title: Nuclear translocation of nuclear factor of activated T cells (NFAT) as a quantitative pharmacodynamic parameter for tacrolimus.
abstract: Nuclear factor of activated T cells (NFAT) is a family of transcription factors involved in regulating the immune response. The canonical NFAT pathway is calcium-dependent and upon activation, NFAT is dephosphorylated by the phosphatase, calcineurin. This results in its translocation from the cytoplasm to the nucleus and transcription of downstream target genes that include the cytokines IL-2, IL-10, and IFNγ. Calcineurin inhibitors including tacrolimus inhibit the NFAT pathway and are used as immunosuppressants in transplant settings to prevent graft rejection. There is, as yet, no direct means to monitor tacrolimus pharmacodynamics. In this study, a rapid, quantitative, image cytometry-based measurement of nuclear translocation of NFAT1 is used to evaluate NFAT activation in T cells and its tacrolimus-induced inhibition. A strong dose-dependent correlation between NFAT1 inhibition and tacrolimus dose is demonstrated in vitro. Time kinetic analysis of NFAT1 inhibition in plasma from stable renal transplant recipients before and after an in vivo dose with tacrolimus correlated with the expected pharmacokinetic profile of tacrolimus. This was further corroborated by analysis of patients' autologous CD4 and CD8 T cells. This is the first report to show that the measurement of NFAT1 activation potential by nuclear translocation can be used as a direct, sensitive, reproducible and quantitative pharmacodynamic readout for tacrolimus action. These results, and the rapid turnaround time for this assay, warrant its evaluation in a larger clinical setting to assess its role in therapeutic drug monitoring of calcineurin inhibitors.
authors.affiliation: Department of Flow and Image Cytometry, Roswell Park Cancer Institute, University at Buffalo, New York.
authors.forename: Orla
authors.initials: O
authors.lastname: Maguire
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Tornatore
authors.forename: Kieran L
authors.initials: KL
authors.lastname: O'Loughlin
authors.forename: Rocco C
authors.initials: RC
authors.lastname: Venuto
authors.forename: Hans
authors.initials: H
authors.lastname: Minderman
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D061207:Calcium Ionophores / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism*; D005434:Flow Cytometry; D006084:Graft Rejection / Q000517:prevention & control; D006801:Humans; D007166:Immunosuppressive Agents / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D007371:Interferon-gamma / Q000097:blood; D015759:Ionomycin / Q000494:pharmacology; D019169:Jurkat Cells; D016030:Kidney Transplantation; D050778:NFATC Transcription Factors / Q000378:metabolism*; D021381:Protein Transport; D015203:Reproducibility of Results; D016559:Tacrolimus / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D061207:Calcium Ionophores; D007166:Immunosuppressive Agents; D050778:NFATC Transcription Factors; D015759:Ionomycin; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate; D016559:Tacrolimus
keywords: imaging flow cytometry; immunosuppression; nuclear factor of activated T cells (NFAT1); tacrolimus