Abstract 914
Cell cycle regulators could be differentially used among self–renewing stem cells, rapidly expanding progenitor cells, and terminally differentiated cells those clonally replicate. ...Cyclin A is a regulatory subunit for cyclin dependent kinase (Cdk) 1 and Cdk2, and it drives S phase progression as well as transition to G2/M phase in cell cycle. We have previously reported that cyclin A2 is not required for fibroblast replication but it is indispensable in maintenance of self-renewing stem cells, including embryonic stem cells and hematopoietic stem cells (HSCs) (Cell 138 2009). The question is whether cyclin A2 plays a role in proliferation of hematopoietic progenitors downstream of the HSC. Here, we further assessed the requirement of cyclin A2 in non-self-renewing hematopoietic progenitors. Quantitative RT-PCR analysis showed that cyclin A2 was expressed in hematopoietic progenitor cells as well as stem cells, and its expression level is highest in lymphoid-committed progenitor stages of both T and B cell lineages. Thus, in order to test the role of cylin A2 in early lymphopoiesis, we crossed cyclin A2 floxed mice with Rag1-Cre knock-in mice. Because recombination activating gene (RAG)-1 is essential for generation of pre-BCRs and pre-TCRs that are critical for expansion of B and T lymphoid progenitor cells, respectively, we hypothesized that the requirement of Cyclin A2 in early lymphopoiesis can be assessed in this system. As we expected, the Rag1-Cre cyclin A2 floxed/floxed mice were viable, and have normal numbers of HSCs and myeloid progenitors. They, however, displayed severe reduction of mature T and B cell numbers that were only 1/100 - 1/10 of wild-type controls. The number of common lymphoid progenitor was unchanged, but there were severely reduced preB cells in bone marrow and T cell progenitors from CD4-CD8- double negative stage in thymus. Furthermore, cell cycle analysis shows that the Cyclin A2 disrupted progenitors are unable to progress from S to G2/M phase, and in vitro culture clearly showed that those progenitors are unable to proliferate and resulted in apoptosis. These findings clearly demonstrate that cyclin A2 is indispensable not only for self-renewing HSCs, but also for proliferation of T and B cell progenitors.
No relevant conflicts of interest to declare.
We report a 72-year-old man with Waldenström’s macroglobulinemia (WM) in whom diffuse large B-cell lymphoma (DLCL) occurred 17 years after the diagnosis of WM. The malignant cells of both DLCL and WM ...expressed CD20 on their surface. CHOP plus anti-CD20 monoclonal antibody, rituximab, were effective for both diseases, and the patient remains disease-free 17 months later.
We report influenza-induced rhabodmyolysis and congestive heart failure after high-dose therapy and hematopoietic stem cell transplantation for malignant lymphoma. Four months after autologous ...peripheral blood stem cell transplantation for the treatment of malignant lymphoma, a 65-year-old Japanese man developed acute congestive heart failure requiring artificial ventilation and rahbdomyolysis. Since influenza A virus was documented from his nasal cavity, he was diagnosed as rhabdomyolysis and congestive heart failure induced by influenza A infection. Neuraminidase inhibitor (oseltamivir 150 mg/day for 5 days) was administrated, and heart failure and respiratory status were improved. Our experience suggests that early treatment with neuraminidase inhibitor may improve the clinical outcome of influenza-induced rhabdomyolysis and congestive heart failure. (Internal Medicine 42: 1127-1130, 2003)
Abstract 518
PU.1, a hematopoietic transcription factor, is indispensable for development of conventional dendritic cells (cDCs) from hematopoietic stem cells. However, the function of PU.1 in mature ...cDC remains unclear. To test the possible role of PU.1 in mature cDCs, we developed mice lacking PU.1 selectively in mature cDCs (DC-PU.1D/D mice) by crossing a PU.1flox mouse line with a transgenic Itgax (CD11c)-Cre strain. In these mice, cDCs were dramatically reduced in spleen, thymus, lymph node, and skin, down to <40%, <25%, <10% and <5% of DCs in control mice respectively, whereas bone marrow cDCs and common dendritic cells progenitors (CDPs) were not affected. Surprisingly, T cell numbers were significantly decreased in DC-PU.1D/D mice, whereas thymic T cell development was normal, suggesting that maintenance of mature T cell pool might be impaired, presumably by dysfunction of PU.1D/D cDCs. In fact, PU.1D/D cDCs failed to efficiently induce ovalbumin-specific T cell response and to produce inflammatory cytokines in response to Toll like receptor (TLR) stimulation both in vitro and in vivo. The intravenous transfer of spleen PU.1D/D cDCs failed to repopulate the spleen of recipient mice, suggesting their poor survival in vivo. Furthermore, the expression of critical molecules for inflammatory responses was downregulated in PU.1D/D cDCs as compared to normal cDCs. These molecules included Myd88 and NFkB that are downstream molecules of TLR signaling, CD86 that is required for T cell stimulation, and CCR7 that is required for cDC migration. These results clearly show that PU.1 is required for development of the functional cDC pool, and the cDC pool plays a critical role in T cell homeostasis.
No relevant conflicts of interest to declare.
Chronic lymphocytic leukemia (CLL) is characterized by consistent expansion of B cells in peripheral lymphoid organs. CLL B cells frequently express CD5 antigen, and have clonal rearrangement of ...immunoglobulin heavy chain (IGH) gene with restricted usage of V1, V3 and V4 of the variant region. CLL has thus been believed to represent retention or proliferation of abnormal B cell clones presumably with anti-apoptotic potential, or with deregulated response to auto-antigens. In this study, we extensively search for CLL-initiating cells by utilizing the NOD/SCID/IL2rgnull (NOG) xenogeneic transplantation system, in which human hematopoietic stem cells (HSCs) can normally develop multi-lineage cells including polyclonal B and T cells. In the NOG xenotransplant system, neither CD34-CD19+ circulating B cells, nor CD34+CD38+ bone marrow (BM) progenitor populations from 12 CLL patients engrafted even after injection of >106 cells. We then transplanted CD34+CD38− BM HSC population from 7 CLL patients into 13 NOG mice. Injection of as few as 103 cells of the CD34+CD38− BM population resulted in multi-lineage reconstitution. Most of these mice, however, died within 12–24 weeks after xenotransplantation. In 9 mice analyzed by multi-color FACS, 7 mice possessed both CD5+ and CD5− B cell populations, and the remaining 2 mice had only CD5− B cells. These CD5+ or CD5− human B cell populations were purified separately by FACS, and tested for the IGH gene rearrangement. Strikingly, 16 out of 20 B cell populations were clonal with single IGH rearrangement irrespective of their CD5 expression, by multiplex PCR analysis. In contrast, CD34+CD38− HSC populations in CLL patients never had IGH rearrangement. We then directly sequenced PCR products of IGH gene in each B cell clones as well as those in the original CLL cells purified directly from patients' blood. Surprisingly, VDJ recombination in B cell clones developed in NOG mice were different from that of the original CLL clones in all 7 CLL cases. Interestingly, all of these clones used only V1, V3 and V4 regions for their VDJ recombination like primary CLL cells. Furthermore, when the CD34+CD38− BM HSC fraction from single CLL patients was transplanted into a set of 3 mice simultaneously, each mouse developed independent B cell clones with different VDJ recombination in all 3 experiments. The fact that CD34+CD38− HSCs from CLL patients but not those from normal individuals give rise to clonal B cell population in our xenograft model strongly suggests that some genetic abnormality for CLL progression is acquired already at the HSC level in CLL patients. HSCs in CLL patients are multipotent, but once they commit to the B cell lineage, they use preferentially the V1, V3 and V4 regions for IGH recombination. Our hypothesis is that such B cell clones may already be abnormal in that they clonally expand in response, for example, to auto-antigens (xeno-antigens in NOG mice), and they may possibly sequentially receive additional mutations to become clinical CLL. Although this xenograft model may not recapitulate full picture of CLL progression, our data clearly show that primary leukemogenic event occurs at the multipotent HSC stage in human CLL.
Abstract 381
Cyclins are regulatory subunits of cyclin-dependent kinase, and are important components of cell cycle engine. The A-type cyclin is generally the S-phase cyclin. Mammalian cells express ...two A-type cyclins, including cyclin A1 that is exclusively expressed in the testis, and cyclin A2 whose expression is ubiquitous. We have recently reported that cyclin A2 is not required for fibroblast proliferation but it is indispensable in maintenance of self-renewal of stem cells, including embryonic stem cells and hematopoietic stem cells (HSCs) (Cell 138 2009). The question is whether cyclin A2 plays a role in proliferation of hematopoietic progenitors downstream of the HSC. Here we further assessed the requirement of A-type cyclin in non-self-renewing hematopoietic progenitors. Quantitative RT-PCR analysis showed that cyclin A2 was expressed in hematopoietic stem and progenitor cells, but its expression level is highest in lymphoid-committed progenitor stages of both T and B cell lineages. Thus, in order to test the role of cylin A2 in early lymphopoiesis, we crossed cyclin A2 floxed mice with Rag1-Cre knock-in mice. Rag1 expression is initiated at the preproB to the proB stages, and the DN1-DN3 stages in the thymus, while their proliferation is dependent at least upon pre-BCR or pre-TCR signal at these stages. Interestingly, the Rag1-Cre cyclin A2 floxed/floxed mice were viable, and have normal numbers of HSCs and myeloid progenitors in the bone marrow. They, however, displayed severe reduction of T and B cell numbers that were only 1/100 - 1/10 of wild-type controls; the number of common lymphoid progenitor was unchanged, but there were almost complete loss of proB and preB cells. Similarly, all thymic T cell progenitor compartments such as CD4-CD8- double negative, and CD4+CD8+ double positive populations were severely reduced. These findings clearly demonstrate that cyclin A2 is indispensable not only for self-renewal of HSCs, but also for proliferation of T and B cell progenitors.
No relevant conflicts of interest to declare.
The prognosis of allogeneic hematopoietic stem cell transplantation (HSCT) for non-remission hematological malignant diseases is usually unfavorable. The most uncontrollable factor is residual ...disease or relapse. To overcome this problem, intensified conditioning regimens- sequential and/or additional chemotherapy to the standard regimen- could be effective. However, increasing the intensity of conditioning might also lead to more complications.
We retrospectively analyzed 81 patients with non-remission disease who received allogeneic HSCT in our institution between 2007 and 2011.
55.6% in 36 myeloablative conditioning patients and 46.7% in 45 reduced-intensity conditioning patients received intensified conditioning. The 5-year probability of overall survival was 35.0% and 17.1% in the standard and intensified group, respectively (
=0.027). Relapse mortality was 30% in the standard regimen group and 36.6% in the intensified regimen group (
=0.54). Transplant-related mortality (TRM) at 30 and 100 days was 5%, 17.1% (
=0.086) and 27.5%, 34.2% (
=0.52) in the standard and intensified group, respectively. There was no difference in TRM between the 2 groups at 30 days and 100 days.
The results of the study confirm the safety of the intensified conditioning regimen. Meanwhile, it could be considered as one of the few methods available to reduce the tumor burden before HSCT for refractory malignant diseases.
Abstract 1471
Poster Board I-494
PU.1, a hematopoietic transcription factor, is indispensable for development of myelo-lymphoid cells from hematopoietic stem cells (HSCs). PU.1-deficient mice fail to ...develop common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs), resulting in complete loss of dendritic cells (DC) in addition to mature myeloid and lymphoid cells. By disrupting PU.1 specifically at the mature DC stage, we here show that PU.1 is necessary for maintenance of mature DC pool and their functions. We crossed PU.1 floxed/floxed mice with a mouse line harboring the Cre transgene driven by the CD11c-BAC. In these mice, PU.1 gene was disrupted in all conventional DCs but not in other hematopoietic cells, including lymphoid cells, myeloid cells and their progenitors. Development of DC precursors such as Lin−c-KitloFLT3+MCSFR+, FLT3+ CLP and FLT3+CMP were not affected. The number of CD11c+B220− DCs, however, significantly reduced in all lymphoid tissues including the thymus, the spleen, the lymph node and the skin, down to <40%, <25%, <10% and <5% as compared with the wild-type control, respectively. Moreover, the number of mature T cells reduced to ∼60% in the spleen as compared to the control. PU.1-deficient DCs displayed impaired functions to induce antigen-driven T cell proliferation, and to produce inflammatory cytokines (TNFa, IL-6, IL-12) in response to Toll like receptor (TLR) stimulation. These results clearly show that PU.1 is required for development of the peripheral DC pool and for maintenance of their immunological functions, which might be required for maintenance of the peripheral T cell pool.
No relevant conflicts of interest to declare.
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