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It is well known that HLA-DRB1 alleles corresponding to HLA-DR15 such as DRB1*1501 and DRB1*1502 are associated with susceptibility to acquired aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH). However, little is known about how these DRB1 alleles contribute to the development of AA. We previously reported that DRB1*1501 is significantly associated with both an increase in the proportion of PNH-type cells and good response to a immunosuppressive therapy (IST) including antithymocyte globulin (ATG) plus cyslosporine (CsA) or CsA alone, while DRB1*1502, whose DR molecules differ from those derived from DRB1*1501 in only one amino acid, is not associated with either of the two factors. To gain insight into the reason for different contributions to the development of AA by the two DRB1 alleles, we determined DRB1 alleles and the proportion of PNH-type cells of 77 patients with recently diagnosed AA and analyzed the relationship of DRB1 alleles and the presence of increased PNH-type cells with response to ATG plus CsA therapy. Fifty-five of 77 patients (71%) improved with ATG plus CsA therapy. When the factors favorably affecting the response to IST in the AA patients were examined under a multivariate analysis, only the presence of PNH-type cells was significantly associated with the response to IST. Kaplan-Meier curves showed that there were significant differences in the probability of the response to IST between the DRB1*1501 + 1502 − patients and either the DRB1*1501 − 1502 + patients (P Figure Figure

Although there is a large body of evidence that immune mechanisms play an essential role in the development of acquired aplastic anemia (AA), knowledge of autoantigens eliciting the immune system attack is limited. In an immune-mediated subset of AA patients characterized by the presence of HLA-DRB1*1501 and an increase in the proportion of paroxysmal nocturnal hemoglobinuria (PNH)-type blood cells, not only T-cell response but also B-cell response to antigens derived from hematopoietic stem cells may take place. To test this hypothesis, we screened sera of such immune-mediated AA patients for the presence of IgG antibodies recognizing a megakaryoblastic leukemia cell line, UT-7, using immunofluorescence staining. Since the sera from several patients stained cytoplasm of UT-7 cells, lysates of the cell line were subjected to Western blotting using the same sera. A clear band of 80 kDa protein was revealed by the sera from several patients. Peptide mass finger printing of the protein extracted from the single band identified this protein as moesin. Moesin is a membrane-cytoskeleton linker protein in cytoplasm and involved in the formation of microvilli, cell adhesion sites, and ruffling membranes. When sera of a large number of AA patients were screened for the presence of antibodies specific to recombinant moesin protein with Western blotting and ELISA, significantly high titer of the antibodies was detected in 35 of 110 (32%) AA patients. The prevalence of anti-moesin antibodies in AA patients showing increased PNH-type cells (41%) was significantly higher than those (12%) without PNH-type cells ( p =0.002). Anti-moesin antibodies were detectable in 15 of 39 (38%) recently diagnosed AA patients before therapy and in 20 of 71 (28%) patients who had a long history of AA. Four of 6 newly diagnosed patients showing anti-moesin antibodies responded to ATG+cyclosporine therapy and restored good hematopoietic function. Since B lymphocytes reportedly secrete moesin as a form of exosome (J Biol Chem, 2003), we examined supernatants of various leukemia cell lines incubated for 1 hour for the presence of moesin. The membrane-linker protein was detectable in the culture supernatant of 4 (UT-7, K562, OUN-1 and NB4) of 10 myeloid leukemia cell lines. These findings indicate that B-cell response to moesin may represent immune pathophysiology of AA and that moesin secreted from hematopoietic progenitor cells as a form of exosome may be efficiently processed by antigen-presenting cells, leading to induction of antibodies and possibly T cells specific to moesin in immune-mediated AA.

To determine the clinical significance of a minor population of glycosylphosphatidylinositol-anchored membrane protein-negative (GPI-AP−) cells in acquired aplastic anemia (AA), we quantified CD55− CD59− granulocytes and red blood cells (RBCs) in peripheral blood from 122 patients with recently diagnosed AA and correlated numbers of GPI-AP− cells and responses to immunosuppressive therapy (IST). The highly sensitive flow cytometry detected 0.005% to 23.1% of GPI-AP− cells in 68.0% of AA patients. Sixty-eight of 83 (91%) patients with an increased ratio of GPI-AP− cells (PNH+) responded to antithymocyte globulin (ATG) + cyclosporin (CsA) therapy, whereas 18 of 39 (48%) without such an increase (PNH−) responded. Failure-free survival rates were significantly higher (64%) among PNH+ than PNH− patients (12%) at 5 years (Figure) although overall survival rates were comparable between the groups. Numbers of GPI-AP− and GPI-AP+ cells increased in parallel among most PNH+ patients who responded to IST, suggesting that these cells are equally sensitive to immune attack. These results indicate that a minor population of GPI-AP− cells represents a reliable marker of a positive IST response and a favorable prognosis among patients with AA. Furthermore, immune attack against hematopoietic stem cells that allows PNH clonal expansion might occur only at the onset of AA. Figure Figure

Modern flow cytometry techniques using monoclonal antibodies (mAbs) specific to glycosylphosphatidylinositol-anchored membrane proteins have revealed an increase in the proportion of paroxysmal nocturnal hemoglobinuria (PNH)-type cells in a varying (30 to 50) percentage of acquired aplastic anemia (AA) patients who do not manifest any signs of hemolysis. However, the clinical significance of such a small proportion of PNH-type cells remains uncertain because previous studies have used a variety of flow cytometric assays with different mAbs and different definitions of an abnormal increase in the proportion of PNH-type cells, and also, few studies have focused on the correlation of presence of increased PNH-type cells with response to immunosuppressive therapy (IST) based on a large number of newly diagnosed AA patients. We previously demonstrated, using two-color flow cytometry capable of precisely determining the proportion of PNH-type cells less than 0.1%, that such a minor population of PNH-type cells represents a reliable marker for presence of immune pathophysiology in refractory anemia patients (Blood,100: 3897–3902, 2002; Blood,102:1211–1216, 2003). To determine if the PNH-type cells serve as a marker for immune pathogenesis in AA as well, we examined peripheral blood in 99 newly diagnosed AA patients (67 severe AA and 32 moderate AA) for presence of CD55−59−CD11b+ granulocytes and CD55−59−glycophorin A+ RBCs and studied correlation of response to ATG + cyclosporine (CsA) therapy with presence of increased PNH-type cells. When the influence of time from blood collection until treatments of cells with mAbs on the outcome of PNH-type-cell detection were examined, the percentages of PNH-type cells remained unchanged for 2 days for granulocytes and for 6 days for RBCs from the time of blood collection. Minor (>0.003% for CD11b+ granulocytes and >0.005% for glycophorin A+ RBCs) population of PNH-type cells were detected in 66 (66.7%) patients. The proportions of PNH-type cells in patients with increased PNH-type cells (PNH+ patients) were 0.005–0.01% in 10 (15.2%) patients, 0.01–0.1% in 20 (30.3%), 0.1–1.0% in 25 (37.9%), and 1.0–8.67% in 10 (15.2%). When clinical characteristics of PNH+ patients were compared with that of PNH− patients, PNH+ patients (mean, 54 year-old) were significantly older than PNH− patients (mean, 42 year-old, [Italic]P[/Italic]=0.042). 55 of 66 (83.3%) PNH+ patients improved with IST and achieved PR or CR whereas 17 of 33 (52.9%) PNH− patients responded. Kaplan-Meier analysis showed that PNH+ patients had a significantly better chance of response to IST than PNH- patients (Figure). These results indicate that a minor population of PNH-type cells defined by the two-color flow cytometry represents a good marker for response to IST in AA patients. Since CD55−59−glycophorin A+ RBCs less than 0.1% remain constant and detectable in blood stored at 4 ?C for up to 6 days, testing blood cells, particularly RBCs, using this sensitive flow cytometry is useful in identifying high responders to IST in AA patients. Figure Figure

Treatment of patients refractory to immunosuppressive therapy is a major problem in the management of aplastic anemia (AA). Some patients who are ineligible for allogeneic stem cell transplantation have been successfully treated with anabolic steroids although little is known about the common characteristics of these patients. In order to characterize high responders to danazol, we retrospectively studied 44 AA (13 male and 31 female) patients who were treated with 300 mg of danazol daily for at least 3 months. All patients had been previously treated with ATG+CsA or CsA alone without appreciable effects before danazol therapy. Peripheral blood of these patients was examined for the presence of PNH-type cells and clonality in granulocytes using sensitive flow cytometry (Blood100: 3897, 2002) and the improved HUMARA assay (Blood102: 1211, 2003). 24 of 44 (55%) patients attained PR or CR according to the response criteria proposed by Camitta. All 3 lineage cells increased in the responders. The rate of response in female patients (21/31, 68%) was significantly higher than that (3/13, 23%) in male patients (P=0.0058). There was no difference in the response rate between older (>50 years) patients (75%) and younger (

A large body of evidence has suggested that acquired aplastic anemia (AA) of patients carrying HLA-DR15 is a kind of organ-specific autoimmune disease where hematopoietic progenitor cells in bone marrow are attacked by CD4+ T cells recognizing endogenous antigens. We recently identified diazepam-binding inhibitor-related protein 1 (DRS-1) as a candidate autoantigen capable of provoking immune system attack against hematopoietic progenitor cells in AA (Blood, 2004). Although in other organ-specific autoimmune diseases such as insulin-dependent diabetes mellitus and primary biliary cirrhosis, cytoplasmic proteins including glutamic acid decarboxylase 65 and pyruvate dehydrogenase complex have been shown to serve as autoantigens and mediate organ damages by CD4+ T cells, it remains unclear whether a peroxisomal protein like DRS-1 can be processed in hematopoietic progenitor cells, presented by HLA-DR15, and eventually serve as a target antigen of specific CD4+ T cells, leading to killing of hematopoietic progenitor cells themselves. To clarify these issues, we established a CD4+ T-cell line specific to a DRS-1 peptide (amino acid residues 191–204) from an AA patient carrying HLA-DR15 who had exhibited a high titer of anti-DRS-1 antibody as well as a high frequency of T-cell precursors specific to DRS-1, and then examined the cytotoxicity of the DRS-1-specific T-cell line against (1) autologous lymphoblastoid cell line (LCL) cells transfected with full length DRS-1 cDNA using a lentiviral vector, (2) myeloid leukemia cell lines carrying HLA-DR15 (KH88 and SAS413) and a leukemia cell line not carrying HLA-DR15 (K562), and (3) CD34+ progenitor cells from normal individuals. When all leukemia cell lines and LCL cells were examined for DRS-1 expression using Western blotting with specific monoclonal antibodies, DRS-1 protein was detected in DRS-1-transfected LCL cells, KH88 and K562, but not in nontransfected LCL cells and SAS413. Overexpression of DRS-1 gene by the CD34+ cells from normal individuals was ascertained by real-time PCR. In the 51Cr release assay, DRS-1-specific T cells showed cytotoxicity against only DRS-1-transfected LCL cells and KH88 in a dose-dependent manner (Figure), indicating that the T cell line requires presence of both DRS-1 and HLA-DR15 on target cells to exert cytotoxicity. When the DRS-1-specific T cells were incubated with CD34+ cells isolated from normal individuals with or without HLA-DR15 at an 10:1 ratio for 4 hours and cultured in a methylcellulose medium supplemented with colony-stimulating factors, the numbers of CFU-GM and BFU-E colonies derived from an HLA-DR15+ individual were 60.0% and 52.9% of a control whereas those derived from an HLA-DR15− individual were 90.1% and 88.2%. These findings indicate that hematopoietic progenitor cells in individuals with HLA-DR15 can present DRS-1 through the DR molecule and a breakdown of immune tolerance to DRS-1 may lead to development of AA. Download : Download high-res image (173KB) Download : Download full-size image

Some patients with aplastic anemia (AA) and approximately 40% of patients with refractory anemia (RA) of myelodysplastic syndrome exhibit karyotypic abnormalities in bone marrow dividing cells. Although some of the patients undergo evolution to acute myeloid leukemia (AML), others follow a clinical course similar to AA patients without chromosomal abnormalities. Except for several abnormalities such as −7 and 5q-, the clinical significance of such chromosomal abnormalities in bone marrow failure patients remains unclear. We recently developed a reliable HUMARA assay capable of detecting a clonal population in granulocytes which constitutes 30% or more of total granulocytes (Blood. 2003;102:1211–1216). Studying correlation between chromosomal abnormalities and the presence of clonality may help in understanding the pathogenetic role of chromosomal abnormalities in AA and RA. We thus analyzed 50 acquired AA and 28 RA female patients who were heterozygous for the HUMARA gene. Chromosomal abnormalities such as add(5)(q13), 9q–9q+ and del(7)(q14q22) were found in 8% of AA and 21% of RA patients. Clonality was detected in 38% of AA patients and 39% of RA patients. Incidence of chromosomal abnormalities in patients with clonality (27%) was higher than that in patients without clonality (4%, p