Prolongation of Cardiac Allograft Survival by a Novel Population of Autologous CD117+ Bone Marrow-Derived Progenitor Cells

doi:10.1111/j.1600-6143.2010.03335.x

Am J Transplant. Author manuscript; available in PMC 2012 January 1.

Published in final edited form as:

Am J Transplant. 2011 January; 11(1): 34–44.

Published online 2010 November 29. doi: 10.1111/j.1600-6143.2010.03335.x

PMCID: PMC3059253

NIHMSID: NIHMS248731

Prolongation of Cardiac Allograft Survival by a Novel Population of Autologous CD117⁺ Bone Marrow-Derived Progenitor Cells¹

Todd J. Grazia,^2,^3,⁴ Robert J. Plenter,^2,³ Helen M. Lepper,^2,³ Francisco Victorino,^2,³ Shelley D. Miyamoto,⁵ Joseph T. Crossno, Jr.,² Biagio A. Pietra,^3,⁵ Ronald G. Gill,^3,^4,⁶ and Martin R. Zamora^2,³

² Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Anschutz Medical Campus, Research 2, Box C272, 9th Floor, Rm 9118, 12700 East 19th Avenue, Aurora, CO 80045

³ Department of Medicine and Immunology, Barbara Davis Center for Childhood Diabetes, Transplantation Immunology, University of Colorado Denver, Anschutz Medical Campus, 1775 Aurora Court, Bldg. M20 Room 3202B, Box B-140, Aurora, CO 80045

⁴ Integrated Department of Immunology, National Jewish Medical and Research Center and University of Colorado Denver, 1400 Jackson St., K830, Denver, CO 80206

⁵ Department of Pediatrics, Division of Cardiology, The Children’s Hospital, University of Colorado Denver, Anschutz Medical Campus, Heart Institute, 13123 East 16th Avenue, Aurora, CO 80045

⁶ Department of Surgery, Colorado Center for Transplantation Care, Research, and Education (CCTCARE) Institute, University of Colorado Denver, Anschutz Medical Campus, 12700 East 19th Avenue, Aurora, CO 80045

Address correspondence to: Todd J. Grazia, M.D., Email: Todd.Grazia/at/ucdenver.edu, University of Colorado Denver, Anschutz Medical Campus, Division of Pulmonary Sciences and Critical Care Medicine, Research 2, Box C272, 9th Floor, Rm 9118, 12700, East 19th Avenue, Aurora, CO 80045, Phone: (303) 724-0550, Fax: (303) 724-6042

The publisher's final edited version of this article is available free at Am J Transplant

Abstract

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.

Keywords: stem cells, transplantation, acute allograft rejection, tolerance induction

Introduction

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.

Materials and Methods

Mice

Female BALB/cByJ (BALB/c H-2^d) and C3H/HeJ (C3H, H-2^k) mice were used as heart transplant donors. Female C57BL/6ByJ (B6, H-2^b) and C57B6-Rag1^tm1/Mom (B6 rag^−/−, H-2^b) mice were used as heart transplant recipients. Aged (12 mos) B6, BALB/c, UBI-GFP/BL6 (B6 gfp, H-2^b), and B6.SJL-Ptprc^a Pepc^b/BoyJ (B6 CD45.1, H-2^b) 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.

CD117 cell-enrichment

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.

Flow cytometry

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-2K^b (clone AF6-88.5), PE-H-2K^d (clone SF1-1.1), APC-CD117 (clone 3C1), and FITC-Foxp3 (clone FJK-16s, per eBioscience intra-cellular kit) was utilized.

Cardiac digestion

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

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.

In vitro and in vivo proliferation assays using carboxyfluorescein succinimidyl ester (CFSE) cell labeling

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).

Cytokines and Antibodies

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.

Heterotopic heart transplantation

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.

Tissue histological examination

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).

Results

Autologous CD117⁺PC do not enhance recipient EC chimerism, but attenuate acute cardiac allograft rejection in a CD117-dependent manner

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-2K^d) versus donor (H-2K^b) 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.

Figure 1

CD117⁺ progenitor cells significantly attenuate acute cardiac allograft rejection in a CD117-dependent, dose-dependent manner

Autologous CD117⁺PC differentially engraft cardiac allografts and peripheral lymphoid tissues

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).

Figure 2

Autologous CD117⁺PC engraft cardiac allografts

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).

Figure 3

Autologous CD117⁺PC differentially engraft cardiac allografts and peripheral lymphoid tissues

Host-derived CD117⁺PC are superior to donor-type cells for attenuating acute cardiac allograft rejection

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).

Table 1

Host-derived CD117⁺PC are superior to donor-type cells for attenuating acute cardiac allograft rejection.

Both CD117⁺ and CD117-depleted bone marrow cells inhibit alloreactivity in vitro

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.

Figure 4

Autologous CD117⁺ progenitor cells, CD117⁻ effluent cells, and unmanipulated bone marrow cells equally inhibit in vitro T-cell proliferation

Figure 5

In vitro inhibition of T-cell proliferation by both CD117⁺PC and CD117⁻effluent cells is dependent on a primarily paracrine mechanism

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).

Autologous CD117⁺PC therapy leads to blunted T-cell allo-responsiveness upon re-stimulation

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).

Figure 6

Conditioned splenocytes are non-specifically hyporesponsive upon re-stimulation ex vivo

Discussion

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.

Supplementary Material

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Abbreviations


PC	progenitor cells

EPC	endothelial progenitor cell

CAC	circulating angiogenic cell

MSC	mesenchymal stem cells

BM	bone marrow

CNI	calcineurin inhibitor

APC	antigen presenting cell

MLN	mesenteric lymph nodes

SPL	spleen

EC	endothelial cell

Footnotes

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

References

1. Taylor DO, Stehlik J, Edwards LB, Aurora P, Christie JD, Dobbels F, Kirk R, Kucheryavaya AY, Rahmel AO, Hertz MI. Registry of the International Society for Heart and Lung Transplantation: Twenty-sixth Official Adult Heart Transplant Report-2009. J Heart Lung Transplant. 2009;28:1007–1022. [PubMed]

2. Johnson MR, Meyer KH, Haft J, Kinder D, Webber SA, Dyke DB. Heart transplantation in the United States, 1999–2008. Am J Transplant. 2010;10:1035–1046. [PubMed]

3. Burt RK, Loh Y, Pearce W, Beohar N, Barr WG, Craig R, Wen Y, Rapp JA, Kessler J. Clinical applications of blood-derived and marrow-derived stem cells for nonmalignant diseases. Jama. 2008;299:925–936. [PubMed]

4. Dimmeler S, Burchfield J, Zeiher AM. Cell-based therapy of myocardial infarction. Arterioscler Thromb Vasc Biol. 2008;28:208–216. [PubMed]

5. Kawamoto A, Losordo DW. Endothelial progenitor cells for cardiovascular regeneration. Trends Cardiovasc Med. 2008;18:33–37. [PMC free article] [PubMed]

6. Bertolini F, Mancuso P, Shaked Y, Kerbel RS. Molecular and cellular biomarkers for angiogenesis in clinical oncology. Drug Discov Today. 2007;12:806–812. [PubMed]

7. Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita Y, Kothari S, Mohle R, Sauvage LR, Moore MA, Storb RF, Hammond WP. Evidence for circulating bone marrow-derived endothelial cells. Blood. 1998;92:362–367. [PubMed]

8. Bhattacharya V, McSweeney PA, Shi Q, Bruno B, Ishida A, Nash R, Storb RF, Sauvage LR, Hammond WP, Wu MH. Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34(+) bone marrow cells. Blood. 2000;95:581–585. [PubMed]

9. Stump MM, Jordan GL, Jr, Debakey ME, Halpert B. Endothelium Grown from Circulating Blood on Isolated Intravascular Dacron Hub. Am J Pathol. 1963;43:361–367. [PubMed]

10. Wu MH, Shi Q, Wechezak AR, Clowes AW, Gordon IL, Sauvage LR. Definitive proof of endothelialization of a Dacron arterial prosthesis in a human being. J Vasc Surg. 1995;21:862–867. [PubMed]

11. Grazia TJ, Pietra BA, Johnson ZA, Kelly BP, Plenter RJ, Gill RG. A two-step model of acute CD4 T-cell mediated cardiac allograft rejection. J Immunol. 2004;172:7451–7458. [PubMed]

12. Hirschi KK, Ingram DA, Yoder MC. Assessing identity, phenotype, and fate of endothelial progenitor cells. Arterioscler Thromb Vasc Biol. 2008;28:1584–1595. [PubMed]

13. Yoder MC, Mead LE, Prater D, Krier TR, Mroueh KN, Li F, Krasich R, Temm CJ, Prchal JT, Ingram DA. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood. 2007;109:1801–1809. [PubMed]

14. Lyman SD, Jacobsen SE. c-kit ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities. Blood. 1998;91:1101–1134. [PubMed]

15. Rohde E, Malischnik C, Thaler D, Maierhofer T, Linkesch W, Lanzer G, Guelly C, Strunk D. Blood monocytes mimic endothelial progenitor cells. Stem Cells. 2006;24:357–367. [PubMed]

16. Zhang SJ, Zhang H, Wei YJ, Su WJ, Liao ZK, Hou M, Zhou JY, Hu SS. Adult endothelial progenitor cells from human peripheral blood maintain monocyte/macrophage function throughout in vitro culture. Cell Res. 2006;16:577–584. [PubMed]

17. Rehman J, Li J, Orschell CM, March KL. Peripheral blood “endothelial progenitor cells” are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation. 2003;107:1164–1169. [PubMed]

18. Rohde E, Bartmann C, Schallmoser K, Reinisch A, Lanzer G, Linkesch W, Guelly C, Strunk D. Immune cells mimic the morphology of endothelial progenitor colonies in vitro. Stem Cells. 2007;25:1746–1752. [PubMed]

19. De Palma M, Venneri MA, Galli R, Sergi Sergi L, Politi LS, Sampaolesi M, Naldini L. Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell. 2005;8:211–226. [PubMed]

20. Zentilin L, Tafuro S, Zacchigna S, Arsic N, Pattarini L, Sinigaglia M, Giacca M. Bone marrow mononuclear cells are recruited to the sites of VEGF-induced neovascularization but are not incorporated into the newly formed vessels. Blood. 2006;107:3546–3554. [PubMed]

21. Jin DK, Shido K, Kopp HG, Petit I, Shmelkov SV, Young LM, Hooper AT, Amano H, Avecilla ST, Heissig B, Hattori K, Zhang F, Hicklin DJ, Wu Y, Zhu Z, Dunn A, Salari H, Werb Z, Hackett NR, Crystal RG, Lyden D, Rafii S. Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes. Nat Med. 2006;12:557–567. [PMC free article] [PubMed]

22. You D, Waeckel L, Ebrahimian TG, Blanc-Brude O, Foubert P, Barateau V, Duriez M, Lericousse-Roussanne S, Vilar J, Dejana E, Tobelem G, Levy BI, Silvestre JS. Increase in vascular permeability and vasodilation are critical for proangiogenic effects of stem cell therapy. Circulation. 2006;114:328–338. [PubMed]

23. Rafii S, Lyden D. Cancer. A few to flip the angiogenic switch. Science. 2008;319:163–164. [PMC free article] [PubMed]

24. Anghelina M, Krishnan P, Moldovan L, Moldovan NI. Monocytes/macrophages cooperate with progenitor cells during neovascularization and tissue repair: conversion of cell columns into fibrovascular bundles. Am J Pathol. 2006;168:529–541. [PubMed]

25. Sood R, Kalloway S, Mast AE, Hillard CJ, Weiler H. Fetomaternal cross talk in the placental vascular bed: control of coagulation by trophoblast cells. Blood. 2006;107:3173–3180. [PubMed]

26. Davie NJ, Crossno JT, Jr, Frid MG, Hofmeister SE, Reeves JT, Hyde DM, Carpenter TC, Brunetti JA, McNiece IK, Stenmark KR. Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: contribution of progenitor cells. Am J Physiol Lung Cell Mol Physiol. 2004;286:L668–678. [PubMed]

27. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. The Journal of clinical investigation. 2002;109:337–346. [PMC free article] [PubMed]

28. Young HE, Steele TA, Bray RA, Hudson J, Floyd JA, Hawkins K, Thomas K, Austin T, Edwards C, Cuzzourt J, Duenzl M, Lucas PA, Black AC., Jr Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec. 2001;264:51–62. [PubMed]

29. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:10344–10349. [PubMed]

30. Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med. 2002;8:403–409. [PubMed]

31. Crossno JT, Jr, Majka SM, Grazia T, Gill RG, Klemm DJ. Rosiglitazone promotes development of a novel adipocyte population from bone marrow– derived circulating progenitor cells. The Journal of clinical investigation. 2006;116:3220–3228. [PMC free article] [PubMed]

32. Ogawa M, Nishikawa S, Yoshinaga K, Hayashi S, Kunisada T, Nakao J, Kina T, Sudo T, Kodama H, Nishikawa S. Expression and function of c-Kit in fetal hemopoietic progenitor cells: transition from the early c-Kit-independent to the late c-Kit-dependent wave of hemopoiesis in the murine embryo. Development. 1993;117:1089–1098. [PubMed]

33. Laing AJ, Dillon JP, Condon ET, Coffey JC, Street JT, Wang JH, McGuinness AJ, Redmond HP. A systemic provascular response in bone marrow to musculoskeletal trauma in mice. J Bone Joint Surg Br. 2007;89:116–120. [PubMed]

34. Patschan D, Krupincza K, Patschan S, Zhang Z, Hamby C, Goligorsky MS. Dynamics of mobilization and homing of endothelial progenitor cells after acute renal ischemia: modulation by ischemic preconditioning. Am J Physiol Renal Physiol. 2006;291:F176–185. [PubMed]

35. Dentelli P, Rosso A, Balsamo A, Colmenares Benedetto S, Zeoli A, Pegoraro M, Camussi G, Pegoraro L, Brizzi MF. C-KIT, by interacting with the membrane–bound ligand, recruits endothelial progenitor cells to inflamed endothelium. Blood. 2007;109:4264–4271. [PubMed]

36. Lupatov AY, Karalkin PA, Suzdal’tseva YG, Burunova VV, Yarygin VN, Yarygin KN. Cytofluorometric analysis of phenotypes of human bone marrow and umbilical fibroblast-like cells. Bulletin of experimental biology and medicine. 2006;142:521–526. [PubMed]

37. Martin-Rendon E, Sweeney D, Lu F, Girdlestone J, Navarrete C, Watt SM. 5-Azacytidine-treated human mesenchymal stem/progenitor cells derived from umbilical cord, cord blood and bone marrow do not generate cardiomyocytes in vitro at high frequencies. Vox sanguinis. 2008;95:137–148. [PubMed]

38. Shih YR, Kuo TK, Yang AH, Lee OK, Lee CH. Isolation and characterization of stem cells from the human parathyroid gland. Cell proliferation. 2009;42:461–470. [PubMed]

39. Grazia TJ, Plenter RJ, Weber SM, Lepper HM, Victorino F, Zamora MR, Pietra BA, Gill RG. Acute cardiac allograft rejection by directly cytotoxic CD4 T cells: parallel requirements for Fas and perforin. Transplantation. 2010;89:33–39. [PMC free article] [PubMed]

40. Grazia TJ, Plenter RJ, Doan AN, Kelly BP, Weber SM, Kurche JS, Cushing SO, Gill RG, Pietra BA. Spontaneous allograft tolerance in B7- deficient mice independent of preexisting endogenous CD4+CD25+ regulatory T-cells. Transplantation. 2007;83:1449–1458. [PubMed]

41. Corry RJ, Winn HJ, Russell PS. Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection. Transplantation. 1973;16:343–350. [PubMed]

42. Schaefer BC, Schaefer ML, Kappler JW, Marrack P, Kedl RM. Observation of antigen-dependent CD8+ T-cell/dendritic cell interactions in vivo. Cell Immunol. 2001;214:110–122. [PubMed]

43. Casiraghi F, Azzollini N, Cassis P, Imberti B, Morigi M, Cugini D, Cavinato RA, Todeschini M, Solini S, Sonzogni A, Perico N, Remuzzi G, Noris M. Pretransplant infusion of mesenchymal stem cells prolongs the survival of a semiallogeneic heart transplant through the generation of regulatory T cells. J Immunol. 2008;181:3933–3946. [PubMed]

44. Ge W, Jiang J, Baroja ML, Arp J, Zassoko R, Liu W, Bartholomew A, Garcia B, Wang H. Infusion of mesenchymal stem cells and rapamycin synergize to attenuate alloimmune responses and promote cardiac allograft tolerance. Am J Transplant. 2009;9:1760–1772. [PubMed]

45. Li XC, Rothstein DM, Sayegh MH. Costimulatory pathways in transplantation: challenges and new developments. Immunological reviews. 2009;229:271–293. [PubMed]

46. Zhang GY, Hu M, Wang YM, Alexander SI. Foxp3 as a marker of tolerance induction versus rejection. Current opinion in organ transplantation. 2009;14:40–45. [PubMed]

47. Sagoo P, Lombardi G, Lechler RI. Regulatory T cells as therapeutic cells. Current opinion in organ transplantation. 2008;13:645–653. [PubMed]

48. Balner H. Persistence of Tolerance Towards Donor–Type Antigens after Temporary Chimerism in Rats. Transplantation. 1964;2:464–474. [PubMed]

49. Kawai T, Cosimi AB, Spitzer TR, Tolkoff-Rubin N, Suthanthiran M, Saidman SL, Shaffer J, Preffer FI, Ding R, Sharma V, Fishman JA, Dey B, Ko DS, Hertl M, Goes NB, Wong W, Williams WW, Jr, Colvin RB, Sykes M, Sachs DH. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med. 2008;358:353–361. [PMC free article] [PubMed]

50. Scandling JD, Busque S, Dejbakhsh-Jones S, Benike C, Millan MT, Shizuru JA, Hoppe RT, Lowsky R, Engleman EG, Strober S. Tolerance and chimerism after renal and hematopoietic-cell transplantation. N Engl J Med. 2008;358:362–368. [PubMed]

51. Alexander SI, Smith N, Hu M, Verran D, Shun A, Dorney S, Smith A, Webster B, Shaw PJ, Lammi A, Stormon MO. Chimerism and tolerance in a recipient of a deceased-donor liver transplant. N Engl J Med. 2008;358:369–374. [PubMed]

52. Soiffer R. Immune modulation and chronic graft-versus-host disease. Bone Marrow Transplant. 2008;42(Suppl 1):S66–S69. [PubMed]

53. Li FX, Zhu JW, Tessem JS, Beilke J, Varella-Garcia M, Jensen J, Hogan CJ, DeGregori J. The development of diabetes in E2f1/E2f2 mutant mice reveals important roles for bone marrow-derived cells in preventing islet cell loss. Proceedings of the National Academy of Sciences of the United States of America. 2003;100:12935–12940. [PubMed]