The rationale of adoptive T cell therapy is based on the attempt to circumvent pre-existing tolerance mechanisms by stimulating potentially tumor-reactive T cells ex vivo. Efforts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of isolating tumor-reactive T cells present in low numbers in peripheral blood of tumor patients. Furthermore, the development of an effective immunotherapy in the autologous context is hampered by the deficit of an effective T cell repertoire against tumor antigens.

We have optimized the techniques for isolating and expanding antigen-specific allogeneic T cells. Following repetitive peptide-driven stimulations with HLA-A*0201 positive dendritic cells the responding HLA-A*0201 negative CD8+ T cells were stained with HLA-A*0201/peptide pentamers. Multimer-positive T cells were sorted and directly cloned by limiting dilution. Using this technique we have succeeded in establishing T cell clones directed against several HLA-A*0201-resticted peptides derived from Ewing Tumor (ET) specific antigens identified via previous DNA microarray analysis and supposed to play a central role in the pathogenesis of this tumor. These T cells not only specifically recognized peptide-pulsed target cells or antigen transfected cells in the context of HLA-A*0201 but also killed HLA-A*0201+ ET expressing the antigen while HLA-A*0201– ET were not affected.

Allogeneic, tumor specific T cells can be easily isolated via Peptide/HLA-multimer technology and may benefit therapeutic strategies in allogeneic stem cell transplantation.