Chimeric Allografts Induced by Short-Term Treatment with Stem Cell Mobilizing Agents Result in Long-term Kidney Transplant Survival without Immunosuppression: I Study in Rats

Abstract

Transplant tolerance allowing the elimination of life long immunosuppression has been the goal of research for 60 years. The induction of mixed chimerism has shown promise and has been successfully extended to large animals and the clinic. However, it remains cumbersome and requires heavy early immunosuppression. Here, we report that 4 injections of AMD3100 (A), a CXCR-4 antagonist, plus 8 injections of low-dose FK506 (F, 0.05mg/kg/day) first week after kidney transplantation extended survival, but death from renal failure occurred at 30–90 days. Repeating the same course of A and F at 1, 2 and 3 months after transplant resulted in 92% allograft acceptance (n=12) at 7 months, normal kidney function and histology with no further treatment. Transplant acceptance was associated with the influx of host stem cells resulting in a hybrid kidney and a modulated host immune response. Confirmation of these results could initiate a paradigm shift in post-transplant therapy.

Introduction

Organ transplantation has developed over the last half century from experimental therapy to lifesaving standard of care for thousands of patients with end stage organ failure. The discovery of potent immunosuppressive drugs led to reduced episodes of early rejection and improved short-term outcomes with one year post kidney transplant survival now over 90%. Long-term outcomes, however, have remained suboptimal: liver and kidney transplant grafts have only a 54% and 44% ten year survival respectively (1). Chronic rejection accounts for most allograft loss after the first year and is largely unresponsive to additional immunosuppression. Besides its inability to prevent late graft loss, conventional immunosuppressive regimens (i.e. steroids, calcineurin inhibitors, and mycophenolate) impart significant morbidity including nephrotoxicity, neurotoxicity, and a greatly increased risk of certain infections and cancers. Methods designed to eliminate the need for conventional immunosuppression using mixed-chimerism approaches (3, 4, 5) have had recent success.

An alternative strategy, termed “reverse chimerism” is suggested by our findings in a rat model of liver transplantation (6, 7) and occurs when a recipient populates a transplanted donor graft (8). This phenomenon was first reported in an aortic allograft model over forty years ago (9, 10) and has been observed to a limited extent when looked for in liver (11–17), kidney (18, 19) and heart (20, 21) transplants in human recipients. Fan et al (22) have also recently reported that bone marrow-derived hematopoietic stem cells and progenitor cells infiltrate allogeneic and syngeneic transplants, confirming these findings. If the limited recipient repopulation of a donor graft that is currently observed could be facilitated, it is possible that conversion to a predominantly host phenotype would permit long-term graft function without immunosuppression. In fact, we have reported in a strongly rejecting rat liver transplantation model that host repopulation of a donor graft was augmented by: transplanting a small liver allograft, initiating a regeneration response; providing low dose immunosuppression (FK506); and a stem cell mobilizing agent (AMD3100) (7). Mechanistically, we discovered that AMD3100 (A) and low-dose FK506 (F) exert potent, synergistic activity in the mobilization of endogenous stem cells, and treatment limited to the first 7 days after surgery led to permanent survival of a small liver allograft. Transplant acceptance was associated with the recruitment of host stem cells resulting in conversion to a predominantly host genotype.

Extension of this strategy to kidney, heart, and other organ transplants raises concerns that mobilizing endogenous host stem cells to replace or repair damaged donor cells may not be effective in organs relying on cells of epithelial origin or having slow turnover rates. The kidney, like most organs, displays bone marrow–derived cells (BMDC) in response to damage (23). However, the lineage of these cells is unclear, their ability to elicit transdifferentiation and conversion to renal tubular cells is controversial and certainly not robust (19,24, 25). Therefore we were not surprised when the brief exposure to A and F was not successful, and changed the protocol to provide additional stem cells at 1,2, and 3 months which resulted in long term survival of 11/12 animals.

Materials and Methods

Rat Strains and Care

Lewis and dark agouti (DA) rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and used at 8–12 weeks of age. The green fluorescent protein (GFP) transgenic Lewis rat strain was obtained from the National Institutes of Health (NIH)-funded Rat Resource and Research Center (RRRC), University of Missouri, Columbia, MO. Animals were maintained in the specific pathogen-free facility of the Johns Hopkins Medical Institutions. Animals were cared for according to NIH guidelines and under a protocol approved by the Johns Hopkins University Animal Care Committee.

Kidney Transplantation

Orthotopic kidney transplantation was performed according to a method previously described (26). Both host kidneys were excised.

Preparation of non-parenchymal cell suspensions from rat kidney with Collagenase D treatment

The kidney was excised and cut gently into small pieces with a scalpel blade. The pieces were put in a beaker containing 5 mL of HEPES buffer (10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2). An additional 100 μL Collagenase D solution was added and the cell suspension was filtered through two layers of sterile gauze (final Collagenase D concentration: 2 mg/mL). A sample of rat kidney was incubated for 45 minutes at 37 degrees under slow continuous rotation. Dissociated tissue was removed and the sample was applied to 100μm mesh placed on a 50 mL tube. 30–40 mL of Hank’s balanced salt solution (HBSS) was added in the sample tube and cell suspension was centrifuged at 400×g for 6 minutes. The supernatant was then aspirated completely. The cell pellet was resuspended in 5 mL HBSS and the cell suspension was applied to a 70 μm mesh placed on a 50 mL tube. The remaining kidney fragments were washed three to four times following the same procedure. The cells in suspension were washed by adding 10 mL of HBSS and centrifugation at 400×g for 6 minutes at 4 degrees. The final pellet was resuspended in dimethyl sulfoxide containing 10% fetal bovine serum (FBS).

Preparation of Peripheral Blood Mononuclear Cells (PBMC)

Mononuclear cells were isolated from peripheral blood by Ficoll-Hypaque (1.077 g/liter, Sigma) density gradient centrifugation. PBMC were harvested from the interface, then washed once with phosphate-buffered saline (PBS) and once with RPMI-1640 (Invitrogen), by centrifugation at 400 g for 5 min.

Mixed Lymphocytes Reaction (MLR)

One-way mixed lymphocytes reaction (MLR) was performed; the responder cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, BioLegend) according to the manufacturer’s instruction. The stimulating cells were inactivated by irradiation with 1500 rads from a source of x-ray and washed with medium, centrifuging at 300 g for 5 min. Responder cells (100 μl/well of 2.5 × 10⁶ cells/ml) were cultured with stimulator cells (100 μl/well of 2.5 × 10⁶ cells/ml) in 96-well flat-bottomed tissue culture plates in triplicate wells in 96-well plate in RPMI 1640 containing 10% FBS, 10 mmol/L HEPES, 50 U/mL penicillin + 5 mg/mL streptomycin, 10 mmol/L MEM sodium pyruvate, 10 mmol/L MEM nonessential amino acids solution). The cell mixture was incubated for 5 days at 37° in a 5% CO2-humidified atmosphere. Cell proliferation was measured using dye (CSFE) dilution by flow cytometry.

Flow Cytometry

Single-cell suspensions (1×10⁶) of blood, spleen and kidney were analyzed for expression of lineage negative Thy-1+, CD34+ and CD133+ stem cell markers, as well as expression of CD4+CD25+Foxp3+ and CD8+Foxp3+ regulatory T cells. All antibodies used were from commercial sources: CD133 (abcom), CD34 (R&D System), Thy-1 (PharMingen), CD3 (FITC), CD4 (PE), CD25 (APC, Biolegend), FoxP3 (PerCP, eBioscience), CD8 (Santa Cruz), Cy3 (Rabbit), FITC (goat), anti-rat CD3, biotin anti-rat CD11b/c (eBioscience). Nonspecific antibody binding was blocked with donkey, mouse, and rat serum (Sigma) for 30 minutes. Cells were incubated with antibodies for 1 hour at 4°C, and the positive cells were counted by flow cytometry (fluorescence activated cell sorting [FACS]) using CELLQuest software (Becton-Dickinson).

Immunohistochemistry

Five μm serially cut, frozen sections were fixed with acetone at (−20°C) for 10 minutes and dried for 1 hour at room temperature. The streptavidin-biotin-peroxidase method with the DAKO Kit (Carpinteria, CA) was used to detect CD34 and CD133 antigens. After inactivation of endogenous peroxidase and blocking of nonspecific antibody binding, the specimens were treated with biotinylated antibodies specific for CD34 (1:100, R&D system) or CD133 (1:100, ab19898, abcam) at 4°C overnight. Subsequently, sections were incubated with streptavidin-biotin-peroxidase complex reagent for 30 minutes at room temperature. Diaminobenzidine tetrahydrochloride was used as the chromogen, and hematoxylin was used for counterstaining.

Immunofluorescence Staining

Frozen sections (5-μm) were fixed with acetone (−20°C) for 10 minutes and dried for 1 hour at room temperature. A Tris-based buffer containing 0.5% casein and 5% normal rat and rabbit serum was used for blocking nonspecific background and for dilution of antibodies. Sections were incubated for 1.5 hours at room temperature with a mixture of a mouse monoclonal antibody specific for Foxp3 (1:100; BioLegend), goat polyclonal antibody specific for CD3 (1:100, Santa Cruz) and rabbit polyclonal antibody specific for c-Met (1:100; Abcam) followed by treatment with Cy3 donkey anti-mouse IgG (1:100; Jackson ImmunoResearch Inc.), Cy3 donkey anti-goat IgG (1:100, Jackson ImmunoResearch Inc.) or Cy3 donkey anti-rabbit IgG (1:100; Jackson ImmunoResearch Inc.) for 1 hour at room temperature. Cell nuclei were stained blue with DAPI. Tissue sections were analyzed by confocal fluorescence microscopy.

Semi-quantitative Reverse Transcription (RT)-PCR Analysis

Total RNA was extracted from kidney tissues and first-strand cDNA synthesis was then performed on 5μg of total RNA using the SuperScript Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Polymerase chain reactions (PCR) contained 1 μL of deoxynucleoside triphosphate mix (10 mM each dNTP), 1 μl of 10 μM each primer, 0.4 μL (5 IU/μL) of Platinum Taq polymerase (Invitrogen, Carlsbad, CA), 1.5 μl of 50 mM MgCl2 and 2 μL total DNA as template in a 50 μL reaction solution. Thermal cycling was started with one cycle at 94°C for 4 minutes. This was followed by 30 cycles at 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and 72°C for final extension for 3 minutes. PCR products were electrophoresed on 1.5% agarose gels and visualized with ethidium bromide staining.

Primer sets for amplification

• SDF-1 5′-tgagatttgccagcacaaag-3′ and 5′-gacagattccttgccgagag-3′

• SCF 5′-gcctacaatggacagcaatg-3′ and 5′-gctgtgaaacctgcactgaa-3′

• c-Met 5′-aagagggcattttggctgt-3′ and 5′-acccaactgtgaaagatctt-3′

• CD133 5′-gaggctgagaaaccccctac-3′ and 5′-cttgcctctttggattctgc-3′

• CXCR-4 5′-gtgcagcaggtagcagtgac-3′ and 5′-gacagacaagtaccggctgc-3′

• Sca-1 5′-tggcagaagttgttgtgagc-3′ and 5′-gcataaacccaaggcactgt-3′

• Foxp3 5′-cccaggaaagacacagcaacctt-3′ and 5′-ctgcttggcagtgcttgagaa-3′

• PD-L1 5′-tgtaccacgtctcccacataaacag-3′ and 5′-accccacgatgaggaacaaa-3′

• IFN-γ 5′-gctagattctggtgacagctggtg-3′ and 5′-caccagctgtcaccagaatctagc-3′

• IL-6 5′-cttccagccagttgccttct-3′ and 5′-gacagcattggaagttgggg-3′

• IL-17 5′-acatgtaaggcagcggtact-3′ and 5′-gctcagagtccagggtgaag-3′

• TGF-β 5′-gagacggaatacagggctttc-3′and 5′-tggagctgaagcagtagttgg-3′

• β-actin 5′-cactgccgcatcctcttcct-3′ and 5′-ctctgtgtggattggtggct-3′

In Situ Imaging of GFP Expression in Kidneys

Kidney grafts were flushed with cold saline (4°C, 10 mL) and fixed with 4% paraformaldehyde via renal artery perfusion. The fluorescence of GFP in kidney grafts was measured by the Xenogen IVIS Imaging system and Living Image software (Xenogen Biosciences).

Genomic DNA Extraction and PCR Analysis for Y Chromosome

Fresh kidney tissue samples obtained from the animals at the time of sacrifice were quick-frozen in liquid nitrogen and stored at −80 °C until use. The tissue was treated with Proteinase K and the genomic DNA (gDNA) was isolated using Phenol-chloroform extraction (Invitrogen). Three 10 μm thick sections from formalin-fixed, paraffin-embedded kidney tissues was deparaffinized in xylene and the gDNA was extracted using QIAamp DNA FFPE Tissue Kit (Qiagen). The gDNA concentration was evaluated using NanoDrop 2000 Spectrophotometer (Nanodrop).

Rat Sry, a sex determining region on Y chromosome and Dax-1 on X chromosome were analyzed using Polymerase Chain Reaction (PCR) on each sample. The primers to detect Sry are: forward 5′-CGCAGAGACTGAAGACCCTACA-3′ and reverse 5′-TGGTTCTTGGAGGACTGGTGT-3′. The primers to detect Dax-1 are forward 5′-AGTGCTGGAGTCTGAACATTGA-3′ and reverse 5′-ATGCTTGCGTGTAGAGGTGG-3. PCR was performed with Platinum Taq polymerase (Invitrogen) on 50 ng of the genomic DNAs. Amplified products were electrophoresed on 2% agarose gels with Nucleic Acid Gel Stain (Cambrex) and visualized under ultraviolet light.

In Situ Hybridization for Y chromosome

The probe is complementary to the rat sex determining region Y (Sry) sequence on Y chromosome. It was generated by Polymerase Chain Reaction (PCR) using male rat genome DNA, with T7 polymerase promoter sequences leaded forward primer 5′-TTTGGGAGCAGTGACAGTTG-3′ and T3 polymerase promoter sequences leaded reverse primer 5′-GCTTTTCTGGTTCTTGGAGG-3′. The amplicon was then subjected to in vitro transcription and Alexa Fluor 488 labeling using FISH Tag RNA Green Kit (Invitrogen) following the manufacture’s instruction. The probe was denatured for 3 min at 80 °C before hybridization. Four μm thick sections from formalin-fixed, paraffin-embedded kidney tissues were deparaffinized in xylene and hydrated through a graded ethanol series. They were then subjected to heat-induced antigen retrieval for 15 min in Tris-EDTA buffer in a steamer, followed by pepsin (Invitrogen) treatment for 2 min at 37 °C. The slides were dehydrated through a graded ethanol series and air-dried. Denaturation was then conducted for 3 min at 95°C, and followed by hybridization with the Fluor 488-labeled, Sry specific probe overnight at 55 °C. Slides were washed and subjected to immunohistochemistry detection using anti-Alexa Fluor 488 antibody (Invitrogen). After applying the secondary antibody, the signal was revealed using Catalyzed Signal Amplification System (DAKO) and counterstained with 3,3′-Diaminobenzidine (Sigma).

Statistic Analysis

Continuous variables were presented as the mean ± SD. Dichotomous variables were presented as both number and percentage values. Data of flow cytometry were analyzed using the Student’s t test (two-tailed), with dichotomous variables analyzed by the Fisher’s exact test (two-tailed). All analyses were performed using SPSS® (SPSS; Chicago, IL). P < 0.05 was considered significant.

Results

Short term treatment with AMD3100 plus low-dose FK506 with repeat dosing results in long term kidney transplant survival in an acute rejection rat model

We performed kidney transplants from dark agouti (DA) rats into green fluorescent protein (GFP) transgenic Lewis rats. We provided low-dose FK506 (F) (0.05mg/kg/day), (<1/10 the immunosuppressive dose) and concomitant CXCR4 antagonism with AMD3100 (A). Transplanted rats were divided into five experimental groups treated with AMD3100 every other day and/or low-dose FK506 daily for the first seven days after transplantation (Fig. 1A). In the three groups that received either no treatment or either drug as monotherapy all kidney allografts were rejected within two weeks after transplantation (Fig. 1B). The combination of A and F treatment prolonged allograft survival, but as suspected, these dual treated animals all eventually died within three months. The creatinine levels increased to about 1mg/dl at 1 month after transplantation. Repeat dosing of the same dual-drug course reduced the serum creatinine levels from 1mg/ml to less than 0.5mg/dl (Supplemental figure 1A). Repeat dosing at 1, 2 and 3 months after transplantation resulted in allograft acceptance in eleven of twelve recipients at 6 months without additional immunosuppression (Fig. 1B). These rats had serum creatinine levels less than 0.5mg/dl at 6 months (Fig. 1C).

Figure 1. Short term treatment with AMD3100 plus low-dose FK506 with repeat dosing results in long term kidney transplant survival in an acute rejection rat model (DA into Lewis).

(A) Experimental design. Transplanted rats were divided into five groups 1. control group treated with saline, 2. AMD3100 (1mg/kg/day) alone, 3. Low dose FK506 (0.05mg/kg/day) alone, 4. a combination of AMD3100 and low dose FK506, 5. repeat dosing at 1, 2 and 3 months. (B) Rat survival after kidney transplantation. (C) Serum creatinine levels. (D) Skin allograft survival. Donor strain (DA) or third party (BN) skin allografts performed three weeks after cessation of drug therapy. (E) H&E staining of kidney allografts at 60, 90 and 180 days after transplantation. Representative photographs of n = 3 or 4 individual samples per group. Images were photographed with a 40× objective.

Immune status

To assay for tolerance, donor strain skin allografts were performed in the A plus F treated rats at 1 month after kidney transplantation. These rats received repeat dosing of dual drug treatment after skin grafting. Interestingly, the three grafts survived for 30–38 days before rejection. There was no disturbance of kidney function or rat survival. Third party grafts were rejected with normal kinetics (Fig. 1D). No donor specific hypo-responsiveness was observed at 4 months after transplantation by using Allo-MLR assays (Supplemental figure 1B).

Graft acceptance was associated with reduced inflammation. All control groups displayed heavy mononuclear infiltrates at seven days (Supplemental figure 1C). There were far fewer inflammatory cells in the kidneys of animals treated with the combination of A and F. Although, there were small areas of tubular atrophy and fibrosis scattered in these kidneys at 6 months, the majority of the kidney tissue displayed lush tubules, normal glomeruli and no fibrosis (Fig. 1E and Supplemental figure 1D).

Recruitment of recipient bone marrow stem cells and regulatory T cells to rat kidney transplants following treatment with dual drug therapy

Using flow cytometry we found that the percentage of lineage negative (Lin-) CD34+ cells significantly increased in the peripheral blood of animals treated with AMD3100, low-dose FK506 or both drugs, while Lin-CD133+ cells significantly increased only in peripheral blood of animals treated with AMD3100 or dual drugs (Fig. 2A). The percentage of Lin-CD34+, Lin-CD133+ cells, CD4+CD25+Foxp3+ and CD8+CD25+Foxp3+ regulatory T cells also significantly increased in cell suspensions made from kidney allografts recovered from animals receiving dual drug treatment compared to all other treatment groups at 7 days after transplantation (Fig. 2B). Lin-CD34+ and Lin-CD133+ cells were also increased in the spleen in animals treated with dual drugs (Supplemental figure 2). Interestingly, AMD3100 treatment alone increased levels of CD8+CD25+Foxp3+ regulatory T cells in peripheral blood.

Figure 2. Recruitment of recipient bone marrow stem cells and regulatory T cells to rat kidney transplants following treatment with the dual drug therapy.

(A) Quantitative analysis of Lineage negative (Lin−) CD34+ and CD133+ cells in blood and kidney allografts by flow cytometry at 7 days after transplantation. Quantitative data are represented as group means (bars) (n=3). *P<0.01 compared to the control group with saline treatment. (B) Characterization of Foxp3+ cells was performed by flow cytometry of cells isolated from blood, and kidney allograft seven days after transplantation. Quantitative data are represented as group means (bars) (n=3). *P < 0.05 and **P < 0.01 compared to the control group with saline treatment. (C) Semi-quantitative PCR analysis of kidney allografts at 7 days after transplantation. Representative graphs of three individual samples.

Semi-quantitative PCR analysis of the kidney transplant revealed that mRNA expression of the attractor molecules stromal cell-derived factor (SDF)-1 and stem cell factor (SCF), and important cellular markers, CD34, CD133, c-Kit, Sca-1 and Foxp3 were all significantly increased in the dual treatment group compared to all other groups at 7 days after transplantation (Fig. 2C). Notably mRNA expression of SDF-1 and CD34 were also significantly increased in the low-dose FK506 treatment group compared to saline and AMD3100 treatment groups. Interestingly, IFN-γ (a Th1 cytokine), IL-17 (a cytokine of Th17), IL-10 and IL-6 (a promoter of Th17 development) were all significantly decreased in the dual treatment group, although TGF-β remained unchanged (Fig. 2C).

Effect of regulatory cells mobilized by dual drug treatment on T cell response

We used allo-MLR to determine the effect of regulatory cells mobilized by dual drug treatment on T cell responses. Lewis host lymphocytes mobilized by A and F treatment were used as responding cells against x-ray irradiated donor DA cells. Different ratios of dual drug mobilized cells to normal responding cells (1/10 to 1/500) tested the potency of suppression. Using CFSE labeling and flow cytometry analysis, control Lewis lymphocytes co-cultured with x-ray irradiated DA lymphocytes showed abundant T cell proliferation. However, when lymphocytes from dual drug treated Lewis rats were studied T cell proliferation significantly decreased (Fig. 3A). Interestingly, the strong control allo-MLR was suppressed when PBMC from dual drug treated animals were present (Fig. 3B). Notably the suppressive effect persisted in allo-MLR even when the mobilized cells were diluted 1/500 with normal effector cells (Fig. 3C).

Figure 3. Dual drug treatment mobilized cells suppress mixed lymphocytes reaction.

(A) Lymphocytes from Lewis rats treated with saline or dual drugs were co-cultured with irradiated DA lymphocytes for 5 days. Lymphocyte reaction was suppressed when lymphocytes from dual drug treated Lewis rats were co-cultured with irradiated DA lymphocytes. (B) Lymphocytes from dual drug treated Lewis rats were added into one-way allo-MLR at different ratios of lymphocytes to responding cells (1/10 to 1/500). (C) Quantitative analysis of lymphocyte proliferation in allo-MLR (n=3 or 4 per group). *p<0.05, **p<0.01.

To determine whether Tregs were chiefly responsible for the immunoregulatory effects of dual drug treatment, mice designed to have GFP labeled Foxp3 cells were treated with A and F. Foxp3 cells were sorted by FACS from peripheral blood and spleens at 3 hours. The number of Foxp3+ T cells doubled in peripheral blood and spleens from mice treated with dual drugs (data not shown). When these sorted Foxp3+ T cells were added into allo-MLR at different ratios, the allo-MLR was significantly suppressed in a dose dependent fashion similar to the A and F mobilized rat PBMC (Supplemental figure 3).

Increased SDF-1+, CD34+ and CD133+ cells in kidney allografts of transplanted rats treated with dual drug therapy

SDF-1+ expression and cells bearing the CD34+ phenotype were significantly increased at day 7 in the animals receiving low-dose FK506 alone (Supplemental figure 4). The number of CD34+ and CD133+ cells and those with surface SDF-1+ was much higher in those receiving dual drug treatment. The SDF-1 positive cells were localized in peritubular areas (Fig. 4A, B). Immunofluorescence double staining demonstrated that many CD34+ cells were host derived (Fig. 4B).

Figure 4. Increased SDF-1+, CD34+ and CD133+ cells in kidney allografts of transplanted rats treated with dual drug therapy.

(A) Detection of SDF-1+ cells in kidney allografts by immunohistochemistry staining. SDF-1+ cells were stained brown with DAB. The number of SDF-1 positive cells was significantly higher in tissue sections from kidney allografts in the dual treatment group at 7 days after transplantation. (B) Immunofluorescence double staining for SDF-1 and CD133. CD133+ cells (red) co-localized with SDF-1+ cells (green). (C) Detection of CD34+ cells in kidney allografts by immunohistochemistry staining. CD34+ cells were stained brown with DAB. The number of CD34 positive cells was significantly higher in tissue sections from kidney allografts in the dual treatment group at 7 days after transplantation. Representative photographs of n = 3 individual transplant samples per group. Images were photographed with a 20× objective.

Chimeric kidney allografts are induced by short-term treatment with A plus F and repeat dosing

To determine the extent of kidney allograft repopulation by host-derived cells, GFP whole-organ fluorescence was measured using the Xenogen system. As expected, the non-transgenic donor DA kidney showed no GFP expression (Supplemental figure 5). However, the transplanted DA graft developed green fluorescence at 30 days after transplantation and a higher degree of fluorescence at 120 days in recipients treated with A and F (Fig. 5A). Fluorescent microscopy revealed that many of the tubules, peritubular capillaries, and glomerular cells were GFP positive at 30 and 120 days after transplantation (Fig. 5B). However, there were still many GFP negative cells. We confirmed the presence of recipient genotypes in the transplant using female DA donors (XX) transplanted to male Lewis recipients (XY). PCR reactions detecting the Y chromosome in kidneys found a majority of host genotype in allografts on day 120 after transplantation (Fig. 5C). In situ hybridization for Y chromosome in kidney tissue sections, demonstrated the presence of the Y chromosome in cells of the tubular epithelium, Bowman’s capsule, glomerulae, and peritubular capillaries at day 120 after transplantation (Fig. 5D and Supplemental figure 5). Notably, recipient derived cells (GFP+ or Y-chromosome+) were not spread evenly. 20–30% cells were GFP or Y-chromosome positive in tissue sections from kidney allografts at 1 month after transplantation. The number of GFP or Y chromosome positive cells increased to 30–50% at 4 months. In a sex mismatched transplantation model, genomic DNA analysis for sex chromosomes (Sry1 and Dax1) by semi-quantitative PCR demonstrated that 30–60% of donor genomic DNA was replaced by the recipient at 4 months after transplantation (Supplemental figure 6). While recipient genomic DNA may also be present in the inflammatory cells, the in situ hybridization studies above showed clearly that sizable numbers of parenchymal cells had the host genome. These results demonstrate that pharmacological mobilization of host endogenous stem cells promoted the conversion of the kidney allograft into a donor-host chimera.

Figure 5. Repopulation of kidney allografts by host-derived stem cells in animals displaying long term acceptance.

(A) The Xenogen imaging system was used to study DA kidneys into GFP Lewis hosts with dual drug treatment after kidney transplantation. The non-transgenic donor DA kidney has no GFP expression. However, the transplanted donor kidney graft shows GFP positive at 30 days and a high degree of fluorescence at 120 days after transplant into a GFP+ Lewis recipient treated with AMD3100 and FK506. (B) Kidney tissue sections analyzed by fluorescent microscopy. GFP positive cells were present in tubules, peritubular capillaries on day 120 after transplantation. (C) The presence of the Y chromosome (Sry1) in allografts was analyzed by PCR 30 days or 120 days after sex mismatched kidney transplantation. X-chromosome (Dax1) expression was used as internal control. PCR analysis showed that kidney allografts were positive for the Y chromosome 30 days after transplantation. The levels of Y chromosome expression in the kidney allografts were significant increased at 120 days after transplantation. (D) A Y-chromosome probe was used to distinguish host cells by in situ hybridization in a sex mismatched female DA (Y-negative) into male Lewis (Y-positive) after kidney transplantation. In situ hybridization demonstrated the presence of the Y chromosome (brown) in tubular epithelial cells, Bowman’s capsule, glomerular cells and peritubular capillaries on day 120 after transplantation. Representative photographs of n = 3 or 4 individual transplant samples. Images were photographed with a 40× or 60× objective.

Discussion

We have developed a novel protocol to enable long-term kidney allograft survival without immunosuppression by using a combination of two FDA approved drugs. AMD3100 (Plerixafor or Mozobil, A), is a CXCR4 antagonist, originally developed as an anti-HIV medicine but found useful chiefly in the mobilization of CD34 and other stem cells from their bone marrow niche. SDF-1α is the ligand in bone marrow that tethers CXCR4 cells, and A blocks this reaction liberating cells to enter the circulation. Currently A is used, often with G-CSF (neupogen), to mobilize hematopoietic stem cells in cases of multiple myeloma and other hematologic malignancies for banking prior to myeloablative chemotherapy. These stem cells are subsequently infused back for hematopoietic reconstitution after cancer treatment. Thus A is well established as a safe and effective agent to mobilize bone marrow stem cells.

F (FK506, Tacrolimus or Prograf), the other component of our therapy, is an immunosuppressive drug widely used after solid organ transplant to eliminate organ rejection. We have found that FK506 at low-dose only (less than 1/10 of effective dosage to prevent rejection), can mobilize stem cells, especially Lin-CD133+ cells and those with macrophage markers in rats and mice (27). Most interestingly, we have demonstrated a potent, synergistic activity of AMD3100 and low-dose FK506 in the mobilization of stem cells (7, 27).

While several studies have shown the presence of marrow derived stem cells in the kidney after various stresses (28–31), no previous studies have reported the existence of large numbers of bone marrow stem cells reported in this study, or that their lineages populated all tissues in the kidney. The obvious difference is our provision of circulating stem cells boosting the opportunity to enter the transplant. However, CXCR4 blockade (AMD3100) alone was not successful, and the addition of low dose FK506 was essential, perhaps exerting its effect via the FK506-binding protein 12 (FKBP12) and not by calcineurin inhibition. Recently, FKBP12 has been reported to promote osteoblastic differentiation by activating the bone morphogenetic protein (BMP) receptor (32–34). Our current studies have shown that F alone increased SDF-1 in the transplant, and we have found in previous studies that cells with macrophage phenotypes are liberated from the bone marrow by FK506 (27). Interestingly these cells carry surface SDF-1 creating a push-pull axis augmenting stem cell graft incorporation. SDF-1 and its receptor CXCR4 play major roles in stem cell motility and development (35). Thus low dose FK506 may promote mobilization and recruitment of stem cells into transplants via increased expression of SDF-1.

CXCR4 is highly expressed on B and T cells (36) and it has been hypothesized that both B and T lymphocytes use the CXCR4/CXCL12 (SDF-1) axis to home to anatomic niches and that, when inhibited, these cells are able to leave their respective niches and accumulate in the peripheral blood. AMD3100 treatment enhanced mobilization of CD4+/CD25^high/CD127^low/FoxP3+ Tregs in the rhesus macaque model (36). Our results confirmed that A treatment alone increased levels of Foxp3+ regulatory T cells in peripheral blood. In addition, at 7 days after transplantation CD4+CD25+Foxp3+ and CD8+CD25+Foxp3+ regulatory T cells significantly increased in the peripheral blood and transplant cell suspensions from animals receiving dual drug treatment compared to all other treatment groups. Further, lymphocytes from peripheral blood in dual drug treated Lewis rats or sorted Foxp3+ T cells from dual drug treated Foxp3 GFP mice significantly suppressed allo-MLR. These results indicate that the Foxp3+ Tregs component of the cells mobilized by combined A and F treatment can suppress allo-immunity. We propose that blocking the SDF-1/CXCR4 reaction with A results in liberating all purpose stem cells while dual treatment was needed to mobilize large numbers of Tregs (Fig. 2A and B). Modest amounts of SDF-1 induced by low dose F in the allograft create a push-pull axis augmenting stem cell and Tregs graft incorporation. However, all measures of components likely to increase allograft acceptance were increased by combined A and F treatment.

In these dual drug treated animals, the creatinine levels were increased to about 1mg/dl at 1 month after transplantation and all eventually died within three months. The renal failure was associated with interstitial fibrosis and tubular atrophy (data not shown). Repeat dosing reduced the serum creatinine level indicating that the mobilized stem cells helped to repair the injured kidney and improved the kidney function and therefore could be benefit to chronic rejection.

We acknowledge that mobilization of Tregs is unlikely to be the only mechanism leading to the immunological state found in this study. Months after transplants of both livers (7) and kidneys we find that host and donor cells reside side-by-side in the graft without signs of injury or inflammation despite a still present ability to recognize and reject donor strain skin. It is possible that donor skin specific antigens induce this response or the rejection of skin allografts is related with innate immunity. NK cells, activated due to missing self-MHC class I molecules on allogeneic cells, are involved in allogeneic skin graft rejection via direct killing of donor cells and through the production of pro-inflammatory cytokines including IFN-γ and TNF-α (37). However, studies in the accompanying paper (pig study) demonstrated that donor skin graft rejection was associated with a kidney graft rejection crisis. While immunomodulation mediated by Treg cells is a likely mechanism responsible for early transplant survival, it does not explain the durability of transplant acceptance without treatment of any sort. A fascinating question is, once established as chimeric neighbors, do the host and donor cells interact to escape rejection? The fact that reactivation of allo-responsiveness was prevented by additional treatment suggests that the number of host cells including Tregs is critical to sustain allograft survival. This finding also has significant clinical implications as a potential therapy for chronic rejection. We propose at intervals after transplantation that rejection injury may exceed repair mechanisms and require a boost of stem cells to rescue graft function and restore donor acceptance.

Supplementary Material

Supp Fig S1

Figure S1. H&E staining of kidney allografts and renal function post repeat dosing.

Supp Fig S2

Figure S2. Stem cells and regulator T cells in spleens and lymph nodes after kidney transplantation.

Supp Fig S3

Figure S3. Dual drug treatment mobilized Foxp3 (gfp+) Tregs suppress allo-MLR in mice.

Supp Fig S4

Figure S4. SDF-1+, CD34+ and CD133+ cells in kidney allografts of transplanted rats treated with dual drug therapy.

Supp Fig S5

Figure S5. Host-derived cells in kidney allografts of animals displaying long term acceptance.

Supp Fig S6

Figure S6. Sex Chromosome analysis in sex mismatched kidney allografts.

Abbreviations

A

AMD3100

BMDC

bone marrow–derived cells

BMP

bone morphogenetic protein

CXCR-4

chemokine (C-X-C motif) receptor 4

F

FK506

FACS

fluorescence activated cell sorting

FKBP

FK506-binding protein

FITC

fluorescein isothiocyanate

Foxp3

forkhead box P3

G-CSF

Granulocyte-colony stimulating factor

GFP

green fluorescent protein

HBSS

Hank’s balanced salt solution

IFN- γ

interferon γ

IL

interleukin

MHC

major histocompatibility complex

MLR

mixed lymphocytes reaction

PCR

polymerase chain reaction

PD-L1

program death ligand 1

PE

phycoerythrin

RRRC

rat resource and research center

Sca-1

stem cell antigen 1

SCF

stem cell factor

SDF1

stromal cell-derived factor 1

TGF-β

Transforming growth factor-β

Tregs

regulatory T cells