Single-Cell Analysis of NFATc1 Amplification in T Cells for Pharmacodynamic Monitoring of Tacrolimus

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IntroductionTherapeutic drug monitoring (TDM) of the calcineurin inhibitor tacrolimus (TAC) is based on blood concentrations that show an imperfect correlation with the occurrence of acute rejection. A pharmacodynamic method that reflects the direct inhibitory effects of TAC may therefore be preferable over traditional pharmacokinetic TDM. Here, we tested whether measuring the amplification of NFATc1, a member of the calcineurin pathway, is suitable for TDM of TAC.Materials and MethodsNFATc1 amplification was monitored in T cells of kidney transplant recipients who received either TAC- (n = 11) or a belatacept (BELA)-based (n = 10) immunosuppressive therapy. Heparinized blood samples were collected at days 0 (pre-transplantation), 4, 30, 90, 180 and 360 after transplantation and stimulated with PMA/ionomycin. In addition, individual drug effects on NFATc1 amplification were studied in vitro, after spiking blood samples of healthy volunteers with either TAC, BELA or mycophenolate mofetil.Results and DiscussionIn TAC- treated patients, at day 30 after transplantation, NFATc1 amplification was significantly inhibited in CD4+ T cells expressing the co-stimulation receptor CD28 (mean inhibition 37%; p = 0.01) and in CD8+CD28+ T cells (mean inhibition 29%; p = 0.02), while this was not observed in CD8+CD28- T cells or in BELA-treated patients. The TAC pre-dose concentrations of these patients correlated inversely with NFATc1 amplification in CD28+ T cells (rs = -0.46; p < 0.01). The in vitro study revealed a dose dependent effect of TAC on NFATc1 amplification in all three tested T cell subsets (mean inhibition 58% at 50 ng/ml TAC; p = 0.02), while belatacept and mycophenolate mofetil did not show an effect.Only one patient under TAC-based therapy suffered from a rejection and, as a consequence, no conclusions could be drawn on the association between NFATc1 amplification and rejection risk.ConclusionIn conclusion, measuring NFATc1 amplification is a promising tool for monitoring the biological effects of TAC on T cell subsets directly, which might be useful for further transplantation diagnostics.

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