Successful Treatment of Hemophagocytic Lymphohistiocytosis Associated with Low-risk Myelodysplastic Syndrome by Azacitidine.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome that occurs as a complication in many clinical settings. Malignancy-associated HLH develops in patients with hematopoietic neoplasms, particularly in those with lymphoma, and its development in those with myelodysplastic syndrome (MDS) is uncommon. We herein report a case of HLH in a patient with low-risk MDS that was successfully treated with azacitidine. The prevalence of immune abnormalities among MDS patients and the immune effects of azacitidine have recently been elucidated, suggesting that MDS-associated HLH occurs as a result of immune impairment, and azacitidine improves this condition by restoring the immune system.

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.

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.

The BALB/c-specific polymorphic SIRPA enhances its affinity for human CD47, inhibiting phagocytosis against human cells to promote xenogeneic engraftment.

It has been shown that in xenotransplantation of human cells into immunodeficient mice, the mouse strain background is critical. For example, the nonobese diabetic (NOD) strain is most efficient, the BALB/c is moderate, and the C57BL/6 is inefficient for human cell engraftment. We have shown that the NOD-specific polymorphism of the signal regulatory protein-alpha (Sirpa) allows NOD SIRPA to bind human CD47, and the resultant "don't eat me" signaling by this binding prevents host macrophages to engulf human grafts, thereby inhibiting rejection. Here we tested whether the efficient xenotransplantation capability of the BALB/c strain is also mediated by the SIRPA-CD47 self-recognition system. BALB/c SIRPA was capable of binding to human CD47 at an intermediate level between those of C57BL/6 SIRPA and NOD SIRPA. Consistent with its binding activity, BALB/c-derived macrophages exhibited a moderate inhibitory effect on human long-term culture-initiating cells in in vitro cultures, and showed moderate phagocytic activity against human hematopoietic stem cells. The increased affinity of BALB/c SIRPA for human CD47 was mounted at least through the BALB/c-specific L29V SNP within the IgV domain. Thus, the mouse strain effect on xenogeneic engraftment might be ascribed mainly to the binding affinity of strain-specific polymorphic SIRPA with human CD47. This information should be useful for developing a novel immunodeficient strain with superior efficiency for xenogeneic transplantation of human cells.

Engulfment of hematopoietic stem cells caused by down-regulation of CD47 is critical in the pathogenesis of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by deregulated engulfment of hematopoietic stem cells (HSCs) by BM macrophages, which are activated presumably by systemic inflammatory hypercytokinemia. In the present study, we show that the pathogenesis of HLH involves impairment of the antiphagocytic system operated by an interaction between surface CD47 and signal regulatory protein α (SIRPA). In HLH patients, changes in expression levels and HLH-specific polymorphism of SIRPA were not found. In contrast, the expression of surface CD47 was down-regulated specifically in HSCs in association with exacerbation of HLH, but not in healthy subjects. The number of BM HSCs in HLH patients was reduced to approximately 20% of that of healthy controls and macrophages from normal donors aggressively engulfed HSCs purified from HLH patients, but not those from healthy controls in vitro. Furthermore, in response to inflammatory cytokines, normal HSCs, but not progenitors or mature blood cells, down-regulated CD47 sufficiently to be engulfed by macrophages. The expression of prophagocytic calreticulin was kept suppressed at the HSC stage in both HLH patients and healthy controls, even in the presence of inflammatory cytokines. These data suggest that the CD47-SIRPA antiphagocytic system plays a key role in the maintenance of HSCs and that its disruption by HSC-specific CD47 down-regulation might be critical for HLH development.