Flexible and sensitive method to functionally validate tumor-specific receptors via activation of NFAT
Journal of Immunological Methods , Volume 280 - Issue 1-2 p. 13- 24
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.
|NFAT, Reporter gene assay, T cell retargeting, Tumor-specific receptors|
|Journal of Immunological Methods|
|Organisation||Department of Medical Oncology|
Schaft, N, Lankiewicz, B, Gratama, J.W, Bolhuis, R.L.H, & Debets, J.E.M.A. (2003). Flexible and sensitive method to functionally validate tumor-specific receptors via activation of NFAT. Journal of Immunological Methods, 280(1-2), 13–24. doi:10.1016/S0022-1759(03)00067-X