Estimation of individual sensitivity to cyclosporin in children awaiting renal transplantation

Jan Dudley1,2,, Carol Truman1, Mary McGraw2, Jane Tizard2, Gausal Haque1 and Ben Bradley1

1 University of Bristol Department of Transplantation Sciences and 2 Department of Paediatric Nephrology, Bristol Royal Hospital for Children, Bristol, UK



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Optimal immunosuppressive drug therapy requires that efficacy be balanced against toxicity. We have performed in vitro assays of cyclosporin (CsA) efficacy in children awaiting renal transplantation.

Methods. Peripheral blood mononuclear cells (PBMC) from 13 children awaiting renal transplantation and 10 healthy paediatric controls (‘responders’) were incubated in the presence of CsA (0–250 ng/ml). Irradiated PBMC from a parent (prospective live donor) were cultured with those of the child in the presence of interleukin 2. Europium-labelled, non-irradiated phytohaemagluttinin-stimulated target cells from the parent were added to the culture after 7 days incubation. Target cell lysis was quantified by time resolved fluorometry. CsA-mediated inhibition of target cell lysis was calculated and used to compare individual responses to the drug. Two-colour flow cytometry was performed to identify activated subsets of lymphocytes at varying concentrations of CsA.

Results. Wide inter-individual variations in per cent lysis and per cent inhibition were observed in patients and controls. Immunophenotyping indicated expansion of CD8+ and CD25+ lymphocyte subsets following allo-stimulation that was inhibited by increasing concentrations of CsA. Eight out of 13 patients and four out of 10 controls were ‘sensitive’ to CsA in vitro in that they achieved 50% inhibition of cell lysis (IC50) at low concentrations of the drug (<50 ng/ml). Eleven patients have subsequently received a renal transplant. Five out of seven of these patients with IC50 <50 ng/ml have suffered problems with infection, nephrotoxicity and graft vasculopathy raising the possibility of ‘over-immunosuppression’.

Conclusion. The data imply a useful role for this model in the prediction of individual response to immunosuppression following allo-stimulation in the pre-transplant setting.

Keywords: allo-specificity; cyclosporin; cytotoxic T lymphocyte; renal transplantation



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Renal transplantation is the preferred treatment for patients with end-stage renal failure (ESRF). However, major factors limit post-transplant outcome including acute rejection, chronic allograft nephropathy (CAN) and complications of powerful immunosuppressive regimes, notably infection, nephrotoxicity and rarely malignancy. Immunosuppressive drug dosing is determined by patient weight or surface area, time post-transplant and bio-availability, as measured by ‘area under the curve’. None of these parameters reflect efficacy of the drug in suppressing donor-specific T lymphocyte-mediated cytotoxicity, a mechanism of prime importance in the acute rejection process [14]. Therapeutic protocols past and present have failed to address the concept of genetically or immunologically determined individual variations in drug susceptibility. We have recently identified the existence of cyclosporin (CsA)-resistant allo-specific cytotoxic T lymphocyte precursors in umbilical cord bloods, suggesting that some drug-resistance may be innately programmed [5]. Immunosuppression itself may limit graft survival, due to nephrotoxicity or CAN [6]. The nephrotoxicity associated with the calcineurin inhibitors may be dose-related, or may reflect individual susceptibility to these drugs. CAN now accounts for more than half of cases of late graft loss in cadaveric transplants [7]. Many authors have suggested that immunosuppressive therapy may contribute to CAN by preferentially inhibiting the Th1 pathway, thus ‘polarizing’ the immune response to the Th2 pathway [812], which is proposed to play a critical role in the initiation of CAN.

The ultimate in vitro model would predict drug efficacy in an individual recipient of a particular transplant, but no such assay exists partly because cellular mechanisms are poorly understood. In this study we have measured CsA efficacy in suppressing allo-specific CTL responses: essentially the Th1 pathway. Assays were conducted in graded concentrations of CsA and their prognostic value in paediatric renal transplant recipients explored.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Parent–offspring pairs
Twenty-six child–parent pairs (13 controls and 13 patients) were enrolled into the study for which ethical approval was granted. The parent ‘stimulator’ for the patients was the mother in 10 cases and the father in three cases, reflecting the prospective live donor. The ‘stimulator’ in all control cases was the mother. The mean age of the patients was 11 years and the male to female ratio was 2.25:1. The mean age of the controls was 9.5 years and male to female ratio was 3:1. Patients were in ESRF as defined by a glomerular filtration rate of <10 ml/min/1.73 m2. Five patients were on twice weekly haemodialysis (HD) at the time of the study. Seven patients were on automated peritoneal dialysis (APD) and one (patient 2) was awaiting a pre-emptive transplant. Two of the patients had received previous renal transplants and had developed antibodies to HLA antigens from their previous grafts. They were not tested against these antigens in the present study. Patient characteristics are detailed in Table 1Go. Renal patients and kidney donors were typed for HLA-A, B, C and DR loci. HLA typing was performed either by serological or DNA methods. Eleven patients received renal transplants and all were treated post-transplant with a standard triple therapy protocol of neoral, prednisone and azathioprine. Neoral was given at 300 mg/m2/daily in two divided doses. Whole blood trough levels were maintained at 200–250 ng/ml throughout the first post-transplant month and 150–200 ng/ml thereafter. Prednisolone was given at 10 mg/m2/twice daily in the immediate post-transplant period tapering to 10 mg/m2 on alternate days by the 12th week. Azathioprine was given at 2 mg/kg daily. The 13 children in the control group were healthy volunteers with normal renal function.


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Table 1.  Patient characteristics

 

Sample collection and processing
Venous blood (20 ml) from children and their parents (50 ml) was collected in preservative-free sodium heparin anticoagulant (25 IU/ml). Bloods from patients were collected in all cases prior to transplantation. For the patients on HD, bloods were collected at least 48 h after an HD session. For those on APD, bloods were collected on the morning after overnight dialysis, ~4 h after disconnection. Peripheral blood mononuclear cells (PBMC) were purified within 12 hours of collection by density gradient centrifugation (Lymphoprep of specific gravity 1.077, Nycomed), washed and suspended in 10% dimethylsulphoxide in complete culture medium (CCM) and cryopreserved in liquid nitrogen until required. On the day of the assay cryopreserved PBMC were thawed and viable cells counted.

In vitro assays
In all assays the ‘responders' were the children and ‘stimulators' were their parents. PBMC were thawed, washed and re-suspended in CCM comprising RPMI 1640 with heat inactivated 10% AB serum (Sigma) and 3 mM glutamine. Responder PBMC were counted and adjusted to a concentration of 0.4x106 PBMC/ml CCM and dispensed into 96-well round-bottomed tissue culture plates (Falcon, 3077) to give 40 000 PBMC/200 µl well. Responder cells were incubated with CCM alone or CsA diluted in CCM (final concentrations 62.5, 125 and 250 ng/ml) for 1 h at 37°C to allow the drug to enter the cells prior to the addition of stimulator cells. Between 12 and 24 replicates were set up for each concentration of CsA. Stimulator PBMC were suspended in CCM (1x106 PBMC/ml) and irradiated (30 Gy) from a cobalt source (IBL 437 C Irradiator), then supplemented with 25 Cetus units/ml of recombinant interleukin-2 (rIL-2) (Euro Cetus, Amsterdam). Stimulator PBMC were then added at a final concentration of 50 000 cells/200 µl well to the 96-well plates containing the responder PBMC. Cultures were incubated at 37°C in 5% CO2 in a humidified atmosphere for 7 days. The assay cultures were fed without disturbing the cells on day 5 by replacing half of the medium of each well with freshly prepared CCM containing 25 Cetus units rIL-2/ml and CsA at the appropriate concentrations.

Preparation of PHA-stimulated target PBMC
Stimulator PMBC were adjusted to 1x106 cells/ml and cultured as targets in CCM containing rIL-2 at 20 Cetus U/ml and purified phytohaemagluttinin (PHA) at 2 mg/ml (Murex diagnostics, UK), in 24-well culture plates (Costar, Cambridge, MA 02140). On days 3 and 5 the PHA-blasts were split and fed with freshly prepared CCM containing rIL-2 at 20 Cetus U/ml. PHA-blasts were incubated for a total of 7 days at 37°C in 5% CO2 in a humidified atmosphere.

Lysis assay
After 7 days incubation, PHA-blasts were harvested and washed twice with phosphate-buffered saline (PBS), then labelled with europium (Eu). Labelling buffer comprised 50 µl of 25 mM diethylene triamino penta-acetate (DTPA) from Sigma and 50 µl of 10 mM europium chloride (EuCl3) from Fluka, Switzerland, in 1 ml HEPES buffer prepared at 4°C (Eu–DTPA). One millilitre of the Eu-DTPA was added to 10x106 PHA-blasts (Eu–PHA-blasts). Dextran sulphate was added at 35 µl/ml of a 10 mg/ml solution to increase cell membrane permeability and facilitate entry of Eu–DTPA into the cells. The mixture was left on crushed ice for 15 min. The reaction was quenched by adding 30 µl of 100 mM CaCl2/ml for a further 10 min. Eu–PHA-blasts were then washed four times with wash buffer containing 79 µl of 1 M CaCl2 in 50 ml RPMI 1640 giving a final concentration of 2 mM CaCl2, then twice with CCM and finally suspended in CCM at 5000 cells/100 µl and gradually warmed to room temperature.

Aliquots of 100 µl of Eu–PHA-blasts were pipetted into the 96-well culture plates containing responder and stimulator PBMC. Plates were given a short spin (200 g) for <1 min at room temperature (room temperature, 20–22°C) and incubated for 3 h at 37°C in 5% CO2 in a humidified incubator. The spontaneous release of Eu–DTPA complex was determined from Eu–PHA-blasts incubated in CCM. Maximum release, i.e. the total releasable fluorescence, was determined from Eu–PHA-blasts incubated in 2% Triton-X-100 (Fluka) in PBS. After 3 h incubation, plates were spun at 600 g for 5 min at room temperature to pellet the cells. Thereafter, 20 µl aliquots of supernatant were pipetted into 96-well flat-bottomed low auto-fluorescence reader plates (Nunc) pre-filled with 200 µl of enhancement solution (Delfia, Finland). These plates were then read, at least 1 h later, in a time resolved Fluorometer (Delfia, Finland) and results expressed as counts per second (c.p.s.).

Data analysis
Data validation. Eu release was calculated and expressed as mean c.p.s. and standard deviation (SD) for each set of replicates. The labelling efficiency of Eu–PHA-blasts was then calculated as:

Efficiency=[(spontaneous release)/(maximum release)]x100.

If release from Eu-PHA-blasts cultured in CCM alone exceeded 30% of the maximum release the assay was discarded.

Per cent lysis, Per cent inhibition and IC50 estimates. The mean c.p.s. values were calculated for each set of replicates and per cent lysis and per cent inhibition was expressed on a scale ranging from 0 to 100%. Thus, per cent lysis = (mean test c.p.s. – spontaneous release)/(maximum release – spontaneous release)x100;

per cent inhibition=100 – (mean test c.p.s. with CsA – spontaneous release) / (mean c.p.s. without CsA – spontaneous release)x100.

From the dose–response plot the IC50 was deduced. IC50 equated to the drug concentration associated with 50% inhibition of cell lysis after fitting a curve to a plot of per cent inhibition against CsA concentration. The Wilcoxon rank sum test was used to compare differences in IC50 between patients and controls.

Flow cytometric analysis of PBMC
This was performed before and after allo-stimulation on patients 2, 4, 5, 9, 10, 11 and 13. Responder PBMC were incubated with 5 µl of anti-human specific antibodies coupled to fluorescein isothyanate (FITC) or phycoerythrin (PE) for 30 min at 4°C. The panel of antibodies comprised PE-conjugated anti-CD3 (UCHT1 DAKO, Denmark), PE-conjugated anti-CD45 (T29/33 DAKO, Denmark), PE-conjugated anti-CD8 (DK25 DAKO, Denmark), FITC-conjugated anti-CD25 (ACT-1 DAKO, Denmark) and FITC-conjugated anti-CD4 (MT310 DAKO, Denmark). Samples were examined on a Coulter Epics XL flow cytometer and data were analysed using Epics Listmode software.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Of the 26 assays (13 patients and 13 controls), three controls failed due to technical reasons including inadequate proliferation of PHA-stimulated blasts (2) and contamination of wells (1). The results of assays performed on the remaining 23 individuals are reported here.

In vitro assays
Per cent lysis. All cases studied were judged technically acceptable by the criterion that spontaneous release from all Eu–PHA-blasts cultured in CCM alone did not exceed 30% of the maximum release.

There was a wide variation between offspring in per cent lysis against their respective parental targets. Addition of CsA to the cultures was associated with an exponential decrease in per cent lysis with increasing concentration, but again there was considerable individual variation (Tables 2Go and 3Go).


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Table 2.  Assay results of 13 patients tested against their parents

 

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Table 3.  Assay results of 10 controls tested against their mothers

 
Per cent inhibition. In most patients and controls per cent inhibition tended to rise to a plateau of maximum inhibition (ICmax), above which increasing concentrations of CsA did not result in increased inhibition of cell lysis.

Estimated IC50. This benchmark corresponded to the concentration of CsA associated with 50% inhibition of cell lysis and was used to compare individual responses to CsA following allo-stimulation. There was a wide inter-individual variation in estimated IC50 (Tables 2Go and 3Go, Figures 1Go and 2Go). Median IC50 was 37 and 60.5 ng/ml for patients and controls, respectively (range 20.5 to >250 and 35 to >250 ng/ml, interquartile range 34 to 88 and 39 to 215 ng/ml in patients and controls respectively). There was a suggestion that patients were more sensitive than controls in that median IC50 was lower in this group, however this was not statistically significant (Wilcoxon rank sum test P=0.09).



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Fig. 1.  Plot showing relationship between per cent inhibition of lysis at different cyclosporin concentrations (patients 1–13).

 


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Fig. 2.  Plot showing relationship between per cent inhibition of lysis at different cyclosporin concentrations (controls 1–10).

 
The reproducibility of the assay was assessed in three patients and two controls at intervals of 9 months to 2 years after initial assay. While similar values were observed for per cent inhibition and IC50, per cent lysis was less reproducible (Tables 2Go and 3Go). This is likely to reflect variable maximum release values yielded by adding Triton X-100 to cells.

Immunophenotyping data
Allo-stimulation was associated with expansion of CD4+, CD8+ and CD25+ lymphocyte subsets. This response was most pronounced in the case of CD25+ cells and was inhibited by increasing concentrations of CsA (Figures 3Go and 4Go). Absolute CTL values varied widely between individuals both before and after allo-stimulation (Figure 3Go).



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Fig. 3.  Plot showing recipient lymphocyte subsets before and after allo-stimulation at different cyclosporin concentrations (patients). Horizontal bars represent median lymphocyte count (i) pre-stimulation, (ii) after allo-stimulation without CsA, (iii) after allo-stimulation CsA 62.5 ng/ml, (iv) after allo-stimulation CsA 125 ng/ml and (v) after allo-stimulation CsA 250 ng/ml. Vertical bars represent range of lymphocyte counts.

 


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Fig. 4.  Plots showing flow cytometric analysis of responder CD8+ and CD25+ lymphocytes pre- and post-allo-stimulation (patient 11). (A) Pre-allo-stimulation. (B) After 7 days incubation with stimulator PBMCs in the absence of CsA. (C) After 7 days incubation with stimulator PBMCs in the presence of CsA 125 ng/ml.

 

Clinical relevance of in vitro assays
During the course of these studies 11 of the 13 patients received renal transplants; in four cases kidneys were donated from the parent and in seven cases from a cadaveric donor. HLA match grades were similar in all cases (Table 4Go). Three patients (3, 6 and 8) experienced minor episodes of acute rejection at 1 and 4 weeks post-transplant and on biopsy were assigned Banff Grades, borderline/1A, 1A and 1B, respectively [17]. The episode was self-limiting in patient 6, in that serum creatinine returned to baseline prior to commencing steroid therapy. The acute rejection episode in patient 8 was associated with a urinary tract infection. All three recipients with rejection were treated with i.v. methylprednisolone (500 mg/m2 for 3 days), with complete recovery of renal function and return of serum creatinine to baseline. The IC50 was <50 ng/ml in two of these (patients 6 and 8) and >50 ng/ml in patient 3. Five out of seven of these patients with IC50 <50 ng/ml have suffered problems with infection, nephrotoxicity and graft vasculopathy raising the possibility of ‘over-immunosuppression’. A further patient in this group (patient 1) was converted to tacrolimus at 16 months post-transplant due to CsA-related gingival hyperplasia and hirsuitism. No patient with an IC50 >50 ng/ml had problems suggestive of over-immunosuppression (Table 4Go).


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Table 4.  Outcome post-transplant

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The salient findings of this study were: a wide inter-individual variation in per cent lysis and inhibition and a potential association between low IC50 in vitro and over-immunosuppression in vivo. These findings raise the tantalising prospect that these assays may offer a versatile method of evaluating an individual's susceptibility to CsA.

The development of an in vitro assay to assess susceptibility to immunosuppressive drugs requires a preconception of the cellular immune mechanisms leading to acute rejection. There is overwhelming evidence for a central role for donor-specific T cells in the rejection of solid organ transplants in experimental animal models. CD4+ T cells play a major part in directing and amplifying responses, while CD8+ T cells have the capacity to specifically lyse donor target cells. In the clinical arena acute rejection may be regarded as being due to mechanisms that are resistant to routinely administered immunosuppressive drugs such as CsA. Thus, the cellular immune mechanisms may be substantially shifted to alternative, CsA-resistant pathways. There is indirect evidence to invoke natural killer (NK) cells [13] and substantial evidence to invoke B cells in this process [14]. Interestingly, both B and NK cells appear highly resistant to therapeutic concentrations of CsA in vitro [15]. Notwithstanding the possibility that several drug-resistant pathways may lead to acute rejection in man we chose to explore and develop assays based on cytotoxic T cell reactions as a starting point for our studies.

The per cent lysis assay measures the lytic function of the mixture of cells activated by parental HLA mismatches and other ‘non-self’ peptides carried by the stimulator cells. The total lytic capacity is considered as being proportional to the summation of the individual clones activated. We have assumed that the bulk of the lytic reaction is mediated by allo-specific CD8+ and CD25+ T cells, however, a contribution from other cells such as cytolytic CD4+ T-cells and NK and NK-T cells cannot be excluded.

Interpretation of per cent lysis results should acknowledge the fact that the 0–100% scale is limited by the mean c.p.s for spontaneous release and maximum release. Both values vary through non-immunological mechanisms that have no clinical significance. Hence, variation in the mean c.p.s. for maximum release was abrogated in the CsA inhibition assays by transforming values into per cent inhibition. The 100% value equates to mean c.p.s. for spontaneous release of the targets in that assay, a value that had already been validated [16].

One of our main objectives was to identify the minimum concentration of drug required to suppress allo-specific lysis. In most cases maximal inhibition of target cell lysis (ICmax) was manifest as a plateau, above which further increases in CsA did not result in further inhibition. The implication of this finding from a clinical standpoint is that patients receiving doses of CsA that are higher than their ICmax may be at risk of the toxic side effects of the drug with no additional benefit in terms of inhibition of donor cell lysis. In order to compare individual response to CsA, we selected as a reference value a drug concentration that inhibited 50% of donor-specific lysis, since this was the most sensitive part of the titration curve. Hence, we derived the IC50 values. IC50 values also have the merit of being adaptable to other cell populations (e.g. NK and B cells) that might be invoked in acute rejection, thereby enabling comparisons of relative CsA resistance between subsets in one individual.

We have reported previously wide inter-individual variations in per cent lysis and inhibition in adults with random HLA mismatches [13]. Such variations in the present study were unexpected in view of the uniform level of HLA mismatch between responder and stimulator. These findings may reflect intrinsic individual differences that are not donor antigen-specific. We have some limited data suggesting that response to immunosuppression may be independent of HLA mismatch, based on experiments with one and two haplotype-mismatched donors. This issue will be further explored by priming recipient PBMCs with pools of HLA mismatched stimulators.

We were unable demonstrate a correlation between IC50 and post-transplant susceptibility to acute rejection; indeed one patient (10) has not experienced any episodes of acute rejection despite being ‘resistant’ to high concentrations of CsA in vitro. It is noteworthy that this patient demonstrated a very low percentage lysis in the absence of CsA (Table 2Go) and this will be re-evaluated using pools of HLA mismatched stimulators.

There were very few episodes of acute rejection in these patients and the significance of Banff borderline and 1A rejection here and in general, is uncertain [17]. The usefulness of our assays as predictors of TH1-mediated acute rejection will need to be assessed in a larger number of patients.

In conclusion, we have shown a wide inter-individual variation in CsA sensitivity in patients and controls. The preliminary data also suggest a possible correlation between high CsA-sensitivity in vitro and subsequent over-immunosuppression in vivo. The prognostic potential of this assay will be tested prospectively in a larger series of live donor transplants using drugs appropriate to the therapeutic protocols employed.



   Acknowledgments
 
We thank Mike Allen, Clinical Project Manager for Wyeth-Ayerst for arranging funding to support Jan Dudley and Carol Truman. Gausal Haque was supported by a Commonwealth Universities scholarship (Commonwealth Ref. no.: CSC-BDM071) and more recently by the Ministry of Health, Government of Bangladesh whom we thank for allowing him to undertake postdoctoral research in the UK. Benjamin Bradley is supported by a grant to the University of Bristol from the Research and Development Central Levy of the Department of Health, National Health Service Executive whom we thank for their continuing support. We also thank the South and West Research and Development Directorate for a project grant for this study.



   Notes
 
Correspondence and offprint requests to: Dr J. Dudley, University of Bristol Department of Transplantation Sciences, Paul O'Gorman Lifeline Centre, Southmead Hospital, Bristol BS10 5NB, UK. Email: jandudley{at}yahoo.com Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 16.10.01
Accepted in revised form: 4. 9.02





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