Donor-Derived Mesenchymal Stem Cells Combined With Low-Dose Tacrolimus Prevent Acute Rejection After Renal Transplantation

A Clinical Pilot Study

Abstract

Background 

The deleterious side effects of calcineurin inhibitors have impaired long-term survival after renal allograft. New immunotherapy regimens that minimize or even eliminate calcineurin inhibitors are required to improve transplantation outcome. Mesenchymal stem cells (MSCs) represent a unique cell population with immunosuppressive function and prolong allograft survival in experimental organ transplant models.

Methods 

In this pilot study, donor-derived bone marrow MSCs combined with a sparing dose of tacrolimus (50% of standard dose) were administered to six de novo living-related kidney transplant recipients. Six other patients who received a standard dose of tacrolimus were enrolled as a control. The safety of MSC infusion, acute rejection, graft function, and patient and graft survival within 12 months after kidney transplantation were observed. The immune profiles were analyzed at different time points after transplantation.

Results 

None of the MSC recipients experienced immediate or long-term toxic side effects associated with MSC infusion. The tacrolimus dose (0.045±0.002 mg/kg) in the MSC group was significantly reduced compared with the control group (0.077±0.005 mg/kg). One acute rejection occurred only in the control group. All patients survived with stable renal function at month 12 and no chimerism was detectable at month 3. Patients in the MSC group showed significantly higher B-cell levels than the control group at month 3.

Conclusion 

These preliminary data suggest that the use of MSCs could provide potential benefits in renal transplantation by reducing the dosage of conventional immunosuppressive drug that is required to maintain long-term graft survival and function.

Kidney transplantation remains the most effective therapy for patients with end-stage renal disease. The major barrier to renal transplantation is acute and chronic rejection of engrafted kidneys caused by the recipient’s immune system. Conventional immunosuppressive drugs are widely used in transplant patients and have proven to be effective in reducing acute rejection and improving short-term outcomes. However, the improvement in long-term graft survival and function remains a major problem after renal transplantation.

Tacrolimus is a calcineurin inhibitor that has proven highly effective in preventing acute rejection and improving short-term survival in renal allograft recipients (¹). However, its inevitable adverse effects, such as nephrotoxicity, diabetogenicity, and neurotoxicity, constrain its long-term utility (^2–4). In modern immunosuppressive regimens, the elimination or minimization of calcineurin inhibitors is required to attain further improved outcomes in kidney transplantation (⁵). At present, it is necessary to implement optimized immunosuppressive regimens to maximize the beneficial effects of induction of transplant tolerance and to minimize the aforementioned adverse effects.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can be induced to undergo rapid proliferation and differentiation into multiple cell lineages (^{6, 7}). In addition, myriad studies have shown that MSCs possess potent immunomodulatory functions, suppressing the proliferation and activation of T cells and natural killer (NK) cells or modulating the maturation and function of antigen-presenting cells such as dendritic cells. The therapeutic application of MSCs has been explored to enhance hematopoietic stem cell engraftment and to treat graft-versus-host disease or autoimmune disease (^8–10). In addition, the immunosuppressive properties of MSCs suggest that they may be applied to inhibit allograft rejection and induce transplant tolerance. In animal transplantation models, many groups observed that MSC infusion significantly prolonged skin and cardiac allograft survival (^11–13). Crop et al. also found that donor MSC infusion could significantly suppress the proliferation of alloactivated T-cell subsets in recipients (¹⁴). These promising results imply that donor-derived MSCs may facilitate the induction of transplant tolerance, thus suppressing renal allograft rejection. Recently, Tan et al. reported that the use of autologous MSCs resulted in a lower incidence of acute rejection, decreased risk of opportunistic infection, and better estimated renal function at 1 year among patients undergoing renal transplantation (¹⁵).

Here, a clinical pilot study was designed to investigate the efficacy and safety of using donor-derived MSC treatment in living-related kidney transplantation to reduce tacrolimus dose and improve transplantation outcome. Because some studies showed that regulatory T cells (Tregs) in recipients are helpful to maintain a stable graft function and reduce acute/chronic rejection (^{16, 17}), patients with stable kidney graft function display an increased frequency of B cells and CD19⁺CD27⁺ memory B cells (^{18, 19}). The immune profiles of the recipients were also constantly monitored, including Treg, B-cell, and memory B-cell counts, to determine whether MSC therapy could take advantage of immune modulation in the clinical setting of living-related kidney transplantation.

RESULTS

Patient Characteristics

Twelve uremia patients were enrolled in this study. The kidney transplantation was performed from September 2009 to January 2011. These patients were divided into an experimental group (MSC group) and a control group (non-MSC group), with six subjects in each. The demographic profiles of both groups are summarized in Table 1. There was no significant difference in baseline parameters between groups. All patients were followed up for 12 months.

TABLE 1: Demographic characteristics of patients

Clinical Outcome After Mesenchymal Stem Cell Infusion

In the experimental group, the arterial injection of MSCs did not result in any obstruction of graft capillaries, and the secondary MSC infusion was performed successfully without any signs of thrombosis, infection, allergy, liver damage, sugar metabolism disorders, or vascular complications during the 12-month follow-up period. Renal allograft function recovered fluently, and no delayed graft function or acute rejection incident was observed.

In the control group, one biopsy-proven acute rejection (Banff Ia) occurred at day 7 and was reversed after pulse therapy with methylprednisolone (0.25 g/day for 3 days) and antithymocyte globulin (100 mg/day for 5 days). The acute rejection rates at months 6 and 12 were 0% in the experimental group and 16.7% in the control group (P>0.05, Fisher’s exact test). There was no significant difference in serum creatinine at months 1, 3, 6, and 12 between these two groups (Fig. 1). All patients and grafts in both groups survived during the 12 months of follow-up. Notably, the average daily doses of tacrolimus within 12 months after kidney transplantation were 0.045±0.002 mg/kg in the experiment group and 0.077±0.005 mg/kg in the control group (P<0.05; Fig. 1). As a result, the trough levels of whole-blood concentration at various time points after transplantation were significantly lower in the MSC group than in the control group (P<0.05). The trough levels were 3.98±1.98 μg/L in the MSC group and 6.78±2.73 μg/L in the control group at month 3, 3.85±0.95 and 6.92±1.67 μg/L at month 6, and 3.92±1.75 and 6.86±1.21 μg/L at month 12, respectively.

FIGURE 1: Serum creatinine level after transplantation. A, serum creatinine levels of each recipient after transplantation detected at day 0 and months 3, 6, and 12. B, serum creatinine levels (mean±SD) of the experimental and control groups at day 0 and months 3, 6, and 12. SD, standard deviation.

Dynamic Changes of Peripheral Blood Lymphocyte Subsets After Mesenchymal Stem Cell Infusion

Peripheral blood lymphocytes were sampled from the renal transplant recipients and analyzed by flow cytometry at four time points: day 0 and months 3, 6, and 12. There were no differences in the proportions of peripheral total T cells (Fig. 2A), CD4⁺ T cells (Fig. 2B), CD8⁺ T cells (Fig. 2C), and NK cells (Fig. 2G) before kidney transplantation or at 3, 6, and 12 months after kidney transplantation between the experimental group and the control group (Fig. 2).

FIGURE 2: Changes in the peripheral lymphocyte populations at different time points after transplantation. Proportions of various lymphocyte populations in all patients’ PBMCs in the experimental and control groups at day 0 and months 3, 6, and 12, including total T cells (A), CD4⁺ T cells (B), CD8⁺ T cells (C), Treg cells (D), total B cells (E), memory B cells (F), and NK cells (G). *P<0.05. P≤0.05, adjusted by time point, was considered significant. NK, natural killer; PBMC, peripheral blood mononuclear cells.

A change in the Treg cell proportions was also observed, but there were no obvious differences between the experimental group and the control group at 3, 6, or 12 months after transplantation (Fig. 2D).

In this study, the proportion of B cells in the experimental group increased significantly at month 3 (12.03% [15.92%]) compared with day 0 (8.85% [9.38%]) but was then reduced to 7.36% (10.47%) at month 6 and 5.41% (4.51%) at month 12. The postoperative B cells proportion in the control group was decreased (4.49% [4.8%]) at month 3 compared with day 0 (10.06% [8.41%]) and decreased further to 3.77% (2.46%) at month 6 and 3.38% (2.08%) at month 12 (Fig. 2E). The frequency of B cells in the experimental and control groups at month 3 was significantly different (Fig. 2E). As shown in Figure 2F, the change in the frequency of CD27⁺ memory B cells was different between the experimental group and the control group. The proportion of memory CD27⁺ B cells increased in the experimental group at month 3 (36.23% [19.15%] vs. 27.46% [14.61%] before operation), decreased at month 6 (23.50% [9.14%]), and finally increased to 29.53% (8.60%) at month 12; the level at month 12 was higher than the pretransplantation level (27.46% [14.62%]). In the control group, the frequency of CD27⁺ memory B cells gradually decreased after the operation to 32.41% (30.20%) at month 3, 26.68% (16.89%) at month 6, and 25.66% (7.52%) at month 12, which was much lower than the pretransplantation level (35.92% [39.88%]; Fig. 2F).

Mixed Lymphocytes Reaction

One-way mixed lymphocytes reaction (MLR) was conducted to compare responses to donor alloantigens before transplantation and at 3, 6, and 12 months after transplantation. As shown in Figure 3, the proliferation of recipients’ peripheral blood mononuclear cells (PBMCs) was stimulated by donors’ PBMCs; in all cases, proliferation was increased at month 3 compared with pretransplantation, decreased at month 6, and then returned to a higher level. The two groups exhibited the same trend in proliferation, but the levels of proliferation were not significantly different between the experimental group and the control group.

FIGURE 3: MLR of recipients to donors at day 0 and months 3, 6, and 12. Recipient PBMCs were isolated before transplantation and at 3 and 6 months after transplantation and labeled with CFSE. Aliquots of 1×10⁵ recipient PBMCs were stimulated with 1×10⁵ donor PBMCs that had been treated with mitomycin C. Cultured cells were harvested after 6 days, and the population distributions of the proliferated cells were measured by FACSCalibur flow cytometry. A, representative result from one patient in the experimental group. B, changes in proliferation in the experimental and control groups at different time points. CFSE, carboxyfluorescein diacetate, succinimidyl ester; MLR, mixed lymphocyte reactions.

Chimerism

The peripheral chimerism of donor leukocytes was measured at months 3 and 12. No chimerism was detectable in any patient.

Intercellular Cytokine Staining

We next detected the proinflammatory/anti-inflammatory cytokine (tumor necrosis factor [TNF]-α, interferon [IFN]-γ, interleukin [IL]-4, and IL-10)–producing cells. The frequency of IL-4–, IL-10–, TNF-α–, and IFN-γ–producing cells was measured before transplantation and at 3, 6, and 12 months after transplantation. At month 3, the proportion of IFN-γ–producing cells increased in the experimental group but decreased in the control patients; the frequency of IFN-γ–producing cells at 6 months after transplantation was lower than that at day 0 and month 3 in both groups (Fig. 4). At month 12, the frequency of IFN-γ–producing cells decreased further in both the experimental group and the control group (Fig. 4). The proportion of TNF-α–producing cells increased in the experimental patients at month 3 but gradually decreased at months 6 and 12. However, the proportion of TNF-α–producing cells in control patients decreased at month 3 and then increased at months 6 and 12 (Fig. 4). The proportions of IL-4–producing cells in both the experimental group and the control group gradually increased in 12 months after kidney transplantation. In both groups, the proportions of IL-10–producing cells gradually increased for 6 months after transplantation and then obviously decreased at month 12 (Fig. 4). However, the variations of these proinflammatory/anti-inflammatory cytokines were not statistically significant.

FIGURE 4: Production of recipients’ proinflammatory/anti-inflammatory cytokines. The recipients’ PBMCs at day 0 and months 3, 6, and 12 were collected, and the frequencies of IFN-γ–, TNF-α–, IL-4–, and IL-10–producing cells were analyzed by flow cytometry. A, representative result from one patient in the experimental group. B, changes in cytokine-producing cell frequencies in the experimental and control groups at different time points. IFN, interferon; IL, interleukin; PBMC, peripheral blood mononuclear cells; TNF, tumor necrosis factor.

DISCUSSION

Since the introduction of calcineurin inhibitors, the acute rejection after renal transplantation has decreased remarkably. Tacrolimus forms a complex with its cytosolic partner, FK506-binding protein 12, which also binds to calcineurin. By blocking calcineurin, tacrolimus prevents the induction of cytokines and their receptors, which are required for the activation and proliferation of lymphocytes (²⁰). However, this inhibition of calcineurin is not specific to immune cells. In addition to immunosuppressive effects, it can also lead to cytotoxicity (²¹), including interstitial fibrosis and tubular atrophy, which is considered to be a significant cause of late dysfunction of transplanted kidneys (²²). Therefore, a reduction of calcineurin inhibitor dose is necessary in clinical kidney transplantation. Because a low dose of tacrolimus is associated with a greater risk of acute rejection in kidney transplantation (^{23, 24}), other adjunctive therapeutic strategies are required to ensure the safety and efficacy of tacrolimus minimization.

The immunomodulatory capacity of MSCs has been demonstrated to be therapeutically valuable. In a recent study, MSCs showed immunosuppressive properties both in vitro and in vivo, and the infusion of donor MSCs effectively prolonged graft survival in animal models (^{12, 14}). Donor-derived MSCs also induced the long-term acceptance of solid organ allografts (²⁵). Thus, we sought to use donor-derived MSCs in combination with low-dose tacrolimus to prevent acute rejection after renal transplantation. Our results suggested that donor-derived MSCs combined with low-dose tacrolimus (~50% of the standard dose) therapy exhibited an effect equivalent to that of standard immunosuppression when used to inhibit acute rejection after living-related renal transplantation. Patients in our MSC group presented with stable vital signs and stable renal function without any signs of acute rejection.

The choice of MSC administration route is important to obtain good outcomes. Experiments in rats have demonstrated that MSCs injected into the renal artery were retained in the glomeruli and both lowered the frequency of glomerulonephritis and prevented acute cellular rejection (^{26, 27}). However, to date, there have been no reports of the intra-arterial injection of MSCs into the human kidney. The direct injection of MSCs into the renal artery may locally depress the inflammatory response and therefore nonspecifically protect the graft. Our studies indicated that the administration of MSCs by both intra-arterial and intravenous routes was feasible and safe, with no evidence of embolism, thrombosis, infection, or any other complications within 12 months after transplantation.

Thus, these preliminary results indicated that MSC infusion in combination with low-dose immunosuppressive drug therapy in renal transplantation patients was feasible and safe. Notably, the posttransplantation serum creatinine levels in the MSC-treated group were higher than those in the control group, although this difference was not statistically significant. The reduction of calcineurin inhibitor dose may not be the cause of this phenomenon; both groups showed similar immune response to donor alloantigens in MLR experiments and the analogous characteristics of T-cell subpopulations measured by flow cytometry. Moreover, the serum creatinine levels in the MSC-treated group remained stable throughout the 12 months of the observation period with no increase in maintenance immunosuppressants. Possible explanations for the sCr level in MSC-treated group may include the following: (a) Donor characteristics. Although the difference was not significant, the mean age of donors in the MSC group was higher than that in control group (43.8±4.36 vs. 38.3±15.31; P=0.431). Furthermore, there were two female donors in the MSC group and one female donor in the control group. The three male donors in the control group were 23, 23, and 29 years old, which may have enabled better recovery after transplantation. (b) Donor and recipient match in body size. To further interpret this phenomenon, the body mass index (BMI) of donors and recipients was calculated. The ratio of donor BMI to recipient BMI, which may reflect the demand of recipients for donor kidney function to some extent, was compared between the two groups. Although there was no significant difference, the ratio in MSC-treated group was lower than that in the control (1.067±0.130 vs. 1.198±0.316; P=0.368).

Previous studies have shown that donor leukocytes can be detected many years after solid organ transplantation in recipients who enjoy long-term graft survival and were able to safely reduce or discontinue immunosuppression therapy (²⁸). In the present study, chimerism was undetectable at months 3 and 12. This implies that MSCs did not contribute to microchimerism.

Recent studies of transplant patients who maintain stable kidney graft function in the absence of immunosuppression drugs showed that these patients have more peripheral B cells (^{18, 19}). In this study, the patients who received MSC infusions in the experimental group had more peripheral B cells than those in the control group at 3 months after transplantation. More research to understand whether the variation of peripheral B cells improves the long-term graft function of transplanted kidneys is required.

MATERIALS AND METHODS

Study Design

Donors and recipients undergoing living-related kidney transplantation procedures in the Second Affiliated Hospital of Guangzhou Medical University were considered for enrolment in this prospective, nonrandomized pilot study. Donor selection complied with the 2004 Amsterdam Forum Guidelines (²⁹) and the 2007 Chinese “Regulation on Human Organ Transplantation” (Order of the State Council No. 491) (³⁰). All candidates met the following inclusion criteria: (a) patients were undergoing primary kidney transplantation, (b) donors and recipients were 18 to 60 years old and ABO compatible, (c) the primary kidney disease was chronic glomerulonephritis, (d) tacrolimus was used as the maintenance immunosuppressive regimen instead of cyclosporine A, and (e) complement-dependent cytotoxicity examination and panel reactive antibody examination were negative (<10%) before kidney transplantation.

Subjects were excluded if (a) kidney transplantation was secondary, multiple, or combined with other allograft organs; (b) recipients had systemic or active infections; (c) recipients had a history of severe cardiovascular or pulmonary dysfunction, malignancy, liver dysfunction, and chronic enteritis; (d) recipients had diabetes mellitus or other glycometabolic disorders; and (e) tacrolimus had to be replaced by other immunosuppressants after kidney transplantation.

The enrolled subjects were consecutive candidates for transplantation that were eligible for the study and were allocated into the experimental group or the control group based on their own choice. As induction therapy, all enrolled recipients were prescribed Cytoxan (200 mg/day) and methylprednisolone (750, 500, 250, and 250 mg/day) from days 0 to 3. Beginning on day 4, patients in the control group received a standard dose of tacrolimus (0.07–0.08 mg/kg/day), whereas patients in the experimental group received a low dose of tacrolimus (0.04–0.05 mg/kg) and two infusions of MSCs. The first infusion of MSCs (5×10⁶ cells) was delivered directly into the renal allograft artery at the time of kidney transplantation. The saline solution containing MSCs (10 mL) was infused within 2 min after reperfusion of renal allografts. The second infusion (2×10⁶ cells/kg) was given intravenously 1 month later. Mycophenolate mofetil (1 g/day) and prednisone were also prescribed to patients in both groups. An oral administration of prednisone was initiated at 30 mg per day at day 4 after kidney transplantation and then tapered by 5 mg every week to the maintenance dose of 15 mg per day.

Patients were followed up for 1 year after kidney transplantation. The acute rejection rates at months 6 and 12 were compared between groups. Acute rejection diagnosis was determined based on clinical manifestations and graft biopsy. Serum creatinine was examined at months 1, 3, 6, and 12. The patient and graft survival at 1 year was also compared.

This study was performed in accordance with the Declaration of Helsinki and was approved by the Ethical Committee of the Second Affiliated Hospital of Guangzhou Medical University. Written informed consent was obtained from all recipients and donors.

Mesenchymal Stem Cell Isolation and Characterization

The mononuclear cell fraction of donor bone marrow was obtained and cultured over eight passages (see Figure S1A, SDC, https://links.lww.com/TP/A728). The resulting cells expressed the surface markers CD29, CD44, CD73, CD90, CD105, and CD166 but not the hematopoietic markers CD45 and CD34 passages (see Figure S1B, SDC, https://links.lww.com/TP/A728). After the sixth passage, the multiple differentiation capacity of MSCs was confirmed by adipogenic passages (see Figure S1C, SDC, https://links.lww.com/TP/A728) and osteogenic phenotype induction passages (see Figure S1D, SDC, https://links.lww.com/TP/A728) as described previously (^{31, 32}). These well-characterized MSCs were used in both clinical and in vitro studies.

Immune Monitoring

White blood cells were routinely counted before transplantation (day 0) and at 3, 6, and 12 months after transplantation. Immunophenotyping for characteristics of T lymphocytes and related subpopulations (CD3⁺CD4⁺CD8^- and CD3⁺CD4^-CD8⁺), Treg (CD4⁺CD25^highCD127^lowFOXP3⁺), naïve B lymphocytes (CD19⁺CD27^-), memory B lymphocytes (CD19⁺CD27⁺), and NK cells (CD56⁺CD3^-NKG2A/NKG2D) was performed using multicolor flow cytometry. Intracellular cytokines, including IL-4, IL-10, TNF, and IFN-r, were stained using standard protocols (all antibodies and isotype-matched control were all from eBioscience, San Diego, CA). Cells were acquired using a multicolor cytometer (FACSCalibur), and data were analyzed with CellQuest Pro software (Becton Dickinson, Franklin Lakes, NJ).

Mixed Lymphocytes Reaction

The one-way MLR was conducted to evaluate the ability of recipients to respond to donor alloantigen challenge. PBMCs were obtained (and frozen) from 12 recipients at day 0 and months 3, 6, and 12 and labeled with carboxyfluorescein diacetate, succinimidyl ester (Sigma-Aldrich, St. Louis, MO). Aliquots of 1×10⁵ recipient PBMCs were plated and stimulated with 1×10⁵ donor PBMCs pretreated with mitomycin C (Sigma, St. Louis, MO). Cultured cells were harvested after 6 days, and proliferation was measured by FACSCalibur flow cytometer (Becton Dickinson).

Chimerism Detection

Chimerism was assessed at months 3 and 12. PCR coamplification of 16 euchromosomal short-tandem repeat loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S317, D16S539, D2S1338, 19S433, vWA, TPOX, D18S51, D5S818, FGA, and AMEL) was performed in a fluorescence-based multiplex reaction using the AmpFLSTR Identifier kit (Applied Biosystems, Foster City, CA). All loci were amplified using a GeneAmp PCR System 9600 (Applied Biosystems). The amplified products were detected by capillary electrophoresis using an ABI 3130XL DNA Genetic Analyzer (Applied Biosystems). Short-tandem repeat profiles were analyzed using GeneScan and Genotyper Analysis Software (Applied Biosystems).

Statistical Analysis

Comparisons between the two groups were performed using the unpaired Student’s t test and the Wilcoxon rank-sum test in SPSS software version 14.0 (SPSS, Chicago, IL). P≤0.05 was considered significant. P≤0.05, adjusted by time point, was considered significant.