Incidence of refractory cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation.

Post-transplant cytomegalovirus (CMV) disease can be almost completely avoided by current infection control procedures. However, CMV reactivation occurs in more than half of patients, and some patients can develop clinically resistant CMV infections. Whether resistance is due to the host's immune status or a viral resistance mutation is challenging to confirm. Therefore, a prospective observational analysis of refractory CMV infection was conducted in 199 consecutive patients who received allogeneic hematopoietic stem cell transplantation at a single institution. Among them, 143 (72%) patients received anti-CMV drugs due to CMV reactivation, and only 17 (8.5%) exhibited refractory CMV infection. These patients had clinically refractory infection. However, viral genome analysis revealed that only one patient exhibited a mutation associated with the anti-CMV drug resistance. Clinical resistance was mainly correlated with host immune factors, and the incidence of resistance caused by gene mutations was low at the early stage after a transplantation.

Azacitidine for the treatment of patients with relapsed acute myeloid leukemia after allogeneic stem cell transplantation.

We retrospectively analyzed 38 patients with AML who received azacitidine (AZA) to treat disease relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients with objective response (OR) (n = 20) after AZA had significantly higher 2-year overall survival (OS) (45.0% vs 5.6%; p = 0.004) than progressive disease. The 2-year OS was significantly higher in the retransplant group (n = 23) than in the nonretransplant group (n = 15) (34.8% vs 13.3%; p = 0.034). We analyzed 167 patients who underwent the second allo-HSCT to clarify the impact of pretransplant AZA after the second allo-HSCT. Patients in the AZA group (n = 21) had significantly higher 2-year disease-free survival (DFS) (32.7% vs 14.5%; p = 0.012) and OS (38.1% vs 17.5%; p = 0.044) than those in the SOC group (n = 146). Our data demonstrate that AZA is an effective and well-tolerated bridging therapy to the second allo-HSCT.

A human SIRPA knock-in xenograft mouse model to study human hematopoietic and cancer stem cells.

In human-to-mouse xenogeneic transplantation, polymorphisms of signal-regulatory protein α (SIRPA) that decide their binding affinity for human CD47 are critical for engraftment efficiency of human cells. In this study, we generated a new C57BL/6.Rag2nullIl2rgnull (BRG) mouse line with Sirpahuman/human (BRGShuman) mice, in which mouse Sirpa was replaced by human SIRPA encompassing all 8 exons. Macrophages from C57BL/6 mice harboring Sirpahuman/human had a significantly stronger affinity for human CD47 than those harboring SirpaNOD/NOD and did not show detectable phagocytosis against human hematopoietic stem cells. In turn, Sirpahuman/human macrophages had a moderate affinity for mouse CD47, and BRGShuman mice did not exhibit the blood cytopenia that was seen in Sirpa-/- mice. In human to mouse xenograft experiments, BRGShuman mice showed significantly greater engraftment and maintenance of human hematopoiesis with a high level of myeloid reconstitution, as well as improved reconstitution in peripheral tissues, compared with BRG mice harboring SirpaNOD/NOD (BRGSNOD). BRGShuman mice also showed significantly enhanced engraftment and growth of acute myeloid leukemia and subcutaneously transplanted human colon cancer cells compared with BRGSNOD mice. BRGShuman mice should be a useful basic line for establishing a more authentic xenotransplantation model to study normal and malignant human stem cells.

Identification of unipotent megakaryocyte progenitors in human hematopoiesis.

The developmental pathway for human megakaryocytes remains unclear, and the definition of pure unipotent megakaryocyte progenitor is still controversial. Using single-cell transcriptome analysis, we have identified a cluster of cells within immature hematopoietic stem- and progenitor-cell populations that specifically expresses genes related to the megakaryocyte lineage. We used CD41 as a positive marker to identify these cells within the CD34+CD38+IL-3RαdimCD45RA- common myeloid progenitor (CMP) population. These cells lacked erythroid and granulocyte-macrophage potential but exhibited robust differentiation into the megakaryocyte lineage at a high frequency, both in vivo and in vitro. The efficiency and expansion potential of these cells exceeded those of conventional bipotent megakaryocyte/erythrocyte progenitors. Accordingly, the CD41+ CMP was defined as a unipotent megakaryocyte progenitor (MegP) that is likely to represent the major pathway for human megakaryopoiesis, independent of canonical megakaryocyte-erythroid lineage bifurcation. In the bone marrow of patients with essential thrombocythemia, the MegP population was significantly expanded in the context of a high burden of Janus kinase 2 mutations. Thus, the prospectively isolatable and functionally homogeneous human MegP will be useful for the elucidation of the mechanisms underlying normal and malignant human hematopoiesis.

Enhanced Reconstitution of Human Erythropoiesis and Thrombopoiesis in an Immunodeficient Mouse Model with Kit(Wv) Mutations.

In human-to-mouse xenograft models, reconstitution of human hematopoiesis is usually B-lymphoid dominant. Here we show that the introduction of homozygous Kit(Wv) mutations into C57BL/6.Rag2(null)Il2rg(null) mice with NOD-Sirpa (BRGS) strongly promoted human multi-lineage reconstitution. After xenotransplantation of human CD34(+)CD38(-) cord blood cells, these newly generated C57BL/6.Rag2(null)Il2rg(null)NOD-Sirpa Kit(Wv/Wv) (BRGSK(Wv/Wv)) mice showed significantly higher levels of human cell chimerism and long-term multi-lineage reconstitution compared with BRGS mice. Strikingly, this mouse displayed a robust reconstitution of human erythropoiesis and thrombopoiesis with terminal maturation in the bone marrow. Furthermore, depletion of host macrophages by clodronate administration resulted in the presence of human erythrocytes and platelets in the circulation. Thus, attenuation of mouse KIT signaling greatly enhances the multi-lineage differentiation of human hematopoietic stem and progenitor cells (HSPCs) in mouse bone marrow, presumably by outcompeting mouse HSPCs to occupy suitable microenvironments. The BRGSK(Wv/Wv) mouse model is a useful tool to study human multi-lineage hematopoiesis.

Calreticulin mutation does not contribute to disease progression in essential thrombocythemia by inhibiting phagocytosis.

Somatic mutations of calreticulin (CALR) have been observed in many cases of essential thrombocythemia (ET) or primary myelofibrosis that harbor non-mutated Janus kinase 2 (JAK2). CALR mainly localizes within the endoplasmic reticulum lumen, but a small fraction of the total CALR pool is distributed over the cell surface. Cell surface CALR is known to transduce prophagocytic "eat me" signals to macrophages and acts as one of the important regulators for macrophage engulfment. In this study, we attempted to clarify whether mutant CALR may affect the threshold for macrophage engulfment and play an integral role in the pathogenesis of CALR-mutated ET. First, we compared the surface expression levels of CALR on hematopoietic stem and progenitor cells (HSPCs) and mature blood cells in patients with myeloproliferative neoplasms and found that the surface expression of mutant CALR did not change. Next, we compared the threshold for macrophage phagocytosis of each HSPC fraction and mature blood cells and found no significant change in the efficiency of macrophage engulfment. Our data suggest that CALR mutation does not affect sensitivity to phagocytosis by macrophages. Finally, we analyzed the phosphorylation statuses of molecules downstream of JAK2 at each HSPC level in patients with ET and found that CALR mutations activated the JAK-STAT pathway in a manner similar to that associated with JAK2 mutations. These results indicate that mutant CALR causes myeloproliferation because of the activation of JAK-STAT pathway and not by the inhibition of phagocytosis, which is similar to the myeloproliferation caused by JAK2 V617F mutation.

A TIM-3/Gal-9 Autocrine Stimulatory Loop Drives Self-Renewal of Human Myeloid Leukemia Stem Cells and Leukemic Progression.

Signaling mechanisms underlying self-renewal of leukemic stem cells (LSCs) are poorly understood, and identifying pathways specifically active in LSCs could provide opportunities for therapeutic intervention. T-cell immunoglobin mucin-3 (TIM-3) is expressed on the surface of LSCs in many types of human acute myeloid leukemia (AML), but not on hematopoietic stem cells (HSCs). Here, we show that TIM-3 and its ligand, galectin-9 (Gal-9), constitute an autocrine loop critical for LSC self-renewal and development of human AML. Serum Gal-9 levels were significantly elevated in AML patients and in mice xenografted with primary human AML samples, and neutralization of Gal-9 inhibited xenogeneic reconstitution of human AML. Gal-9-mediated stimulation of TIM-3 co-activated NF-κB and ß-catenin signaling, pathways known to promote LSC self-renewal. These changes were further associated with leukemic transformation of a variety of pre-leukemic disorders and together highlight that targeting the TIM-3/Gal-9 autocrine loop could be a useful strategy for treating myeloid leukemias.

Diffuse panbronchiolitis after humanized anti-CCR4 monoclonal antibody therapy for relapsed adult T-cell leukemia/lymphoma.

We present the case of a 62-year-old Japanese woman with relapsed adult T-cell leukemia/lymphoma (ATLL) who was treated with humanized anti-CCR4 monoclonal antibody (KW-0761). Although this antibody was highly effective against refractory ATLL, 6 months after the final KW-0761 infusion, the patient complained of hypoxia due to diffuse panbronchiolitis. Physicians should remain vigilant to the possibility of such previously unreported late-onset adverse effects associated with KW-0761 therapy.