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First published online March 15, 2007
Stem Cells Vol. 25 No. 6 June 2007, pp. 1448 -1455
doi:10.1634/stemcells.2007-0013; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Fas Ligand Enhances Hematopoietic Cell Engraftment Through Abrogation of Alloimmune Responses and Nonimmunogenic Interactions

Michal Pearl-Yafea, Esma S. Yolcub, Jerry Steina, Ofer Kaplanc, Isaac Yaniva, Haval Shirwanb, Nadir Askenasya

aFrankel Laboratory, Center for Stem Cell Research, Department of Pediatric Hematology-Oncology, Schneider Children's Medical Center of Israel, Petach Tikva, Israel;
bInstitute for Cellular Therapeutics and Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA;
cDepartment of Surgery, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Key Words. Adult stem cells • Apoptosis • Hematopoietic stem cell transplantation • Fas

Correspondence: Nadir Askenasy, M.D., Ph.D., Frankel Laboratory, Center for Stem Cell Research, Schneider Children's Medical Center of Israel, 14 Kaplan Street, Petach Tikva, Israel 49202. Telephone: 972-3921-3954; Fax: 972-3921-4156; e-mail: anadir{at}012.net.il

Received January 17, 2007; accepted for publication March 7, 2007.
First published online in STEM CELLS EXPRESS   March 15, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Early after transplantation, donor lineage-negative bone marrow cells (lin– BMC) constitutively upregulated their expression of Fas ligand (FasL), suggesting an involvement of the Fas/FasL axis in engraftment. Following the observation of impaired engraftment in the presence of a dysfunctional Fas/FasL axis in FasL-defective (gld) donors or Fas-defective (lpr) recipients, we expressed a noncleavable FasL chimeric protein on the surface of donor lin– BMC. Despite a short life span of the protein in vivo, expression of FasL on the surface of all the donor lin– BMC improved the efficiency of engraftment twofold. The FasL-coated donor cells efficiently blunted the host alloimmune responses in primary recipients and retained their hematopoietic reconstituting potential in secondary transplants. Surprisingly, FasL protein improved the efficiency of engraftment in syngeneic transplants. The deficient engraftment in lpr recipients was not reversed in chimeric mice with Fas– stroma and Fas+ BMC, demonstrating that the host marrow stroma was also a target of donor cell FasL. Hematopoietic stem and progenitor cells are insensitive to Fas-mediated apoptosis and thus can exploit the constitutive expression of FasL to exert potent veto activities in the early stages of engraftment. Manipulation of the donor cells using ectopic FasL protein accentuated the immunogenic and nonimmunogenic interactions between the donor cells and the host, alleviating the requirement for a megadose of transplanted cells to achieve a potent veto effect.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Activation-induced cell death (AICD) is crucial to the maintenance of immune homeostasis. One of the major regulatory mechanisms by which AICD occurs is the Fas receptor, the trimerization of which signals apoptosis and inhibits cell proliferation [1, 2]. Binding of the Fas ligand (FasL) to its cognate receptor controls the size of the pool of reactive immunocytes on the one hand [1, 2] and serves as an effector molecule of cytolytic functions on the other [3, 4]. Exploitation of this pathway in the transplant setting has been extensively explored as an immunomodulatory approach to prevent allograft rejection [5–7]. By physical elimination and inhibition of the proliferation of reactive immune cells, FasL disrupts the physiological chain of alloimmune response, improving graft survival. FasL overexpression in splenocytes, dendritic cells, bone marrow cells (BMC), and endothelium specifically counterattacks the reactive responses in allogeneic transplants [8–14]. Presentation of FasL in the context of alloantigens results in donor-specific anergy with preservation of responses to third-party antigens [10–13]. Conversely, graft-versus-host (GVH) effector cells can be specifically eliminated ex vivo upon stimulation of the donor T cells with host antigens in conjunction with FasL [15]. Recent evidence from our laboratory indicates that these beneficial effects achieved by FasL-mediated AICD may be mediated by the differential sensitivities of T regulatory and T effector cells to activation of this apoptotic pathway (A. Kaminitz, M.P.-Y, E.S.Y., J.S., O.K., I.Y., H.S., N.A., unpublished data).

Research on the role of the Fas/FasL axis in the context of hematopoietic stem and progenitor cell (HSPC) transplants has focused on the Fas-mediated activities of immune cells, either in support of HSPC engraftment or graft versus host disease [9–22]. For example, FasL-defective (gld) BMC have impaired engraftment potential as compared with wild-type cells, resulting in mixed chimerism (survival of residual host cells) [16, 20]. This engraftment deficiency can be overcome by the use of more aggressive ablative pretransplant conditioning [21]. Engraftment was similarly impaired in Fas-defective (lpr) recipients [22, 23], and induction of effective tolerance required the presence of a functional Fas receptor in the recipient [18]. These data suggest that physiological FasL expression by donor cells plays a role in engraftment through interaction with the Fas+ cells in the host.

We recently found that the Fas receptor and its ligand are coexpressed by murine HSPC in the early phases of homing to the bone marrow, suggesting that this molecular interaction plays a role in early engraftment (submitted manuscript). As opposed to reports that the Fas receptor suppresses donor cell activity [24–30], we found that Fas receptor expression and its activation did not accentuate the apoptotic pathway in HSPC. The insensitivity of the donor HSPC to Fas-mediated apoptosis questioned the role of FasL in hematopoietic cell engraftment. There is evidence that hematopoietic stem and progenitor cells exert a veto effect that suppresses alloreactive responses in vivo and in vitro in a manner that is analogous to the inhibitory effect of immunocytes described above [31–33]. The mechanism of suppression appears to involve deletion of host alloresponsive cells rather than anergy of the host alloreactive responses and involves members of the tumor necrosis factor superfamily [34]. Consistent with these data, induced expression of noncleavable FasL in donor HSPC and dendritic cells improved engraftment efficiency and induced donor antigen-specific immune nonresponsiveness [35, 36].

In this study we characterized the functional involvement of FasL in HSPC engraftment. The upregulation of Fas and FasL expression on bone marrow-homed donor HSPC indicated that this molecular interaction is relevant to the very early stages of engraftment. Therefore, we used a FasL protein that has a very short lifetime in vivo [13, 37] and is diluted during the differentiation myeloid progenitors that may generate immunogenic activities [38, 39]. The protein aggregates spontaneously to deliver potent apoptotic signals through trimerization of the Fas receptor and also may be adhered to the surface of cells via biotinylation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Animal Preparation and Transplantation
Mice used in this study were C57Bl/6J (B6, H2Kb, CD45.2), B6.SJL-Ptprca Pepcb/BoyJ (H2Kb, CD45.1), B6.MRL-Fas-lpr/J (lpr, H2Kb, CD45.2), B6Smn.C3-Fasl-gld/J (gld, H2Kb), BALB/c (H2kd), B10.BR-H2k-H2-T18a/SgSnJJrep (C57Bl/BR, H2Kk), and C57BL/6-TgN(ACTbEGFP)1Osb (GFP, H2kb), purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). The mice were housed in a barrier facility. All of the procedures were approved by the Institutional Animal Care Committee. Recipients were conditioned using total body irradiation delivered by an x-ray irradiator (Rad Source 2000; Rad Source Technologies Inc., Alpharetta, GA, http://www.radsource.com) at a rate of 106 rad/minute. Irradiation was routinely performed 18–24 hours before transplantation. In all experiments, sublethal irradiation and lethal irradiation were administered at 850 rad and 950 rad total body irradiation (TBI), respectively. Donor cells were suspended in 0.2 ml of phosphate-buffered saline (PBS) and infused into the lateral tail vein. In secondary transplants, half of the cellular content of one femur was transplanted into irradiated recipients.

Cell Isolation, Characterization, and Staining
Whole BMC were harvested from femurs and tibia of donors, and low-density cells were collected as previously described [13, 40–42]. For immunomagnetic separation of lineage-negative (lin–) BMC, the cells were incubated for 45 minutes at 4°C with saturating amounts of biotinylated anti-mouse monoclonal antibodies (mAb) specific for CD5, B220, TER-119, Mac-1, Gr-1, and NK1.1. All antibodies were obtained from hybridoma cell cultures, except Ter-119 and NK1.1 (eBioscience, Santiago, CA, http://www.ebioscience.com). mAb-coated cells were washed twice with PBS containing 2% fetal calf serum and were incubated with sheep-anti-rat IgG conjugated to M-450 magnetic beads at a ratio of four beads per cell (Dynal Inc., Lake Success, NY, www.invitrogen.com). The lineage-positive (lin+) cells conjugated to beads were precipitated by exposure to a magnetic field, and supernatant containing lin– BMC was collected. The efficiency of the lin– cell separation procedure was reassessed by flow cytometry using a cocktail of primary labeled mAb against the lineage markers listed above. The average yield of this procedure was 4%–5% lin– BMC, and the viability was >95% as determined by the trypan blue and propidium iodide exclusion. To achieve a higher degree of purity (>95%), the immunomagnetic separation was repeated in some cases.

Flow Cytometry
Donor chimerism was determined from the percentage of donor and host peripheral blood lymphocytes. Blood was collected in heparinized serum vials in 200 µl of M199 and centrifuged over 1.5 ml of lymphocyte separation medium (Cedar Lane, Hornby, ON, Canada, http://www.cedarlanelabs.com). Before analysis, red blood cells (RBC) were lyzed using ammonium chloride solution for 4 minutes at room temperature, and the process was arrested by addition of excess cold solution. The nucleated cells were incubated for 45 minutes at 4°C with phycoerythrin-anti-H2Kb (clone CTKb; Caltag Laboratories, Burlingame, CA, http://www.caltag.com) and fluorescein isothiocyanate-anti-H2Kd mAb (clone SF-1.1; BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml). Measurements were performed with a Vantage SE flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Positive staining was determined on a log scale and normalized with control cells stained with isotype control mAb. Cell death and apoptosis were determined in cells incubated with 5 mg/ml 7-aminoactinomycin-D and Annexin-V (IQ-Products, Groningen, The Netherlands, http://www.iqproducts.nl). The Fas receptor was identified with a primary labeled antibody (eBioscience), and FasL was stained with a biotinylated MFL4 mAb (BD Pharmingen) and fluorochrome-labeled streptavidin (eBioscience).

Adsorption of FasL Protein on the Surface of Cells
Nucleated BMC and splenocytes harvested under aseptic conditions were suspended in 5 µM freshly prepared EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL, http://www.piercenet.com) in PBS for 30 minutes at room temperature [13, 37]. After washing twice with PBS, the cells were incubated with streptavidin-FasL chimeric protein (100 ng of protein per 1 x 106 cells in PBS). After two washes, the efficiency of adsorption was evaluated by flow cytometry using anti-streptavidin and anti-FasL antibodies.

Apoptotic Challenge Using FasL Protein
A20 murine lymphoblastoma cells and splenocytes served as controls for the apoptotic activity of the chimeric protein. Bone marrow cells were incubated (5 x 106 cells per milliliter) for 24 hours in {alpha}-minimal essential medium culture medium supplemented with StemPro Nutrient Supplement (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), 2 mM L-glutamine, 50 µM 2β-mercaptoethanol (ME), 10 ng/ml stem cell factor, and 100 ng/ml thrombopoietin. All factors were purchased from Peprotech (Rocky Hill, NJ, http://www.peprotech.com). The cells were challenged by addition of 75–250 ng/ml streptavidin-FasL chimeric protein for 18–24 hours. Apoptosis and death were monitored by flow cytometry.

Mixed Lymphocyte Reaction
Splenocytes were labeled with 2.5 µM of the intracellular dye 5-(and-6-)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Carlsbad, CA, http://probes.invitrogen.com), and 50 x 106 cells were plated on Petri dishes for 45 minutes to enrich for lymphocytes. After 45 minutes, nonadherent cells were collected, washed, and incubated with irradiated (2,000 rad) stimulator splenocytes in Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 13.6 µM folic acid, 270 µM L-asparagine, 548 µM L-arginine, 10 mM HEPES, 50 µM 2-ME, 100 mg/ml streptomycin, 100 U/ml penicillin, 5% heat-inactivated fetal bovine serum, and 1% heat-inactivated mouse serum. All of the ingredients were purchased from Biological Industries (Kibbutz Beit Haemek, Israel, http://www.bioind.com) and Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com). Controls included irradiated syngeneic and third party cells. Cultures were harvested after a 5 days incubation period, and the dilution of CFSE was analyzed by flow cytometry by gating on the live lymphocytes. Data were analyzed using CellQuest software (Becton Dickinson). All mixed lymphocyte reaction (MLR) assays were performed in triplicates and are representative of a minimum of three animals per group.

Organ Harvesting
Spleen, lung, and liver were harvested after intracardiac perfusion of 30 ml of cold PBS containing 100 units of heparin. The tissues were sectioned into pieces and processed; the lung was digested in 380 U/ml collagenase type V (Sigma) for 60 minutes at 37°C, and the liver was digested in 1,500 U/ml collagenase for 20 minutes at 37°C. All tissues, including spleen, were filtered over a 40-µm mesh, and cell suspensions were washed twice with PBS.

Long-Term Culture of Bone Marrow Stroma
B6.lpr mice irradiated with 850 rad TBI were transplanted with 107 whole BMC from wild-type syngeneic mice (CD45.1->CD45.2). After 3 weeks, donor chimerism was measured in peripheral blood, and the BMC were harvested from transplanted and naïve mice (control). Following RBC lysis, the cells were plated at a density of 106 cells per cm2 in murine mesenchymal medium (Stem Cell Technologies) supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. The medium was replaced twice per week to eliminate the nonadherent cells, and the culture was maintained until attaining confluency (approximately 3 weeks). The adherent cells were then detached by trypsinization and stained with rat anti-mouse mAb against CD11b, CD45.1, and CD45.2 (eBioscience). The donor (CD45.1) and host (CD45.2) origin of the cultured stromal cells was determined in flow cytometry by gating out the CD11C+ cells.

Statistical Analysis
Data are presented as means ± SD for each experimental protocol. Results in each experimental group were evaluated for reproducibility by linear regression of duplicate measurements. Differences between the experimental protocols were estimated with a post hoc Scheffe t test, and significance was considered at p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The Fas Receptor and Ligand Play a Role in Hematopoietic Cell Engraftment
Two days after transplantation of allogeneic lin– BMC into irradiated hosts (H2Kd->H2Kb), the bone marrow-homed cells were harvested and analyzed for the expression of Fas and FasL. Whereas the residual BMC of the host displayed a moderate upregulation in the expression of these molecules, the donor cells showed a remarkable upregulation of Fas and FasL within hours after homing to the bone marrow (Fig. 1A). Approximately 20% of the transplanted cells displayed lineage markers during the first 2 days after transplantation, and approximately 75% of these cells displayed the Fas receptor and ligand. Fas and FasL expression was also pronounced in the lin– subset of bone marrow-homed donor cells. The constitutive joint expression of the Fas receptor and ligand was likely the result of the skewed cytokine environment in the irradiated bone marrow and upregulation of these molecules by cycling bone marrow cells [24–30]. To determine whether the expression of FasL by the donor cells was of physiological significance, lin– BMC were transplanted into wild-type and Fas-defective (lpr) allogeneic hosts (Fig. 1B). The deficient engraftment in lpr recipients (p < .05) suggested a role for this molecular interaction in the process of hematopoietic cell engraftment.


Figure 1
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  		Figure 1. FasL plays a role in allogeneic cell engraftment. (A): Mice conditioned with 850 rad total body irradiation (TBI) were injected with 107 allogeneic lin– bone marrow cells (BMC) (H2Kd->H2Kb). After 48 hours, the bone marrow of the recipient mice was harvested, and the expression of Fas and FasL were determined in reference to the donor-host origin and lineage marker expression (n = 8). (B): Wild-type and lpr mice conditioned with 850 rad TBI were transplanted with 2 x 106 lin– BMC from BALB/c donors. The levels of donor chimerism were measured in the peripheral blood at 3 weeks post-transplantation (n = 6). (C): Radiation-conditioned recipients (850 rad) were injected with 106 naïve lin– BMC in conjunction with 106 lin– BMC or splenocytes coated with FasL protein and a control group transplanted with 2 x 106 naïve lin– BMC (n = 5). The levels of donor chimerism determined in the peripheral blood at 3 weeks post transplantation. Abbreviation: FasL, Fas ligand; spl, splenocytes; X, 106 naïve lin– bone marrow cells.

 
Ectopic Expression of FasL Enhances the Engraftment of Allogeneic Hematopoietic Cells
The enforced expression of FasL in bone marrow and dendritic cells improves the efficiency of engraftment [35, 36]. We expressed the FasL chimeric protein on the surface of splenocytes and lin– BMC via biotinylation and transplanted these cells along with 106 naïve lin– BMC into irradiated allogeneic hosts (H2Kd->H2Kb). The levels of engraftment were significantly improved by expression of the FasL protein on the lin– BMC (p < .001) and were further improved by its expression on donor splenocytes (p < .001), as compared with a double number of naïve lin– BMC (Fig. 1C). These data suggested that inhibition of the alloresponses was beneficial to donor hematopoietic cell engraftment. However, it remained unclear whether the engrafted cells were within the naïve or the FasL-coated subsets of lin– BMC.

To determine whether expression of FasL on all the grafted lin– BMC cells would impact engraftment, the protein was adsorbed on the surface of donor cells with almost absolute efficiency (Fig. 2A). Transplantation of 1.5 x 106 FasL-decorated allogeneic lin– BMC resulted in superior levels (p < .001) of donor hematopoietic chimerism at 3 weeks as compared with unmodified cells (Fig. 2B). All mice proceeded to develop full chimerism at 16 weeks post-transplantation, indicating that transient display of the ectopic protein did not influence the eventual establishment of durable hematopoietic chimerism (Fig. 2C). To test whether the improved early engraftment did not cause extinction of the stem cells, we performed secondary transplants [43, 44]. Transplantation of lin– BMC from the full chimeras into secondary myeloablated hosts showed no significant differences in engraftment (Fig. 2D). Thus, expression of the FasL protein on donor cells was well tolerated, improved their short-term engraftment, and did not impair their long-term hematopoietic reconstituting potential.


Figure 2
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  		Figure 2. Expression of FasL on all donor cells improves allogeneic cell engraftment. (A): Streptavidin-FasL chimeric protein was efficiently adsorbed on the surface of bone marrow cells (BMC) via biotinylation. The protein was detected with an anti-FasL monoclonal antibody in flow cytometry. (B): B6 recipients conditioned with 850 rad total body irradiation were injected with 106 naïve (n = 7) and FasL-decorated (n = 10) lin– BMC from BALB/c donors. Chimerism was determined in the peripheral blood by flow cytometry at 3 weeks post-transplantation. (C): Mice transplanted with unmanipulated and FasL-coated lin– BMC proceeded to develop full chimerism at 16 weeks post-transplantation (n = 10). (D): At 14 weeks after primary transplantation, the chimeric mice served as donors of whole BMC to secondary irradiated hosts (n = 5). Abbreviations: FasL, Fas ligand; PE, phycoerythrin.

 
Expression of FasL Protein Blocks Alloreactivity in a Fas-Dependent Manner
The likely mechanism in support of allogeneic cell engraftment is AICD-mediated inhibition of the alloresponses by the overexpressed FasL protein. To provide direct evidence for the immunoinhibitory role of FasL, cells coated with the protein were injected intraperitoneally into allogeneic hosts. The responses of recipient splenocytes were assayed after 7 days in a mixed lymphocyte reaction. FasL-decorated lin– BMC and splenocytes specifically blocked alloreactive responses, and the responses to third party antigens remained intact (Fig. 3A, 3B). FasL-decorated splenocytes were more effective than lin– BMC in their inhibition of alloreactive T-cell responses. This may be due to the prevalence of professional antigen-presenting cells in the spleen, capable of effectively activating alloreactive T cells that become sensitive to FasL-mediated killing through AICD [5–7, 35]. Taken together, these data demonstrated a very early involvement of FasL in the process of hematopoietic cell engraftment, which was in part mediated by systemic inhibition of the alloimmune responses against the graft. This was apparent in the superior homing of syngeneic versus allogeneic donor cells [42].


Figure 3
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  		Figure 3. FasL chimeric protein abrogates alloreactivity early after transplantation. (A): B6 mice (H2Kb) injected with 8 x 106 naïve or FasL-coated allogeneic lin– bone marrow cells (BMC) (H2Kd) were evaluated in a 5-day mixed lymphocyte reaction (MLR) assay at 7 days post-transplantation (n = 6). Splenocytes of B10.BR mice (H2Kk) served as third-party antigens. (B): The spleens of mice injected with 8 x 106 naïve or FasL-coated allogeneic splenocytes (H2Kd->H2Kb) were evaluated in MLR (n = 5). (C): Irradiated (2,000 rad) lin– BMC from green fluorescent protein mice coated with FasL chimeric protein were injected into allogeneic BALB/c hosts irradiated with 650 rad. Five days later, the organs were harvested, and the presence of FasL on the donor cells was assayed by flow cytometry (n = 5). Abbreviations: Allo, allogenic; FasL, Fas ligand; Spleno, splenocytes; Syn, syngeneic.

 
We determined the time frame of FasL protein activity on the surface of lin– BMC in view of the short survival of allogeneic splenocytes in vivo [13, 14, 37]. To avoid dilution of the protein by cell division, the donor lin– GFP+ BMC were decorated with FasL, irradiated, and then transplanted into sublethally irradiated allogeneic hosts (B6.GFP->BALB/c). Approximately 15% of the FasL-coated cells were recovered from the bone marrow, spleen, liver, and lung at 5 days after injection, and 30%–50% of these cells retained the FasL protein on their surface, as determined with an anti-streptavidin antibody (Fig. 3C). Loss of the protein from the surface of donor cells recovered from these organs was consistent with an apparent half-life time of approximately 4 days, suggesting that FasL is particularly important in the very early stages of hematopoietic cell engraftment.

Transient Display of Ectopic FasL Protein Improves Syngeneic Cell Engraftment
The veto activity of FasL-decorated lin– BMC [31, 34] may operate at the systemic level, where it prevents destruction of prehomed BMC by the host immune system, or in the bone marrow, where it may remove the residual host cells that survived irradiation. The residual host cells include immunocytes, HSPC, and bone marrow stroma. To determine whether the engraftment-facilitating advantage of cells expressing FasL in allogeneic hosts operated only through the abrogation of alloreactive responses (systemic and intra-bone marrow), we performed syngeneic transplants, where immune modulation would not be expected to be a relevant variable in the engraftment process. Surprisingly, decoration of BMC with FasL improved (p < .005) early engraftment in the syngeneic setting (Fig. 4A), and the mice proceeded to develop full donor chimerism at 16 weeks post-transplantation (Fig. 4B). These results reinforced the observation that engrafting BMC are apparently insensitive to FasL-induced apoptosis and that expression of this protein improves short-term engraftment without harming long-term repopulating cells. To test whether modulation of engraftment was attributed to specific effects of FasL, and not to secular effects of protein decoration on the surface of cells, we performed syngeneic transplants using FasL-defective gld donors. The engraftment of gld (FasL–) cells in wild-type recipients was deficient as compared with that of unprocessed wild-type (FasL+) and FasL-coated lin– BMC (Fig. 4C). These data showed that both the constitutive upregulation of FasL and its ectopic expression supported hematopoietic cell engraftment. Taken together, these results provided evidence that the Fas/FasL interaction played a nonimmunogenic role in the early stages of hematopoietic cell engraftment.


Figure 4
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  		Figure 4. FasL chimeric protein enhances syngeneic cell engraftment. (A): Mice conditioned with 850 rad total body irradiation were transplanted with 5 x 105 naïve and FasL-coated lin– BMC from syngeneic donors (CD45.1->CD45.2). Donor chimerism was measured in the peripheral blood at 3 weeks post-transplantation. (B): After syngeneic transplants, the short-term engraftment was superior in recipients of FasL-coated cells, and all the mice proceeded to develop full donor chimerism at 16 weeks post-transplantation (n = 11). (C): Wild-type recipients were transplanted with 5 x 105 syngeneic (CD45.2->CD45.1) lin– BMC from FasL-defective (gld) donors and from wild-type donors with and without FasL protein expression (n = 6). Abbreviations: BMC, bone marrow cells; FasL, Fas ligand.

 
The Primary Target of the Donor Cell FasL Is the Bone Marrow Stroma
To ascertain that the engraftment-facilitating effect of FasL operated through Fas signaling, we performed transplants of cells from wild-type mice into Fas-defective lpr recipients (n = 8). The engraftment advantage achieved by expression of the FasL protein on the surface of the cellular allografts was lost, indicating that the mechanism involved a competent Fas signaling pathway in the host (Fig. 5A). These results were further proof that the enhanced engraftment of FasL decorated cells was a specific effect of FasL and not an artifact of ex vivo cell manipulation. As expected, syngeneic transplants of unmanipulated and FasL-coated cells into lpr mice led to similar levels of engraftment (Fig. 5B).


Figure 5
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  		Figure 5. The primary target of donor cell FasL is the host marrow stroma. (A): B6/lpr recipients conditioned with 850 rad were injected with 1.5 x 106 naïve and FasL-coated lin– BMC from allogeneic donors (H2Kd->H2Kb). Chimerism was determined in the peripheral blood by flow cytometry at 3 weeks post-transplantation (n = 6). (B): Expression of FasL in 5 x 105 lin– BMC transplanted into syngeneic lpr recipients (CD45.1->CD45.2) had no significant effect on engraftment at 3 weeks (n = 6). (C): Transplantation of 107 whole BMC from GFP donors into myeloablated lpr recipients resulted in full donor (GFP+) chimerism in bone marrow at 6 weeks post-transplantation (n = 8). (D): Stromal cultures of the GFP/lpr chimeras were predominantly of the lpr (GFP–) host phenotype after gating out CD45+ and CD11c+ cells. The data are representative of cultures from five transplanted mice. (E): Full GFP/lpr chimeras (Fas+GFP+ BMC and Fas–GFP– stroma) served as recipients of naïve and FasL-coated lin– BMC from CD45.1 mice (n = 6). Chimerism was determined in the peripheral blood at 3 weeks post-transplantation. Abbreviations: BMC, bone marrow cells; FasL, Fas ligand; GFP, green fluorescent protein.

 
The evidence of a nonimmunogenic mechanism involved in the engraftment-supporting effect of FasL led us to seek the specific cellular targets of FasL-decorated cells in the host (stroma or residual host HSPC and immunocytes that survived irradiation). We generated a chimeric mouse with Fas– stroma and Fas+ hematopoietic cells. This mouse was created by transplantation of 107 whole BMC from GFP donors into myeloablated (950 rad TBI) lpr recipients. At 6 weeks post-transplantation, these mice displayed full donor chimerism in the peripheral blood and the bone marrow (Fig. 5C). To ascertain that the marrow stroma was of the lpr host phenotype, the marrow aspirate was plated in long-term cultures. The predominant phenotype of the cells that grew in culture was of the lpr host (GFP–) origin, after gating out the CD11c+ and CD45+ cells (Fig. 5D). Sublethally-irradiated (850 rad) chimeras served as recipients of a second transplant of syngeneic CD45.1 cells. The levels of hematopoietic chimerism were similar after transplantation of naïve and FasL-coated lin– BMC (Fig. 5E), similar to the loss of FasL-mediated engraftment advantage in lpr recipients (Fig. 5B). By elimination, these data indicated that the primary target of donor cell FasL was the mouse stroma, rather than veto activity on residual hematopoietic cells in the host bone marrow.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
The results of this study unravel the complex involvement of the Fas/FasL interaction in the early stages of hematopoietic stem and progenitor cell engraftment. The robust constitutive expression of the receptor and its ligand in donor cells suggested that this interaction is an important event in early engraftment. We recently demonstrated that the bone marrow-homed donor cells that upregulated Fas expression were insensitive to apoptosis mediated by this receptor (submitted manuscript). Thus, Fas was not involved in suppression of donor cell activity and viability, as had been previously reported [24–29]. The insensitivity of lineage-negative donor cells to Fas-mediated apoptosis allowed them to counterattack the host alloreactive immune cells using constitutively expressed FasL. This activity was markedly enhanced by enforced expression of the noncleavable FasL protein on the surface of cells. This engraftment advantage is mediated, at least in part, by FasL-mediated interaction between the donor cells and the host marrow stroma through nonimmunogenic mechanisms. Thus, FasL augmented hematopoietic cell engraftment by at least two distinct mechanisms.

The immunogenic mechanism of FasL-mediated effects involved counterattack of the host alloreactive responses by presentation of the donor antigens in conjunction with FasL as an apoptotic trigger [5–14]. As demonstrated by the blunted MLR responses of splenocytes to donor antigens presented in conjunction with FasL, this immunomodulatory approach was efficient in abrogation of the alloimmune responses at the systemic level. Our data are consistent with the deficit in engraftment in the absence of a functional Fas/FasL pathway [20–23], spontaneous recovery of host hematopoiesis after transplantation of FasL-defective cells [21], and the role attributed to this molecular interaction in transplant tolerance [16–18]. In parallel to the activity of the constitutively expressed FasL, the expression of an ectopic membrane-bound FasL chimeric protein amplified the process of AICD to induce peripheral tolerance to the grafted cells. Although the engraftment of wild-type cells was only moderately superior to that of Fas-defective cells, enforced expression of the FasL protein improved engraftment by 80%. Elaboration of a full immune response against the graft requires 2–3 days. By contrast, the half-life of the chimeric FasL protein on the surface of engrafting cells was apparently 4 days. This suggests that the Fas/FasL effect is a proximal event in the process of HSPC engraftment. An important feature of FasL-mediated immunomodulation is its antigen selectivity, which preserves intact responses to third-party antigens [12, 13, 35] and makes this approach attractive for clinical applications.

The early immunomodulation achieved by expression of FasL protein on the surface of HSPC in concert with the insensitivity of these cells to Fas-mediated apoptosis endows the hematopoietic progenitors themselves with a potent regulatory activity against the host alloreactive responses, as previously suggested [31–34]. The early timing of this veto activity preceded the eventual early evolution of donor-derived myeloid cells with immune capacity [38, 39]. Thus, it is the hematopoietic precursors themselves that are involved in presentation of the donor antigens, primarily the major histocompatibility antigens in the fully allogeneic transplants [34]. A higher immunosuppressive efficacy of splenocytes versus hematopoietic progenitors was likely caused by the superior number of professional antigen-presenting cells in the spleen [5, 6, 8–13, 35]. Although there is evidence that veto effects in selected conditions do not require the activity of perforin or FasL [45, 46], ligands of the tumor necrosis factor (TNF) family are involved in inhibitory activities in the immune system [3–7, 16–19, 34, 39]. The enhanced expression of a chimeric FasL protein on the surface of hematopoietic progenitors alleviated the need for large numbers of cells that use TNF-{alpha} and FasL to blunt immunogenicity and achieve transplant tolerance [31–33, 47]. These characteristics make the HSPC attractive targets for interventions aiming to achieve immunomodulatory effects [15].

In addition to potent immune regulatory activity, the donor cell FasL was also involved in nonimmunogenic interactions in the host bone marrow. We have no evidence to support the possibility that the donor cells removed residual host bone marrow cells to free niches for engraftment [48, 49]. The veto effect on residual host immune cells may have occurred in the bone marrow as well as at the systemic level; however, this was not a parameter that affected the efficiency of engraftment. Notably, the observed effects of FasL preceded in time the possible involvement of this molecular pathway in mediating GVH disease-induced bone marrow-hypoplasia [22]. Reconstitution of lpr mice with wild-type bone marrow cells created a chimera with Fas– stroma and Fas+ hematopoietic cells. In these chimeras, the engraftment deficit observed in the lpr mice was not restored. Thus, FasL apparently supported engraftment in the syngeneic setting by interaction with the marrow stroma. This interaction involves distinct mechanisms from the suppression of graft rejection in the allogeneic transplants.

Fas signaling appears to be involved in several functions of the marrow stroma that host engraftment and likely converges with additional cytokine-mediated signals in directing and regulating the activity of the grafted cells [48]. Although the stromal niche that hosts engraftment in the host bone marrow has been intensively studied, the information on the reciprocal influences of engrafting cells on the host marrow stroma is scarce [50]. We recently demonstrated that the expression of the Fas receptor and ligand on engrafting lin– BMC was specifically upregulated by the bone marrow as compared with other tissues, and that a brief incubation of hematopoietic progenitors in femoral preparations ex vivo was sufficient to upregulate these molecules (unpublished data). It is likely that the communication between the hematopoietic grafts and the marrow stroma includes multiple chemokines and cellular elements [51]. For example, adhesive interactions mediated by stroma-derived factor-1 and the cognate receptor CXCR4 induce the expression of TNF-{alpha} [51], a potent stimulator of Fas receptor expression [24–30]. Subsequently, activation of the Fas receptor and TNF-{alpha} induce the expression of the interferon-{gamma} (INF-{gamma}) receptor [52], and both TNF-{alpha} and INF-{gamma} upregulate the expression of the Fas receptor [24–30]. It is also possible that these complex stem cell-stroma interactions also involve cytokine-mediated communication with myeloid intermediates in the bone marrow [51]. The molecular mechanisms underlying the consequences of stromal Fas binding and the specific cellular targets should be scrutinized in mice deficient for expression of specific elements of these cytokine pathways. Nonapoptotic signaling through the Fas receptor [53] has been attributed to significant stimulatory roles in various cellular components of the bone marrow stroma, including fibroblasts, osteoclasts, and endothelial cells [54–58]. Recent data from our laboratory indicate that FasL stimulates the activity of stem cells and early progenitors under conditions of stress hematopoiesis and transplantation. These findings converge with information from other model systems that demonstrated a supportive effect of the Fas/FasL interaction in liver regeneration [59], and expression of FasL by virtually all the embryonic stem cells generated hematopoiesis in immunocompromised mice [60].

In summary, the proposed regulatory mechanisms mediated by FasL include abrogation of allorejection, interaction with the bone marrow stroma, apoptosis, and stimulatory signals in hematopoietic cells. The insensitivity of the donor cells and bone marrow cellular components of the host to Fas-mediated apoptosis make FasL an attractive approach to augment the success of stem cell transplants. The use of a protein with a limited life span in vivo markedly enhanced engraftment and alleviated the need of large numbers of donor cells to achieve efficient immunomodulation and hematopoietic reconstitution.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
H.S. and E.S.Y. own stock in, have acted as consultants to, have performed contract work for, served as officers or members of the Board for, and have financial interests in ApoImmune Inc. (Louisville, KY).


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This work was funded by Grants from the United States-Israel Binational Science Foundation (2003276 to N.A., I.Y., H.S., E.S.Y.), the Frankel Trust for Experimental Bone Marrow Transplantation (I.Y., J.S., N.A.), JDRF Innovative Grant 5-2005-1102 (to N.A.), ADA 1-05-JF-56, NIH R21: HL080108-01 (to E.S.Y.), and NIH R01:AI47864, NIH R21:AI057903 (to H.S.). The chimeric FasL protein technology used in this manuscript is licensed from the University of Louisville by ApoImmune Inc., Louisville, KY, for which H.S. serves as CSO and H.S. and E.S.Y. have significant equity in the company.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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M. Pearl-Yafe, J. Stein, E. S. Yolcu, D. L. Farkas, H. Shirwan, I. Yaniv, and N. Askenasy
Fas Transduces Dual Apoptotic and Trophic Signals in Hematopoietic Progenitors
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