Allogeneic HLA-A2-Restricted WT1-Specific T Cells From Mismatched Donors Are Highly Reactive but Show Potentially Hazardous Promiscuity.

Abstract 4081

Poster Board III-1016

T cells recognizing self antigens that are overexpressed in malignant cells such as the Wilms tumor protein (WT1) have been proposed to contribute to the anti-tumor reactivity after both allogeneic and autologous stem cell transplantation. The WT1 gene is expressed in human acute leukemia and chronic myeloid leukemia at much higher levels than in normal mononuclear blood cells and CD34+ hematopoietic progenitors. Various groups have attempted to use WT1 as a target in cancer immunotherapy. HLA-A2-restricted WT1-reactive CD8+ T cells that recognize peptide-loaded HLA-A2+ target cells have been isolated from both HLA-A2 negative and positive donors, but recognition of primary leukemic cells has only consistently been demonstrated with WT1-reactive CD8+ T cells generated in the mismatched setting.

We hypothesized that HLA-A2-restricted WT1-specific CD8+ T cells generated from HLA-A2 positive donors are likely to be specific for the WT1 peptide, but may show only low reactivity against targets presenting endogenous WT1 antigen in the context of HLA-A2 due to thymic deletion of high affinity T cells. In contrast, HLA-A2-restricted WT1-specific T cells generated from HLA-A2 negative donors may be highly reactive against these targets, but with increased risk for cross reactivity.

To test this hypothesis, WT1-specific T cells were generated from HLA-A2 positive and negative donors by stimulating purified CD8+ T cells with the HLA-A2 binding WT1_126-134 peptide (RMFPNAPYL) loaded onto autologous dendritic cells in the case of HLA-A2 positive donors, or onto autologous HLA-A2 transduced EBV-LCL in the case of HLA-A2 negative donors. WT1-specific CD8+ T cell clones were isolated by flowcytometric cell sorting using WT1/A2 tetramers, and expanded. All T cell clones selected for functional analysis stained brightly with WT1/A2 tetramers, but not with irrelevant HLA-A2 control tetramers (PR1, PRAME, CMVpp65, HA-1), indicating specificity for the WT1 peptide presented in HLA-A2.

Functional analysis was conducted using interferon-gamma (IFNg) ELISA and flowcytometric measurement of cytokine production and degranulation using HLA-A2+ EBV-LCL or T2 cells as target cells. WT1-reactive clones isolated from HLA-A2+ donors only recognized HLA-A2+ targets loaded with high (>10nM) concentrations of exogenous WT1 peptide, whereas most clones generated from HLA-A2 negative individuals showed high reactivity against the HLA-A2+ targets, even in the absence of exogenously loaded WT1 peptide, indicating cross-reactivity.

To investigate antigen specificity, we constructed artificial antigen-presenting beads coated with HLA-A2 monomers containing different ratios of WT1 and CMVpp65 peptides, and used these beads to stimulate the WT1-reactive T cell clones. Dose dependent recognition of WT1, but not of the control pp65 peptide, was found for WT1 reactive T cell clones isolated from HLA-A2 positive donors as well as for clones from HLA-A2 negative donors. In response to stimulation with the various concentrations of WT1 loaded beads, IFNg production by WT1-reactive T cell clones from HLA-A2 negative donors was higher than production by clones from HLA-A2 positive donors, indicating that WT1-reactive clones from HLA-A2 negative donors had higher affinity for the HLA-A2/WT1 complex.

To further examine and quantify the potential promiscuity of the WT1-reactive clones, we tested their capacity to bind a panel of 300 randomly selected HLA-A2 tetramers. Whereas clones generated from HLA-A2 positive individuals only recognized the WT1 tetramer, different WT1-reactive T cell clones generated from HLA-A2 negative individuals recognized approximately 5% of the tetramers, indicating that they were highly promiscuous in their peptide recognition. Competition experiments using HLA-A2+ target cells loaded with various concentrations of exogenous WT1 peptide indicated differential recognition of the HLA-A2 target peptides by different WT1-reactive T cell clones generated from HLA-A2 negative individuals.

In conclusion, allogeneic HLA-A2-restricted WT1-specific T cells isolated from mismatched donors may be more tumor-reactive than their autologous counterparts, but show potentially hazardous promiscuity.