Flexible and sensitive method to functionally validate tumor-specific receptors via activation of NFAT.
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)alphabeta or a chimeric (ch-)TCRalphabeta, 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 TNFalpha 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.
Schaft
Niels
N
Department of Medical Oncology, Laboratory of Medical and Tumor Immunology, Erasmus MC-Daniel den Hoed, Groene Hilledijk 301, 3075 EA Rotterdam, The Netherlands.
Lankiewicz
Birgit
B
Gratama
Jan Willem
JW
Bolhuis
Reinder L H
RL
Debets
Reno
R
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D017930:Genes, Reporter; D005821:Genetic Techniques / Q000706:statistics & numerical data; D006801:Humans; D019169:Jurkat Cells; D050778:NFATC Transcription Factors; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha