Background and objectives: Calcineurin inhibitors (CNIs) are widely used in organ transplantation (Tx). Due to the narrow therapeutic window, regular drug monitoring is necessary to balance sufficient efficacy with minimal toxicity. Until now, the monitoring has mainly been based on measurements of drug concentrations. However, despite concentration guided dosing of CNIs, patients still suffer from acute or chronic rejection and CNI related toxicity during CNI therapy. Pharmacokinetic (PK) measurements do not necessarily reflect the CNI effect on immune cells and pharmacodynamic (PD) monitoring of the nuclear factor of activated T cells (NFAT)-regulated gene expression has been suggested as a promising supplementary tool for individualizing CNI therapy.
The objective of the present project was to develop and validate an assay to examine NFAT regulated genes as PD biomarkers of CNI response in blood samples from renal transplant recipients.
Materials and methods: Heparinized peripheral whole blood samples with and without addition of different immunosuppressive drugs were stimulated with phorbol 12 myristate13 acetate (PMA) and ionomycin for up to 72 hours at 37C. Samples from renal allograft recipient were drawn before and 1.5 hours after tacrolimus (Tac) dosing and stimulated with PMA and ionomycin for 3 hours at 37C. Following cell lysis, total RNA was isolated with the automated MagNA Pure LC instrument and reverse transcribed using Transcriptor First Strand cDNA Synthesis Kit (Roche). The expression of NFAT regulated genes, interleukin 2 (IL2), interferon (IFNG) and colony-stimulating factor 2 (granulocyte-monocyte) (CSF2) was determined by real time PCR on the LightCycler® 480 instrument (Roche). The calculations included normalization to the expression of three reference genes, 5-aminolevulinate synthase 1 (ALAS1), beta-2-microglobulin (B2M) and ribosomal protein L13A (RPL13A), and PCR efficiency correction.
Results: SYBR Green I analysis, gel electrophoresis and Sanger sequencing demonstrated selective amplification of the target gene sequences. Standard curves were linear over a range corresponding to starting concentrations of 107 to 102 cDNA templates per reaction. The between and within assay coefficient of variability (CV) was ≤19.8% and ≤14.8%, respectively. The maximum response to PMA and ionomycin was observed after 3 hours incubation, resulting in 120 000, 20 000 and 8000 fold increases of IL2, IFNG and CSF2 expression, respectively. Ex vivo exposure to different immunosuppressive drugs and concentrations, demonstrated that inhibition of NFAT-regulated gene expression seemed to be relatively CNI specific and inversely correlated to CNI concentration. There was a large variability of NFAT-regulated gene expression between patients before Tx. About one week after Tx, 4 of 5 patients demonstrated residual gene expression ≤ 13%. Three of these patients demonstrated Tac C0 levels within the therapeutic target range.
Conclusions: Several studies support the potential of NFAT-regulated gene expression as a PD tool for further individualization and improvement of CNI therapy. A reverse transcription (RT) and real-time PCR assay was developed and validated to determine IL2, IFNG and CSF2 gene expressions in whole blood samples. The preliminary results of the present study support the use of NFAT-regulated gene expression as PD biomarker of Tac response. However, the value of monitoring NFAT regulated gene expression during Tac therapy needs to be determined in prospective intervention trials with larger patient cohorts.