immunotherapy and transplantation to cure type 1 diabetes
Huurman, V.A.L.
Citation
Huurman, V. A. L. (2009, March 4). T cell immunity to islets of Langerhans : relevance for immunotherapy and transplantation to cure type 1 diabetes.
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CHAPTER 6
T cell immunity after tapering of
immunosuppression in islet transplantation
CHAPTER 6
Increase in highly avid alloreactive cytotoxic T lymphocyte frequency during tapering of immunosuppression after islet cell transplantation
V.A.L. Huurman1,2,4, J.H.L. Velthuis1,4, R. Hilbrands3,4, P.M.W. van der Meer-Prins1,4, G. Duinkerken1,4, F.K. Gorus3,4, F.H.J. Claas1,4, B. Keymeulen3,4, D.L. Roelen1,4, D.G. Pipeleers3,4 and B.O. Roep1,4
1 Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands
2 Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands;
3 Diabetes Research Center, Brussels Free University-VUB, Brussels, Belgium;
4 JDRF Center for Beta Cell Therapy in Diabetes
Submitted for publication
ABSTRACT
BACKGROUND
Transplantation of isolated islet of Langerhans cells has considerable potential as a cure for type 1 diabetes but continuous immune suppressive therapy often causes considerable side effects. Tapering of immunosuppression in successfully transplanted patients would lower patients’ health risk. We studied the effect of tapering on islet auto- and alloimmune reactiv- ity in vitro.
METHODS
In vitro T cell auto- and alloreactivity of five transplant recipients was determined during tapering using lymphocyte stimulation tests to test autoreactivity and cytotoxic T lympho- cyte precursor (CTLp) assays to define alloreactivity against the islet graft during tapering of immune suppression. Graft-specific cytokine responses were measured using Luminex technology. Avidity of CTLs was determined by addition of anti-CD8. The influence of im- munosuppression was mimicked by in vitro addition of tacrolimus and MPA, the active metabolite of MMF.
RESULTS
T cell autoreactivity increased in two out of five patients during tapering. Overall alloreactive CTLp frequencies did not change, but their avidity was significantly higher after tapering than during maintenance immunosuppressive therapy (p=0.002). In each individual patient, C-Peptide levels over time followed the immune profile. In vitro addition of tacrolimus but not MPA was able to neutralize increased CTLp frequencies during tapering to baseline levels and led to a significant increase of graft-specific IL10 production.
CONCLUSIONS
Tapering of immunosuppression is characterized by diverse immune profiles that relate to plasma C-peptide levels. The proportion of highly avid allospecific CTLs, which associate strongly with rejection, increased during tapering. Increasing alloreactivity due to decreas- ing tacrolimus levels may relate to graft function. In the future this may guide tapering of immunosuppression after islet cell transplantation.
T cell immunity after tapering of immunosuppression in islet transplantation 171
INTRODUCTION
Transplantation of islet of Langerhans cells isolated from a donor pancreas is a promising treatment for type 1 diabetes (T1D) patients with large serum glucose variation1-3. The propor- tion of patients becoming independent from exogenous insulin or preserving endogenous insulin production after transplantation has increased in recent years. The vast majority of transplant recipients show graft function and experience a significant decrease of serum glucose variation which ameliorates their quality of life4,5. Nevertheless, the need for continu- ous immunosuppressive therapy to prevent graft rejection and recurrence of autoimmunity imposes a heavy burden impairing functionality of the immune system in general. This may lead to opportunistic infections, post-transplant lymphoproliferative disease and increased risk of carcinoma6-8. Additionally, the functionality of regulatory T cells promoting long-term graft acceptance may be impaired9. Eventually these disadvantages may be more detrimen- tal to the patient’s condition and quality of life than regular intensive insulin treatment. In the European JDRF Center for Beta Cell Therapy in Diabetes, immunosuppressive medication is therefore routinely tapered off in recipients starting one year after transplantation.
Measures of auto- and alloreactivity may change upon tapering of immunosuppression, either by increased reactivity or preferably by the induction of operational tolerance. While demonstrated in kidney transplantation10, operational graft tolerance in the absence of immunosuppression has not yet been described in islet transplantation, where tolerance to both auto- and alloreactivity must be achieved. We previously demonstrated that β-cell mass and autoimmune T cell reactivity determine clinical outcome in patients transplanted under anti-thymocyte globulin (ATG) induction and tacrolimus/mycophenolate mofetil (MMF) maintenance immunosuppression11. Cytokine production in response to islet donor HLA associated with outcome12, while graft-specific CD8+ cytotoxic T cells (CTLs) only proved associated with graft failure in immunosuppressive regimes containing sirolimus13. In case of kidney transplantation, the amount of CTLs with high avidity for donor HLA (resistant to in vitro CD8 blockade) was able to discern permissible from non-permissible HLA mismatches14 and may prove more informative than the total amount of alloreactive CTLs with regard to graft loss.
The aim of the current pilot study was to identify immune parameters correlating with islet graft function in islet transplantation during tapering of immune suppressive therapy, that may guide tapering in the future. A cohort of five patients transplanted under ATG- tacrolimus-MMF immunosuppression was selected in whom immunosuppression was tapered differentially. The patterns of auto- and alloreactive parameters during tapering were evaluated blinded from clinical outcome and β-cell function and subsequently related to clinical and metabolic follow-up.
MATERIALS AND METHODS
PATIENTS AND CLINICAL FOLLOW-UP
Five patients were studied that received islet cell grafts characterized for cellular composition as described before4. All patients received ATG induction and tacrolimus/MMF maintenance immunosuppressive therapy and all had been included in the cohort previously studied for immune factors influencing graft survival in the first year after transplantation11. ATG (Fresenius, Fresenius Hemocare, WA, USA) was given as a single infusion of 9 mg/kg and subsequently at 3 mg/kg for 6 days except when T-lymphocyte count was under 50/mm³.
Tacrolimus (Prograft, Fujisawa/Pharma Logistics) was dosed according to trough level: 8-10 ng/ml in the first three months post transplantation, 6-8 ng/ml thereafter). MMF (Roche) was initially dosed at 2000 mg/day and already decreased at the end of the first year in some patients (clinical and immunological patient characteristics in week 0-52 after transplanta- tion are shown in Table 1).
After 52 weeks, patients continued to be followed up regularly regarding plasma C-peptide level (at glycemia between 120-200 mg/dl) and % HbA1c. Decision to taper off immunosup- pression was based on clinical parameters for each patient individually. If needed, insulin treatment was reintroduced in islet allograft recipients if two consecutive HbA1c measure- ments exceeded 7.0%.
TABLE 1 Patient characteristics.
Parameter 36 40 41 38 29
Age (yr) 45 42 46 52 46
Gender (M/F) M F M M M
Body weight (kg) 70 65 72 65 90
Duration of disease (yr) 33 30 21 28 24
Age at onset (yr) 12 12 25 24 22
HbA1c (%) before transplantation 8.10 6.50 7.00 8.80 7.80
Insulin dose (IU/kg/d) before transplantation 0.54 0.74 0.86 0.66 0.84 Mean fasting glycemia (mg/dl) before transplantation 98 235 138 63 135
Number of transplants 1 1 2 2 3
Total injected β-cells (106 per kg) 2.6 2.3 3.8 4.1 2.5
Each transplant >2x106 β-cells per kg y y n n n
No. of donors 4 3 6 3 7
HLA class I mismatches tested (in CTLp) 12/12 9/9 12/15 7/7 19/20
HLA class II mismatches tested (in MLC) 2/2 2/4 5/6 5/6 8/10
Total ATG dosage (mg/kg) 20.8 24.0 19.6 20.9 21.1
Median Tacrolimus level (ng/ml). 0-12 months 7.5 9.0 9.0 9.0 6.0
Median MMF dosage (mg/day) 0-12 months 2000 2000 2000 2000 2000
Pre-transplant cellular autoimmunity - n n - y
Post-transplant cellular autoimmunity 0-52 weeks - y y - y
Post-transplant cellular alloreactivity 0-52 weeks - y n y n
Insulin independent y y y n n
Average plasma C-peptide level 0- 52 weeks (ng/ml) 2.02 2.09 1.39 0.64 0.90
T cell immunity after tapering of immunosuppression in islet transplantation 173
FOLLOW-UP OF CELLULAR AUTOREACTIVITY
Cellular immune responses were determined blinded from clinical results. Cellular autore- activity was determined at regular intervals during tapering, as described before15. Briefly, 150.000 fresh PBMCs/well were cultured in 96 well round-bottomed plates in Iscove’s Modi- fied Dulbecco’s Medium (IMDM) with 2 mMol/l glutamine (Gibco, Paisley, Scotland) and 10%
pooled human serum in the presence of antigen, IL-2 (35U/ml) or medium alone in triplicates.
After 5 days 3H-thymidine (0.5 μCi per well) was added and 3H-thymidine incorporation was measured after 16 hours on a β plate counter. Antigens analyzed included IA-2 (10 μg/ml), GAD65 (10 μg/ml), insulin (25 μg/ml) and tetanus toxoid (‘third party’ antigen, 1,5LF/ml).
Results were interpreted as stimulation index (SI) compared to medium value.
FOLLOW-UP OF ALLOREACTIVE BY CYTOTOXIC T LYMPHOCYTE PRECURSOR (CTLP) ASSAY AND MIXED LYMPHOCYTE CULTURE (MLC)
The CTLp assay to determine CD8+ T cell mediated cytotoxic alloreactivity was described be- fore16. For this study the assay was performed on cryopreserved PBMC drawn from patients before, during and after tapering. Briefly, PBMC were cultured in a limiting dilution assay (40.000 to 625 cells/well, 24 wells per concentration) with three to four different irradiated stimulator PBMCs expressing HLA class I antigens also expressed on the injected β-cell grafts (50.000 cells/well). Cells were cultured for seven days at 37°C in 96-well round-bottomed plates in RPMI 1640 medium with 3 mM L-glutamine, 20 U/ml IL-2 and 10% pooled human serum. All culture plates were duplicated at day 7 by pipetting half the cells from each well and transferring them to a new plate. One plate was used for determination of the high avidity CTLp frequency by adding culture media + anti-CD8 (3.0 μg/ml final concentration) for 1 hour, while in the other plate only culture media was added in that hour to measure the total CTLp frequency. Next, Europium-labelled graft HLA-specific target cells (5.000 cells/
well) were added to all stimulator/responder combinations for 4 hours. Wells were scored positive if the Europium release through target cell lysis exceeded spontaneous release +3 SD. Quantification of CTLp frequencies was performed by computer software developed by Strijbosch et al.17.
For MLC experiments determining graft-specific CD4+ T cell-mediated proliferation, the same stimulator and responder cells were used as for CTLp assays. One-way MLC were set up in triplicates in 96-well V-bottomed plates (Costar, Cambridge, MA, USA) in 150μl RPMI with 2 mMol/L l-glutamine (Gibco, Paisley, Scotland) and 10% pooled human serum. Responder cells (40.000) were incubated with 50.000 irradiated stimulator cells (irradiated at 3000 rad) per well at 37 °C / 5%CO2. After 5 days, 3H-thymidine (1.0 μCi per well) was added and 3H- thymidine incorporation was measured on a β plate counter after 16 hours. Proliferation in response to phyto-haemagglutinin was used as positive control. Results were interpreted as stimulation index (SI) compared to background value (responder only + stimulator only). The fraction of recipient-donor mismatches that was tested is shown per patient in Table 1.
To test the influence of immunosuppression on in vitro alloreactivity, mycophenolate acid (MPA, the active metabolite of MMF) was added in CTLp and MLC assays at 250 ng/ml and tacrolimus at 0.3 ng/ml. These concentrations were determined on basis of earlier ex- periments14 as well as titration experiments using mismatched healthy panel donor PBMC as stimulator and responders. In each subsequent experiment, a healthy donor control sample was included to validate the inhibitory capacity.
HLA ANTIBODIES
Screening for the presence of HLA class I and class II-specific antibodies was performed on all available samples by ELISA (LAT class I & II, One Lambda, CA). When positive, the specific- ity of HLA Class I antibodies was determined by complement-dependent cytotoxicity assay against a selected panel of 52 HLA typed donors.
ANALYSIS OF CYTOKINE PRODUCTION IN MLC
Production of different cytokines was measured with Luminex technology using a human Th1/Th2 Bio-plex cytokine kit (BioRad, Veenendaal, the Netherlands), including IL2, IL4, IL5, IL10, IL12p70, IL13, GM-CSF, IFNγ and TNFα, according to the manufacturer’s protocol. Briefly, antibody-coated cytokine-specific beads were added to 96-well Millipore plates (BioRad).
Supernatants taken at day 5 from graft-specific mixed lymphocyte cultures were added for 45 min at room temperature in the dark under 300 Hz shaking, allowing cytokines to bind to the cytokine-specific beads. Subsequently, plates were washed and incubated with biotiny- lated anti-cytokine detection antibody for 30 min. After washing, streptavidin PE was added for 10 min. to bind to the detection antibody. Fluorescence labelling of cytokine-specific beads was analyzed by a double-laser Bio-plex reader (Bio-Rad). Cytokine concentration was determined based on a standard curve included in each plate, using cytokine standards provided by the manufacturer.
STATISTICS
Wilcoxon signed rank test was used to analyze paired observations of continuous variables.
Friedman test was used to analyze paired observations of multiple groups with correction for multiple comparison by Dunn’s post-hoc test. Statistical analyses were performed using GraphPad Prism version 4.0. p < 0.05 was considered significant.
FIGURE 1 Overview of clinical and cellular immune parameters of five islet cell transplant recipients during tapering of immunosuppression. Shown are A) immunosuppression levels: tacrolimus trough level (full line), MMF dosage (dotted line); B) plasma C-peptide level; C) Stimulation index of autoreactive proliferative response to IA-2 (full line) and GAD (dotted line); D) alloreactive CTLp frequency in response to different graft- specific stimulator cells; E) High avidity alloreactive CTLp frequency (after addition of anti-CD8).
T cell immunity after tapering of immunosuppression in islet transplantation 175
RESULTS
GRAFT FUNCTION DURING TAPERING
Three out of five patients studied showed stable graft function and were insulin independent at the start of tapering of immunosuppression (Figure 1). One patient required exogenous insulin but showed stable graft function (C-peptide>0.5 ng/ml), while another patient had no detectable islet graft function remaining. In this patient (#29) tapering was started 125 weeks after transplantation.
Tacrolimus trough levels were decreased by at least 67% in all patients. MMF was tapered off only in the patient without graft function. All patients with remaining graft function displayed a gradual decrease in plasma C-peptide levels. This pattern largely followed the decrease in tacrolimus trough levels (Figure 1). All patients had to resume insulin injection during the tapering period.
IMMUNE RESPONSES DURING TAPERING
Immunological follow-up during tapering showed differential patterns between patients (Figure 1). Two patients (#36 and #41) showed a marked increase in cellular autoimmune reactivity to GAD and IA-2. In the other patients no increases in autoimmune response were observed. No significant changes in in vitro immune reactivity were observed in response to whole insulin and tetanus toxoid (not shown). Overall, alloreactive CTLp frequencies increased in two patients (#38 and #41) during tapering and decreased in two patients (#36 and #40), remaining largely unchanged in patient #29. No graft HLA-specific alloantibodies were observed at any time in any of the recipients.
FIGURE 2 Alloreactive CTLp frequency before, during (median if multiple values) and after tapering. Data represent CTLp frequencies in response to different graft-specific stimulator cells A) without addition of anti-CD8 and B) with addition of anti-CD8 (only high avidity CTLs). C) % inhibition by anti-CD8 significantly decreases after tapering (p=0.002 by Wilcoxon signed rank test), indicating a relative increase in highly avid CTLs. Horizontal lines represent median values.
T cell immunity after tapering of immunosuppression in islet transplantation 177
In individual patients, changes in graft function followed changes in immune reactivity. De- creasing C-peptide production of patient #36 was accompanied by increased autoreactivity and of patient #41 by increases in both auto- and alloreactivity. In this patient autoreactivity was already present before tapering started. In patient #40, alloreactivity eventually de- creased but remained elevated in the early tapering phase, possibly explaining the decrease in C-peptide production. In patient #38, the low remaining C-peptide production decreased further, while alloreactive CTLp frequency increased. In patient #29, no change in auto- or alloreactivity was observed, but C-peptide production had been lost before tapering.
All CTLp assays were also performed with addition of anti-CD8 to determine the frequency of high-avidity CTLs (Figure 1E), as these T-cells display the strongest association with allograft rejection in different transplantation settings. When analyzing all stimulator-responder combinations with complete data (n=15) before, during and after tapering, no significant changes in total or highly avid CTLp frequency were observed (Figure 2A and B). However, the percentage inhibition by anti-CD8 significantly decreased after tapering (p=0.002, Figure
FIGURE 3 Inhibition of alloreactivity by addition of tacrolimus and/or MMF of CD4+ T cell proliferation (A), CD8+ CTLs (B) and highly avid CTLs (C). Data represent stimulator-responder combinations of five patients before, during (median if multiple values) and after tapering of immunosuppression (mean+SEM). * p<0.05, **
p<0.01 by Friedman test. In all significant tests, the difference between MPA and tacrolimus/MPA remains after Dunn’s correction for multiple comparison. Control experiments were performed using an HLA-mismatched combination of stimulator and responder cells from healthy volunteers. The same control samples were used for each experiment.
2C), indicating a relatively higher proportion of highly avid alloreactive CTLs. MLC prolifera- tion and graft-specific cytokine production did not change significantly (not shown).
IN VITRO EFFECT OF IMMUNOSUPPRESSION ON ALLOREACTIVITY
To test a possible relationship between changing alloreactivity and reduced immune sup- pression, tacrolimus and/or MPA were added in pharmacological concentrations in vitro.
These experiments were performed on stimulator-responder combinations that previously displayed clear changes in CTLp frequency over time.
Addition of tacrolimus and tacrolimus/MPA led to inhibition of the majority of the al- loreactive response while MPA alone was less able to inhibit (Figure 3). This difference was observed both in donor HLA Class II-restricted mixed lymphocyte reaction (MLC) and in total CTLp frequency before and during, but not after tapering. Regarding high-avidity CTLs, a significant difference was only observed during the tapering period but not before or after.
The percentage inhibition did not change significantly over time. Control MLC and CTLp reactions were included in each experiment using only cells of healthy panel donors (HLA mismatched between stimulators and responders). In these experiments the same pattern of limited inhibition by MPA only and higher inhibition by tacrolimus or tacrolimus/MPA was seen (Figure 3). Interestingly, the percentage inhibition by MPA of highly avid CTLs tended to be higher in these control samples than in the combined patient samples (Figure 3C, median inhibition 89.8 and 53.7% respectively, p=0.07 by Mann-Whitney U test).
In the two patients with increasing high avidity CTLp alloreactivity (#41 and #38), tacroli- mus was able to completely neutralize the increase to pre-tapering levels (Figure 4), while MMF was less able to do so. Analysis of cytokine production in MLC supernatants from all five patients showed that addition of tacrolimus and tacrolimus/MPA, but not MPA alone, led to increased graft-specific IL10 production regardless of the stage of tapering, while IL2 production decreased (Figure 5). A trend was observed towards higher production of IL10 during tapering when no immunosuppression was added (p=0.18 by Friedman test).
FIGURE 4 Inhibition of increasing alloreactivity in patients 41 (A) and 38 (B) by in vitro immunosuppression.
T cell immunity after tapering of immunosuppression in islet transplantation 179
DISCUSSION
This pilot study aimed to identify changes in immune reactivity after islet transplantation during reduction of daily dosage and/or trough level of immunosuppression. In a detailed analysis of five patients, different patterns of immune reactivity could be observed after ta- pering that in each case could explain changes in graft function. The increase in the amount of highly avid CTLs implies influence of tapering on the type of reactivity and suggests a future role for such analyses for tapering guidance. In vitro experiments indicate that tacroli- mus is a potent inhibitor of increasing alloreactivity and may induce increased graft-specific IL10 production.
A large variety of factors may influence long-term islet graft survival. These factors can be both auto- and alloimmune-related as well as β-cell related (e.g. to graft composition, insulin-producing capacity and graft size)5,18,19. Furthermore, immunosuppression itself can be unfavourable for islet cell engraftment20 and function21,22. However, the correlation of tacrolimus trough levels with plasma C-peptide levels that was observed in the current study is striking and suggests a considerable role for tacrolimus to retain rather than inhibit graft function.
It was shown previously that autoimmune T cell reactivity may be insufficiently affected by immunosuppressive strategies currently employed in clinical islet transplantation11. In
FIGURE 5 Graft-specific production of IL10 after addition of tacrolimus and/or MPA. Data represent production of IL10 (A) and IL2 (B) of five patients’ PBMC in reponse to graft-specific stimulator cells before, during (median if multiple values) and after tapering of immunosuppression (mean+SEM). ** p<0.01, ***
p<0.001 by Friedman test. For all comparisons significant differences remain after Dunn’s correction for multiple comparison: for IL10, pre and post tapering the differences No IS -tacrolimus and MPA - tacrolimus remain significant and during tapering the difference No IS - tacrolimus/MPA remains significant. For IL2, pre tapering the difference No IS - tacrolimus, during tapering the difference No IS - tacrolimus and tacrolimus - MPA, and post tapering the difference MPA - tacrolimus/MPA remains significant.
this study, the pattern of autoreactivity over time was not predicted by its presence prior to tapering. Furthermore, autoreactivity remained low in patients that were not autoreactive before tapering. While these findings might imply that the applied immunosuppression is not responsible for inhibition of autoreactive T cells, clear increases in autoreactivity were observed in two patients that at least in one case may have contributed to loss of graft func- tion during tapering. Furthermore, in patients with low remaining graft function or β-cell mass, islet autoreactivity may no longer be detectable in the circulation.
Alloreactivity showed different patterns during tapering as well, and in most patients fol- lowed C-peptide levels and tacrolimus trough levels. In patients with decreasing alloreactivity this would indicate that the amount of alloreactive CTLs decreases as the graft is destroyed, but this cannot explain increased alloreactivity found in patients with little remaining graft function. The different patterns may explain why the amount of total as well as highly avid CTLs does not change significantly when patients are analyzed collectively. However, the increasing percentage of highly avid CTLs confirms earlier observations that harmful CTLs are sufficiently inhibited under full tacrolimus/MMF immunosuppression13 and suggests that they become more apparent after tapering.
The direct inverse relationship between alloreactivity and immunosuppression was assessed by in vitro addition of immunosuppressive agents. Increasing alloreactivity could only be partly inhibited by MPA in a concentration that was titrated beforehand on naïve panel donors. Such titration was needed because MPA levels used in vitro and found in human serum reported in the literature differ considerably23,24. The incomplete inhibition by MPA may be a consequence of the minimal tapering of MMF in vivo, so its effect could persist in PBMCs when tested in vitro. This possibility is supported by the fact that MPA led to im- proved inhibition of highly avid CTLs in control samples. Alternatively, it could be argued that transplanted patients harbour more highly avid CTLs than naïve donors and MPA alone is not able to suppress these.
In most patients primarily tacrolimus was tapered. The tacrolimus concentration in vitro (0.3 ng/ml) resembles 6 to 15 ng/ml in vivo since 95 to 98% of tacrolimus is bound to eryth- rocytes in the circulation25. These concentrations of tacrolimus could completely inhibit the increasing alloreactivity regardless of addition of MPA. We speculate that alloreactivity would have remained at baseline levels in those patients if tacrolimus had not been tapered.
This observation is corroborated by the fact that tacrolimus decreases graft-specific produc- tion of pro-inflammatory IL2 and increases production of the regulatory cytokine IL10. In contrast, production of IL10 does not change overall while this has been one of the few indicators of graft success in the first year after transplantation12.
Tapering of immunosuppression in transplantation is presently guided mainly by clinical markers of sustained graft function since immune markers for long-term clinical graft ac- ceptance are not sufficiently reliable26. Tapering management could be improved by earlier
T cell immunity after tapering of immunosuppression in islet transplantation 181
detection of processes leading to deteriorating graft function. In islet cell transplantation, both auto- and alloreactivity need to be inhibited and should therefore be monitored. This pilot study underscores that the earlier suggested tolerance marker IL10 is associated with lower alloreactivity after addition of tacrolimus, but as yet does not appear representative for graft function during tapering. Knowledge on long-term graft acceptance after tapering may increase by assessment in larger patient cohorts and analysis of other suggested toler- ance markers such as TGFβ, FoxP3 and CD4+/CD25bright T cells27. The current data suggest that tapering of tacrolimus leads to differential changes in immune reactivity with an increasing role for high-avidity alloreactive CTLs, which may be responsible for loss of graft function.
Follow-up of immune parameters therefore is useful to guide tapering of especially tacroli- mus and to achieve a stable state where the loss of graft function due to immunological causes is minimal while the burden of immunosuppressive medication can be decreased.
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