Clinical Trial on the Safety and Tolerability of Personalized Cancer Vaccines Using Human Platelet Lysate-Induced Antigen-Presenting Cells.

Research and development of personalized cancer vaccines as precision medicine are ongoing. We predicted human leukocyte antigen (HLA)-compatible cancer antigen candidate peptides based on patient-specific cancer genomic profiles and performed a Phase I clinical trial for the safety and tolerability of cancer vaccines with human platelet lysate-induced antigen-presenting cells (HPL-APCs) from peripheral monocytes. Among the five enrolled patients, two patients completed six doses per course (2-3 × 107 cells per dose), and an interim analysis was performed based on the immune response. An immune response was detected by enzyme-linked immunosorbent spot (ELISpot) assays to HLA-A*33:03-matched KRASWT, HLA-DRB1*09:01-compliant KRASWT or G12D, or HLA-A*31:01-matched SMAD4WT, and HLA-DRB1*04:01-matched SMAD4G365D peptides in two completed cases, respectively. Moreover, SMAD4WT-specific CD8+ effector memory T cells were amplified. However, an attenuation of the acquired immune response was observed 6 months after one course of cancer vaccination as the disease progressed. This study confirmed the safety and tolerability of HPL-APCs in advanced and recurrent cancers refractory to standard therapy and is the first clinical report to demonstrate the immunoinducibility of personalized cancer vaccines using HPL-APCs. Phase II clinical trials to determine immune responses with optimized adjuvant drugs and continued administration are expected to demonstrate efficacy.

Expandable Sendai-Virus-Reprogrammed Human iPSC-Neuronal Precursors: In Vivo Post-Grafting Safety Characterization in Rats and Adult Pig.

One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.

Derivation of Sendai-Virus-Reprogrammed Human iPSCs-Neuronal Precursors: In Vitro and In Vivo Post-grafting Safety Characterization.

The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.

Selective vulnerability of human-induced pluripotent stem cells to dihydroorotate dehydrogenase inhibition during mesenchymal stem/stromal cell purification.

The use of induced mesenchymal stem/stromal cells (iMSCs) derived from human induced pluripotent stem cells (hiPSCs) in regenerative medicine involves the risk of teratoma formation due to hiPSCs contamination in iMSCs. Therefore, eradicating the remaining undifferentiated hiPSCs is crucial for the effectiveness of the strategy. The present study demonstrates the Brequinar (BRQ)-induced inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in de novo pyrimidine biosynthesis, selectively induces apoptosis, cell cycle arrest, and differentiation; furthermore, it promotes transcriptional changes and prevents the growth of 3-dimensional hiPSC aggregates. Contrastingly, BRQ-treated iMSCs showed no changes in survival, differentiation potential, or gene expression. The results suggest that BRQ is a potential agent for the effective purification of iMSCs from a mixed population of iMSCs and hiPSCs, which is a crucial step in successful iMSC-based therapy.

The Detection of Immunity against WT1 and SMAD4P130L of EpCAM+ Cancer Cells in Malignant Pleural Effusion.

Malignant pleural effusion (MPE) provides a liquid tumor microenvironment model that includes cancer cells and immune cells. However, the characteristics of tumor antigen-specific CD8+ T cells have not been investigated in detail. Here, we analyzed MPE samples taken from a patient with pancreatic cancer who received a dendritic cell vaccine targeting Wilms' Tumor 1 (WT1) antigen over the disease course (two points at MPE1st and 2nd, two months after MPE1st). Epithelial cell adhesion molecule (EpCAM)+ cancer cells (PD-L1- or T cell immunoglobulin mucin-3, TIM-3-), both PD-1 or TIM-3 positive CD8+ T cells, and CD14+CD68+CD163+TIM-3+ macrophages increased from the MPE1st to MPE2nd. The ratio of WT1-specific cytotoxic lymphocytes (WT1-CTLs) to MPE CD8+ T cells and IFN-γ secretion of WT1-CTLs were reduced with disease progression. Coincidentally, the fraction of central memory T (TCM) of WT1-CTLs was decreased. On the other hand, CD8+ T cells in response to SMAD4P130L, which is homogeneously expressed in EpCAM+ cancer cells, were detected using in vitro expansion with the HLA-A*11:01 restrictive SVCVNLYH neoantigen. Furthermore, the CD8+ T cell response to SMAD4P130L was diminished following remarkably decreased numbers of CD8+ TCM in MPE samples. In conclusion, CD8+ T cells responding to WT1 or SMAD4P130L neoantigen expressed in EpCAM+ pancreatic cancer cells were detected in MPE. A tumor antigen-specific immune response would provide novel insight into the MPE microenvironment.

Different In Vitro-Generated MUTZ-3-Derived Dendritic Cell Types Secrete Dexosomes with Distinct Phenotypes and Antigen Presentation Potencies.

Human dendritic cell (DC) dexosomes were evaluated for their function and preclinical validation for vaccines. Dexosomes are small DC-secreted vesicles that contain absorbing immune signals. Vaccine manufacturing requires a significant number of monocyte-derived DCs (Mo-DCs) from donor blood; thus, Mo-DC dexosomes are expected to serve as novel materials for cancer vaccination. In this study, we characterized a potential dexosome model using immature and mature MUTZ3-derived DCs (M-imIL-4-DC, M-imIFN-DC, M-mIL-4-DC, and M-mIFN-DC) and their dexosomes (M-imIL-4-Dex, M-imIFN-Dex, M-mIL4-Dex, and M-mIFN-Dex). Despite the lack of significant differences in viability, M-mIFN-DC showed a significantly higher level of yield and higher levels of maturation surface markers, such as CD86 and HLA-ABC, than M-mIL-4-DC. In addition, M-mIFN-Dex expressed a higher level of markers, such as HLA-ABC, than M-mIL-4-Dex. Furthermore, M-mIFN-Dex exhibited a higher level of antigen presentation potency, as evaluated using a MART-1 system, than either M-imIFN-Dex or M-mIL-4-Dex. We found that M-mIFN-Dex is one of the four types of MUTZ3-derived DCs that harbor potential immunogenicity, suggesting that DC dexosomes could be useful resources in cancer immunotherapy.

Quality Verification with a Cluster-Controlled Manufacturing System to Generate Monocyte-Derived Dendritic Cells.

Dendritic cell (DC) vaccines for cancer immunotherapy have been actively developed to improve clinical efficacy. In our previous report, monocyte-derived DCs induced by interleukin (IL)-4 with a low-adherence dish (low-adherent IL-4-DCs: la-IL-4-DCs) improved the yield and viability, as well as relatively prolonged survival in vitro, compared to IL-4-DCs developed using an adherent culture protocol. However, la-IL-4-DCs exhibit remarkable cluster formation and display heterogeneous immature phenotypes. Therefore, cluster formation in la-IL-4-DCs needs to be optimized for the clinical development of DC vaccines. In this study, we examined the effects of cluster control in the generation of mature IL-4-DCs, using cell culture vessels and measuring spheroid formation, survival, cytokine secretion, and gene expression of IL-4-DCs. Mature IL-4-DCs in cell culture vessels (cluster-controlled IL-4-DCs: cc-IL-4-DCs) displayed increased levels of CD80, CD86, and CD40 compared with that of la-IL-4-DCs. cc-IL-4-DCs induced antigen-specific cytotoxic T lymphocytes (CTLs) with a human leukocyte antigen (HLA)-restricted melanoma antigen recognized by T cells 1 (MART-1) peptide. Additionally, cc-IL-4-DCs produced higher levels of IFN-γ, possessing the CTL induction. Furthermore, DNA microarrays revealed the upregulation of BCL2A1, a pro-survival gene. According to these findings, the cc-IL-4-DCs are useful for generating homogeneous and functional IL-4-DCs that would be expected to promote long-lasting effects in DC vaccines.

Identification of CD56dim subpopulation marked with high expression of GZMB/PRF1/PI-9 in CD56+ interferon-α-induced dendritic cells.

As the sentinels of innate and adaptive immune system, dendritic cells (DCs) have been considered to hold a great promise for medical application. Among the diverse types of DCs, monocyte-derived DCs (mo-DCs) generated in vitro have been most commonly employed. We have been improving the culture protocol and devised a protocol to produce mature interferon-α-induced DCs (IFN-DCs), hereinafter called (mat)IFN-DCs. While exploring the relationship between the expression of CD56 and the cytotoxic activity of (mat)IFN-DCs, we unexpectedly found that sorting of (mat)IFN-DCs with CD56 antibody-coated microbeads (MB) resulted in fractionating cells with tumoricidal activity into the flow-through (FT) but not MB-bound fraction. We uncovered that the FT fraction contains cells expressing low but substantial level of CD56. Moreover, those cells express granzyme B (GrB), perforin (PFN), and serpin B9 at high levels. By employing a specific inhibitor of PFN, we confirmed that direct tumoricidal activity relies on the GrB/PFN pathway. We designated subpopulation in FT fraction as CD56dim and that in CD56 positively sorted fraction as CD56bright , respectively. This is the first time, to our knowledge, to identify subpopulations of CD56-positive IFN-DCs with distinct tumoricidal activity which is ascribed to high expression of the components of GrB/PFN pathway.

Interferon-α-Induced Dendritic Cells Generated with Human Platelet Lysate Exhibit Elevated Antigen Presenting Ability to Cytotoxic T Lymphocytes.

Given the recent advancements of immune checkpoint inhibitors, there is considerable interest in cancer immunotherapy provided through dendritic cell (DC)-based vaccination. Although many studies have been conducted to determine the potency of DC vaccines against cancer, the clinical outcomes are not yet optimal, and further improvement is necessary. In this study, we evaluated the potential ability of human platelet lysate (HPL) to produce interferon-α-induced DCs (IFN-DCs). In the presence of HPL, IFN-DCs (HPL-IFN-DCs) displayed high viability, yield, and purity. Furthermore, HPL-IFN-DCs displayed increased CD14, CD56, and CCR7 expressions compared with IFN-DCs produced without HPL; HPL-IFN-DCs induced an extremely higher number of antigen-specific cytotoxic T lymphocytes (CTLs) than IFN-DCs, which was evaluated with a human leukocyte antigen (HLA)-restricted melanoma antigen recognized by T cells 1 (MART-1) peptide. Additionally, the endocytic and proteolytic activities of HPL-IFN-DCs were increased. Cytokine production of interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α was also elevated in HPL-IFN-DCs, which may account for the enhanced CTL, endocytic, and proteolytic activities. Our findings suggest that ex-vivo-generated HPL-IFN-DCs are a novel monocyte-derived type of DC with high endocytic and proteolytic activities, thus highlighting a unique strategy for DC-based immunotherapies.

Dendritic Cells Pre-Pulsed with Wilms' Tumor 1 in Optimized Culture for Cancer Vaccination.

With recent advances in cancer vaccination therapy targeting tumor-associated antigens (TAAs), dendritic cells (DCs) are considered to play a central role as a cell-based drug delivery system in the bioactive immune environment. Ex vivo generation of monocyte-derived DCs has been conventionally applied in adherent manufacturing systems with separate loading of TAAs before clinical use. We developed DCs pre-pulsed with Wilms' tumor (WT1) peptides in low-adhesion culture maturation (WT1-DCs). Quality tests (viability, phenotype, and functions) of WT1-DCs were performed for process validation, and findings were compared with those for conventional DCs (cDCs). In comparative analyses, WT1-DCs showed an increase in viability and recovery of the DC/monocyte ratio, displaying lower levels of IL-10 (an immune suppressive cytokine) and a similar antigen-presenting ability in an in vitro cytotoxic T lymphocytes (CTLs) assay with cytomegalovirus, despite lower levels of CD80 and PD-L2. A clinical study revealed that WT1-specific CTLs (WT1-CTLs) were detected upon using the WT1-DCs vaccine in patients with cancer. A DC vaccine containing TAAs produced under an optimized manufacturing protocol is a potentially promising cell-based drug delivery system to induce acquired immunity.

In Vivo Administration of Recombinant Human Granulocyte Colony-Stimulating Factor Increases the Immune Effectiveness of Dendritic Cell-Based Cancer Vaccination.

Significant recent advances in cancer immunotherapeutics include the vaccination of cancer patients with tumor antigen-associated peptide-pulsed dendritic cells (DCs). DC vaccines with homogeneous, mature, and functional activities are required to achieve effective acquired immunity; however, the yield of autologous monocyte-derived DCs varies in each patient. Priming with a low dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 16-18 h prior to apheresis resulted in 50% more harvested monocytes, with a significant increase in the ratio of CD11c+CD80+ DCs/apheresed monocytes. The detection of antigen-specific cytotoxic T lymphocytes after Wilms' tumor 1-pulsed DC vaccination was higher in patients treated with rhG-CSF than those who were not, based on immune monitoring using tetramer analysis. Our study is the first to report that DC vaccines for cancer immunotherapy primed with low-dose rhG-CSF are expected to achieve higher acquired immunogenicity.

Soluble ST2 suppresses IL-5 production by human basophilic KU812 cells, induced by epithelial cell-derived IL-33.

BACKGROUND: Epithelial cell-derived IL-33 has an important role in the initiation and activation of innate allergic inflammation. IL-33 acts as a cytokine through the ST2 receptor (ST2L) and it stimulates the production of Th2 cytokines. Soluble ST2 (sST2) may regulate Th2 responses by neutralizing the activity of IL-33. Basophils express ST2L and produce IL-5 in response to IL-33. However, the role of the epithelial cell-basophil interaction and sST2 in IL-5 production remains unclear. METHODS: Cultured human bronchial epithelial (hBE33) cells, that contained the human IL-33 gene (i.e., hBE33 cells) and a human basophilic cell line, KU812 cells, were used to study the epithelial cell-basophil interaction in the production of IL-5 induced by HDM. RESULTS: At 15 min after incubation, HDM stimulated the rapid release of IL-33 from cultured hBE33 cells. IL-33 and the supernatant of HDM-treated hBE33 cells stimulated IL-5 production from KU812 cells. Anti-IL-33 antibody and anti-ST2 antibody treatment of KU812 cells suppressed IL-5 production, which had been induced by the supernatant of HDM-treated hBE33 cells. The hBE33 cells secreted sST2 in a time-dependent manner. The production of sST2 by KU812 cells co-cultured with hBE33 cells was significantly increased, compared with KU812 cells cultured with the supernatant of hBE33 cells. Soluble ST2 suppressed IL-5 production by KU812 cells, which was induced by the supernatant of HDM-treated hBE33 cells. CONCLUSIONS: Epithelial cell-derived IL-33 promoted IL-5 production by KU812 cells. The subsequently produced sST2 has important roles in regulating Th2 responses.

Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs.

The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)-mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis.

A mechanism of interleukin-25 production from airway epithelial cells induced by Japanese cedar pollen.

IL-25 likely has vital roles in initiating and activating type-2 immune responses in AR. We hypothesized that the molecules produced IL-25 by allergen-producing organisms such as JC is involved in the pathogenesis of AR. Participants included 13 patients with Japanese cedar pollinosis and 10 HCs. We measured the IL-25 protein concentration in nasal secretions and in culture supernatants of PNECs. NHBE cells were stimulated with pharmacological and immunological agents and JC. The IL-25 concentration in nasal secretions was significantly higher in patients with Japanese cedar pollinosis than in HCs. JC stimulated IL-25 production from PNECs. TNF-α, IL-4, and IL-13 significantly enhanced JC-induced IL-25 production; their activation by serine proteases was sufficient to enhance IL-25 production. Furthermore, the NADPH oxidase activity, including JC enhanced IL-25 production. A better understanding of JC-induced IL-25 production by epithelial cells may allow the development of novel therapeutic and preventive strategies for Japanese cedar pollinosis.

The effect of calprotectin on TSLP and IL-25 production from airway epithelial cells.

BACKGROUND: Calprotectin is a heterodimer complex of the S100A8 and S100A9 proteins, and has various functions as an innate mediator at the sites of inflammation. The aim of this study was to elucidate the roles of calprotectin in the eosinophilic chronic rhinosinusitis (ECRS). METHODS: Allergen-induced production of calprotectin was evaluated in cultured normal human bronchial epithelial (NHBE) cells by ELISA and RT-PCR. We then examined the roles of calprotectin on Alternaria alternata (Alternaria)-induced production of thymic stromal lymphopoietin (TSLP) and IL-25 in NHBE cells. The extracellular concentration and allergen-induced secretion of calprotectin in cultured primary nasal epithelial (PNE) cells were examined and compared between patients with ECRS and non-eosinophilic chronic rhinosinusitis (NECRS). RESULTS: Alternaria, house dust mites, protease from Staphylococcus aureus, papain, trypsin, polyinosinic:polycytidylic acid and lipopolysaccharide stimulated calprotectin production in the cultured NHBE cells. The combination of calprotectin and ATP stimulated the production of TSLP and IL-25 in NHBE cells, and calprotectin stimulated Alternaria-induced production of TSLP and IL-25, which was suppressed by blocking P2 purinergic receptors and by treatment with siRNA for S100A8, S100A9 or calprotectin receptors (Toll-like receptor 4 or receptor for advanced glycation end products). Allergen-induced calprotectin production was significantly stimulated in PNE cells from patients with ECRS. CONCLUSIONS: These results indicate that calprotectin enhances the allergen-induced Th2-type inflammatory responses in airway epithelial cells via the secretion of TSLP and IL-25, and that calprotectin secreted by the epithelial cells may be involved in the pathogenesis of ECRS.

Endogenous Protease Inhibitors in Airway Epithelial Cells Contribute to Eosinophilic Chronic Rhinosinusitis.

RATIONALE: Cystatin A and SPINK5 are endogenous protease inhibitors (EPIs) that may play key roles in epithelial barrier function. OBJECTIVES: To investigate the roles of EPIs in the pathogenesis of chronic rhinosinusitis (CRS). METHODS: We examined the expression of cystatin A and SPINK5 in the nasal epithelial cells of patients with CRS. Additionally, the in vitro effects of recombinant EPIs on the secretion of the epithelial-derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin in airway epithelial cells, and the in vivo effects of recombinant EPIs in the nasal epithelium of mice exposed to multiple airborne allergens (MAA) were examined. MEASUREMENTS AND MAIN RESULTS: Compared with control subjects and patients with noneosinophilic CRS, patients with eosinophilic CRS showed significantly lower protein and mRNA expression of cystatin A and SPINK5 in the nasal epithelium. Allergen-induced production of IL-25, IL-33, and thymic stromal lymphopoietin in normal human bronchial epithelial cells was inhibited by treatment with recombinant cystatin A or SPINK5. Conversely, the production of these cytokines was increased when cystatin A or SPINK5 were knocked down with small interfering RNA. Chronic MAA exposure induced goblet cell metaplasia and epithelial disruption in mouse nasal epithelium and decreased the tissue expression and nasal lavage levels of cystatin A and SPINK5. Intranasal instillations of recombinant EPIs attenuated this MAA-induced pathology. CONCLUSIONS: Cystatin A and SPINK5 play an important role in protecting the airway epithelium from exogenous proteases. The preservation of EPIs may have a therapeutic benefit in intractable airway inflammation, such as eosinophilic CRS.

HMGB1-TLR4 signaling contributes to the secretion of interleukin 6 and interleukin 8 by nasal epithelial cells.

BACKGROUND: Alarmins play important roles in the pathogenesis of inflammatory and autoimmune diseases. However, the role of the alarmin protein high-mobility group box 1 (HMGB1) in upper airway inflammation is unclear. OBJECTIVE: To determine if HMGB1 is present in the nasal mucosa and, if so, to elucidate its role in upper airway inflammation. METHODS: Nasal secretions were collected from a total of 32 patients with chronic rhinosinusitis with nasal polyp, allergic rhinitis, and control subjects. The concentration of HMGB1 in nasal secretions and its tissue and cellular localization were examined by enzyme immunoassays and immunofluorescent staining of nasal polyps and cultured nasal epithelial cells. We then examined whether nasal epithelial cells secrete HMGB1 after inflammatory stimulation by tumor necrosis factor (TNF) α. The effects of HMGB1 on the production and secretion of interleukin (IL) 6 and IL-8 were also examined in cultured nasal epithelial cells. RESULTS: Significantly higher concentrations of HMGB1 were found in nasal secretions from patients with chronic rhinosinusitis with nasal polyp or allergic rhinitis compared with the control subjects. HMGB1 expression was localized in the nuclei of epithelial cells and other constitutive cells in nasal polyps and in the nuclei of cultured nasal epithelial cells. TNF-α stimulated the production and secretion of HMGB1 by cultured nasal epithelial cells. HMGB1 stimulated the production and secretion of IL-6 and IL-8 by cultured nasal epithelial cells, and anti-toll-like receptor 4 blocking antibody significantly inhibited HMGB1-induced secretion of IL-6 and IL-8. CONCLUSIONS: Nasal secretions contain substantial amounts of HMGB1. TNF-α stimulates the production of HMGB1, which, in turn, upregulates the production and secretion of IL-6 and IL-8 by nasal epithelial cells via toll-like receptor 4, which indicated that HMGB1 plays an important role in the pathogenesis of upper airway inflammation.

Epithelial Cell-Derived Cytokines Contribute to the Pathophysiology of Eosinophilic Chronic Rhinosinusitis.

The epithelial cell-derived cytokines, thymic stromal lymphopoietin (TSLP), interleukin (IL)-25, and IL-33 induce T helper 2 type immune responses. In the present study, we investigate the role of these cytokines in the pathophysiology of eosinophilic chronic rhinosinusitis (ECRS). Nasal tissue specimens from chronic rhinosinusitis patients were assayed for the expression of TSLP, IL-25, IL-33, protease-activated receptor (PAR)-2, and P2Y2 receptor (P2Y2R). Cytokine production in cultured nasal epithelial cells (PNECs) was also examined. The mRNA levels of TSLP and IL-25 and the concentrations of IL-25 and IL-33 increased in PNECs from ECRS patients. Immunohistological staining demonstrated that TSLP, IL-25, and IL-33 were localized in the epithelial cells of nasal polyps, and that their expression was increased in ECRS. The mRNA levels of TSLP and IL-25 correlated with the clinical severity of ECRS, as indicated by the computed tomography score. The TSLP mRNA levels and IL-33 protein concentration correlated with the number of eosinophils in the nasal polyps of patients with ECRS. Airborne allergen-induced cytokine production increased in PNECs of these patients. Expression levels of the PAR-2 and P2Y2R increased in cultured PNECs and nasal polyps from patients with ECRS. The results indicate that increased induction and expression of TSLP, IL-25, and IL-33 from nasal epithelial cells contribute to the pathophysiology of ECRS.

SS18-SSX, the Oncogenic Fusion Protein in Synovial Sarcoma, Is a Cellular Context-Dependent Epigenetic Modifier.

The prevalence and specificity of unique fusion oncogenes are high in a number of soft tissue sarcomas (STSs). The close relationship between fusion genes and clinicopathological features suggests that a correlation may exist between the function of fusion proteins and cellular context of the cell-of-origin of each tumor. However, most STSs are origin-unknown tumors and this issue has not yet been investigated in detail. In the present study, we examined the effects of the cellular context on the function of the synovial sarcoma (SS)-specific fusion protein, SS18-SSX, using human pluripotent stem cells (hPSCs) containing the drug-inducible SS18-SSX gene. We selected the neural crest cell (NCC) lineage for the first trial of this system, induced SS18-SSX at various differentiation stages from PSCs to NCC-derived mesenchymal stromal cells (MSCs), and compared its biological effects on each cell type. We found that the expression of FZD10, identified as an SS-specific gene, was induced by SS18-SSX at the PSC and NCC stages, but not at the MSC stage. This stage-specific induction of FZD10 correlated with stage-specific changes in histone marks associated with the FZD10 locus and also with the loss of the BAF47 protein, a member of the SWI/SNF chromatin-remodeling complex. Furthermore, the global gene expression profile of hPSC-derived NCCs was the closest to that of SS cell lines after the induction of SS18-SSX. These results clearly demonstrated that the cellular context is an important factor in the function of SS18-SSX as an epigenetic modifier.

Mutant IDH1 Dysregulates the Differentiation of Mesenchymal Stem Cells in Association with Gene-Specific Histone Modifications to Cartilage- and Bone-Related Genes.

Somatic mutations in the isocitrate dehydrogenase (IDH)1/2 genes endow encoding proteins with neomorphic activity to produce the potential oncometabolite, 2-hydroxyglutarate (2-HG), which induces the hypermethylation of histones and DNA. The incidence of IDH1/2 mutations in cartilaginous tumors was previously shown to be the highest among various types of tumors, except for those in the central nervous system. Mutations have been detected in both benign (enchondromas) and malignant (chondrosarcomas) types of cartilaginous tumors, whereas they have rarely been found in other mesenchymal tumors such as osteosarcomas. To address this unique tumor specificity, we herein examined the effects of IDH1 R132C, which is the most prevalent mutant in cartilaginous tumors, on the differentiation properties of human mesenchymal stem cells (hMSCs). The induction of the IDH1 R132C gene into MSCs markedly increased the amount of 2-HG and up-regulated global histone methylation. The induction of IDH1 R132C promoted the chondrogenic differentiation of hMSCs by enhancing the expression of SOX9 and COL2A1 genes in association with an increase in the active mark (H3K4me3), but disrupted cartilage matrix formation. On the other hand, IDH1 R132C inhibited expression of the ALPL gene in association with an increase in the repressive mark (H3K9me3), and subsequently inhibited the osteogenic properties of hMSCs and human osteosarcoma cells. Since osteogenic properties are an indispensable feature for the diagnosis of osteosarcoma, the inhibitory effects of IDH1 R132C on osteogenic properties may contribute to the lack of osteosarcomas with the IDH1 R132C mutation. These results suggested that IDH1 R132C contributed to the formation of cartilaginous tumors by dysregulating the chondrogenic and osteogenic differentiation of hMSCs via gene-specific histone modulation.