support cord blood‐derived primitive human CD34‐negative severe combined immunodeficiency‐repopulating cells. The HSC‐supportive actions of DP MSCs were partially carried out by soluble factors, including IGF‐2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34‐positive (CD34+) as well as CD34‐negative (CD34−) HSCs was important for the support/maintenance of the CD34+/− HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo. Stem Cells 2015;33:1554–1565

Cellular Adjuvant Properties, Direct Cytotoxicity of Re-differentiated Vα24 Invariant NKT-like Cells from Human Induced Pluripotent Stem Cells

Vα24 invariant natural killer T (iNKT) cells are a subset of T lymphocytes implicated in the regulation of broad immune responses. They recognize lipid antigens presented by CD1d on antigen-presenting cells and induce both innate and adaptive immune responses, which enhance effective immunity against cancer. Conversely, reduced iNKT cell numbers and function have been observed in many patients with cancer. To recover these numbers, we reprogrammed human iNKT cells to pluripotency and then re-differentiated them into regenerated iNKT cells in vitro through an IL-7/IL-15-based optimized cytokine combination. The re-differentiated iNKT cells showed proliferation and IFN-γ production in response to α-galactosylceramide, induced dendritic cell maturation and downstream activation of both cytotoxic T lymphocytes and NK cells, and exhibited NKG2D- and DNAM-1-mediated NK cell-like cytotoxicity against cancer cell lines. The immunological features of re-differentiated iNKT cells and their unlimited availability from induced pluripotent stem cells offer a potentially effective immunotherapy against cancer.