Immunosuppressive Pharmacology Research Program, Translational Pharmacology Research Center, NYS Center of Excellence in Bioinformatics and Life Sciences, Department of Pharmacy Practice, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, New York
Division of Nephrology, Department of Medicine, Erie County Medical Center, School of Medicine and Biomedical Sciences; University at Buffalo, Buffalo, New York
Department of Flow and Image Cytometry, Roswell Park Cancer Institute, University at Buffalo, New York
Correspondence to: Hans Minderman, Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Cancer Cell Center, Elm and Carlton Streets, Buffalo, NY, USA. E-mail: firstname.lastname@example.org
Pharmacologic immunosuppression is essential for solid organ transplantation as a countermeasure to allograft rejection. Tacrolimus, a calcineurin inhibitor (CNI) prescribed post-transplant, binds to the immunophyllin FK-506 binding protein, FKBP12 [1-4]. This complex inhibits the calcium-dependent phosphatase, calcineurin, preventing the dephosphorylation required for nuclear translocation of the transcription factor, nuclear factor of activated T cells (NFAT) [1, 2, 5, 6]. Inhibition of NFAT nuclear translocation in CD4+ and CD8+ T cells reduces transcription of its target genes including interleukin-2 (IL-2) and interferon-gamma (IFNγ), thus inhibiting the immune response [1, 2, 5].
Currently, there is no clinically applicable, direct measurement of inhibition of cellular targets for immunosuppression. Consensus dosing of tacrolimus aims at achieving target troughs (lowest drug concentration predose) according to multicenter clinical trials balancing immunosuppression with adverse effects . In clinical practice, tacrolimus dosing modifications are currently based on monitoring of predose trough concentrations and in response to clinical symptoms such as rejection of the graft (under-immunosuppression) or infections (over-immunosuppression) [1-4]. Calcineurin phosphatase activity and NFAT regulated gene expression of cytokines have the potential to serve as biomarkers for the pharmacodynamic activity of CNIs [7-12]. NFAT regulated gene expression of IL-2, IFNγ, and granulocyte–monocyte colony stimulating factor (GM-CSF) in peripheral blood mononuclear blood cells (PBMCs) from renal and liver transplant patients receiving CNIs has been correlated with over-immunosuppression [12-16]. However, the correlation of this approach with trough concentrations of CNIs has not been studied [1, 4, 17, 18]. Importantly, these analyses are labor intensive and time consuming and focus on downstream targets of the NFAT pathway, cytokines, whose expression may be affected by other CNI-independent factors. Cytoplasmic to nuclear translocation of transcription factors such as NF-κB and NFAT are correlated with their signaling activity and image cytometry has previously been applied to quantify these intracellular redistributions [19-21].
This study describes direct measurement of NFAT1 activation by quantifying its nuclear localization in CD4+ and CD8+ T cells using a flow cytometry-based image analysis approach. There are currently five known members of the NFAT transcription factor family. NFAT1 was chosen for this study since this biomarker is known to be sensitive to tacrolimus [reviewed in ]. This method provides an innovative approach to obtain quantitative information on the prevalence of NFAT1 in heterogeneous cell populations and its specific cytoplasmic and nuclear localization which is linked to its activation.
All analysis files mentioned are available upon request.
Cell Culture and Treatments
The Jurkat human T cell line was maintained at exponential growth at 37°C in a humidified atmosphere of 5% CO2 in air in RPMI-1640 media (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (PAA Laboratories, Queensland, Australia), 2 mM l-glutamine (Mediatech, Manassas, VA), 20 U mL−1 penicillin and 20 μg mL−1 streptomycin (Mediatech, Manassas, VA).
For treatments of Jurkat cells, cell densities were adjusted to 1 × 106 cells mL−1 and phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Invitrogen, Carlsbad, CA) were added to achieve final concentrations of 20 ng mL−1 and 1.5 µM, respectively and incubated for 30 min at 37°C. To test tacrolimus inhibition, cells were pretreated with 1 pM–10 µM tacrolimus (LKT Labs, St. Paul, MN) for 1.5 h at 37°C, and PMA/Ionomycin was added with continued incubation with tacrolimus for 30 min. Control samples at each concentration were incubated without stimulants. Following activation, cells were fixed for 10 min in 4% methanol-free formaldehyde (Polysciences, Warrington, PA) and stained for NFAT1 (below).
In Vitro NFAT1 Activation in Healthy Donor Whole Blood
One sodium heparin tube was collected by venous puncture and allowed to “rest” at room temperature for at least 1 h. Collection protocol was approved by the Institutional Review Board (IRB) at Roswell Park Cancer Institute. PMA/Ionomycin (Invitrogen, Carlsbad, CA) were added to achieve final concentrations of 200 ng mL−1 and 15 µM, respectively to 500 µL whole blood for 30 min. To test PMA/Ionomycin serial dilutions, the reagent mix was prepared prior to serial dilution with 1× phosphate buffered saline (PBS). To test IFNγ expression, cells were cotreated with PMA/Ionomycin and Brefeldin A (BFA) at a final concentration of 2.5 µg mL−1. To test tacrolimus inhibition, cells were pretreated with 1 nM–10 µM tacrolimus for 1.5 h at 37°C with continued incubation with tacrolimus for 30 min. Control samples at each concentration were incubated without stimulants. Following activation, cells were immunophenotyped for CD4+ and CD8+ markers, fixed, red blood cells lysed, and stained for NFAT1 (below).
Clinical Study and Selected Renal Transplant Recipients
Three stable renal transplant recipients who participated in a nonrandomized clinical pharmacokinetic study were included in this study. The clinical study was approved by UB Health Sciences IRB with IRB# PHP0720608B and adhered to the Declaration of Helsinki. Patients provided written informed consent prior to study participation. Patients received tacrolimus and enteric coated mycophenolate sodium (EC-MPS) at steady-state conditions for at least 6 months with no dosage adjustments for 7 days prior to the study.
At time 0 (predose tacrolimus trough), blood was collected for NFAT1 evaluation, tacrolimus trough, metabolic, renal, and hepatic function tests and with complete blood count using heparinized and EDTA tubes. Oral tacrolimus and EC-MPS were then administered and additional blood samples collected at 1, 2, 3, 4, and 6 h after taking the immunosuppressive drugs for tacrolimus concentrations and NFAT1 assessment. Samples collected for NFAT1 analysis were stored at room temperature and processed within 8 h of collection. Tacrolimus clinical troughs were analyzed within 24 h and timed patient samples were analyzed in batch at ECMC Clinical Laboratory using ARCHITECT tacrolimus assay (Abbott, Abbott Park, IL), a chemiluminescent microparticle immunoassay. The lower limit of detection was 1.5 ng mL−1 and intraday assay variability was <7%.
Tacrolimus Plasma Concentration vs. NFAT1 Activation
Jurkat cells, at a cell density of 1 × 106 cells mL−1 (two parallel tubes/time: one served as an unstimulated control while the other one was used for assessment of PMA/Ionomycin effect), were resuspended in 500 µL plasma and incubated at 37°C for 1.5 h. PMA/Ionomycin was added to one of the paired cultures to achieve final concentrations of 200 ng mL−1 and 15 µM, respectively, with continued incubation in plasma for 30 min. Cells were fixed for 10 min in 4% methanol-free formaldehyde (Polysciences, Warrington, PA) and stained for NFAT1 (below).
Following fixation, nonspecific binding was eliminated by blocking with 20 µL Mouse IgG in 100 µL permeabilization wash buffer (PWB) consisting of 0.1% Triton X-100 (EMD Biosciences, San Diego, CA) in 1× PBS for 20 min. For NFAT1; mouse monoclonal FITC conjugated-NFAT1 antibody (BD Biosciences, San Diego, CA) was diluted 1:50 in PWB and added to cells with incubation for 20 min in the dark at room temperature. Cells were washed in 1× PBS to remove unbound antibody, then resuspended in 100 μL 1× PBS. Following the completion of the presented studies we have implemented a change in NFAT1 antibody which improved the interexperiment variability. This information is presented in Supporting Information Figure 1. For our ongoing studies we are now using an unconjugated rabbit anti human-NFAT1 antibody (Cell Signaling) which is diluted 1:50 in PWB and added to cells with incubation for 20 min in the dark at room temperature. A FITC-conjugated F(ab')2 fragment donkey anti rabbit IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) is then used to visualize NFAT1. This secondary stain is diluted 1:200 in PWB and added to cells with incubation for 20 min in the dark at room temperature. Cells are then washed in 1× PBS to remove unbound antibody and re-suspended in 100 μL 1× PBS. For IFNγ; mouse monoclonal FITC conjugated- IFNγ antibody (BD Biosciences, San Diego, CA) was diluted 1:5 in PWB and added to cells with incubation for 20 min in the dark at room temperature. Cells were washed in 1× PBS to remove unbound antibody, then resuspended in 100 μL 1× PBS. To visualize the nucleus, DAPI (Invitrogen, Carlsbad, CA) was added at a final concentration of 0.5 µg mL−1 prior to acquisition.
CD4 and CD8 immunophenotyping in whole blood samples was performed before fixation. Following activation, samples were preincubated with normal mouse IgG for 10 min on ice, and then stained with CD4-APC (BD Biosciences) and CD8-PE (BD Biosciences). Red blood cells were subsequently lysed and white blood cells fixed according to the manufacturer's recommendations for Lyse/Fix Buffer (BD Phosflow, BD Biosciences).
Data acquisition was performed on an imaging flow cytometer (ImagestreamX; Amnis/EMD Millipore, Seattle, WA) between years 2011 and 2012. Images acquired include a brightfield image (Channel 1 and 9; 430–480 nm), FITC (Channel 2; 480–560 nm), PE (Channel 3; 560–595 nm), DAPI (Channel 7; 430–505 nm), and APC (Channel 11; 660–740 nm). FITC and PE were excited by a 488-nm laser with an output of 100 mW, DAPI was excited by a 405-nm laser with an output of 10 mW, and APC was excited by a 658-nm laser with an output of 120 mW. Cell classifiers were applied to eliminate debris (minimum–maximum area), cells without a nucleus (minimum intensity in Channel 7), and cells with a high scatter profile (maximum intensity scatter channel 6). For each sample, bright field, NFAT1-FITC, CD8-PE, CD4-APC, and DAPI (nuclear stain) images were simultaneously collected for 20,000 events which in all cases resulted in at least 400 analyzable target cells (e.g., CD4+ or CD8+) to minimize sample-size related variability of the Rd value parameter according to manufacturer recommendations (Supporting Information Fig. 2).
In each experiment single color controls were stained for all fluorochromes and 500 events were collected with all relevant lasers on for each individual control. All channels were on, with brightfield LEDs and scatter laser off to accurately observe fluorescence overlap in all channels. Only those events exhibiting a positive signal in the channel of interest were collected (e.g., the NFAT1-FITC control was positive in channel 2). Each single color control file was then merged to generate a compensation matrix (an example of which is shown in Supporting Information Fig. 3), and all sample files were processed with this matrix applied.
Following compensation for spectral overlap, image analysis was performed with IDEAS® software (Amnis/EMD Millipore, Seattle, WA) as described previously . The gating and masking strategy are shown in Supporting Information Figure 4. Briefly, the CD4+ and CD8+ populations were hierarchically gated for single cells, focus quality and their respective relevant fluorescence intensity. To assess nuclear NFAT1 translocation, the corresponding nuclear (DAPI) image and NFAT1 (FITC) image of each cell was compared and a similarity score (SS) was assigned for individual cells. The SS is a Fisher Z transform of Pearson's correlation of the pixel intensity values of the DAPI and NFAT1 images within the nuclear area of each cell. The SS is assigned a positive or negative signature based on the value of the slope of the first order linear regression. If NFAT1 is nuclear, then its image will be similar to the DAPI image resulting in a DAPI/NFAT1 SS with a high positive value. Cells with predominantly cytoplasmic NFAT1 would have anti-similar DAPI and NFAT1 images, and a negative or low SS. Because the SS are calculated for each cell, a SS distribution histogram is created for each sample population (see Fig. 1A). The Fisher Discriminant ratio (Rd value) is the difference between the mean Similarity Score of two populations (nonstimulated and stimulated, respectively), divided by the sum of the standard deviations of their respective distributions.
All analysis graphs (unless otherwise stated) and corresponding statistics were performed using GraphPad Prism Version 6 for Windows (GraphPad Software, La Jolla, CA). Results are expressed as mean and standard error. Single measurement comparisons between two groups were tested using paired student's t tests. Statistical significance is indicated by *P < 0.05; **P < 0.01.
PMA/Ionomycin Concentration-Dependent Stimulation of NFAT1 Nuclear Translocation Quantified by Imaging Flow Cytometry
Nuclear translocation is essential for the activation of NFAT1. The human Jurkat T cell line was incubated in absence/presence of PMA/Ionomycin for 0.5 h. The time-point was based on previous optimizations of the assay (data not shown). Ionomycin increases intracellular calcium allowing NFAT dephosphorylation by calcineurin and translocation . PMA activates Protein Kinase C (PKC), which in turn up-regulates members of the transcriptional machinery allowing transcription of downstream targets . The distribution of NFAT1 in the Jurkat cells moves into the nucleus upon activation (Fig. 1A). Untreated cells have an average Similarity score of 1.02 ± 0.68, while treated cells have an average Similarity score of 2.48 ± 0.79, resulting in an Rd value of 0.99. To measure sensitivity of the assay, the dose-dependent change in nuclear translocation of NFAT1 in CD4+ and CD8+ T cells in healthy donor whole blood following exposure to a titration range of PMA/Ionomycin was assessed. The 1× concentration was set at 200 ng mL−1 and 15 µM (final) based on previous optimizations (not shown). This was diluted 2–64× and NFAT1 translocation measured in CD4+ and CD8+ T cells (Fig. 1B). Maximal activation, with Similarity scores above 2, is maintained with up to fourfold dilution. An 8 fold dilution of PMA/Ionomycin shows stimulation beginning to decline, with a sharp decline in nuclear localization of NFAT1 in the 16 fold dilution. At higher dilutions the Similarity score is similar to that of unstimulated cells (0). Representative images of cells in each of the distribution profiles are also shown (Fig. 1C). Images on the left clearly show a cytoplasmic localization of NFAT1 (red) with their corresponding Similarity scores, that moves into the nucleus upon treatment (right images with Similarity scores).
Functional Correlation Between NFAT1 Nuclear Translocation and Cytokine Production
To determine whether measurement of nuclear translocation of NFAT1 by imaging flow cytometry correlates with production of its downstream targets, healthy donor whole blood was treated with PMA/Ionomycin for varying time-points up to 24 h (Fig. 2). To measure the accumulation of IFNγ protein within the cells, 2.5 µg mL−1 Brefeldin A was added to inhibit transport of proteins from the endoplasmic reticulum. NFAT1 nuclear translocation occurs quickly peaking at 0.5 h. Nuclear localization is maintained up to 3 h and gradually decreases near to baseline at 24 h. IFNγ accumulation, measured by mean fluorescence intensity of the protein, gradually increases with highest peak at 24 h. Later time-points could not be accurately measured as protein accumulation causes cellular stress leading to cell death.
Dose-Dependent Inhibition of NFAT1 Nuclear Translocation Potential by Tacrolimus In Vitro
The human Jurkat T cell line and healthy donor whole blood were treated with PMA/Ionomycin in the presence or absence of increasing tacrolimus concentrations. In Jurkat cells, the highest Rd value is observed with PMA/Ionomycin activation without tacrolimus. A dose-dependent inhibition of nuclear translocation of NFAT1 was observed with increasing tacrolimus concentrations up to 1 µM (1,000 nM); higher concentrations induced cellular toxicity and resulted in cell death. Tacrolimus dose-dependent inhibition of nuclear NFAT1 translocation was also observed in CD4+ T cells of healthy donors again maximal at 1 µM tacrolimus exposure (Fig. 3B). The relatively lower level of NFAT1 inhibition in CD4+ cells in whole blood compared to the Jurkat cell line is likely associated with the known high drug binding to red blood cells and plasma proteins accounting for reduced unbound tacrolimus [4, 26].
The Patient Plasma Levels of Tacrolimus are Sufficient to Inhibit NFAT1 Nuclear Translocation in Cells Naive to Tacrolimus Treatment
To demonstrate the sensitivity of this assay and its correlation with the whole blood concentration of tacrolimus currently measured in patients, NFAT1 nuclear translocation was analyzed in the Jurkat cell line incubated with patient plasma obtained before and after receiving an oral dose of tacrolimus in vivo. Whole blood was collected from three stable renal transplant patients at predose troughs (time 0), then 1, 2, 3, 4, and 6 h after oral tacrolimus administration. The whole blood tacrolimus concentrations were measured using the ARCHITECT tacrolimus assay. Plasma was isolated from these samples and Jurkat cells were incubated with 500 µL plasma obtained at each time point in the presence and absence of PMA/ionomycin for 2 h. NFAT1 translocation potential (Rd values between the similarity score distributions with and without PMA/Ionomycin) and the measured tacrolimus concentrations versus time profile over the 6-h time interval are shown (Fig. 4). Tacrolimus concentrations were sufficient to inhibit NFAT1 nuclear translocation in Jurkat cells and the pharmacodynamics (NFAT1 inhibition) follows the tacrolimus concentration vs. time profiles in renal transplant recipients.
Tacrolimus-induced Inhibition of NFAT1 Nuclear Translocation can be Measured In Vivo
To demonstrate clinical applicability, the correlation between timed steady-state tacrolimus pharmacokinetics and inhibition of NFAT1 translocation was analyzed in the same three stable renal transplant patients (Fig. 5). Whole blood was collected as above and inhibition of NFAT1 translocation was determined by the NFAT1 translocation potential (Rd value between paired PMA/ion stimulated and nonstimulated samples) as single assessments at each time point. Inhibition of NFAT1 translocation potential (effect) was evident in the autologous CD4+ T cells among all three renal transplant recipients. For patient A, the NFAT1 nuclear translocation potential was highest at time zero (tacrolimus trough) before drug administration. A time dependent inhibition was observed up to 3 h following drug administration. This pharmacodynamic response of NFAT1 corresponded with the timing of the measured peak tacrolimus concentration. Inhibition of NFAT1 then rebounded as the tacrolimus concentrations declined. In case of patient B, NFAT1 nuclear translocation decreased following drug administration but the tacrolimus-induced inhibition was not as pronounced and more variable compared to patient A. For patient C, the pharmacodynamic response for NFAT1 inhibition again appears to present the inverse of tacrolimus concentration vs. time profile as noted in patients A and B. Similar results for all patients were observed in CD8+ T cells (data not shown).
Pharmacologic inhibition of the NFAT signal transduction pathway by calcineurin inhibitors (CNIs) is central to post-transplant immunosuppression with notable improvement in allograft survival achieved [1, 3, 4]. In clinical practice, therapeutic drug monitoring of tacrolimus troughs is essential due to interpatient variability in pharmacokinetic and pharmacodynamics of this drug. As a cellular target for tacrolimus action, intracellular NFAT1 translocation has the potential to be a direct quantitative pharmacodynamic parameter of response for transplant recipients to overall tacrolimus exposure. To our knowledge, the current study is the first to quantify the dose-dependent inhibition of NFAT1 translocation by tacrolimus in immunophenotypically defined target cells using imaging flow cytometry. Importantly, the effects of tacrolimus can be quantitatively measured within hours, as opposed to measuring downstream events such as the decreased expression of cytokines, which usually requires several days. Thus this approach has the potential to be applicable in a clinical setting of therapeutic monitoring.
Tacrolimus inhibition of NFAT1 translocation in vitro is dose-dependent in cell lines and whole blood from healthy donors and is inhibitory of the activation of the immunologic pathway (activation potential) following stimulation with PMA/Ionomycin. The maximum inhibition of NFAT1 translocation observed in the Jurkat cell line was at 1 µM of tacrolimus. Inhibition at higher tacrolimus concentrations could not be measured as cellular toxicity occurred. The larger variation of the data observed with the healthy donor samples compared to the Jurkat cell line is likely due to interindividual differences between the four donors. Targeted tacrolimus trough concentrations range from 5 to 15 ng mL−1 (∼10 nM) in transplant patients [4, 27]. Tacrolimus is extensively bound to erythrocytes (∼75–85%) and plasma proteins (14–16%), thus only a small fraction is available for intracellular distribution to account for the inhibition of calcineurin [4, 26, 28]. Relatively small differences in peripheral blood pharmacokinetics could therefore account for significant changes in pharmacodynamic effect from chronic CNI therapy. Another contributing factor to variable pharmacodynamic effect of tacrolimus would be the variable availability of the target immunophilins in T cells [7, 29].
To demonstrate clinical feasibility for the measurement of NFAT1 inhibition by tacrolimus, nuclear translocation was measured in three stable renal transplant recipients. Because tacrolimus concentrations are known to achieve a peak between 1 and 3 h following oral administration, serial samples were drawn from patients up to 6 h after the tacrolimus dose to observe the effects of the peak concentrations and drug distribution. These patients were clinically stable and greater than 12-month post-transplant but immunologic reactivity is variable between these patients based upon donor and recipient immunologic matches which may be reflective in the NFAT responses.
To ensure that the tacrolimus concentrations measured by the immunoassay were bio-active, the Jurkat T cell line was exposed to plasma from these patient samples and a time-pharmacodynamic inhibition was observed in all samples that correlated strongly with the tacrolimus concentration vs. time profile. In all three cases, inhibition of NFAT1 activation potential in the Jurkat cells was notably greater than that observed for the matching CD4+ T cells of the patients. A tentative explanation may be that these patients received chronic tacrolimus treatment with CD4+ T cells exposed to steady-state concentrations for months while the Jurkat cells were naïve to tacrolimus. The different measured peak tacrolimus concentrations in these three patients did result in corresponding different degrees of maximum inhibition of NFAT1 activation reflecting the interpatient variability described with CNI . The combined variables that could affect tacrolimus pharmacologic response should favor the incorporation of this direct pharmacodynamic assessment in therapeutic drug monitoring of tacrolimus. Because a number of clinical covariates have been found to influence the variability of tacrolimus pharmacokinetics and pharmacodynamics, these factors including gender, race, lipoprotein concentrations, hematocrit, concurrent medications, renal function, and P-glycoprotein function should be included with future evaluation of the clinical value of this approach [4, 27, 30].
It is noteworthy that the pharmacodynamic inhibition of NFAT1 appears as an inverse response to the tacrolimus concentration vs. time profiles with almost no time delay. This has implications for future clinical studies with limited sampling points with regards to evaluating the importance of maximum inhibition (at peak drug concentrations) versus steady state inhibition (at trough concentrations). However, the purpose of the inclusion of patient tacrolimus profiles was to demonstrate feasibility of the application in a clinical setting. Clearly, a larger clinical study should be performed to obtain conclusive evidence for these purported observations. Because the pharmacodynamic responses in the autologous CD4+ and CD8+ T cells appear more variable than observed in the Jurkat cells (compare Figs. 4 and 5) replicate measurements will be performed in future clinical studies to ensure accuracy of measurement which will enable the determination if the observed interpatient variability is due to a true biological variability between time points.
Combined, these in vitro and in vivo studies provide support for the NFAT1 activation potential as a tacrolimus pharmacodynamic biomarker. Because tacrolimus is the most frequently prescribed CNI for combination immunosuppressive regimens post-transplant in the US , this assay may provide a prospective diagnostic test to directly identify patients who would benefit from CNI dose modification. However, before this test would be applicable as an overall assessment for clinical immunosuppression following CNI-based treatments, it will be necessary to investigate the effects of other factors involved in the immunosuppressive combination regimen. Many transplant recipients are treated concurrently with mycophenolic acid (MPA). MPA inhibits the proliferation of T and B cells by inhibiting purine synthesis in DNA replication . The impact of MPA alone on NFAT activation has not been well studied, but several reports have shown that MPA affects the activation of the closely related transcription factor, NF-κB [33, 34]. In addition, the limited prescribing of the glucocorticoid, prednisone, in high risk transplant recipients has also been shown to impact the activity of other transcription factors [35, 36]. It would be imperative to investigate the additional impact of these immunosuppressive agents on the NFAT response.
Applicability of this assay in future clinical studies will require thorough validation of the methods used with regards to understanding the inter assay precision and robustness as well as the stability of the samples in such a clinical setting. Current data indicates interexperiment variability of the NFAT1 activity in the Jurkat cell line with Rd values ranging from ∼1 (Fig. 1a) to ∼0.4 (Fig. 3). This variability was primarily found to be attributable to two factors; the interexperimental condition of the Jurkat cells—standardizing the use of the cells to the exponential growth phase minimized this variability; and the conjugated antibody used—changing the antibody has also minimized variability (Supporting Information Fig. 1). However, day to day assay variability could also have contributed thus to correct for this in our ongoing clinical longitudinal studies we are including a singly prepared batch of fixed Jurkat cells from which aliquots are analyzed at each time point which enables normalization for day-to day assay variability. This study was centered on the NFAT1 isoform of the NFAT family. There are five NFAT family members. NFAT5 is regulated by osmotic stress and is not calcium dependent and therefore not calcineurin dependent [37, 38]. NFAT1-NFAT4 are calcium-dependent and highly homologous. There is a certain level of functional redundancy between these four isoforms; NFAT1 and NFAT2 are essential for cytokine production in T cells , and NFAT3 and NFAT 4 are important in vascular development . NFAT1 is the most studied isoform in the context of immune function but the activity of the other calcium-dependent isoforms may be relevant and should be considered in future studies.
In conclusion, this report presents the potential for a novel approach to measure pharmacodynamic responses to tacrolimus-induced immunosuppression. The combined data warrant further evaluation of NFAT response as a compliment to therapeutic drug monitoring tool for tacrolimus post-transplant.
The authors wish to acknowledge Drs Sherree Friend, Ph.D and Vidya Venkatachalam, Ph.D (Amnis Corp/EMD Millipore) for providing Supporting Information Figure 2 and their continued application support with regards to the ImageStream data acquisition and analysis.