Leukemia-Reactive Cytotoxic CD8+ and CD4+ T-Cells Specific for Novel WT-1 Epitopes Are Generated In Vitro by Sensitization with Overlapping Pentadecapeptides (15-mers) Spanning the Wilms Tumor Protein.

Abstract

The Wilms Tumor protein, WT-1 is differentially expressed in 60–80% of acute leukemias and CML. Evidence also suggests WT-1 expression in clonogenic leukemia stem cells, making WT-1 an attractive target for T-cell therapy. However, to date, only a limited number of immunogenic epitopes have been identified. To address this limitation, we employed a pool of 15-mers with 10 amino acid overlaps spanning the complete sequence of WT-1 loaded on autologous monocyte derived dendritic cells or EBV transformed BLCL to sensitize T-cells from normal donors of varied HLA genotypes. Specific 15-mers eliciting WT-1 specific responses were identified by quantitating IFNγ+ CD8+ or CD4+ T-cell responses to secondary stimulation with 15-mer subpools organized in a mapping grid with single overlaps. Phenotypes of T-cells producing IFNγ+ in response to identified specific 15-mers were determined by FACS analysis. We identified the HLA class I or II allele presenting each epitope by assessing the cytolytic activity and/or cytokine production of the pooled-peptide sensitized T-cells in response to 2° stimulation with a panel of PHA blasts loaded with the identified 15-mer, each sharing a single HLA allele with the T-cell donor. T-cells from each of 15 donors tested generated WT-1 peptide reactive T-cells in response to the WT-1 peptide pool. Of 23 epitopes identified that elicited T-cell responses, 19 were heretofore unrecognized epitopes presented by HLA A0201(N=4), A0301(N=2), A2402 (N=2), B3501(N=3), B0701(N=1), B4402(N=2), B3808(N=1), DRB, 0401(N=2), DRB10402(N=1) and DRB1 0701(N=1). Each of the 15 new epitopes presented by class I alleles elicited IFNγ+ CD8+ T-cells that were also cytotoxic against leukemic blasts sharing the restricting allele. All four epitopes presented by class II HLA alleles elicited IFNγ+ CD4+ T-cell responses, of which 2 were also cytotoxic against HLADR+ leukemia blasts expressing the restricting allele. Responses to these newly identified epitopes were comparable to or exceeded those elicited by the known immunogenic peptide epitope 126–134RMFPNAPYL presented by HLAA0201. Like RMF-specific T-cells, T-cells specific for 4 of the new epitopes tested to date do not exhibit activity against normal hematopoietic progenitor cells. Thus, by sensitizing T-cells from unselected normal donors, with autologous DCs loaded with a pool of overlapping 15-mers spanning WT-1, we have generated T-cells specific for a series of new epitopes of WT-1, each, presented by a distinct class I or II HLA allele, which also exhibit HLA-restricted cytotoxic against WT-1+ leukemic cells. Clinical trials of adoptive therapy with WT-1 specific T-cells generated by this approach are being initiated for adoptive therapy.