WT1 peptide mix used for in vitro generation of WT1 CTL (41.29)

Abstract

We developed a method to in vitro prime and expand WT1-specific CD8+ T cells using a WT1 peptide mix which could facilitate the expansion of WT1 specific T cells from donors of different HLA backgrounds. Monocyte-derived dendritic cells or PBMC isolated from 4 normal donors were pulsed with WT1 overlapping peptides prior to T cell stimulation. CD8 selected T cells were primed using WT1 peptide pulsed dendritic cells and then re-stimulated 11 days later using WT1 peptide pulsed autologous PBMC. IL-7 and IL-2 were added to the T cell cultures 4 days after the first stimulation to promote CTL differentiation. 7 days after the second CD8 stimulation, WT1 specificity was measured by tetramer staining. All donors exhibited tetramer positive T cells (donor 1=3.11%, donor 2=6.32%, donor 3=2.17%, donor 4=1.08%). WT1-specific cytotoxicity measured by 51Cr-release assays was also assayed 7 days post the second stimulation; however, WT1-specific cytotoxicity was not observed. WT1-specific CD8 T cells from 3 of the 4 donors were cloned and enriched by limited dilution. Clones were removed from OKT3 and WT1-specific cytotoxicity was measured in 51Cr-release assays. At least 1 clone from each of the 3 donors exhibited WT1 specific cytotoxicty (donor 1=54%, donor 2=20%, donor 3=26%). In conclusion, CD8 selected PBMC stimulated by antigen presenting cells pulsed with a WT1 peptide mix stained positive for HLA-A2 WT1-tetramers and exhibited WT1-specific cytotoxicity after cloning and subsequent expansion.