A high-avidity WT1-reactive T-cell receptor mediates recognition of peptide and processed antigen but not naturally occurring WT1-positive tumor cells

Wilms tumor gene 1 (WT1) is an attractive target antigen for cancer immunotherapy because it is overexpressed in many hematologic malignancies and solid tumors but has limited, lowlevel expression in normal adult tissues. Multiple HLA class I and class II restricted epitopes have been identified in WT1, and multiple investigators are pursuing the treatment of cancer patients with WT1-based vaccines and adoptively transferred WT1-reactive T cells. Here we isolated an HLA-A∗0201-restricted WT1-reactive T-cell receptor (TCR) by stimulating peripheral blood lymphocytes of healthy donors with the peptide WT1:126-134 in vitro. This TCR mediated peptide recognition down to a concentration of ∼0.1 ng/mL when pulsed onto T2 cells as well as recognition of HLA-A∗0201⁺ target cells transfected with full-length WT1 cDNA. However, it did not mediate consistent recognition of many HLA-A∗0201⁺ tumor cell lines or freshly isolated leukemia cells that endogeneously expressed WT1. We dissected this pattern of recognition further and observed that WT1:126-134 was more efficiently processed by immunoproteasomes compared with standard proteasomes. However, pretreatment of WT1⁺ tumor cell lines with interferon gamma did not appreciably enhance recognition by our TCR. In addition, we highly overexpressed WT1 in several leukemia cell lines by electroporation with full-length WT1 cDNA. Some of these lines were still not recognized by our TCR suggesting possible antigen processing defects in some leukemias. These results suggest WT1:126-134 may not be a suitable target for T-cell based tumor immunotherapies.