Abstract
The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of WT1 which could be useful for immunotherapy. Potential HLA-A*01 epitopes predicted by a MHC binding algorithm were screened for recognition by peripheral blood mononuclear cells (PBMC) from patients with spontaneous T cell responses using intracellular cytokine cytometry. Epitope processing was shown by proteasomal cleavage. Epitope-specific T cells were generated from CD4+CD25+ regulatory T cell-depleted PBMC. One of five predicted HLA-A*01-binding candidate epitopes showed high immunogenicity as 5 of 14 patients with hematologic malignancies had WT1.317-327-reactive T cells ranging from 0.4% to 1.5% of CD3+CD8+ T cells. Proteasomal degradation assays indicated the cleavage of WT1.317-327. The depletion of regulatory T cells from PBMCs enabled the rapid expansion of WT1.317-327-specific CTL, whereas no CTL could be generated from unfractionated PBMC. WT1.317-327-specific CTL efficiently lysed an autologous WT1-expressing tumor cell line but not HLA-A*01-negative WT1-expressing tumor cells. Immunogenicity of the epitope across histologies was verified by the demonstration of spontaneous ex vivo WT1.317-327-specific T cell responses in two of six patients with HLA-A*01-positive melanoma or lung cancer. In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*01-restricted T cell epitope of the tumor antigen WT1, which is of considerable interest for use in vaccination trials.
Highlights
The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies
The search for new T cell epitopes in known tumor antigens using the classical ‘‘reverse immunology’’ strategy has led to the identification of numerous T cell epitopes, most of them restricted to HLA-A*0201 [22]
We have identified a new HLA-A*01 epitope and have thereby evaluated a modified reverse immunology strategy to identify immunogenic T cell epitopes from WT1 combining the following steps: (a) prediction of potential epitopes by the SYFPEITHI algorithm, (b) screening of potential epitopes for recognition by T cells from tumor patients likely to exhibit spontaneous T cell responses against the target antigen, (c) in vitro degradation by constitutive and immunoproteasome to show natural epitope processing, and (d) generation of WT1 epitope – specific CTL from CD4+CD25+ regulatory T cell – depleted peripheral blood mononuclear cells (PBMC) to show cytolysis of WT1-expressing tumor cells
Summary
The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies. The reverse immunology approach includes the prediction of potential T cell epitopes from known tumor antigens, their analysis for MHC-binding, followed by the in vitro generation of peptide-reactive T cells, and their testing for target cell recognition. This strategy is rather laborious, because of the need for T cell induction against multiple peptides, including many that may not be processed. Epitope processing can be verified by in vitro proteasomemediated digestion pattern analysis This approach has reliably identified HLA-A*02 – binding epitopes from tumor and viral proteins using 25- to 30-mer peptides encompassing the putative epitope [27, 28]
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