Flt3 ligand promotes myeloid dendritic cell differentiation of human hematopoietic progenitor cells: possible application for cancer immunotherapy.

Current in vitro culture systems allow the generation of human dendritic progenitor cells (CFU-DCs). The aim of this study was to assess the effect of Flt3 ligand (FL) on the proliferation of human peripheral blood-derived myeloid CFU-DCs and their differentiation into more committed precursor cells (pDCs) using in vitro culture systems. Immunomagnetically separated CD34+ cells were cultured in serum-free, as well as in serum-containing, liquid suspension cultures to investigate the expansion and/or proliferation/differentiation of CFU-DCs, pDCs, and more mature dendritic cells (DCs). FACS-sorted CD34+Flt3+/- cells were cultured in methylcellulose to assay hematopoietic progenitors, including CFU-DCs. In the clonal cell culture supplemented with granulocyte/macrophage (GM) colony-stimulating factor (CSF), interleukin-4, and tumor necrosis factor alpha, the frequency of CFU-DCs was significantly higher in the CD34+Flt3+ fraction than in the CD34+Flt3- population, thus suggesting functional Flt3 expression on CFU-DCs. Serum-free suspension culture of CD34+ cells revealed the potent effect of FL on the expansion of CFU-DCs in synergy with GM-CSF and thrombopoietin (TPO). In addition, FL strongly induced the maturation of CFU-DCs into functional CD1a+ pDCs in serum-containing liquid suspension culture. Moreover, these FL-generated pDCs showed remarkable potential to differentiate into mature DCs with surface CD83/CD86 expression, which induced a distinct allogeneic T-cell response. These results clearly demonstrate that FL supports not only the proliferation of early hematopoietic progenitor cells, but also the maturation process of committed precursor cells along with the DC-lineage differentiation. Therefore, it is possible to develop a more efficient DC-based cancer immunotherapy using this specific cytokine combination, GM-CSF+TPO+FL in vitro in the near future.

Successful induction of clinically competent dendritic cells from granulocyte colony-stimulating factor-mobilized monocytes for cancer vaccine therapy.

Recent studies have suggested that dendritic cell (DC)-based immunotherapy is one promising approach for the treatment of cancer. We previously studied the clinical toxicity, feasibility, and efficacy of cancer vaccine therapy with peptide-pulsed DCs. In that study, we used granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood monocytes as a cell source of DCs. However, previous investigations have suggested that G-CSF-mobilized peripheral blood monocytes produce reduced levels of proinflammatory cytokines such as interleukin (IL)-12 and tumor necrosis factor (TNF)-alpha. These T helper (Th)-1-type cytokines are thought to promote antitumor immune response. In this study, we assessed the functional abilities of DCs generated from G-CSF-mobilized monocytes obtained from 13 patients with CEA-positive advanced solid cancers. Peripheral blood mononuclear cells were obtained from leukapheresis products collected before and after systemic administration of G-CSF (subcutaneous administration of high-dose [5-10 microg/kg] human recombinant G-CSF for five consecutive days). In vitro cytokine production profiles after stimulation with lipopolysaccharide (LPS) were compared between monocytes with and without G-CSF mobilization. DCs generated from monocytes were also examined with respect to cytokine production and the capacity to induce peptide-specific T cell responses. Administration of G-CSF was found to efficiently mobilize peripheral blood monocytes. Although G-CSF-mobilized monocytes (G/Mo) less effectively produced Th-1-type cytokines than control monocytes (C/Mo), DCs generated from G/Mo restored the same level of IL-12 production as that seen in DCs generated from C/Mo. T cell induction assay using recall antigen peptide and phenotypic analyses also demonstrated that DCs generated from G/Mo retained characteristics identical to those generated from C/Mo. Our results suggest that G-CSF mobilization can be used to collect monocytes as a cell source for the generation of DCs for cancer immunotherapy. DCs generated in this fashion were pulsed with HLA-A24-restricted CEA epitope peptide and administered to patients safely; immunological responses were induced in some patients.

Mobilization of peripheral blood stem cells (PBSCs) after etoposide, adriamycin and cisplatin therapy, and a multimodal cell therapy approach with PBSCs in advanced gastric cancer.

The EAP combination of etoposide (ETP), doxorubicin (ADM) and cisplatin (CDDP) has been reported to be highly active for advanced gastric cancer. However, it is associated with severe myelotoxicity, and its use has declined. We examined whether peripheral blood stem cells (PBSCs) could be mobilized during hematopoietic recovery after EAP, and assessed the possibility of using multimodal cell therapy with PBSCs for the treatment of advanced gastric cancer. Five men with advanced gastric adenocarcinoma were enrolled. All patients were chemotherapy-naïve. EAP (ETP, 360 mg/m2; ADM, 40 mg/m2; CDDP, 80 mg/m2) was given to each patient, and myelotoxicity was carefully monitored. Granulocyte colony-stimulating factor was administered after the neutrophil nadir, and PBSCs were collected by leukapheresis during hematopoietic recovery. The median nadir of the neutrophil count after EAP was 225/ml, occurring between day 17 and 20. Sufficient numbers of PBSCs [CD34(+) cells, CFU-GM] could be mobilized in 4/5 patients. A 45-year-old patient with extended lymph node metastasis received high-dose EAP with peripheral blood stem cell transplantation (PBSCT), followed by cancer vaccine therapy with dendritic cells (DCs), induced from cryopreserved PBSCs. Both high-dose EAP with PBSCT and DC-based immunotherapy was safely performed for the first time against gastric cancer. Although associated with severe myelotoxicity, EAP can mobilize sufficient numbers of PBSCs during hematopoietic recovery. Multimodal cell therapy combining high-dose chemotherapy with PBSCT and DC-based immunotherapy is feasible and can be a reasonable approach in advanced gastric cancer.

Streptococcal preparation OK432 promotes functional maturation of human monocyte-derived dendritic cells.

Streptococcal preparation OK432 is an immunomodulatory agent extensively used as adjuvant therapy for gastric cancer in Japan. OK432 augments the cytotoxic activity of various effector cells such as lymphocytes, macrophages and (natural killer) NK cells and induces the production of multiple cytokines. Dendritic cells (DC) are professional antigen-presenting cells (APC) that can be used for cancer vaccine therapy. In the present study, we investigated the effect of OK432 on the activation of DC. Here we report that OK432 induced phenotypic and functional maturation of human monocyte-derived DC. In vitro culture of immature DC generated from adherent peripheral blood mononuclear cells (PBMC) using granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) with OK432 at various doses (0.01 to 0.1 KE/ml) for 2 days resulted in increased cell surface expression of CD80, CD83, CD86 and ICAM-1 in a dose-dependent manner. The expression of CD83, a selective marker of mature DC, on DC activated by OK432 (OK-DC) was maximally enhanced after 3 days of incubation. Assay of cytokine production in OK-DC after 2 days in culture revealed that OK432 was a strong inducer of IL-12 and interferon-gamma (IFN-gamma). OK432 efficiently augmented the primary allogeneic T-cell responses by DC. This distinct phenotypic profile and allostimulatory capacity of OK-DC was stable for at least 48 h of additional culture in the absence of any cytokines. Moreover, the antiviral cytotoxic T lymphocytes (CTL) response in vitro was also enhanced by the addition of OK432 to the cultures. These findings suggest that OK432 is a potent stimulator of DC, and that stimulated DC are strong inducers of the T helper 1 (Th1)-type response. We conclude that OK-DC are likely candidates for use as an adjuvant for DC-based cancer immunotherapy.

SCID-repopulating cell activity of human cord blood-derived CD34- cells assured by intra-bone marrow injection.

Precise analysis of human CD34-negative (CD34(-)) hematopoietic stem cells (HSCs) has been hindered by the lack of a simple and reliable assay system of these rare cells. Here, we successfully identify human cord blood-derived CD34(-) severe combined immunodeficiency (SCID)- repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) technique. Lineage-negative (Lin(-)) CD34(-) cells did not show SRC activity by conventional tail-vein injection, possibly due to their low levels of homing receptor expression and poor SDF-1/CXCR4- mediated homing abilities, while they clearly showed a high SRC activity by IBMI. They generated CD34(+) progenies not only in the injected left tibia but also in other bones following migration. Moreover, they showed slower differentiating and reconstituting kinetics than CD34(+) cells in vivo. These in vivo-generated CD34(+) cells showed a distinct SRC activity after secondary transplantation, clearly indicating the long-term human cell repopulating capacity of our identified CD34(-) SRCs in nonobese diabetic (NOD)/SCID mice. The unveiling of this novel class of primitive human CD34(-) SRCs by IBMI will provide a new concept of the hierarchy in the human HSC compartment and has important implications for clinical HSC transplantation as well as for basic research of HSC.

Role of human interleukin-9 as a megakaryocyte potentiator in culture.

OBJECTIVE: This study investigated the effect of interleukin-9 (IL-9) on the proliferation and differentiation of human colony-forming unit megakaryocytic progenitor cells (CFU-Meg). MATERIALS AND METHODS: Peripheral blood-derived CD34(+)IL-6R(-) cells were sorted and cultured in the presence of IL-9, erythropoietin (Epo), stem cell factor (SCF), and thrombopoietin (TPO) alone or in combination. The number of pure and mixed megakaryocyte colonies, the size of pure megakaryocyte colonies, the ploidy distribution of megakaryocytes, and proplatelet formation were investigated. RESULTS: Apart from TPO, no single factor could support CFU-Meg-derived colony formation, but each two-factor combination among IL-9, Epo, and SCF supported a few CFU-Meg colonies. Interestingly, the combination of Epo+SCF+IL-9 induced four to six times as many CFU-Meg colonies as any of the two-factor combinations. Neutralizing monoclonal antibodies (mAbs) for IL-9 receptor and c-kit completely abolished this synergistic effect. In contrast, addition of neutralizing anti-c-Mpl or anti-CXCR4 Abs did not influence colony formation, indicating that this synergistic effect was independent of TPO or SDF-1. Moreover, the endogenous production of TPO by cultured CD34(+)IL-6R(-) cells in the presence of Epo+SCF+IL-9 was ruled out by reverse transcriptase polymerase chain reaction for TPO mRNA. Interestingly, the combination of TPO, Epo, SCF, and IL-9 supported the largest number of pure and mixed megakaryocyte colonies, suggesting that this combination of cytokines might recruit primitive megakaryocytic as well as multipotential progenitors. This combination also potently enhanced proplatelet formation compared with TPO alone or a combination of Epo, SCF, and IL-9. CONCLUSION: This study demonstrated for the first time that human IL-9 can potentiate human megakaryocytopoiesis in the presence of Epo and/or SCF.