RT Journal Article
SR Electronic
T1 T-Cell Receptor-Stimulated Calcineurin Activity Is Inhibited in Isolated T Cells from Transplant Patients
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 602
OP 607
DO 10.1124/jpet.109.154096
VO 330
IS 2
A1 James A. Tumlin
A1 Brian R. Roberts
A1 Kenneth E. Kokko
A1 Osama El Minshawy
A1 Jennifer L. Gooch
YR 2009
UL http://jpet.aspetjournals.org/content/330/2/602.abstract
AB The addition of calcineurin inhibitors, including cyclosporine A (CsA) and FK-506 (tacrolimus), to transplant protocols has markedly reduced acute allograft rejection and prolonged patient survival. Although monitoring of serum drug levels has been shown to be a poor indicator of efficacy, there is little data on calcineurin enzymatic activity in humans. Therefore, we measured calcineurin in isolated CD3+/4+ T cells from 81 non-transplant controls and 39 renal allograft patients by using a 32PO4-labeled calcineurin-specific substrate. A gender difference was observed in the control cohort, with activity in males significantly higher than that in females (1073 ± 134 versus 758 ± 75 fmol/μg/min, respectively). Activity of both groups was comparably inhibited by 5 ng/ml tacrolimus (27 ± 4 versus 30 ± 4%). Calcineurin is a downstream target of the T-cell receptor (TCR). Therefore, activity was measured in isolated T cells after incubation with anti-CD3/CD28 antibodies to stimulate the TCR. Calcineurin activity increased significantly from 1214 ± 111 to 1652 ± 138 fmol/μg/min; addition of either tacrolimus or CsA (500 ng/ml) blocked CD3/CD28 stimulation. Despite therapeutic levels of tacrolimus and CsA (mean 11.4 and 172 ng/ml), basal calcineurin activity was significantly higher among renal transplant recipients than controls (1776 ± 175 versus 914 ± 78 fmol/μg/min). In contrast, anti-CD3/CD28 antibodies failed to stimulate calcineurin activity in transplant subjects. Finally, we found that basal and stimulated calcineurin activities are inversely related. Consistent with this finding, basal activity in resting T cells rose over time after transplant but stimulation fell (r2 = 0.785, p < 0.05). These data suggest that examination of TCR-stimulated calcineurin activity after renal transplantation may be useful for monitoring immunosuppression of individual patients. U.S. Government work not protected by U.S. copyright