T-cell receptor-stimulated calcineurin activity is inhibited in isolated T cells from transplant patients.
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 (32)PO(4)-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/microg/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/microg/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/microg/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 (r(2) = 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.
Department of Internal Medicine, University of Tennessee College of Medicine, Chattanooga, Tennessee, USA.
James A
JA
Tumlin
Brian R
BR
Roberts
Kenneth E
KE
Kokko
Osama
O
El Minshawy
Jennifer L
JL
Gooch
D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002469:Cell Separation; D015331:Cohort Studies; D016572:Cyclosporine / Q000627:therapeutic use; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000627:therapeutic use; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D016030:Kidney Transplantation / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D011948:Receptors, Antigen, T-Cell / Q000037:antagonists & inhibitors / Q000502:physiology*; D016176:T-Lymphocyte Subsets / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D016559:Tacrolimus / Q000627:therapeutic use
D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D011948:Receptors, Antigen, T-Cell; D016572:Cyclosporine; D019703:Calcineurin; D016559:Tacrolimus