HLA-A0201 Restricted Killing Activity of WT1-Specific CTL Generated From the Peripheral Blood Lymphocytes of Normal Healthy Donors

• Published in: Blood 2009-11, Vol.114 (22), p.3026-3026
• Main Authors: Krishnadas, Deepa Kolaseri, Stamer, Mindy, Dunham, Kim, Bao, Lei, Lucas, Kenneth
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V114.22.3026.3026

Abstract 3026 Poster Board II-1002 The Wilms' tumor antigen (WT1) is over-expressed on several human leukemia and solid tumors, and thus is considered as a potential target for cancer immunotherapy. Combating leukemia by targeting WT1 expressing leukemic cells using in vitro generated WT1-specific CTL is one potential approach, but it is difficult to generate an immune response against WT1 due to low T cell precursor frequency in normal healthy individuals. Earlier studies have shown the generation of WT1-A*0201 peptide specific CTL from CD8+ T cells by cloning. Another study reported the production of IFN- γ by WT-1 specific CD8+ T cells. However, the cytolytic killing ability of these IFN- γ producing cells was not further characterized. Here, we demonstrate the generation of WT1-A*0201 specific CTL from the peripheral blood lymphocytes (PBL) of normal healthy donors using CD137 selection. The PBL were stimulated once with RMFPNAPYL (WT1-A*0201 peptide) pulsed autologous dendritic cells and twice with WT1-A*0201 peptide pulsed irradiated peripheral blood mononuclear cells (PBMC). Following three stimulations, the PBL were selected for CD137+ expression and rapidly expanded with OKT3 and IL-2. The WT1-A*0201 specific CTL showed killing of target cells and production of IFN-γ in an antigen-specific manner. The percent killing of WT1-A*0201 peptide pulsed T2 cells (TAP−, HLA- A2+) and autologous B blast (BB) were significantly higher when compared with their control targets. T2 cells and BB either pulsed with an irrelevant A*0201 peptide or un-pulsed served as the control. We have observed similar results with WT1-A*0201 specific CTL generated from normal donor CD8+ cells. However, the efficiency of WT1-A*0201 CTL generated from PBL to kill target cells and produce IFN- γ was higher than CTL from CD8+ cells. The CTL generated from PBL killed BA25, a WT1 expressing A2+ leukemia cell line but failed to kill Molt-4, a WT1 expressing A2− cell line, clearly indicating HLA-A2 restricted CTL activity. The specificity of the generated CTL were further confirmed by staining with WT1-HLA-A*0201 tetramer. The percentage of WT1-specific CD3+CD8+Tetramer+ cells either remained same or higher in CTL generated from PBL when compared with those generated from CD8+ cells. CD137 selection leads to the generation of significant number of CTL in a shorter time when compared to conventional cloning methods. In addition, generation of WT1-A*0201 specific CTL from PBL avoids CD8+