Hiroyasu Inoue

Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells.

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34+ cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-γ production. However, NK cell cytotoxicity and IFN-γ production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-γ Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-γ production plays an important role in Fas-mediated cytotoxicity.

Tyrosine Phosphorylation of I-κB Kinase α/β by Protein Kinase C-Dependent c-Src Activation Is Involved in TNF-α-Induced Cyclooxygenase-2 Expression

The signaling pathway involved in TNF-α-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src kinase inhibitor (PP2) attenuated TNF-α- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity. TNF-α- or TPA-induced I-κB kinase (IKK) activation was also blocked by these inhibitors, which reversed I-κBα degradation. Activation of c-Src and Lyn kinases, two Src family members, was inhibited by the PKC, tyrosine kinase, or Src kinase inhibitors. The dominant-negative c-Src (KM) mutant inhibited induction of COX-2 promoter activity by TNF-α or TPA. Overexpression of the constitutively active PKCα (PKCα A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects were inhibited by the dominant-negative c-Src (KM), NF-κB-inducing kinase (NIK) (KA), or IKKβ (KM) mutant. The dominant-negative PKCα (K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIK overexpression. In coimmunoprecipitation experiments, IKKα/β was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF-α or TPA treatment. Two tyrosine residues, Tyr188 and Tyr199, near the activation loop of IKKβ, were identified to be crucial for NF-κB activation. Substitution of these residues with phenylalanines attenuated COX-2 promoter activity and c-Src-dependent phosphorylation of IKKβ induced by TNF-α or TPA. These data suggest that, in addition to activating NIK, TNF-α also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK and converge at IKKα/β, and go on to activate NF-κB, via serine phosphorylation and degradation of IκB-α, and, finally, to initiate COX-2 expression.

Bradykinin B2 Receptor Mediates NF-κB Activation and Cyclooxygenase-2 Expression via the Ras/Raf-1/ERK Pathway in Human Airway Epithelial Cells

In this study, we investigated the signaling pathways involved in bradykinin (BK)-induced NF-κB activation and cyclooxygenase-2 (COX-2) expression in human airway epithelial cells (A549). BK caused concentration- and time-dependent increase in COX-2 expression, which was attenuated by a selective B2 BK receptor antagonist (HOE140), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), an NF-κB inhibitor (pyrrolidine dithiocarbate), and an IκB protease inhibitor (l-1-tosylamido-2-phenylethyl chloromethyl ketone). The B1 BK receptor antagonist (Lys-(Leu8)des-Arg9-BK) had no effect on COX-2 induction by BK. BK-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the κB site deletion of COX-2 construct. BK-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser338 by BK was inhibited by manumycin A and GW 5074. BK-induced ERK activation was inhibited by HOE140, manumycin A, GW 5074, and PD 098059. Stimulation of cells with BK activated IκB kinase αβ (IKKαβ), IκBα phosphorylation, IκBα degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-κB-specific DNA-protein complex, and κB-luciferase activity. BK-mediated increase in IKKαβ activity and formation of the NF-κB-specific DNA-protein complex were inhibited by HOE140, a Ras dominant-negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Our results demonstrated for the first time that BK, acting through B2 BK receptor, induces activation of the Ras/Raf-1/ERK pathway, which in turn initiates IKKαβ and NF-κB activation, and ultimately induces COX-2 expression in human airway epithelial cell line (A549).

Allogeneic haematopoietic cell transplantation from alternative donors with a conditioning regimen of low-dose irradiation, fludarabine and cyclophosphamide in Fanconi anaemia

A pilot study was undertaken using a fludarabine‐based conditioning regimen to improve haematopoietic cell transplantation (HCT) from alternative donors in 27 Fanconi anaemia (FA) patients. Patients were conditioned with 150–180u2003mg/m2 of fludarabine, 40u2003mg/kg of cyclophosphamide, 5–10u2003mg/kg of antithymocyte globulin, and 300–450u2003cGy of thoracoabdominal/total body irradiation. One patient who received unrelated cord blood transplantation failed to engraft, another patient died of sepsis. The 1‐year overall survival was 96·3% (95% CI, 89–100). This conditioning regimen exerted an immunosuppressive effect that enabled durable engraftment in alternative donor HCT without severe toxicity.

The Kinetics of Immune Reconstitution after Cord Blood Transplantation and Selected cd34+ Stem Cell Transplantation in Children: Comparison with Bone Marrow Transplantation

The present study compares immune reconstitution after allogeneic cord blood transplantation (CBT) and CD34+ stem cell transplantation (CD34-SCT) with that after bone marrow transplantation (BMT). Eighty-eight children who underwent CBT (20 patients), BMT (58), and CD34-SCT (10) were enrolled, and lymphocytes and T-, B-, and natural killer—lymphocyte subsets were monitored for more than 5 years after transplantation. CBT recipients showed significant increases in (1) total lymphocyte counts (P < .001), (2) CD4+/CD8+ cell ratios (P < .01), (3) CD4+ and CD4+CD45RA+ cells (P < .001), (4) CD8+CD11b+ cells (P < .001), and (5) CD19+ and CD19+CD5+ cells (P < .0001) and marked decreases in the frequencies of CD8+ and CD8+CD11b- cells (P < .0001). CD34-SCT recipients showed lower lymphocyte counts in the first 6 months and an emergence of lymphocyte and CD4+CD45RA+ cells at approximately 9 months and 1 year. Both CBT and CD34-SCT recipients showed increased frequencies of CD56+ cells at 1 month (CD34-SCT versus BMT, P < .001) but decreased frequencies after 6 months (CBT versus BMT, P < .001). Lymphoproliferative responses to exogenous interleukin 2 were constantly lower in CBT and CD34-SCT recipients than in BMT recipients.These results suggest that the delay in immune reconstitution after CBT in the early phase was mainly qualitative and related to the immaturity of cells, whereas the delay in CD34-SCT was mainly quantitative in the first several months. Int J Hematol. 2003;77:399-407.

Human umbilical cord blood NK T cells kill tumors by multiple cytotoxic mechanisms

Natural killer (NK) T cells are restricted by CD1d and play an important role in the rejection of malignant tumors, but how kill these tumors is unclear. To investigate this, we cultured Valpha24+CD4+ NK T cells in human umbilical cord blood, which was enriched by immunomagnetic beads. In short-term (4 h) cytotoxicity assays, the NK T cells could kill only those targets expressing CD1d. In longer cytotoxicity assays (20 h), however, the NK T cells were able to kill all the tumors, regardless of CD1d expression. When each of the perforin, Fas-FasL, and TNF-alpha cytotoxic mechanisms were blocked, it was apparent that perforin killing dominated in both the short- and long-term assays. In the short-term assay, perforin killing required that the targets expressed CD1d, but killing was more efficient if Fas was present because then the Fas-FasL mechanism was also used. Thus, cells that lacked Fas and CD1d and were not killed in the 4-h assay, were instead lysed in 20-h assay through a combination of perforin and TNF-alpha killing. NK T cells can kill tumor targets by perforin, Fas-FasL, and TNF-alpha mechanisms. TNF-alpha killing requires longer contact between effectors and targets, suggesting that TNF-alpha acts by enhancing perforin killing.

Changes in thyroid function after bone marrow transplant in young patients.

Abstract Background :u2002Changes in thyroid function among young patients who received bone marrow transplantation (BMT) were evaluated.

Unmanipulated HLA-haploidentical bone marrow transplantation for the treatment of fatal, nonmalignant diseases in children and adolescents.

Fetomaternal microchimerism has been demonstrated, and immunologic tolerance to unshared HLA antigens between mother and offspring may be suggested. We used T-cell-repleted bone marrow transplantation (BMT) from their HLA-haploidentical mothers to treat 6 patients with fatal nonmalignant diseases. The number of mismatched HLA loci in the graft -versus-host disease (GVHD) direction was 3 in 4 patients and 2 in 2 patients. The number in the host-versus-graft direction was 3 in 4 patients, 2 in 1 patient, and 1 in 1 patient. Microchimerism of inherited paternal antigens was demonstrated in 5 donors, and microchimerism of noninherited maternal antigens was detected in 3 recipients. GVHD prophylaxis consisted of short-course methotrexate, tacrolimus, and mycophenolate mofetil (3 patients) or short-course methotrexate, tacrolimus, and methylprednisolone (1 patient). Engraftment was achieved in 5 patients who had received preconditioning, and T-cell engraftment was confirmed in 1 patient with severe combined immunodeficiency. Acute GVHD developed in 3 patients: grade 1 in 2 patients and grade 2 in 1 patient. Chronic GVHD was observed in 5 patients: localized type in 3 patients and extended type in 2 patients. Five patients were alive 11 to 30 months after BMT and 1 patient died of chronic GVHD. Unmanipulated haploidentical BMT from a maternal donor may be the treatment of choice of poor-prognosis nonmalignant diseases.

Killing activity of human umbilical cord blood-derived TCRValpha24+ NKT cells against normal and malignant hematological cells in vitro: a comparative study with NK cells or OKT3 activated T lymphocytes or with adult peripheral blood NKT cells

Abstract.Purpose: We aimed to determine the effects of human umbilical cord blood (UCB)-derived natural killer T (NKT) cells as immunological effectors against hematological malignancies, as well as auto- or allo-dendritic cells (DCs) or EB transformed cell lines (EBCLs). Materials: TCRVα24+ Vβ11+ UCB- or PB-NKT cells were isolated by sorting and activated by α-galactosylceramide-pulsed autologous DCs. UCB-NK cells were induced from CD34+ cells by stem cell factor plus IL-15. UCB-T cells were primarily activated by anti-CD3 monoclonal antibody. All those effectors were cultured with IL-2 (100xa0U/ml), and their cytotoxic activities were evaluated by 51Cr-release assay. UCB-NKT cells were cultured with IL-12, IL-18 or higher dose of IL-2 (1000xa0U/ml), and again tested for the cytotoxicity against selected targets. Results: UCB-NKT cells exhibited a pattern of killing activity against various hematological malignancies similar to that of UCB-NK cells, but could not kill K562, which was a vulnerable target for NK cells. The level of activity was quite similar to that of PB-NKT cells. In contrast, OKT-3-activated UCB-T lymphocytes showed a stronger and wider spectrum of killing compared with UCB-NK or NKT cells. IL-12, IL-18 or a higher dose of IL-2 upregulated the activity; however several targets, including fresh leukemic cells, still remained resistant. NKT cells killed auto- or allo-DCs at a level similar to that of T cells, but could not kill allo-EBCLs, which were efficiently killed by T cells. While NK cells showed only marginal or no killing against DC or EBCLs. Discussion: The anti-cancer activity of human NKT cells depends on the concentrations or the combination of Th1-cytokines. Basically, those cells might not be contributing to the immune surveillance of hematological malignancies, as shown by a relatively low cytotoxicity against malignant cells, together with the quite strong killing against auto-DCs.

Extensive and long-term ex vivo production of dendritic cells from CD34 positive umbilical cord blood or bone marrow cells by novel culture system using mouse stroma

We previously developed a system using murine strome (HESS-5), which could expand umbilical cord blood (UCB) stem and progenitor cells, especially CD34+/38- cells, in the presence of human recombinant cytokines. In this study, the ability of expanded UCB- or bone marrow (BM)-CD34+ cells to differentiate into dendritic cells (DCs) was examined. DCs could be induced either from short or long term cultured CD34+ cells after switching the cytokines from Flk-2/Flt-3 ligand, stem cell factor (SCF), thrombopoietin (TPO) to granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) (immature type) plus tumor necrosis factor (TNF)-alpha with stimulation by CD40L transfectant (mature type). Each immature or mature UCB-DCs showed a dextran uptake or a potent allo-T lymphocytes proliferative ability, respectively. Furthermore, those DCs from BM significantly stimulated auto-T lymphocytes in an antigen (varicella zoster virus) specific manner. In conclusion, a novel culture system using HESS-5 is useful to support a rapid and sustained generation of primitive myeloid cells which can develop into functional DCs.