Abstract
It is unclear how a paroxysmal nocturnal hemoglobinuria (PNH) clone expands and bone marrow failure (BMF) occurs in PNH patients, although an immunologic mechanism by human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes (CTLs) has been suggested. It has been also reported that immunization with HLA-binding peptides of Wilms’ tumor gene (WT1) in hematopoietic cells induces a WT1 peptide-specific CTL response, and WT1 RNA is highly expressed in BM mononuclear cells (MNCs) in PNH patients (Shichishima T et al., Blood, 2002). In this study, to clarify some roles of WT1 peptide-specific and HLA-restricted CTLs, the frequencies of peripheral blood (PB) WT1 peptide-specific and HLA-A*2402-restricted CTLs by flow cytometric tetramer analysis and WT1 peptide-stimulated interferon (IFN)-γ-producing MNCs by enzyme-linked immunospot assay in 5 PNH patients with the HLA-A*2402 allele were examined. We also investigated cytotoxicity of WT1 peptide-specific and HLA-A*2402-restricted CTL clone (TAK-1) cells on BM MNCs by 51Cr-releasing assay, colony forming-unit granulocyte-macrophage colony formation of CD34+CD59+ and CD34+CD59− cells, and CD59 expression in viable 7AAD−CD34+ cells by flow cytometry in those patients, and expression of IFN-γ in TAK-1 cells by flow cytometry, after co-incubation of BM cells from them with TAK-1 cells. As controls, 8 healthy volunteers (HV) with the HLA-A*2402 allele and 2 PNH patients and HV without the allele were examined. We found that the frequencies of PB WT1 peptide-specific and HLA-A*2402-restricted CD8+ cells (p<0.005) and WT1 peptide-stimulated IFN-γ-producing MNCs (p<0.02) were significantly higher in PNH patients with the HLA-A*2402 allele (0.255 ± 0.164% and 25.2 ± 15.4 / 5 x 105 cells, respectively) than HV with the allele (0.052 ± 0.025% and 6.6 ± 6.8 / 5 x 105 cells, respectively). In PNH patients or HV, TAK-1 cells significantly killed BM MNCs, suppressed colony formations of CD34+CD59+ and CD34+CD59− cells, and expressed IFN-γ in the absence and presence of a WT1 peptide or only in the presence of the peptide, respectively, in an HLA-A*2402-restricted manner. Reduction rates of colony formation of CD34+CD59− cells from the patients with the HLA-A*2402 allele by TAK-1 cells were significantly less than those of CD34+CD59+ cells in PNH patients, in the absence (38.3 ± 23.0% and 59.0 ± 28.0%, respectively, p<0.01) and presence (74.7 ± 12.8% and 90.6 ± 11.1%, respectively, p<0.002) of a WT1 peptide. After co-incubation of BM MNCs from the patients with TAK-1 cells, proportions of viable CD34+CD59− cells from PNH patients significantly increased in the absence (62.87 ± 27.29%; p<0.01) and presence (62.32 ± 25.73%; p<0.01) of a WT1 peptide compared with those of the controls incubated without TAK-1 cells (52.40 ± 24.58%) in an HLA-A*2402-restricted manner. In conclusion, WT1 peptide-specific and HLA-restricted CTLs may play important roles in the expansion of a PNH clone during immunologic selection and in the occurrence of BMF via IFN-γ in PNH.
The importance of flow cytometry in the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria
