Kazuhiro Mochizuki

Inhibition of histone methylation arrests ongoing graft-versus-host disease in mice by selectively inducing apoptosis of alloreactive effector T cells.

Histone methylation is thought to be important for regulating Ag-driven T-cell responses. However, little is known about the effect of modulating histone methylation on inflammatory T-cell responses. We demonstrate that in vivo administration of the histone methylation inhibitor 3-deazaneplanocin A (DZNep) arrests ongoing GVHD in mice after allogeneic BM transplantation. DZNep caused selective apoptosis in alloantigen-activated T cells mediating host tissue injury. This effect was associated with the ability of DZNep to selectively reduce trimethylation of histone H3 lysine 27, deplete the histone methyltransferase Ezh2 specific to trimethylation of histone H3 lysine 27, and activate proapoptotic gene Bim repressed by Ezh2 in antigenic-activated T cells. In contrast, DZNep did not affect the survival of alloantigen-unresponsive T cells in vivo and naive T cells stimulated by IL-2 or IL-7 in vitro. Importantly, inhibition of histone methylation by DZNep treatment in vivo preserved the antileukemia activity of donor T cells and did not impair the recovery of hematopoiesis and lymphocytes, leading to significantly improved survival of recipients after allogeneic BM transplantation. Our findings indicate that modulation of histone methylation may have significant implications in the development of novel approaches to treat ongoing GVHD and other T cell-mediated inflammatory disorders in a broad context.

Delta-Like Ligand 4 Identifies a Previously Uncharacterized Population of Inflammatory Dendritic Cells That Plays Important Roles in Eliciting Allogeneic T Cell Responses in Mice

Graft-versus-host disease (GVHD) reflects an exaggerated inflammatory allogeneic T cell response in hosts receiving allogeneic hematopoietic stem cell transplantation (HSCT). Inhibition of pan-Notch receptor signaling in donor T cells causes reduction of GVHD. However, which Notch ligand(s) in what APCs is important for priming graft-versus-host reaction remains unknown. We demonstrate that δ-like ligand-4 (Dll4) and Dll4-positive (Dll4high) inflammatory dendritic cells (i-DCs) play important roles in eliciting allogeneic T cell responses. Host-type Dll4high i-DCs occurred in the spleen and intestine of HSCT mice during GVHD induction phase. These Dll4high i-DCs were CD11c+B220+PDCA-1+, resembling plasmacytoid dentritic cells (pDCs) of naive mice. However, as compared with unstimulated pDCs, Dll4high i-DCs expressed higher levels of costimulatory molecules, Notch ligands Jagged1 and Jagged2, and CD11b, and produced more Ifnb and Il23 but less Il12. In contrast, Dll4-negative (Dll4low) i-DCs were CD11c+B220−PDCA-1−, and had low levels of Jagged1. In vitro assays showed that Dll4high i-DCs induced significantly more IFN-γ– and IL-17–producing effector T cells (3- and 10-fold, respectively) than Dll4low i-DCs. This effect could be blocked by anti-Dll4 Ab. In vivo administration of Dll4 Ab reduced donor-alloreactive effector T cells producing IFN-γ and IL-17 in GVHD target organs, leading to reduction of GVHD and improved survival of mice after allogeneic HSCT. Our findings indicate that Dll4high i-DCs represent a previously uncharacterized i-DC population distinctive from steady state DCs and Dll4low i-DCs. Furthermore, Dll4 and Dll4high i-DCs may be beneficial targets for modulating allogeneic T cell responses, and could facilitate the discovery of human counterparts of mouse Dll4high i-DCs.

Ezh2 Regulates Transcriptional and Posttranslational Expression of T-bet and Promotes Th1 Cell Responses Mediating Aplastic Anemia in Mice

Acquired aplastic anemia (AA) is a potentially fatal bone marrow (BM) failure syndrome. IFN-γ–producing Th1 CD4+ T cells mediate the immune destruction of hematopoietic cells, and they are central to the pathogenesis. However, the molecular events that control the development of BM-destructive Th1 cells remain largely unknown. Ezh2 is a chromatin-modifying enzyme that regulates multiple cellular processes primarily by silencing gene expression. We recently reported that Ezh2 is crucial for inflammatory T cell responses after allogeneic BM transplantation. To elucidate whether Ezh2 mediates pathogenic Th1 responses in AA and the mechanism of Ezh2 action in regulating Th1 cells, we studied the effects of Ezh2 inhibition in CD4+ T cells using a mouse model of human AA. Conditionally deleting Ezh2 in mature T cells dramatically reduced the production of BM-destructive Th1 cells in vivo, decreased BM-infiltrating Th1 cells, and rescued mice from BM failure. Ezh2 inhibition resulted in significant decrease in the expression of Tbx21 and Stat4, which encode transcription factors T-bet and STAT4, respectively. Introduction of T-bet but not STAT4 into Ezh2-deficient T cells fully rescued their differentiation into Th1 cells mediating AA. Ezh2 bound to the Tbx21 promoter in Th1 cells and directly activated Tbx21 transcription. Unexpectedly, Ezh2 was also required to prevent proteasome-mediated degradation of T-bet protein in Th1 cells. Our results demonstrate that Ezh2 promotes the generation of BM-destructive Th1 cells through a mechanism of transcriptional and posttranscriptional regulation of T-bet. These results also highlight the therapeutic potential of Ezh2 inhibition in reducing AA and other autoimmune diseases.

The histone methyltransferase Ezh2 is a crucial epigenetic regulator of allogeneic T-cell responses mediating graft-versus-host disease.

Posttranscriptional modification of histones by methylation plays an important role in regulating Ag-driven T-cell responses. We have recently drawn correlations between allogeneic T-cell responses and the histone methyltransferase Ezh2, which catalyzes histone H3 lysine 27 trimethylation. The functional relevance of Ezh2 in T-cell alloimmunity remains unclear. Here, we identify a central role of Ezh2 in regulating allogeneic T-cell proliferation, differentiation, and function. Conditional loss of Ezh2 in donor T cells inhibited graft-versus-host disease (GVHD) in mice after allogeneic bone marrow (BM) transplantation. Although Ezh2-deficient T cells were initially activated to proliferate upon alloantigenic priming, their ability to undergo continual proliferation and expansion was defective during late stages of GVHD induction. This effect of Ezh2 ablation was largely independent of the proapoptotic molecule Bim. Unexpectedly, as a gene silencer, Ezh2 was required to promote the expression of transcription factors Tbx21 and Stat4. Loss of Ezh2 in T cells specifically impaired their differentiation into interferon (IFN)-γ-producing effector cells. However, Ezh2 ablation retained antileukemia activity in alloreactive T cells, leading to improved overall survival of the recipients. Our findings justify investigation of modulating Ezh2 as a therapeutic strategy for the treatment of GVHD and other T cell-mediated inflammatory disorders.

Nonmyeloablative stem cell transplantation for nonmalignant diseases in children with severe organ dysfunction

Allogeneic stem cell transplantation (SCT) can cure several nonmalignant diseases in children. However, patients frequently have significant morbidity before transplantation and there is a high transplant-related mortality. Nonmyeloablative SCT might achieve the same goals but with less toxicity. Six pediatric patients with nonmalignant diseases underwent nonmyeloablative SCT from different stem cell sources. All patients were conditioned with fludarabine/melphalan with additional anti-thymocyte globulin for haploidentical grafts and prophylaxis for graft-versus-host disease (GVHD) consisting of tacrolimus and methotrexate with additional prednisolone for haploidentical grafts. Hematopoietic stem cells were neither T-cell depleted nor purged. All patients had severe organ dysfunction that precluded transplantation with conventional conditioning. Five of the six are alive and in complete disease resolution at a median of 19 months (range, 7–53 months) after SCT. One patient died of bacteremia before engraftment. Three patients achieved complete donor chimerism. Two patients remained stable mixed chimerism. Short-term toxicities were minimal. Acute and chronic GVHD were not seen. In summary, the fludarabine-based nonmyeloablative regimen followed by SCT provides a good approach for children with nonmalignant diseases. Even patients with severe organ dysfunctions had adequate engraftment with acceptable toxicities.

Successful unrelated cord blood transplantation for chronic granulomatous disease: a case report and review of the literature.

Abstract:  CGD is a rare inherited immunodeficiency disorder that is caused by disability of oxidative killing. We presented a two‐yr‐old boy with CGD who was suffering from multiple systemic abscesses. He received the first BMT from his HLA‐haploidentical mother after conditioning with Flu, melphalan, and ATG. Although the maximum of 42% donor chimerism was achieved, it disappeared 73 days after the BMT. Then, we performed 5/6‐matched unrelated cord blood re‐transplantation after conditioning with Flu, Bu, and TBI (2 Gy). Engraftment and complete donor chimerism were achieved on days 18 and 19, respectively. The patient is now free from infection and maintains complete donor chimerism without GVHD 36 months after the cord blood re‐transplantation. We postulate that the unrelated CBT has a potential to be an alternative strategy and might be beneficial for patients with CGD who do not have an HLA‐identical donor.

The Notch Ligand DLL4 Defines a Capability of Human Dendritic Cells in Regulating Th1 and Th17 Differentiation

Notch signaling regulates multiple helper CD4+ T cell programs. We have recently demonstrated that dendritic cells (DCs) expressing the Notch ligand DLL4 are critical for eliciting alloreactive T cell responses and induction of graft-versus-host disease in mice. However, the human counterpart of murine DLL4+ DCs has yet to be examined. We report the identification of human DLL4+ DCs and their critical role in regulating Th1 and Th17 differentiation. CD1c+ DCs and plasmacytoid DCs (pDCs) from the peripheral blood (PB) of healthy donors did not express DLL4. In contrast, patients undergoing allogeneic hematopoietic stem cell transplantation had a 16-fold more DLL4+CD1c+ DCs than healthy donors. Upon activation of TLR signaling, healthy donor-derived CD1c+ DCs dramatically upregulated DLL4, as did pDCs to a lesser extent. Activated DLL4+ DCs were better able to promote Th1 and Th17 differentiation than unstimulated PB DCs. Blocking DLL4 using a neutralizing Ab decreased Notch signaling in T cells stimulated with DLL4+ DCs, and it reduced the generation of Th1 and Th17 cells. Both NF-κB and STAT3 were crucial for inducing DLL4 in human DCs. Interestingly, STAT3 directly activated DLL4 transcription and inhibiting STAT3 alone was sufficient to reduce DLL4 in activated PB DCs. Thus, DLL4 is a unique functional molecule of human circulating DCs critical for directing Th1 and Th17 differentiation. These findings identify a pathway for therapeutic intervention for inflammatory disorders in humans, such as graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, autoimmunity, and tumor immunity.

Therapeutic transfusions of granulocytes collected by simple bag method for children with cancer and neutropenic infections: results of a single-centre pilot study.

Background and Objectives  Granulocyte transfusion therapy (GTX) can be effective for life‐threatening infections unresponsive to conventional antimicrobial therapies in severely neutropenic children with cancer. We developed a new granulocyte collection method, named the ‘bag method’, in which apheresis, hydroxyethyl starch (HES) or dexamethasone are not used. We undertook a pilot study to determine the feasibility and the safety of GTX collected by the bag method for children with cancer and life‐threatening infections.

Feasibility of tacrolimus, methotrexate, and prednisolone as a graft-versus-host disease prophylaxis in non-T-cell-depleted haploidentical hematopoietic stem cell transplantation for children

Mochizuki K, Kikuta A, Ito M, Sano H, Akaihata M, Kobayashi S, Ohto H, Hosoya M. Feasibility of tacrolimus, methotrexate, and prednisolone as a graft‐versus‐host disease prophylaxis in non‐T‐cell‐depleted haploidentical hematopoietic stem cell transplantation for children. 
Clin Transplant 2011: 25: 892–897.

Hemolytic disease of the newborn due to anti-Dib : a case study and review of the literature

BACKGROUND:  The severity of hemolytic disease of the newborn (HDN) due to Diegob (Dib) mismatch ranges from no symptoms to severe jaundice that requires exchange transfusion (ET). The clinical significance of anti‐Dib is incompletely recognized.