Flexible and sensitive method to functionally validate tumor-specific receptors via activation of NFAT

• Published in: Journal of immunological methods 2003-09, Vol.280 (1), p.13-24
• Main Authors: Schaft, Niels, Lankiewicz, Birgit, Gratama, Jan Willem, Bolhuis, Reinder L.H., Debets, Reno
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - metabolism; Antineoplastic agents; Biological and medical sciences; Cytotoxicity, Immunologic; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Genes, Reporter; Genetic Techniques - statistics & numerical data; Humans; Immunotherapy; Jurkat Cells; Medical sciences; Molecular immunology; Neoplasms - genetics; Neoplasms - metabolism; NFAT; NFATC Transcription Factors; Nuclear Proteins; Pharmacology. Drug treatments; Receptors, Antigen, T-Cell, alpha-beta - genetics; Receptors, Antigen, T-Cell, alpha-beta - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Reporter gene assay; Sensitivity and Specificity; T cell retargeting; T-Lymphocytes - immunology; T-Lymphocytes - metabolism; Techniques; Transcription Factors - genetics; Transcription Factors - metabolism; Transduction, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha - biosynthesis; Tumor-specific receptors
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(03)00067-X

Tumor-specific receptors may provide effective tools for anti-tumor immunogene therapy. However, the functional analysis of primary human T cells engrafted with tumor-specific receptors is laborious and emphasizes the need for a fast and sensitive method to validate such receptors. To this end, we have set up a Jurkat T cell-based reporter gene assay, and tested receptors with various formats, i.e., receptors based on either a monoclonal antibody (mAb), a full-length T cell receptor (fl-TCR)αβ or a chimeric (ch-)TCRαβ, and various antigen specificities for their ability to mediate tumor-specific activation of nuclear factor of activated T cells (NFAT). The mAb-based receptor specifically mediates NFAT activation after stimulation with tumor antigen-positive target cells. The observed receptor-mediated NFAT responses were validated by the use of ligand- and receptor-specific mAbs, as well as cyclosporin A (CsA) and a dominant negative mutant of NFAT. Furthermore, anti-TCR mAbs, peptide-loaded tumor cells and antigen-positive tumor cells all resulted in specific NFAT activation in TCR/CD8 co-transduced Jurkat T cells, irrespective of the TCR format used. Importantly, receptor-mediated NFAT responses parallel tumor-specific cytolysis and TNFα production of receptor-transduced primary human T lymphocytes. In fact, inhibition of NFAT activation compromises the immune responses of primary human T lymphocytes, pointing to a central involvement of NFAT in anti-tumor T cell responses. Taken together, receptor-mediated activation of NFAT constitutes a representative measure of anti-tumor T cell responses, and the genetically modified Jurkat T cells provide a flexible and sensitive tool with which to select rapidly tumor-specific (chimeric) receptors for immunogene therapy.