The Role of Wilms’ Tumor Gene (WT1) Peptide-Specific Cytotoxic-T Lymphocytes in Selection of a Paroxysmal Nocturnal Hemoglobinuria (PNH) Clone.
Authors: Ikeda Kazuhiko , Shichishima Tsutomu , Shichishima-Nakamura Akiko , Yasukawa Masaki , Noji Hideyoshi , Akutsu Kazuko , Maruyama Yukio 
Published: April 2020

Abstract
It is known that the expression of WT1 increases in leukemic cells from patients with acute and chronic leukemia, and that in leukemic patients, the WT1 elicits WT1 peptide-specific cytotoxic T-lymphocytes (CTLs) in an HLA-restricted manner, suggesting the possibility that immune therapy targeting WT1 may be effective. Also, the WT1 is highly expressed in bone marrow (BM) cells from paroxysmal nocturnal hemoglobinuria (PNH) patients (Shichishima et al, Blood, 2002). However, the significance of high expression of the WT1 in PNH remains unknown. To clarify some roles of WT1 peptide-specific CTLs of PNH patients in the expansion of a PNH clone in this study, we studied the frequencies of WT1 peptide-specific CD8+ CTLs in peripheral blood (PB) mononuclear cells (MNCs) from 4 PNH patients with the HLA-A*2402 allele by flow cytometry (FCM) using a WT1 peptide-specific and HLA-A*2402-restricted tetramer. Subsequently, we examined cell cytotoxicity of a WT1 peptide-specific and HLA-A*2402-restricted CTL clone (TAK-1; Ohminami et al, Blood, 2000) to BM MNCs from the patients using 51Chromium-releasing assay, of which the results were presented at effector versus target ratios (E:T) of 1:1 and 3:1. We also investigated the changes in the proportions of CD59− cells of 7-amino-actinomycin D-negative, viable, CD34+ cells from the patients by FCM after coincubation of BM MNCs with the TAK-1 at an E:T of 1:1. Tetramer analysis showed that the frequencies of WT1 peptide-specific and HLA-restricted CD8+ T cells in PNH patients (mean ± standard deviation; 12.138 ± 2.090 / 104 cells) with the HLA-A*2402 allele were higher than those in HVs (n=10; 3.498 ± 2.591 / 104 cells) (p&lt;0.001) with the allele. 51Chromium-releasing assay showed that the TAK-1 spontaneously killed BM MNCs from the PNH patients with the HLA-A*2402 allele, but not from the HVs (n=2) with the allele, in the absence of a WT1 peptide. Subsequently, in the presence of a WT1 peptide, the TAK-1 killed BM MNCs from both the PNH patients and HVs with the HLA-A*2402 allele. The TAK-1 did not kill BM MNCs from both one PNH patient and one HV without the HLA-A*2402 allele even in the presence of a WT1 peptide. FCM analysis of BM MNCs from PNH patients with the HLA-A*2402 allele showed that the proportions of viable CD34+CD59− cells after coincubation with the TAK-1 were higher in the absence (52.54 ± 26.14 %; p&lt;0.02) and presence (49.90 ± 26.33 %; p&lt;0.01) of a WT1 peptide compared with those after incubation without the TAK-1 (43.52 ± 26.16 %). In contrast, coincubation with the TAK-1 did not affect the proportions of viable CD34+CD59− cells from one PNH patient without the HLA-A*2402 allele. These findings suggest that WT1 peptide-specific CTLs are clearly elicited, probably, by an intrinsic WT1 peptide highly expressed in hematopoietic precursor cells from PNH patients. Nevertheless, CD59− hematopoietic precursor cells from PNH patients have a tendency to relatively survive against attacks of WT1 peptide-specific CTLs in an HLA-restricted manner, resulting in expansion of a PNH clone.