Zhirong Qi

Origin and fate of blood cells deficient in glycosylphosphatidylinositol‐anchored protein among patients with bone marrow failure

Peripheral blood from 489 recently diagnosed patients with aplastic anaemia (AA) and 316 with refractory anaemia (RA) of myelodysplastic syndrome was evaluated to characterize CD55−CD59− [paroxysmal nocturnal haemoglobinuria (PNH)]‐type blood cells associated with bone marrow (BM) failure. PNH‐type cells were detected in 57% and 20% of patients with AA and RA, respectively. The percentages of PNH‐type granulocytes ranged from 0·003% to 94·2% and the distribution was log‐normal with a median of 0·178%. Serial analyses of 75 patients with PNH‐type cells over 5 years revealed that the percentage of PNH‐type cells constantly increased in 13 (17%), persisted in 44 (59%), disappeared in the remaining 18 (24%) although even in the ‘Disappearance’ group, PNH‐type granulocytes persisted for at least 6 months. A scattergram profile of PNH‐type cells unique to each patient persisted regardless of the response to immunosuppressive therapy and only single PIGA mutations were detected in PNH‐type granulocytes sorted from four patients. These findings suggest that the PNH‐type cells in patients with BM failure are derived from single PIGA mutant haematopoietic stem cells even when their percentages are <1% and their fate depends on the proliferation and self‐maintenance properties of the individual PIGA mutants.

Anti-Moesin Antibodies in the Serum of Patients with Aplastic Anemia Stimulate Peripheral Blood Mononuclear Cells to Secrete TNF-α and IFN-γ

Moesin is an intracellular protein that links the cell membrane and cytoskeleton, while also mediating the formation of microtubules and cell adhesion sites as well as ruffling of the cell membrane. To determine the roles of anti-moesin Abs derived from the serum of patients with aplastic anemia (AA) in the pathophysiology of bone marrow failure, we studied the expression of moesin on various blood cells and the effects of anti-moesin Abs on the moesin-expressing cells. The proteins recognized by anti-moesin mAbs were detectable on the surface of T cells, NK cells, and monocytes from healthy individuals as well as on THP-1 cells. The peptide mass fingerprinting of the THP-1 cell surface protein and the knock-down experiments using short hairpin RNA proved that the protein is moesin itself. Both the anti-moesin mAbs and the anti-moesin polyclonal Abs purified from the AA patients’ sera stimulated THP-1 cells and the PBMCs of healthy individuals and AA patients to secrete 60–80% as much TNF-α as did LPS 100 ng/ml. Although the polyclonal Abs induced IFN-γ secretion from the PBMCs of healthy individuals only when the PBMCs were prestimulated by anti-CD3 mAbs, the anti-moesin Abs were capable of inducing IFN-γ secretion from the PBMCs of AA patients by themselves. Anti-moesin Abs may therefore indirectly contribute to the suppression of hematopoiesis in AA patients by inducing myelosuppressive cytokines from immunocompetent cells.

Hydroxyurea upregulates NKG2D ligand expression in myeloid leukemia cells synergistically with valproic acid and potentially enhances susceptibility of leukemic cells to natural killer cell-mediated cytolysis.

(Cancer Sci 2010; 101: 609–615)

Autoantibodies specific to hnRNP K: a new diagnostic marker for immune pathophysiology in aplastic anemia

To identify a new diagnostic marker for the immune pathophysiology of aplastic anemia (AA), we screened sera of immune-mediated AA patients for the presence of antibodies (Abs) specific to proteins derived from a leukemia cell line UT-7 using two-dimensional electrophoresis followed by immunoblotting. The target proteins were identified by peptide mass fingerprinting. Heterogeneous nuclear ribonucleoprotein (hnRNP) K was identified as a novel autoantigen. An enzyme-linked immunosorbent assay revealed high titers of anti-hnRNP K Abs in 85 (31%) of 273 patients with AA. Sixty-four patients received antithymocyte globulin and cyclosporine after undergoing screening for anti-hnRNP K Ab, anti-DRS-1 Ab, anti-moesin Ab, and paroxysmal nocturnal hemoglobinuria (PNH)-type cells. Twenty (87%) of 23 patients with the presence of anti-hnRNP K Abs responded to the immunosuppressive therapy (IST), while 19 (46%) of 41 patients without the presence of anti-hnRNP K Abs responded. A multivariate analysis showed only PNH-type cells and anti-hnRNP K Abs to be significant factors for the prediction of a good response to IST. The detection of anti-hnRNP K Abs as well as PNH-type cells may therefore be useful for diagnosing the immune pathophysiology of AA.

Expansion of donor-derived hematopoietic stem cells with PIGA mutation associated with late graft failure after allogeneic stem cell transplantation

A small population of CD55(-)CD59(-) blood cells was detected in a patient who developed donor-type late graft failure after allogeneic stem cell transplantation (SCT) for treatment of aplastic anemia (AA). Chimerism and PIGA gene analyses showed the paroxysmal nocturnal hemoglobinuria (PNH)-type granulocytes to be of a donor-derived stem cell with a thymine insertion in PIGA exon 2. A sensitive mutation-specific polymerase chain reaction (PCR)-based analysis detected the mutation exclusively in DNA derived from the donor bone marrow (BM) cells. The patient responded to immunosuppressive therapy and achieved transfusion independence. The small population of PNH-type cells was undetectable in any of the 50 SCT recipients showing stable engraftment. The de novo development of donor cell-derived AA with a small population of PNH-type cells in this patient supports the concept that glycosyl phosphatidylinositol-anchored protein-deficient stem cells have a survival advantage in the setting of immune-mediated BM injury.

GPI‐anchored protein‐deficient T cells in patients with aplastic anemia and low‐risk myelodysplastic syndrome: implications for the immunopathophysiology of bone marrow failure

Glycosylphosphatidylinositol‐anchored protein‐deficient (GPI‐AP−) T cells can be detected in some patients with bone marrow failure (BMF), but the link between these cells and BMF pathophysiology remains to be elucidated. To clarify the significance of GPI‐AP− T cells in BMF, peripheral blood from 562 patients was examined for the presence of CD48−CD59−CD3+ cells using high‐resolution flow cytometry (FCM), and the GPI‐AP− T cells were characterized with regard to their phenotype and sensitivity to inhibitory molecules, including herpesvirus entry mediator (HVEM) and a myelosuppressive cytokine, TGF‐β. A multi‐lineage FCM analysis detected CD48−CD59−CD3+ T cells in 72 (12.8%) of the patients, together with GPI‐AP− myeloid cells. Unexpectedly, 12 patients (10 with aplastic anemia and 2 with myelodysplastic syndrome‐refractory anemia, 2.1%), who showed clinical features similar to those of other BMF patients with GPI‐AP− myeloid cells, such as a good response to immunosuppressive therapy, displayed 0.01–0.3% GPI‐AP− cells exclusively in T cells. The CD48−CD59− T cells consisted of predominantly effector memory (EM) and terminal effector cells, while CD48−CD59− T cells from non‐BMF patients who had received anti‐CD52 antibody only showed EM and central memory phenotypes. TGF‐β and HVEM capable of inhibiting T‐cell proliferation via its GPI‐AP CD160 ligation suppressed the in vitro proliferation of GPI‐AP+ T cells more potently than that of GPI‐AP− T cells from the same patients. The presence of GPI‐AP− T cells, as well as GPI‐AP− myeloid cells, may therefore reflect the immunopathophysiology of BMF in which cytokine‐mediated suppression of hematopoietic stem cells via GPI‐AP‐type receptors takes place.