Reiko Namikawa

Generation of primary antigen-specific human T- and B-cell responsesin immunocompetent SCID-hu mice

Generation of primary antigen-specific human T- and B-cell responses in immunocompetent SCID-hu mice

HUMAN HEMATOLYMPHOID CELLS IN SCID MICE

The severe combined immunodeficient C.B.-17 scid/scid (SCID) mouse has been widely used to study the normal processes of murine lymphoid differentiation. To create an in vivo model of the human hematolymphoid system, this mouse strain has been engrafted with human organ systems (the SCID-hu mouse) or with human peripheral blood mononuclear cells (the hu-PBL-SCID mouse). These mouse models have now been characterized and used to analyze human infectious diseases, hematopoiesis, malignancies and vaccines.

Pharmacologic Expansion of Donor-Derived, Naturally Occurring CD4+Foxp3+ Regulatory T Cells Reduces Acute Graft-versus-Host Disease Lethality Without Abrogating the Graft-versus-Leukemia Effect in Murine Models

Adoptive transfer of regulatory T cells (Tregs) prevents graft-versus-host disease (GVHD) in mouse models, indicating a pivotal role for Tregs in controlling GVHD. The present study demonstrates the efficacy of Tregs pharmacologically induced in vivo in GVHD prevention. A single i.v. administration of a liposomal formulation of α-galactosylceramide (RGI-2001) at the time of allogeneic bone marrow transplantation with spleen cells significantly prolonged the survival of mice experiencing lethal acute GVHD. RGI-2001 expanded donor-derived CD4(+)Foxp3(+) Tregs in the spleen, lymph nodes, and bone marrow in a dose-dependent manner. On day 15 posttransplantation, the spleens of mice treated with RGI-2001 (1 μg/kg) contained 5-fold higher percentages or 10-fold higher numbers of CD4(+)Foxp3(+) Tregs compared with the spleens of untreated mice. Host-specific immunosuppression was introduced in treated mice, whereas the responsiveness to third-party alloantigens and leukemia cells was maintained. Using Foxp3:GFP reporter mice as donors, it was clearly shown that RGI-2001 expanded the pre-existing naturally occurring Tregs (nTregs) in donor spleen cells. Finally, RGI-2001 synergized with a subtherapeutic dose of rapamycin in nTreg expansion and further prolonged survival. Our results provide the first demonstration of the efficacy of nTregs pharmacologically expanded in vivo in preventing acute GVHD without abrogation of the beneficial graft-versus-leukemia effect.

Regulatory Roles of the Ligand for Flk2/Flt3 Tyrosine Kinase Receptor on Human Hematopoiesis

The biological activities of the ligand for the Flk2/Flt3 receptor tyrosine kinase (FL) on human hematopoietic cells are reviewed. In in vitro studies, FL shows relatively few effects by itself on the proliferation and differentiation of hematopoietic cells, but exhibits a potent costimulatory activity in enhancing the proliferation of progenitor cells of multiple lineages. FL promotes the growth of clonogenic myeloid progenitor cells in the presence of other cytokines known to be active on myeloid progenitors, including GM‐CSF, interleukin 3 (IL‐3), kit ligand (KL), M‐CSF and G‐CSF. In addition, FL synergizes with IL‐7 in inducing the proliferation of pro‐B cells, whereas FL has little effect on the growth of clonogenic erythroid progenitors. Furthermore, FL induces the in vitro expansion of the high proliferative potential colony‐forming cells (HPP‐CFC) and stimulates the proliferation of long‐term culture‐initiating cells (LTC‐IC), suggesting an activity on the proliferation of putative stem cells. Thus, FL plays important roles in regulating the proliferation of hematopoietic progenitor cells and, therefore, may have therapeutic applications.

Direct Evaluation of Radiation Damage in Human Hematopoietic Progenitor Cells In Vivo

We have developed techniques by which normal functional elements of human bone marrow can be implanted into immunodeficient C.B-17 scid/scid (SCID) mice. Afterward, long-term multilineage human hematopoiesis is sustained in vivo. We evaluated the effect of irradiation on the function of human bone marrow with this in vivo model. After whole-body X irradiation of the engrafted animals, it was determined that the D0 value of human committed progenitor cells within the human marrow was 1.00 +/- 0.09 (SEM) Gy for granulocyte-macrophage colony-forming units (CFU-GM) and 0.74 +/- 0.12 Gy for erythroid burst-forming units (BFU-E). The effects of irradiation on the hematopoietic elements were reduced when the radioprotective agent WR-2721 was administered prior to irradiation. After low-dose irradiation, recovery of human myelopoiesis was accelerated by treatment with human granulocyte colony-stimulating factor (G-CSF). This small animal model may prove amenable for the analysis of the risk of the exposure of humans to radiation as well as for the development of new modalities for the prevention and treatment of radiation-induced hematopoietic damage.

Progress in the Ex Vivo Expansion of Hematopoietic Progenitors

In this review we describe how studies on the cytokine-stimulated growth of murine bone marrow (BM) progenitors have lead to the observations that large increases in progenitor numbers can be achieved in short-term cytokine-stimulated liquid cultures. Transplantation of these ex vivo expanded murine BM cells was shown to decrease the number of BM cells required to confer radioprotection and to increase the recovery rate of both myeloid and erythroid peripheral blood cells. The ex vivo expansion of murine BM cells does not however, markedly diminish stem cells capable of long-term hematopoietic reconstitution. Investigations on the expansion of human BM, peripheral blood, umbilical cord blood and fetal hematopoietic progenitors have demonstrated that clinically useful increases in progenitor numbers from these tissues are possible. Thus, ex vivo progenitor expansion may soon be of use in transplantation protocols to accelerate hematopoietic reconstitution and in gene therapy protocols if hematopoietic stem cells can be maintained during ex vivo culture.

Ex vivo manipulations alter the reconstitution potential of mobilized human CD34 + peripheral blood progenitors

The phenotype and functions of CD34+ cells isolated from peripheral blood (PB) of steady-state healthy volunteers (ssPB-CD34), and of patients or healthy volunteers after mobilization (mPB-CD34) were investigated. ssPB-CD34+ cells contain a lymphoid cell population that co-express T or B cell markers, while mPB-CD34+ cells lack this population. After 5-day culture, significantly higher levels of expansion in cell, CD34+ cell, and HPP-CFC numbers were induced in ssPB-CD34+ cells, as compared to mPB-CD34+ cells. Hematopoietic reconstitution potential of these ex vivo manipulated CD34+ PBPC was evaluated in SCID-hu mice. It was found that ssPB-CD34+ cells retained the potential to reconstitute human bone marrow (BM), as well as thymus implanted in SCID animals. In contrast, only very low levels of reconstitution were detected in human hematopoietic tissues injected with cultured mPB-CD34+ cells. Reconstitution was restricted to myeloid cells, and no B cell reconstitution in bone marrow, or T cell reconstitution in thymus was achieved by these cells. The loss of B cell reconstitution potential of mPB-CD34+ cells was shown to be induced in a time-dependent manner during culture. These results indicate that mPB-CD34+ cells have different phenotypic and functional properties from ssPB-CD34+ cells. This may affect the efficacy of cell and gene therapy with mobilized PBPC.

Serological analysis of cell surface antigens of HL-60 cells before and after treatment with a phorbol ester tumor promoter

The human HL-60 cell line derived from acute promyelocytic leukemia, consisting of promyelocytic type of cells, was able to differentiate into adherent cells with monocytemacrophage features by the treatment with 12-0-tetradecanoyl phorbol-13-acetate (TPA). Cell surface antigens of HL-60 cells before and after TPA treatment were studied with monoclonal antibodies and four hybridoma clones producing IgM antibodies were established. Two antibodies (HL-21 and HL-47) reacted only with the immunizing TPA-treated HL-60 cells, and HL-1 antibody produced against untreated cells was reactive with both TPA-treated and untreated cells, but HL-5 antibody reacted predominantly with the immunizing untreated cells. Serological reactivity against various types of normal hematopoietic cells and acute leukemias (diagnosed by the French-American-British classification) was studied by immune adherence assay and immuno-electron microscopy. HL-21 antibody was reactive with monocytes and most cases of M4 and M5 types of acute non-lymphocytic leukemia cells. HL-47 antibody did not react with the cells of myelocyte-monocyte lineage or mature lymphocytes, but it did react with one-third of acute lymphocytic leukemia (L1 and L2) cases. Since all HL-47+ cases were included in the group of common ALL antigen positive cases, it was estimated that HL-47 is a differentiation antigen present on lymphocyte precursors, from which null-cell type acute lymphocytic leukemia cells generally originate. HL-1 antibody reacted with the cells of myelocyte-monocyte lineage as well as those of most acute non-lymphocytic leukemias. HL-5 antibody reacted with granulocytes and M2 type of acute myelocytic leukemia cases, and also with M5 type of acute monocytic leukemia cases. Serological studies of these antibodies revealed that TPA can induce to differentiate HL-60 cells not only into HL-21+ macrophage-like cells, but also into HL-47+ lymphoid stem cells. In addition, these antibodies were demonstrated to be very valuable for differential diagnosis of acute leukemias.

Administration of Flk2/Flt3 ligand induces expansion of human high-proliferative potential colony-forming cells in the SCID-hu mouse.

The effects of Flk2/Flt3 ligand (FL) administration on human hematopoiesis were investigated using SCID-hu mice transplanted with human fetal bone fragments. Treatment with recombinant human FL induced significant increases in the frequencies of the high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in steady-state human bone marrow. FL also promoted the expansion of high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in the human bone marrow during the recovery phase after irradiation, which was evident in increases in the frequencies as well as in the absolute numbers of colony-forming cells. Furthermore, higher percentages of CD33+ CD15- cells were found in the marrows treated with FL as compared to that of controls, indicating that FL hastened the recovery of at least some aspect of myelopoiesis after irradiation. These results indicate that FL induces the expansion of primitive hematopoietic progenitor cells in vivo and, therefore, may be useful in treating patients to promote an early hematopoietic recovery after cytoablative therapies.

Human T-Cell Development in SCID-hu Mice

Thymic development proceeds in stages whereby immature T-cell precursors lacking T-cell receptor (TCR) and CD4 and CD8 expression (double negative: DN), progress through a TCRlo CD4+CD8+ (double positive: DP) stage before differentiating into mature TCRhi CD4+CD8− or CD4−CD8+ (single positive: SP) thymocytes.1 DP thymocytes are subjected to positive and negative selection.2 Positive selection results in the development of a peripheral T-cell population capable of responding to antigen presented in the context of self major histocompatibility complex (MHC). Negative selection eliminates T cells with potentially harmful self-reactivity. This process is defined as clonal deletion. DP thymocytes which are not clonally deleted progress to a mature SP stage.