The ultimate challenge in organ transplantation is to achieve transplant tolerance. Although studies describing this condition after clinical kidney transplantation (KTx) are already extremely rare (1–3), the development of tolerance in humans remains elusive. Nevertheless, the involvement of and even cell therapy with CD4+CD25bright+ regulatory T cells (Tregs) has been frequently suggested (3–8).
Associations between tolerance and regulatory T cells were found in immunosuppression-free liver transplant recipients from whom the proportion and number of Tregs was elevated (5, 7). Also, in a small group of operational tolerant renal recipients the level of peripheral Tregs and the transcription factor for regulatory cells, FoxP3, was higher when compared with patients with chronic rejection (9, 10).
Although these findings suggest an association between transplant tolerance and the presence of Tregs, data providing evidence for the presence of functional donor-specific Tregs after transplantation are only available from stable immunosuppressed kidney transplant recipients (11–13). However, it remains to be demonstrated that Tregs also play a role in the induction of donor-specific hyporesponsiveness in patients after transplantation.
Unlike experimental animals, kidney transplant patients receive lifelong immunosuppression to prevent graft rejection. Because these regimens influence T cells (14), they may also affect the induction and function of Tregs (15). Particularly because most of these drugs target the IL-2 pathway, which is crucial for the function, homeostasis, and survival of CD4+CD25+FoxP3+ T cells (16–19). Thus, these immunosuppressive drugs may interfere with the development of donor-specific Tregs thereby impairing a potential key player responsible for graft acceptance.
Therefore, we performed a prospective study on 79 fully immunosuppressed kidney transplant patients to determine whether donor-specific Tregs are induced in the first year after transplantation. Understanding the dynamic features of antigen-specific regulatory T cells will contribute to our understanding of the role of these cells in anti-donor reactivity.
MATERIALS AND METHODS
The medical ethics committee of Erasmus Medical Centre approved the study protocol, and all patients provided informed consent (medical ethics committee number 2004-264). As part of a multicenter trial (20), patients were enrolled from March 2004 until March 2006 and follow-up was performed for 1 year. We included 79 patients, who were equally randomized to treatment, arm 1 with tacrolimus/rapamycin (n=39) or arm 2 with tacrolimus/mycophenolate mofetil (MMF, n=40, Table 1). There were no significant differences in patient characteristics between the two arms of treatment at baseline. In both arms of treatment, patients received prednisone for the first 4 to 6 weeks. The dosing and aimed whole-blood trough levels of the study medication are summarized in Table 2. Peripheral blood samples were obtained within 24 hr before and 3, 6, and 12 months after KTx. Blood samples before KTx were obtained before patients received immunosuppressive medication. In addition, blood was obtained from 17 healthy controls (HC), consisting of 10 men and 7 women with a mean age of 52±8.6 years. These characteristics of our HC were comparable with our patient population.
Flow Cytometric Analysis
Blood samples were collected in heparinized tubes and analyzed for the presence of T-cell subsets by four-color flow cytometry using monoclonal antibodies (mAbs) directly conjugated to fluorescein (FITC), phycoerythrin (PE), allophycocyanin (APC), or peridinin chlorophyll protein (PerCP). One hundred microliters of blood was incubated with 10 μL of the dual mAb combinations CD45-FITC/CD14-PE; IgG1-FITC/IgG2b-PE; and IgG1-PerCP/IgG1-APC as isotype control. Furthermore, we used the mAb CD3-FITC, CD4-PerCP, CD8-APC, and CD25-PE. To further determine how Tregs evolve, we added a combination of CD4-PerPC/CD25-PE/CD45RO-APC/CCR7-FITC to 100 μL whole blood. The antibodies were purchased from BD Biosciences (San Jose, CA,) and R&D Systems (Abingdon, UK). After 30 min of incubation at room temperature, red blood cells were lysed with fluorescence-activated cell sorter lysing solution (BD Biosciences) during 10 min. Cells were then washed twice and analyzed on a flow cytometer (FACSCalibur, BD Biosciences) using SimulSet and CELL Quest Pro software (BD Biosciences). To establish an analysis gate that included at least 90% of the lymphocytes, the CD45/CD14 reagent was used. At least 20,000 gated lymphocyte events were acquired from each tube. Cells with a CD45RO− phenotype were considered to be naive cells and cells with a CD45RO+ phenotype memory cells.
Expression of FoxP3 and CD127
FoxP3 is a transcription marker for regulatory cells, and in July 2006 it was shown that the expression of CD127 inversely correlates with FoxP3 expression and the suppressive function of Tregs (21). We began the experiments on our study cohort using fresh materials before the anti-FoxP3 antibody became available for analysis (eBioscience, San Diego, CA) and before its correlation with CD127 was reported. Therefore, to gain insight into the expression profile of FoxP3 and CD127 in our patient materials; we stained peripheral blood samples of an additional cohort of patients (n=34). These samples were taken 24 hr pre KTx and stained with CD4-PerCP (BD Biosciences), CD25-PE (epitope B, BD PharMingen, San Diego, CA), CD127-FITC (eBioscience), and FoxP3-APC (clone PCH101, eBioscience). Patient characteristics from this additional cohort were comparable with our study population from Table 1.
Isolation of Peripheral Blood Lymphocytes
Peripheral blood mononuclear cells (PBMC) were isolated from 49-mL heparinized peripheral blood by density gradient centrifugation using Ficoll-Paque (density, 1.077 g/mL; Amersham, Uppsala, Sweden). PBMC were collected from the interphase, washed twice in Roswell Park Memorial Institute 1640 (BioWhittaker, Verviers, Belgium) and resuspended in human culture medium (HCM) consisting of Roswell Park Memorial Institute 1640-Dutch Modification (Gibco, BRL, Scotland, UK) supplemented with 10% heat inactivated pooled human serum, 4 mM l-Glutamine (Gibco BRL), 100 IU/mL penicillin (Gibco BRL), and 100 μg/mL streptomycin (Gibco BRL).
Isolation of CD25bright+ Cells
After isolation, PBMC were washed once and resuspended in 45 μL MACS-buffer/10×106 PBMC prepared according to the manufacturer’s protocol (Miltenyi, Bergisch Gladbach, Germany). The CD25bright+ cells were depleted from PBMC by incubating PBMC with anti-CD25 microbeads (Epitope A, Miltenyi Biotec) followed by a positive selection (POSSELD-program) on the autoMACS (Miltenyi). Cells not selected by the microbeads were referred to as the CD25neg/dim fraction (11). To control for the autoMACS procedure, 6×106 PBMC were treated by the same protocol in the absence of anti-CD25 microbeads.
Purity of the fractions was measured by flow cytometry using CD3-FITC, CD4-PerCP, CD8-APC (BD Bioscience), and CD25-PE (epitope B, BD PharMingen). Phenotypical analysis of both fractions demonstrated that the average proportion of CD4+ cells in the CD25bright+ fraction was 95% and in the CD25neg/dim fraction was 62% (Fig. 1A, B). The proportion of CD4+CD25bright+ cells in the CD25bright+ fraction was 72% (Fig. 1C), and the proportion of CD4+CD25neg/dim cells in the CD25neg/dim fraction was 96% (Fig. 1D). These proportions were not different over time and comparable with proportions measured in samples from HC (Fig. 1A–D).
Mixed Lymphocyte Reactions
In the mixed lymphocyte reactions (MLR), 5×104 freshly isolated patient-PBMC and CD25neg/dim cells were stimulated with 5×104 irradiated (40 Gy) donor PBMC (donor-Ag) and 5×104 (40 Gy) human leukocyte antigen (HLA) A, B, and DR fully mismatched third party PBMC. Because it has been described that improved histocompatibility between recipient and donor enhances immune regulation and graft survival after KTx (22); we also stimulated patient-PBMC and CD25neg/dim cells with 5×104 (40 Gy) fourth party PBMC. Fourth party PBMC have the same number of mismatches at the HLA A, B, and DR level to the recipient as the donor to the recipient, but the mismatches are based on different antigens. The same third and fourth party PBMC were used for an individual patient at all analyzed time points. The MLR was performed in HCM, in triplicate, in a 96-wells round bottom plate for 7 days. At day 6, 3H-thymidine 0.5 μCi/well was added to the culture; and 16 hr later samples were harvested and radioactivity was measured in counts per minute (CPM) using a β-counter (PerkinElmer, Oosterhout, The Netherlands).
Regulation of Allo-Ag Stimulated Responder Cells by CD25bright+ Cells
Regulation of proliferation by CD25bright+ cells was quantified both by their depletion from PBMC and in co-culture experiments with the CD25neg/dim responder cells. After depletion the increase in proliferation reflects the regulatory capacity of the CD25bright+ cells. To compare the effect of depletion over time, we calculated the percentage of increase (%increase) in those cultures where the effect of depletion was positive.
In the MLR, isolated CD25bright+ cells were added to CD25neg/dim responder cells at a ratio of 1:5, 1:10, 1:20, and 1:40. The effect was calculated as the percentage of inhibition (%IH), when the proliferative response of alloactivated CD25neg/dim cells was more than 1000 CPM; and the effect of depletion was positive.
Proliferation of Mitogen-Stimulated Cells
We determined the capacity of PBMC and CD25neg/dim cells (5×104) to proliferate on stimulation with 1 μg/mL Phytohaemagglutinin (PHA; Murex Biotech Ltd., Kent, UK). All cultures were performed in HCM, in triplicate in a 96-wells plate for 3 days. At day 2, 3H-thymidine 0.5 μCi/well was added to the culture, 16 hr later the samples were harvested, and radioactivity was counted.
All calculations were performed using GraphPad Prism 4.0 or SPSS 11.5. On the basis of the distribution of the data, we performed parametric or nonparametric testing. For paired analysis the paired t test was performed; and to compare data from patients versus HC, we used the unpaired t test. To determine if a certain parameter changed significantly over time, One-Way ANOVA was used. To analyze several variables at a fixed time point, Cox or linear regression analysis was performed. A P value less than 0.05 is marked with *, P less than 0.01 with **, and P less than 0.001 with ***. For each analysis, statistics are described more specifically in the appropriate tables and figure legends.
Of 79 randomized patients receiving a kidney transplant, 62 (78%) completed the study and 17 (22%) were withdrawn due to adverse events. There was no difference between patients treated in arm 1 (tacrolimus/rapamycin) or arm 2 (tacrolimus/MMF) for adverse events (8 vs. 9), patient survival (97% vs. 98%), graft survival (97% vs. 93%), rejection incidence (13% vs. 10%), or renal function (serum creatinine 119 μmol/L vs. 130 μmol/L) at one year. Blood trough levels of the medication were within target range. The trough levels of tacrolimus were higher in arm 2 than in arm 1, which was consistent with the study protocol (Table 2). All the patients who had rejection were treated with antirejection therapy and are therefore described separately.
Characterization of CD4+CD25bright+ Regulatory T Cells
Analysis of whole blood samples from patients and healthy controls (HC) was performed for lymphocyte subsets, including Tregs defined as the CD4+CD25bright+ T-cell population in combination with slightly less CD4 expression (Fig. 2A). Flow cytometry showed that the absolute number of Tregs and their proportion, decreased within the first year after KTx (Table 3 and Fig. 2B, P<0.05 and P<0.001, respectively).
We also analyzed the expression of CD45RO, CCR7, FoxP3, and CD127 by Tregs. These results revealed that the absolute number of CD4+CD25bright+CD45RO+ cells (Table 3) and their proportion decreased after KTx (P<0.05 and P=0.06, respectively). The absolute number of CD4+CD25bright+CCR7+ cells did not change in the first year after transplantation (Table 3), whereas their proportion increased (P<0.01).
As described in Materials and Methods we determined the expression of FoxP3 and CD127 by Tregs on peripheral blood samples from an additional cohort of patients before KTx. Based on the gate in Figure 2(A), the average percentage of Tregs that expressed FoxP3 was 72%. This finding is in line with the results described in an article by Liu et al. (21). The average percentage of FoxP3+ Tregs with a CD127neg/low phenotype was 87%.
Proliferation of PBMC
The average proliferation of patient-PBMC to the mitogen PHA was more than 51,000 CPM at all tested time points and comparable with proliferation of PBMC from HC (57,000 CPM). Before transplantation, proliferation of patient-PBMC to donor-Ag, third and fourth Party-Ag was significantly lower as compared with proliferation of PBMC from HC to alloantigens (Fig. 3, all P<0.001). After transplantation, proliferation of PBMC to donor-Ag remained low whereas increasing proliferation to third and fourth Party-Ag, was measured (Fig. 3, P<0.001 and P<0.05, respectively). Thus, we observed a proportional hyporesponsiveness toward donor-Ag.
The Suppressive Function of CD25bright+ Cells
The effect of depletion of CD25bright+ cells from PBMC on direct alloresponses was determined in MLR. Because of the limited amount of peripheral blood available from our patients, we could not analyze the effect of depletion of CD25bright+ cells in cultures stimulated with fourth Party-Ag. After depletion of the CD25bright+ fraction, we observed an overall increase of the proliferative response in cultures stimulated with donor-Ag and third Party-Ag before and after transplantation (Fig. 4A). Before transplantation, the average effect of depletion on proliferative responses of alloreactive cells was 51% on stimulation with donor-Ag and 57% on stimulation with third Party-Ag (Fig. 4B). After transplantation, the effect of depletion increased significantly in co-cultures stimulated with donor-Ag and less so with third Party-Ag (Fig. 4B, P<0.01 and P=0.07, respectively). Furthermore, at 6 and 12 months after transplantation the % increase was higher in cultures stimulated with donor-Ag than with third Party-Ag (Fig. 4B, P<0.05 and P=0.09, respectively).
The suppressive capacity of the isolated CD25bright+ cells on a per cell basis was determined in co-culture experiments with CD25neg/dim responder cells. The isolated CD25bright+ cells did not proliferate on stimulation with allo-Ag. Co-culture experiments proved that the suppressive effect of CD25bright+ cells on CD25neg/dim responder cells is a dose-dependent phenomenon (Fig. 4C,D). Before transplantation, the capacity of CD25bright+ cells to suppress donor-Ag or third Party-Ag stimulated CD25neg/dim responder cells (1:10 ratio) was significantly lower when compared with HC (Fig. 4E, both P<0.05). After transplantation, the average suppressive capacity of CD25bright+ T cells improved significantly in cultures stimulated with donor-Ag (Fig. 4E, 51%–75%, P<0.001), but not with third Party-Ag (50%–57%, P=0.49). Furthermore, at 6 and 12 months after transplantation, the capacity of CD25bright+ cells to suppress donor-Ag stimulated CD25neg/dim cells was significantly higher than on stimulation with third Party-Ag (Fig. 4E, P<0.01 and P<0.001, respectively). The results on the %IH at a 1:10 ratio were comparable with the ratio of 1:5, 1:20, and 1:40, but significance was lost at 1:20 and 1:40.
Rejectors Versus Nonrejectors
In this study, 9 of 79 patients (11%) had a rejection episode. All rejections occurred in the first month after transplantation (median, 12 days; range, 3–28). Antirejection therapy consisted of high dose solumedrol. At baseline, no differences were found between rejectors and nonrejectors for clinical characteristics, flow cytometric results, proliferation of PBMC, and the suppressive function of CD25bright+ cells. Therefore, we found that none of these factors are a predictor for rejection before transplantation.
We analyzed the suppressive capacity of CD25bright cells from rejectors at 12 months after transplantation. Our results show that this suppressive capacity in co-cultures of CD25neg/dim responder cells stimulated with donor-Ag or third Party-Ag was not different from nonrejectors at month 12. This is in line with a study from Demirkiran et al. (23) on liver transplant recipients.
Immunosuppressive Drugs: Tacrolimus/MMF Versus Tacrolimus/Rapamycin
We compared the two arms of treatment to determine whether therapy with tacrolimus/MMF versus tacrolimus/rapamycin affected Tregs differently. No difference was observed for any of the phenotypical or functional Treg-characteristics analyzed in this study between these arms of treatment.
In a multivariate analysis the factors like gender, recipient age, dialysis type, time on dialysis, origin of donor kidney, first KTx/more than first, number of HLA mismatches, primary kidney disease, blood group, cytomegalovirus status, and the level of panel reactive antibodies before KTx were not associated with the number, proportion, or function of CD25bright+ T cells on a fixed time point or over time.
To investigate whether donor-specific CD4+CD25bright+ regulatory T-cell function is generated in de novo kidney transplant recipients; we prospectively analyzed their suppressive capacity in the first year after transplantation. In the MLR, depletion of CD25bright+ cells from PBMC, and their capacity to suppress the proliferation of CD25neg/dim cells, demonstrated improved Treg function in the first year. We also found donor-specific hyporesponsiveness, whereas Treg activity was significantly more donor-directed compared with third Party-Ag.
Data from in vitro and animal studies indicated that immunosuppressive drugs have detrimental effects on Tregs (15, 17, 24). However, the development of donor-specific Tregs in the present study shows that the immune system can bypass these unfavorable effects in vivo to a certain extent. Apart from the restored kidney function (25), an explanation might be that immunosuppressive drugs like cyclosporin and tacrolimus, do not inhibit the transcription of IL-2 (14), an important cytokine for the function and survival of CD4+CD25bright+FoxP3+ T cells (16–19). However, the pivotal role for this cytokine was not always observed (26), as other members of the IL-2 family may compensate for the absence of IL-2 (18, 27). This probably results from their shared signaling through the common gamma chain (i.e., CD132), which activates the signal transducer and activator of transcription factor 5 (STAT 5), and therefore induces the expression of the transcription factor for regulatory cells FoxP3 (8, 28, 29).
The observed donor-specific hyporesponsiveness as compared with the reactivity against third and fourth Party-Ag, did not result from better histocompatibility between donor and recipient (22). Regulation could be another explanation. Indeed, the suppressive function of Tregs from our patients became increasingly potent to donor-Ag stimulated cultures after transplantation. It has been reported that Tregs respond dynamically to their antigenic environment in a transgenic mouse model, which showed that these regulatory T cells proliferated in response to T-cell receptor engagement (30). In the transplantation setting, the continuous presence of donor-antigen could therefore stimulate the peripheral proliferation and accumulation of Tregs. Moreover, it has been reported that operationally tolerant patients have an unexpected strongly altered T-cell receptor Vβ usage and high T-cell receptor transcript accumulation in selected T cells (31). This may explain why we found generation of donor-specific regulatory T-cell function and not higher Treg numbers. Also, development of potent Tregs might be favored by the lymphopaenic state of transplant patients (Table 3), because stimuli that originate from lymphopaenia favor their homeostatic proliferation and enhance their suppressor function (32).
In the present study, we measured donor-specific hyporesponsiveness in the direct pathway of allorecognition, which was mediated by CD4+CD25bright+ Tregs. In contrast, a cross-sectional study from Game et al. (33) stated that Tregs do not contribute to the direct pathway of hyporesponsiveness in stable transplant patients. The difference between their findings and the present study may be explained by differences in immunosuppressive strategies, the lower number of patients studied (n=12 vs. n=79) and the time after transplantation (2–20 years vs. ≤1 year, respectively). Especially the latter may be essential, because the indirect pathway becomes more important in the long term (4). In addition, other mechanisms could be envisioned that contribute to the measured donor-specific hyporesponsiveness, including anergy, ignorance, and clonal deletion of donor-specific effector T cells.
The generation of donor-specific Treg function occurred in the presence of immunosuppressive agents that have the potential to hamper their development and suppressive function (15, 24, 27). Therefore, the individual effect of these drugs or their combinations may still have influenced the dynamics by which donor-specific Treg function is generated. Several studies indicated that rapamycin does not interfere with the suppressive activity of CD4+CD25bright+FoxP3+ T cells and favors their expansion in vivo (34–36), whereas MMF and calcineurin inhibitors (CNI) for example, tacrolimus prevent the expansion of these cells (14, 34). Here, we did not observe a difference in the effect of treatment with tacrolimus/rapamycin or tacrolimus/MMF on the number and function of Tregs. These findings can be explained by the dominant effect of tacrolimus in both arms of treatment. Especially, because calcineurin inhibitor-based treatment is associated with decreased numbers of Tregs and possibly impairs their functional development (15, 24, 34, 37).
Another explanation for the observed changes in the peripheral compartment may be an increased recruitment of Tregs to secondary lymphoid tissues and the transplanted organ (26, 30, 35, 38–40). CCR7 is a homing marker for the lymphoid tissues, and in this study we demonstrated that the proportion of Tregs that expressed CCR7 significantly increased. This suggests an increased potential of the peripheral Treg compartment to home to lymphoid tissues (41, 42). Also CCR7 is expressed by naive T cells (41, 42) and indeed flow cytometric analysis revealed a decreased proportion of Tregs with a memory phenotype, indicating an increased proportion of naive Tregs. Because it has been demonstrated that especially naive Tregs give rise to potent Ag-specific Tregs (43), their strong proportional increase may have favored the development of the observed donor-specific Treg function.
In summary, we prospectively analyzed the development of peripheral CD4+CD25bright+ T cells from kidney transplant recipients in the first year after transplantation. Our results demonstrated that even in the presence of full immunosuppression potent donor-specific CD4+CD25bright+ regulatory T-cell function is generated in these patients.
The authors thank Dr. Nicolle Litjens for her help with the flow cytometric measurements, and her advice for interpretation of the data.
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