Flexible and sensitive method to functionally validate tumor-specific receptors via activation of NFAT
- 作者：Schaft ; Niels ; Lankiewicz ; Birgit ; Gratama ; Jan Willem ; Bolhuis ; Reinder L.H. ; Debets ; Reno
- 关键词：NFAT ; Reporter gene assay ; T cell retargeting ; Tumor-specific receptors ; BCS ; bovine calf serum ; CsA ; cyclosporin A ; dnNFATmut ; dominant negative NFAT mutant ; fl ; full length ; GaM ; goat-anti-mouse ; mAb ; monoclonal antibody ; MFI ; mean fluorescence intensit
- 刊名：Journal of Immunological Methods
- 出版年：2003
- 期刊代码：57_00221759
- 类别：imm
- 出版时间：September, 2003
- 卷：280
- 期：1-2
- 页码：13-24
- 文件大小：221 K
摘要
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.