Shunichi Kato

Functional Human T Lymphocyte Development from Cord Blood CD34+ Cells in Nonobese Diabetic/Shi-scid, IL-2 Receptor γ Null Mice

An experimental model for human T lymphocyte development from hemopoietic stem cells is necessary to study the complex processes of T cell differentiation in vivo. In this study, we report a newly developed nonobese diabetic (NOD)/Shi-scid, IL-2Rγ null (NOD/SCID/γcnull) mouse model for human T lymphopoiesis. When these mice were transplanted with human cord blood CD34+ cells, the mice reproductively developed human T cells in their thymus and migrated into peripheral lymphoid organs. Furthermore, these T cells bear polyclonal TCR-αβ, and respond not only to mitogenic stimuli, such as PHA and IL-2, but to allogenic human cells. These results indicate that functional human T lymphocytes can be reconstituted from CD34+ cells in NOD/SCID/γcnull mice. This newly developed mouse model is expected to become a useful tool for the analysis of human T lymphopoiesis and immune response, and an animal model for studying T lymphotropic viral infections, such as HIV.

Unification of Hematopoietic Stem Cell Transplantation Registries in Japan and Establishment of the TRUMP System

There are 4 registries of hematopoietic cell transplantation in Japan; the Japan Society for Hematopoietic Cell Transplantation (JSHCT), Japanese Society of Pediatric Hematology, Japan Marrow Donor Program, and Japan Cord Blood Bank Network; each play an important role in society by reporting the number and outcomes of transplantations and contributing new findings obtained from studies on individual topics. However, there have been a number of issues with the difficulty of analyzing data in overlapping registries and multiple databases at centers affiliated with each of the 4 registry organizations. JSHCT was pivotal in orchestrating the computerization and unification of hematopoietic stem cell transplantation registries for the purpose of resolving these issues and providing a more accurate awareness of hematopoietic stem cell transplantations being performed in Japan. JSHCT played a central role in developing the “Transplant Registry Unified Management Program (TRUMP)” to enable transplantation institutes to manage patient information with emphases on convenience to institutes, safety of patient information, and quality of data management. While enhancing domestic registries, the program seeks to coordinate with other hematopoietic cell transplantation registries around the world to contribute to the development of registries throughout Asia.

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.

HLA mismatch combinations associated with decreased risk of relapse: implications for the molecular mechanism

The finding that the risk of relapse in hematologic malignancy decreases after allogeneic hematopoietic stem cell transplantation (HSCT) has lead to the concept of a graft-versus-leukemia (GVL) effect. However, this beneficial effect is considered to be frequently offset by graft-versus-host disease (GVHD). Thus, improving HSCT outcomes by separating GVL from GVHD is a key clinical issue. This cohort study registered 4643 patients with hematologic malignancies who received transplants from unrelated donors. Six major human leukocyte antigen (HLA) loci were retrospectively genotyped. We identified 4 HLA-Cw and 6 HLA-DPB1 mismatch combinations responsible for a decreased risk of relapse; of these, 8 of 10 combinations were different from those responsible for severe acute GVHD, including all 6 of the HLA-DPB1 combinations. Pairs with these combinations of HLA-DPB1 were associated with a significantly better overall survival than were completely matched pairs. Moreover, several amino acid substitutions on specific positions responsible for a decreased risk of relapse were identified in HLA-Cw, but not in HLA-DPB1. These findings might be crucial to elucidating the mechanism of the decreased risk of relapse on the basis of HLA molecule. Donor selection made in consideration of these results might allow the separation of GVL from acute GVHD, especially in HLA-DPB1 mismatch combinations.

Functional CD5+ B cells develop predominantly in the spleen of NOD/SCID/γcnull (NOG) mice transplanted either with human umbilical cord blood, bone marrow, or mobilized peripheral blood CD34+ cells

OBJECTIVEnHuman CD5+ B cells are the major B cell subset in fetal spleen and umbilical cord blood (CB), and their number gradually diminishes in both spleen and peripheral blood from infancy through childhood while conventional B cells increase. In this study, we investigated whether CD5+ cells differentiate from adult hematopoietic stem cells (HSCs) as well as fetal ones in immunodeficient mice.nnnMETHODSnIn our system, NOD/SCID/gammac(null) (NOG) mice were transplanted with CD34+ cells from CB (hCB model), adult bone marrow (hBM model), and mobilized peripheral blood (hMPB model).nnnRESULTSnIn these model mice, a high proportion of CD19+IgM+CD5+ mature B cells appeared in the spleen, regardless of the CD34+ cell origin, 4 to 8 weeks after transplantation, while the majority were CD19+IgM-CD5- immature B cells in BM. The CD19+CD5- BM cells showed to express CD5 after the coculture with NOG spleen cells. In the sera of immunized hCB model mice with DNP-KLH, antigen-specific IgM but not IgG was enhanced.nnnCONCLUSIONnOur results show that adult CD34+ cells develop into functional CD5+ B cells in NOG spleen as much as fetal CD34+ cells do.

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.

Chronic graft-versus-host disease following umbilical cord blood transplantation: retrospective survey involving 1072 patients in Japan

We have little information on chronic graft-versus-host disease (GVHD) after cord blood transplantation (CBT). We investigated its clinical features in 1072 Japanese patients with hematologic malignancies who received a transplant through the Japan Cord Blood Bank Network. The primary end point was to investigate the incidence of any chronic GVHD. Median age of the patients was 33 years (range, 0-79 years). The cumulative incidence of chronic GVHD 2 years after transplantation was 28%. Chronic GVHD was fatal in 29 patients. Multivariate analysis demonstrated that development of chronic GVHD was favorably associated with both overall survival and event-free survival. Multivariate analysis identified risk factors of chronic GVHD: higher patient body weight, higher number of mismatched antigens for GVHD direction, myeloablative preparative regimen, use of mycophenolate mofetil in GVHD prophylaxis, and development of grades II to IV acute GVHD. Although chronic GVHD is a significant problem after CBT, it is associated with improved survival, perhaps due to graft-versus-malignancy effects.

Cardiovascular toxicity of cryopreserved cord blood cell infusion.

Although infusion of cryopreserved bone marrow or peripheral blood stem cell is associated with a variety of symptoms, there have been no reports detailing the data of infusion-related toxicities of cryopreserved cord blood (CB) units. We prospectively evaluated the incidence and significance of infusion-related toxicities in 34 adult patients undergoing unrelated CB transplantation. Cryopreserved CB units were thawed and immediately infused, unfiltered, through a central intravenous catheter without further manipulation. Heart rate, blood pressure, oxygen saturation and clinical symptoms were recorded during and after infusion. Twenty-four percent of patients experienced non-cardiovascular toxicities related to infusion. The incidence of systolic and diastolic hypertension and bradycardia was 58, 64 and 32%, respectively. Although three patients (9%) with severe systolic hypertension after the infusion required treatment with antihypertensive agents, no patients experienced life-threatening side effects or needed discontinuation of CB unit infusion. Patient or transplant characteristics had no effect on the hypertension and bradycardia related to the infusion of CB. These data suggest that infusion of cryopreserved CB without further manipulation after thawing is safe and well tolerated. However, cardiovascular toxicities including hypertension and bradycardia were frequently observed.

Hemophagocytic lymphohistiocytosis after hematopoietic stem cell transplantation in children: A nationwide survey in Japan

Hemophagocytic lymphohistiocytosis (HLH) is associated with hypercytokinemia in children. Although HLH can be also observed after hematopoietic stem cell transplantation (HSCT), the incidence and clinical features of HLH after HSCT remain obscure.

Growth after bone marrow transplantation in children.

Growth of the patients with hematological malignancies, aplastic anemia, Fanconis anemia, and Wiscott-Aldrich syndrome who had been treated with bone marrow transplantation (BMT) was studied. Fourteen out of 21 patients showed suppression of linear growth after BMT. Recovery of the growth velocity after 1-2 years tended to occur if BMT was performed at younger age. Six of eight patients with chronic graft-versus-host-disease (CGVHD) had impaired growth after BMT, whereas eight of 13 (61%) without CGVHD did. Provocative tests for growth hormone (GH) performed 5-72 months after BMT revealed three boys who showed poor response to more than two different stimuli. Two of these three boys had prolonged suppression of growth. Neither the age at BMT, difference in disease, nor presence of posttransplant growth retardation gave significant difference in the response of GH to provocative tests. It was concluded that approximately two-thirds of marrow-grafted children experienced transient decrease in growth velocity after BMT.