Hirohito Yamazaki

Single-cell-resolution PIGA sequencing showed that rare glycosylphosphatidylinositol-deficient cells in healthy individuals are polyclonal, comprising long-lived hematopoietic stem/progenitor-derived clones and nonpersistent populations. Those in acquired aplastic anemia reflect the selective expansion of multipotent PIGA-mutant stem/progenitor cells across myeloid and lymphoid lineages.

We studied the pathophysiology of aplastic anaemia (AA) in six different pairs of relatives without a family history of hematologic disorders or congenital AA. Five and four of the six pairs shared the HLA-DRB1*15:01 and B*40:02 alleles, respectively. Glycosylphosphatidylinositol-anchored protein-deficient blood cells were detected in eight of the 10 patients evaluated. In a mother-daughter pair from one family, flow cytometry detected leukocytes lacking HLA-A2 due to loss of heterogeneity in chromosome 6p. Whole-exome sequencing of the family pair revealed a missense mutation in MYSM1. These results suggest that genetic inheritance of immune traits might underlie familial AA in some patients.

Objectives: Eltrombopag, a thrombopoietin-receptor agonist, stimulates hematopoiesis in patients with acquired aplastic anemia (AA). Cytomorphologic changes in bone marrow after eltrombopag administration are still unclear. This study examined the effect of eltrombopag on cytomorphologic findings using data from prior phase 2 studies (E1201 and E1202). Methods: Microscopic examinations were performed in 31 patients with AA (E1201 [n = 21], E1202 [n = 10]). The relationship between hematologic improvement and morphologic findings was also investigated. Results: In 5 patients (E1201 [n = 3], E1202 [n = 2]), the bone marrow blast count increased after initiation of eltrombopag treatment compared with screening values. The blast count was less than 5%, and the increase in bone marrow blasts was transient in all 4 patients who had bone marrow examinations at follow-up. In 8 patients (E1201 [n = 5], E1202 [n = 3]), dysplastic forms of megakaryocytes were found in the bone marrow following treatment initiation. Dysmegakaryopoiesis of 10% or more was found in 3 patients. None of the patients revealed micromegakaryocytes. Ten patients showed an increase in bone marrow blasts and/or dysmegakaryopoiesis following treatment initiation. Nine of 10 patients showed hematologic improvement in 1 or more lineages. Conclusions: Dysmegakaryopoiesis without micromegakaryocytes and a transient increase of less than 5% in bone marrow blast count may be signs of hematologic improvement with eltrombopag for patients with AA.

To determine whether antigen presentation by HLA-DR on hematopoietic stem progenitor cells (HSPCs) is involved in the development of acquired aplastic anemia (AA), we studied the HLA-DR expression on CD45^dimCD34⁺CD38⁺ cells in the peripheral blood of 61 AA patients including 23 patients possessing HLA-class I allele-lacking (HLA-class I[−]) leukocytes. HLA-DR-lacking (DR[−]) cells accounted for 13.0–57.1% of the total HSPCs in seven (11.5%) patients with HLA-DR15 who did not possess HLA-class I(–) leukocytes. The incubation of sorted DR(–) HSPCs in the presence of IFN-γ for 72 h resulted in the full restoration of the DR expression. A comparison of the transcriptome profile between DR(–) and DR(+) HSPCs revealed the lower expression of immune response-related genes including co-stimulatory molecules (e.g., CD48, CD74, and CD86) in DR(–) cells, which was not evident in HLA-class I(–) HSPCs. DR(–) cells were exclusively detected in GPI(+) HSPCs in four patients whose HSPCs could be analyzed separately for GPI(+) and GPI(–) HSPCs. These findings suggest that CD4⁺ T cells specific to antigens presented by HLA-DR15 on HSPCs may contribute to the development of AA as well as the immune escape of GPI(–) HSPCs in a distinct way from CD8⁺ T cells recognizing HLA-class I-restricted antigens.

Although a glycosylphosphatidylinositol-anchored protein (GPI-AP) CD109 serves as a TGF-β co-receptor and inhibits TGF-β signaling in keratinocytes, the role of CD109 on hematopoietic stem progenitor cells (HSPCs) remains unknown. We studied the effect of CD109 knockout (KO) or knockdown (KD) on TF-1, a myeloid leukemia cell line that expresses CD109, and primary human HSPCs. CD109-KO or KD TF-1 cells underwent erythroid differentiation in the presence of TGF-β. CD109 was more abundantly expressed in hematopoietic stem cells (HSCs) than in multipotent progenitors and HSPCs of human bone marrow (BM) and cord blood but was not detected in mouse HSCs. Erythroid differentiation was induced by TGF-β to a greater extent in CD109-KD cord blood or iPS cell-derived megakaryocyte–erythrocyte progenitor cells (MEPs) than in wild-type MEPs. When we analyzed the phenotype of peripheral blood MEPs of patients with paroxysmal nocturnal hemoglobinuria who had both GPI(+) and GPI(−) CD34⁺ cells, the CD36 expression was more evident in CD109⁻ MEPs than CD109⁺ MEPs. In summary, CD109 suppresses TGF-β signaling in HSPCs, and the lack of CD109 may increase the sensitivity of PIGA-mutated HSPCs to TGF-β, thus leading to the preferential commitment of erythroid progenitor cells to mature red blood cells in immune-mediated BM failure.