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Autologous CD117+ progenitor cells (PC) have been successfully utilized in myocardial infarction and ischemic injury, potentially through the replacement/repair of damaged vascular endothelium. To date, such cells have not been used to enhance solid organ transplant outcome. In this study, we determined whether autologous bone marrow-derived CD117+PC could benefit cardiac allograft survival, possibly by replacing donor vascular cells. Autologous, positively selected CD117+PC were administered post-transplantation and allografts were assessed for acute rejection. Although significant generation of recipient vascular cell chimerism was not observed, transferred PC disseminated both to the allograft and to peripheral lymphoid tissues and facilitated a significant, dose-dependent prolongation of allograft survival. While CD117+PC dramatically inhibited alloreactive T-cell proliferation in vitro, this property did not differ from non-protective CD117− bone marrow populations. In vivo, CD117+ PC did not significantly inhibit T cell alloreactivity or increase peripheral regulatory T cell numbers. Thus, rather than inhibiting adaptive immunity to the allograft, CD117+ PC may play a cytoprotective role in prolonging graft survival. Importantly, autologous CD117+PC appear to be distinct from bone marrow-derived mesenchymal stem cells (MSC) previously used to prolong allograft survival. As such, autologous CD117+PC represent a novel cellular therapy for promoting allograft survival.
Cardiac transplantation is the accepted therapy for patients with end-stage cardiomyopathy. Despite several decades studying rejection, results remain unsatisfactory with survival being 50 – 56% at 10 years (1, 2) and 20% at 20 years (1). Clearly, new insight into the mechanisms of cardiac rejection and tissue repair must be identified. Importantly, novel therapeutic modalities are required to improve survival and to diminish reliance on non-specific immunosuppressants with their associated morbidities. Studies utilizing endothelial progenitor cells (EPC) have shown benefit for tissue repair following myocardial infarction and ischemic injury (3–6). Although a precise mechanism was not determined, studies show that EPC (7–10) contribute to the re-endothelialization of vascular grafts. Relevance to transplantation is the finding that the allograft endothelial cell (EC) is likely the target of acute CD4 T-cell mediated rejection (11), making modification of donor EC a candidate means of attenuating acute rejection.
Currently, no clear EPC-specific marker exists (12). However, Yoder and coworkers have defined an EPC as CD34+, CD45−, CD31+, CD133−, CD14−, CD115−, VEGFR2+, and positive for other EC markers such as vWF and eNos (12, 13). Interestingly, there exists a subset of monocytic cells expressing high levels of EC markers, thought previously to represent EPC, termed Circulating Angiogenic Cells (CAC). These cells are hematopoietic progenitor-derived (14) co-expressing CD45, CD11b, CD11c, CD14, and CD68 and ingest bacteria in vitro (13, 15–17). Importantly, these cells contribute to neo-angiogenesis (16–18), local vascular remodeling (19–24), and may incorporate into vessels as ‘EC-like’ cells (25). Additionally, other bone marrow-derived progenitor cells (PC) identified by the cell surface marker c-kit, give rise to EC, smooth muscle cells, adipocytes, and hematopoietic cells (26–31). C-kit is a transmembrane tyrosine kinase receptor (CD117) for stem cell factor (32) that is mobilized from the bone marrow and tracks to atherosclerotic lesions (30), sites of cardiac infarct (29), and areas of ischemic injury (33, 34). C-kit+ cells contain cellular sub-populations that have the potential to give rise to EPC and CAC and c-kit expression on PC is critical for homing to injured vasculature during neo-angiogenesis (35). C-kit+ PC are distinct from mesenchymal stem cells (MSC) which are considered to be both CD117 and CD45 negative (14, 36–38).
In the current study, our initial objective was to determine if autologous CD117+ (c-kit+) PC would abrogate acute cardiac allograft rejection by replacing damaged allograft vasculature with recipient-derived cells, thus limiting the consequence of direct alloreactivity (11). While results show that autologous CD117+PC do not increase recipient EC chimerism, they do attenuate acute allograft rejection in a dose-dependent manner. In addition, CD117+PC inhibit T-cell alloreactivity in vitro via a largely paracrine mechanism, and appear to dampen late T-cell responsiveness when re-stimulated ex-vivo. Importantly, CD117+PC represent a unique population of bone marrow-derived stem cells than have therapeutic potential for enhancing allograft survival.
Female BALB/cByJ (BALB/c H-2d) and C3H/HeJ (C3H, H-2k) mice were used as heart transplant donors. Female C57BL/6ByJ (B6, H-2b) and C57B6-Rag1tm1/Mom (B6 rag−/−, H-2b) mice were used as heart transplant recipients. Aged (12 mos) B6, BALB/c, UBI-GFP/BL6 (B6 gfp, H-2b), and B6.SJL-Ptprca Pepcb/BoyJ (B6 CD45.1, H-2b) mice were used as cell donors. All mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). Animals were housed under pathogen-free conditions at the University of Colorado Center for Comparative Medicine, according to NIH guidelines. All studies were approved by Institutional Animal Care and Use Committee.
Cell donor femurs, tibias, and humeri were aseptically removed and flushed with 1xHBSS and a 25-gauge needle. Cells were strained (70um), centrifuged (500g for 5 min), and resuspended in 10 ml autoMACS Buffer with 5% BSA (Miltenyi). Cells were overlaid on Lympholyte-M (Cedarlane) and centrifuged at 800g for 20 min. Cells were counted, resuspended at 80ul/107 cells in autoMACS Buffer and incubated with anti-CD117 MicroBeads (Miltenyi) for 15 min per manufacturer’s protocol. Cells were rinsed, passed over a MidiMACS positive selection LS column (Miltenyi), rinsed, and run over a second column per manufacturer’s protocol. Effluent cells were obtained via flow-through from the first column. Cells were kept at 4°C at all times.
Analysis was performed using a FACSCalibur cytometer (Becton Dickinson), with cell staining performed as published (11). A 1:100 dilution of PE-CD25 (clone PC61), PerCP-CD4 (clone RM4-5), PerCP-CD8 (clone 53-6.7), APC/FITC-CD45.1 (clone A20), PE/APC-CD31 (clone 390), PE/PerCP-CD45 (clone 30-F11), FITC-H-2Kb (clone AF6-88.5), PE-H-2Kd (clone SF1-1.1), APC-CD117 (clone 3C1), and FITC-Foxp3 (clone FJK-16s, per eBioscience intra-cellular kit) was utilized.
Control and allograft hearts were digested for flow cytometric analysis as previously published (11). Separated cells were prepared for flow cytometry as above.
Mixed lymphocyte reactions (MLR) with recipient-type B6 lymph node responder cells and BALB/c or C3H stimulator splenocytes were performed as previously published (39, 40). B6 CD117+PC, B6 CD117− effluent cells, or B6 bone marrow cells were added (at BM rations of 1:1 – 1:256 with responders). Differences within a given quadruplicate culture assay were assessed with the unpaired T-test via InStat statistical software.
Single cell suspensions of RBC-depleted B6 CD45.1 splenocytes (5×106 cells/ml in 1×PBS + 0.1% FCS) were CFSE-labeled in 5mM CFSE (1:2000) for 10 min at 37°C. Cells were washed twice with RPMI + 20%FCS and resuspended in media (EMEM +10% FCS, 10−5 M 2-Me, and antibiotics). CFSE-labeled splenocytes (2×106) in 0.3ml of media were then added to the each main well of the transwell plate (CoStar, Cat# 3422). Gamma-irradiated (2500 rads) BALB/c splenocytes (3×106) were then added to each main well in 0.15ml of media. Media, 2×106 B6 CD117+PC or 2×106 B6 CD117− effluent cells were added to the appropriate main wells in a total of 0.15ml media. Finally, media, 2×105 B6 CD117+PC, or 2×105 B6 CD117− effluent cells were added to the corresponding transwell in a total volume of 0.1ml. CFSE-labeled B6 CD45.1+ responders were harvested on day 4 for flow cytometry (CD45.1, CD4, CD8, and CFSE).
For tracking cell proliferation in vivo, CFSE labeling of B6 CD45.1 splenocytes was accomplished as above and 107 were injected retro-orbitally (RO) on the day of transplantation (BALB/c → B6). On day +1, 107 B6 CD117+PC, 107 B6 CD117− effluent cells, or no cells were injected RO. Heart-grafted mice were sacrificed on day +4 with harvest of the allograft, peripheral blood, mesenteric lymph nodes, and spleen for flow cytometry (CD45.1, CD4, CD8, and CFSE).
Recombinant transforming growth factor-β 1 (R&D Systems, cat# 240-B) at 10 ng/mL, anti-mouse/bovine/human transforming growth factor-β 1 (R&D Systems, clone 1D11) at 75 ug/mL, recombinant mouse interleukin-10, (Pharmingen, cat# 550070) at 3 ug/mL, and anti-mouse interleukin-10R (Pharmingen, clone 1B1.3a) at 50 ug/mL were utilized for in vitro CFSE assays and MLR.
Cardiac allografts from BALB/c or C3H donor mice were transplanted heterotopically into B6 or B6 rag−/− recipients by standard microsurgical techniques (41). Allograft survival was assessed by daily palpation with rejection defined as loss of palpable beating that was confirmed at laparotomy. Survival differences were determined using Kaplan Meier Analysis via MedCalc statistical software.
Transplanted hearts were fixed (10% formalin) and paraffin-embedded. Sections were cut (5μm), deparaffinized, rehydrated, washed (1xPBS), and blocked for 1hr in TSA blocking buffer. Primary antibody was mixed in TSA blocking buffer and sections were incubated (anti-GFP@1:1000, Abcam ab6673) overnight. Sections were washed, incubated (1hr) with the secondary antibody (Cy3@1:250 in 1xPBS), washed and mounted (30% glycerin).
We first tested the hypothesis that CD117+PC would prolong cardiac allograft survival by increasing recipient EC chimerism by determining the degree of initial allograft EC chimerism induced by autologous CD117+PC transfer. Heart allografts were transplanted followed by host injection with 107 B6 CD117+PC on day +1 (or received no treatment). Allografts were harvested on day+7 and analyzed by flow cytometry for relative host (H-2Kd) versus donor (H-2Kb) MHC class I expression by both CD31+CD45− cells (putative EC) and CD31+CD45+ cells (putative CAC). This day 7 time point was chosen since it encompassed initial ischemia-reperfusion injury as well as initial innate and adaptive reactivity and as such represented a period in which potential signals for PC homing to the allograft vasculature were present. Additionally, this time point allowed us to assess if initial recipient-type chimerism occurred following initial host reactivity. Interestingly, we found no significant increase in host-type chimerism following CD117+ PC transfer in either potential EC or CAC cell compartments (supplemental Fig 1s). In parallel, allograft recipients were treated with subsequent transfer of 2×106 or 107 B6 CD117+PC on day +1, +5, +9, and +15. As described above, we chose CD117 as a PC marker since it’s expression is required for homing to sites of ischemic injury (35) and is frequently co-expressed on EPC (28) as well as CAC (27). Additionally, we utilized freshly isolated CD117+ cells to avoid bias toward any potential terminally-differentiated progenitor cell and to allow the allograft environment (ischemia-reperfusion and allo-immunity) to direct homing and differentiation in vivo. Subsequently, CD117+PC infusions were administered through the estimated period of maximal ischemia-reperfusion-injury and acute allo-immune inflammation. Positive selection of CD117+ donor bone marrow resulted in ≥ 80% CD117+ donor cells (Fig 1A). The CD117-depleted effluent cells were used as a control population (≤ 0.5% CD117+, Fig 1B). Results demonstrated pronounced dose-dependent allograft prolongation using autologous CD117+PC therapy vs. controls (p<0.0002, Fig 1C). Importantly, allograft prolongation was CD117-dependent since CD117-depleted effluent cells did not prolong allograft survival versus controls (Fig 1C). Taken together, results show that despite a lack of potential recipient EC chimerism, autologous CD117+PC therapy leads to robust, dose-dependent cardiac allograft prolongation.
Since allograft prolongation by autologous CD117+PC was not associated with increased recipient EC chimerism, we determined whether CD117+PC localized to the allograft. To accomplish this, we utilized B6GFP+ transgenic mice (42) as CD117+PC cell donors. GFP+CD117+ cells (2 x 106) were injected RO on day +1 post-transplantation. On day +7, allografts were harvested for immunohistochemistry. Results demonstrated that GFP+ cells (PC-derived) were found in allografts in both peri-vascular and intra-luminal locations (Fig 2B–D).
To determine if the presence of GFP+ cells in allografts (and peripheral lymphoid tissues) demonstrated any specificity or rather were randomly distributed, we performed paired experiments whereby BALB/c → B6 heart transplants were treated with 107 GFP+CD117+PC or 107 GFP+CD117− effluent cells on day +1 (from the same cell donors to control for viability from a given cell prep). Allografts and peripheral lymphoid tissues were then analyzed by flow cytometry on day +7. Triplicate experiments demonstrated a large increase in the number of GFP+ cells in the spleen (SPL), mesenteric lymph nodes (MLN), and bone marrow (BM) when the transplant recipient received GFP+CD117+ PC as compared to recipients of GFP+CD117− control cells in equivalent numbers (Fig 3). This strongly suggested that either homing or increased survival of CD117+PC-derived cells occurred at these sites of inflammation. Importantly, we also examined the distribution of GFP+CD117+PC and GFP+CD117− effluent cells in the allograft versus the native heart on day +15 post-transplantation (as CD117− control treated allografts all reject by day +15). Numbers of GFP+ control CD117− effluent cells did not differ between the allograft and the native heart (not shown). However, there was a large relative increase in the number of GFP+ cells in the CD117+PC treated allografts versus the native hearts (Supplemental Fig 2s). Finally, recipients of 2 x 106 GFP+CD117+PC demonstrated persistence of the BM cells within the allograft even at the time of rejection whereas very few GFP+ cells were found in the corresponding native hearts (<1%) (not shown).
Results imply that allograft prolongation by CD117+PC was not related to MSC within the donor cell inoculum. That is, previous studies indicated that MSC are CD117− CD45− (14, 36–38) whereas the cells used in this study are < 0.5% CD117− CD45− (Fig 1A). Importantly, host-type CD117− effluent cells, 30% of which are potential CD117−CD45− MSC (Fig 1B), do not prolong cardiac allografts (Fig 1C).
Moreover, previous murine cardiac studies indicated that donor and recipient-type MSC are equally capable of promoting allograft survival, but only under either semi-allogeneic conditions (43) or with concomitant Sirolimus therapy (44). As we found that host-type CD117+PC robustly prolonged allograft survival under fully allogeneic conditions (Fig 1C), we also tested whether donor-type CD117+PC would prolong cardiac allograft survival. Results demonstrated that donor-type CD117+PC resulted in modest allograft prolongation versus untreated controls (Table 1). Interestingly, donor-type CD117+PC were significantly less effective than autologous cells for inducing allograft prolongation (Table 1).
As increased allograft survival with autologous CD117+PC did not appear related to MSC, we investigated whether CD117+PC had unique immunomodulatory properties. Firstly, we determined whether co-cultured B6CD117+PC, B6CD117− effluent cells, and unmanipulated B6 BM cells, (at 1:1 with responders), inhibited a standard MLR in vitro. Results demonstrated that the addition of CD117+PC, as well as both control populations, strongly inhibited the proliferation of allo-specific T-cells (Fig 4A). As inhibition of T-cell proliferation appeared to be non-specific, we next performed titrations of both CD117+PC and CD117− effluent cells (1:1 – 1:256) to determine if there existed a difference in the potency of inhibition between the two cellular populations. Results demonstrated that CD117+ PC inhibited T-cell proliferation significantly better than CD117− effluent cells at high ratios with responder T-cells (out to 1:2, p<0.0002, unpaired T-test), but that there was no significant difference in their abilities to inhibit T-cell proliferation out beyond 1:2 (Fig 4B). Importantly, significant in vitro T-cell inhibition by both CD117+ PC and CD117− effluent was lost at 1:32 with responders (Fig 4B). Given these results, we next sought to elucidate if the inhibitory effect of CD117+PC or other BM populations on T-cell proliferation was contact-dependent and/or paracrine in vitro. To accomplish this, we performed in vitro CFSE proliferation assays utilizing transwell culture plates (See Methods). Results demonstrated that co-culture with either CD117+ or CD117-depleted (effluent) BM-derived cells resulted primarily in profound paracrine inhibition of alloreactive T-cells (Fig 5). Finally, combined inhibition of TGF-β and IL-10 resulted in moderate reversal of T-cell inhibition by B6 CD117−effluent but not B6 CD117+PC (Supplemental Figure 3s), suggesting a potential parallel pathway between TGF-β and IL-10 for in vitro inhibition of T-cell proliferation by control CD117−effluent cells. However, IL-10 and TGF-β do not appear to be significantly involved mechanistically for in vitro inhibition of T-cell proliferation by CD117+PC.
We next looked at the effect of CD117+PC on in vivo T-cell inhibition. CFSE-labeled B6CD45.1 splenocytes (107) were injected RO on day 0 relative to BALB/c → B6 heart transplantation. On day+1, 107 B6 CD117+PC or 107 B6 CD117− effluent cells were injected RO in experimental animals (paired control recipients were left untreated). Results demonstrated no statistically significant difference in T-cell proliferation in the MLN, SPL, or allograft in CD117+PC treated or CD117− effluent treated allograft recipients (not shown).
Finally, as previous studies have demonstrated donor-type MSC to increase CD4+CD25+Foxp3+ regulatory T-cells (Tregs) in vivo (43, 44), we determined if CD117+PC treatment also led to an increase in Tregs. BALB/c → B6 heart transplant recipients received 107 B6 CD117+PC (or no cells) RO on day +1 and were sacrificed on day +7 for analysis. Results show no statistically significant increase in CD4+CD25+Foxp3+ Tregs in any compartment (not shown). Additionally, we also looked at BALB/c → B6 heart transplant recipients that received 107 B6 CD117+PC on days +1, +5, +9, and +15 at the time of rejection and found no significant increase in Tregs in any compartment out to >50 days post transplantation (not shown).
Given that CD117+PC were shown to inhibit T-cell proliferation in vitro but not initial proliferation in vivo, we determined if autologous CD117+PC impacted the late responsiveness of T-cells restimulated ex-vivo. To accomplish this, we harvested ‘conditioned’ splenocytes, from recipients of BALB/c → B6 heart transplants that received 107 autologous CD117+PC on days +1,+5,+9, and +15 and whose allografts survived >30 days, and utilized these for in vitro re-stimulation experiments. Results demonstrated a non-specific blunting of the conditioned T-cell proliferative response to both BALB/c and third party C3H stimulators compared to controls (Fig 6A).
The need for alternative therapies in solid organ transplantation has been obvious for quite some time. Despite standard immunosuppression, rates of acute and chronic rejection, and as a consequence survival, have not been satisfactory (1). Unfortunately, new therapies for allograft prolongation in humans have been mostly unsuccessful, in part due to the fact that many of these therapies rely on T-cell dependent mechanisms (45) including regulatory T-cells (Tregs) (46, 47). A significant obstacle has been the fact that standard immunosuppression with CNI is inhibitory to T-cells - both effector and regulatory (45). Recently, donor-specific bone marrow transplantation (BMT) (48–51) has demonstrated tolerance induction in animal models and humans. However, this therapy is donor-derived and therefore carries the risk of graft versus host disease (GVHD) (52), as well as other serious immune reactions (49, 51). Consequently, autologous therapy could form an attractive safe alternative to donor cell therapy. In two studies, autologous MSC were used to treat fully allogeneic cardiac allografts with no allograft prolongation seen in the first (43) and robust prolongation only with concomitant Sirolimus therapy in the second (44). Studies with donor-derived MSC also demonstrate an increase in CD4+CD25+Foxp3+ Tregs, which was interpreted as indicating that there may be a mechanistic requirement for Tregs in tolerance induction by MSC (43). As an alternative approach, we investigated a different autologous progenitor cell population with the hypothesis that we would attain allograft prolongation through the repair/replacement of donor vascular endothelium. We initially hypothesized that CD117+PC would differentiate into vascular cells and incorporate into ischemia-reperfusion/allo-immune injured intra-graft vessels, thus partially ‘hiding’ the allograft by limiting the primary target of the acute alloresponse (11).
Results demonstrated significant dose-dependent, CD117-dependent cardiac allograft prolongation by CD117+PC despite a lack of increased recipient vascular chimerism. CD117+PC differentially engrafted cardiac allografts and peripheral lymphoid tissues in vivo and profoundly inhibited T-cell proliferation in vitro. However, in vitro results demonstrated relatively equal inhibition of T-cell proliferation by autologous CD117+PC, CD117− effluent cells, and unmanipulated BM cells, demonstrating that multiple BM populations can inhibit T-cell proliferation in vitro. Interestingly, neither CD117+PC nor CD117− effluent cells significantly effected early T-cell proliferation in vivo. Despite this, recipient splenocytes, conditioned by CD117+PC and prolonged exposure to BALB/c allografts, were significantly diminished in their proliferative capacity to ex-vivo re-stimulation with donor-type and third party allo-APCs, suggesting CD117+PC lead to a dampened or ‘sluggish’ late cellular allo-immune response. At present, we would propose that the benefit to the allograft afforded by CD117+ PC may not be via a clear inhibition of adaptive reactivity per se, but rather by an unexpected cytoprotective property of the CD117+ PC. We would note that this type of pro-survival benefit of autologous BM-derived cells was demonstrated by Li et al. who found that severe pancreatic injury in E2f1/2 deficient mice was rescued by syngeneic, wild-type bone marrow (53). Clearly, future mechanistic studies will need to include investigation into pro-survival factors that might potentially be involved in the in vivo mechanism of action of CD117+ PC and their ability to prolong cardiac allograft survival.
A potential caveat to these findings is the possibility that a small number of contaminating MSC could be responsible for allograft prolongation (< 0.5% of the CD117+PC prep is CD117−CD45−). However, it is quite unlikely that this is the case. Importantly, treatment with CD117-depleted (effluent) cells which contain approximately 30% CD117−CD45−cells as potential MSCs did not lead to increased cardiac allograft survival, thus arguing against a contribution by MSC and demonstrating a requirement for CD117 expression. Also, CD117+PC therapy does not lead to an increase in CD4+CD25+Foxp3+ Tregs as previously found with MSC therapy. Thus, while MSCs form an important cell-based approach to modifying allograft reactivity, it appears that CD117+PC represent an additional progenitor cell population that has the potential to promote allograft survival.
A surprising finding was in the sometimes large number of GFP+ cells present at day +7 within the spleens of animals receiving GFP+CD117+PC (Fig 3A). In triplicate paired experiments, consistent absolute fold changes between GFP+CD117− control and GFP+CD117+PC-treated recipient spleens were noted (ranging from 13–22 fold increase with CD117+PC-treatment, p < 0.02, paired T-test, not shown). Interestingly, in some cases greater than 40% of total spleen cells were GFP+ after GFP+CD117+PC cell transfer. Given that only a total of 107 GFP+CD117+ cells were injected, it seems likely that expansion of PC-derived cells occurred, although we did not specifically assess for proliferation of these cells. Such potential cell expansion may not be surprising given the enrichment of stem cells and progenitor cells in the donor cell population.
It will be important in future studies to determine the eventual differentiated cell fate(s) responsible for the efficacy of autologous CD117+PC therapy in prolonging allograft survival. As discussed, EPC and CAC are likely candidates. Thus far, immunohistochemical and flow cytometric data did not show any significant difference in the relative allograft content of GFP+CD45+CD31+ (potential CAC) or GFP+CD45−CD31+ (potential EPC) cells in allografts treated with either GFP+CD117+PC or GFP+CD117− effluent cells (not shown). Future studies to selectively remove potential CAC and EPC populations from the injected CD117+PC cell prep should demonstrate any contribution from either of these two candidate progenitors.
In conclusion, results of these studies demonstrate the novel finding that autologous CD117+PC abrogate acute cardiac allograft rejection in a dose-dependent, CD117-dependent fashion and that the allograft promoting effects appear to be unique from those of previously studied MSC. Importantly, as autologous cells, CD117+PC pose presumably limited risk to the host and as such possess the potential to form an adjunct role with other tolerance-promoting agents to promote allograft survival. It will be intriguing to determine if CD117+PC play a general cytoprotective role that promotes survival of tissue vascular and/or parenchymal cells under the duress of transplantation and alloreactivity.
1This work was supported by American Heart Association Grant, 09 BGIA 2190055 (TG), National Institutes of Health Grant, K08 HL077503 (TG), and NIH DERC Grant, P30-DK057516 (Barbara Davis Center)
Disclosure: The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation