Application of mesenchymal stromal cells (MSC) has been proposed for solid organ transplantation based on their potent immunomodulatory effects. Since side effects from the injection of large cells cannot be excluded, the hypothesis rises that extracellular vesicles (EV) may cause immunomodulatory effects comparable to MSC without additional side effects. We used MSC-derived EV in a rat renal transplant model for acute rejection. We analysed peripheral blood leukocytes (PBL), kidney function, graft infiltrating cells, cytokines in the graft, and alloantibody development in animals without (allo) and with EV application (allo EV). There was no difference in kidney function and in the PBL subpopulation including Tregs between allo and allo EV. In the grafts T- and B-cell numbers were significantly higher and NK-cells lower in the allo EV kidneys compared to allo. TNF-
Life-long drug-based immunosuppression still is the standard regime to induce clinical allograft acceptance, however, at the price of significantly reduced overall well-being of transplanted patients. Therefore, research into alternative treatment approaches is warranted to decrease the need for immunosuppressive medication, improve long-term graft survival, and ideally induce tolerance. In the highly complex pathophysiology of acute renal allograft rejection, several components of the immune system are involved leading to vascular, glomerular, and tubular injuries. While pharmacological interventions often target one aspect only, cell-based therapies have the potential to influence multiple pathophysiological mechanisms.
Mesenchymal stromal cells (MSC) are of special therapeutic interest because of their capacity to enhance tissue repair by secreting bioactive molecules that (a) inhibit apoptosis and limit the extent of damage or injury, (b) inhibit fibrosis or scarring at sites of injury, (c) protect the microvasculature and stimulate angiogenesis to improve perfusion, and (d) stimulate the mitogenesis of tissue-intrinsic progenitor cells [
Recently, extracellular vesicles (EV) have been shown to play important roles in intercellular communications [
Very few studies of EV from different sources are available in SOT in animal models [
The aim of our study was to analyze the effect of recipient derived EV from MSC on allograft rejection in a rat renal transplant model with exclusive and complete MHC disparity.
In this experimental setting, we used isogeneic MSC. Bone marrow was procured from Lewis (LEW) rats (recipient strain) by flushing femurs and tibiae. Cells were resuspended in DMEM/Hams-F12 medium supplemented with 20% preselected fetal bovine serum (both, Biochrom, Germany) and 2 mol/L L-glutamine (Gibco, Germany) and seeded in tissue culture flasks (Greiner, Germany). Plastic adherent cells were grown to near confluency, passaged, and stored in liquid nitrogen as passages 3-4 and used as working cell bank. Expanded MSC were characterized for their phenotype using flow cytometry and differentiation capability into adipogenic, osteogenic, and chondrogenic lineages as described previously [
Expanded MSC at a confluency of 80% were fed with medium containing EV-depleted FCS (ultracentrifugation for 12 hours at 4°C with 100,000 g). Thereafter, MSC were cultured for 16 hours in DMEM/Hams-F12 medium supplemented with 0.5% EV-depleted bovine serum albumin (Sigma, Germany) and 2 mol/L L-glutamine. EV from the supernatant were collected after depletion of cells and cell debris by centrifugation for 20 min at 4°C with 2,000 g, depletion of apoptotic bodies for 20 min at 4°C with 12,000 g, and concentration by ultracentrifugation for 70 min at 4°C with 100,000 g with swinging buckets in a final volume of 1 mL in PBS. The MSC from the expansion cell cultures were counted with Trypan blue and the amount of EV correlated to the cell number from which the EV were harvested.
Animal experiments were approved by The Local Ethical Committee (number 49/09) and performed according to local and EU guidelines. Male LEW rats (LEW,
Life-sustaining RTx was performed as previously described [
As a control group, rats received a kidney graft from the same strain (isogeneic control (iso) group; LEW → LEW,
After a fully MHC-mismatched kidney transplantation (LEW.1U → LEW) rats received either medium (allo,
Serum creatinine (SCr) was analyzed after nephrectomy and on day 7 with Reflovet Plus (Roche Diagnostics, Switzerland; detection limit 0.5 mg/dL). Body weight and general condition were monitored daily.
Peripheral blood lymphocytes were stained with the following antibodies (all, Biolegend, San Diego, California, USA): CD3 (1F4), CD4 (W3/25), CD8 (Ox-8), CD45 (Ox-1), CD161 (10/78), CD25 (Ox-39), FoxP3 (150D), CD45RA (Ox-33), and polyclonal Goat anti-Rat IgG and IgM (Dianova, Hamburg, Germany).
Peripheral blood lymphocytes were obtained by treatment of EDTA blood samples with commercially available erythrocyte lysis buffer (Ortho Diagnostics, Neckargemuend, Germany). Samples of 0.5–1.0 × 106 cells were washed twice and incubated with 50
Fluorescence analysis was performed on a FACScanto (BD Bioscience, San Jose, California, USA). 1 × 104 cells measured with a standardized lymphocyte live gate were accumulated on logarithmic scales and analyzed using a FlowJo computer program (Ashland, Orlando, USA).
Differential blood counts were performed to calculate the numbers of lymphocyte subpopulation per
For immunohistochemistry on frozen sections, the following mAbs were used: R73 (rat TCR constant determinant; Biolegend, San Diego, California, USA), ED1 (rat tissue macrophages, monocytes, and dendritic cells), 10/78 (CD161, NK-cells) (both, Serotec, Germany), Ki-B1R (rat pan B-cell marker; Dianova, Hamburg, Germany), and 3.4.1 (CD8, BD Biosciences, San Jose, California, USA). Single staining techniques were performed as described previously [
Graft infiltrating cells within the renal cortex were counted in ten 400-fold high power fields (hpf) per section. Five representative animals of each group were analyzed for graft infiltrating cells by an independent investigator.
Total RNA from harvested kidneys was isolated and purified using RNeasy Mini Kit (Qiagen, Germany). Quantity and quality of RNA were determined using Infinity M200 (Tecan, Germany). Complementary DNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen, Germany).
Quantitative real-time PCR (qRT-PCR) for interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF
Representative samples of renal cortex of each group were used: iso
To analyze the presence of circulating donor-specific anti-MHC antibodies, sera of transplanted animals were incubated with peripheral blood lymphocytes of donor rat strain (LEW.1U) and recipient strain (LEW) as a control as described previously [
Samples were analyzed using a FACScanto (BD Bioscience, San Jose, California, USA) and results were evaluated using FlowJo computer program (Ashland, Orlando, USA).
Statistical analyses were performed using GraphPad Prism version 6.04 for Windows, (GraphPad Software, USA). Unpaired two-tailed
In both allo groups we observed a severely impaired kidney function. SCr in the allo groups on day 7 was not different in animals with and without EV application. The mean SCr in the allo group was 3.6 ± 0.7 g/dL and was 3.8 ± 1.0 g/dL (
Serum creatinine (SCr) at day 7 after transplantation. There was no significant difference between the allo and the allo EV group.
Analyzing the peripheral blood cell composition of transplanted animals on day 7 after transplantation, no difference in the percentage of CD3+/CD4+ T-cells was detected. CD4+/CD25+ cells were also not different (both not shown). CD4+/FoxP3+ regulatory T-cells were decreased in both allo groups compared to the iso animals, however, without reaching statistically significant difference between the allo and the allo EV group (data not shown). The number of B-cells in the allo EV group was lower compared to allo animals (not shown), however, at a nonsignificant level. CD8 T-cells and NK-cells were slightly increased in the allo EV group, not reaching statistical significance, too.
In kidney grafts harvested on day 7 after transplantation, there was a massive cell infiltrate of mononuclear cells in both allo groups while there were only very few infiltrating lymphocytes in the iso group (data not shown). In allotransplanted grafts macrophages and T-cells were the most prominent infiltrating cells. No difference regarding the infiltration of macrophages (Figure
Cell infiltrates in kidney grafts. Graft infiltrates of macrophages ((a): ED1), T-cells ((b): TCR), B-cells ((c): KiB1), and NK-cells ((d): 10/78) in the allo group compared to allo EV as cells per high power field. While macrophages were not different (a), T- and B- cells ((b), (c)) were significantly more frequent in kidneys from the allo EV group while NK-cells were reduced compared to allo (d).
Macrophages (ED1)
T-cells (TCR)
B-cells (KiB1)
NK-cells (1078)
Median TNF
Gene expression in transplanted kidney grafts (
TNF
IL-10
Sera of representative allo transplanted animals were tested for donor-specific MHC antibodies before transplantation and on day 7. While prior to transplantation (day 0) none of the sera were positive for antibodies against LEW.1U cells, and all tested sera became positive on day 7 (allo group
This is the first report on the results of immunomodulation with MSC-derived EV in a renal transplantation model with exclusive and complete MHC disparity.
EV are known to be an essential mediator in cell-cell communication by horizontal transfer of lipids, proteins, mRNAs, and microRNAs [
It has previously been shown that application of MSC and MSC-derived EV can ameliorate ischemia-reperfusion injury (IRI) [
The kidneys in our experiment were analyzed 7 days after transplantation. It is likely that at this time point IRI is already overruled by the adaptive alloimmune response. In allotransplantation, TNF
IL-10 as an anti-inflammatory or tolerogenic cytokine was significantly higher expressed in allo transplanted kidneys compared to the iso controls. But comparing both allo groups, there was no difference regarding IL-10 in the kidneys or Tregs in the blood of the animals. We need to admit that we did not stain Tregs in the grafts, which might be influenced differently in comparison to the peripheral blood lymphocytes.
Studies are available describing an inhibitory effect of MSC and EV on B-cell activity [
We propose that T- and B-cell activation in a strong reactive allotransplantation model cannot be ameliorated by EV, since MHC dependent mechanisms might be much stronger compared to nonimmunological injury in IRI.
It is remarkable that we saw a reduced NK-cell number and nearly no TNF
It needs to be discussed that we used recipient type (LEW) derived MSC for production of EV, while other authors used donor derived cells [
We conclude that EV can be administered safely in a rat renal transplantation model. No fatal side effects have been seen in the rats. There was no clinical difference regarding kidney function between the allo and the allo EV group. We could not prove an effect of EV on T- or B-cell mediated acute rejection, but we demonstrated different pattern of graft infiltrating lymphocytes and cytokines in the grafts induced by EV.
Suggesting a dominant effect of recipient derived EV on the innate immune system, but not an adequate suppression of the adaptive immunity, additional immunosuppression might be needed to induce significant modulation of allograft rejection with EV.
Mesenchymal stromal cells
Extracellular vesicle
Ischemia-reperfusion injury
Renal transplantation
Serum creatinine
High power field.
All authors of this paper have no conflict of interests to disclose.
The authors wish to thank C. Gossler and S. Christiansen for expert technical assistance.