Expression of Fas and Fas-ligand in donor hematopoietic stem and progenitor cells is dissociated from the sensitivity to apoptosis

Experimental Hematology 35 (2007) 1601–1612

Expression of Fas and Fas-ligand in donor hematopoietic stem and progenitor cells is dissociated from the sensitivity to apoptosis Michal Pearl-Yafea, Esma S. Yolcub, Jerry Steina, Ofer Kaplanc, Haval Shirwanb, Isaac Yaniva, and Nadir Askenasya a Frankel 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, Ky., USA; cDepartment of Surgery, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

(Received 3 May 2007; revised 2 July 2007; accepted 12 July 2007)

Objective. The interaction between the Fas receptor and its cognate ligand (FasL) has been implicated in the mutual suppression of donor and host hematopoietic cells after transplantation. Following the observation of deficient early engraftment of Fas and FasL-defective donor cells and recipients, we determined the role of the Fas–FasL interaction. Methods. Donor cells were recovered after syngeneic (CD45.1/CD45.2) transplants from various organs and assessed for expression of Fas/FasL in reference to lineage markers, carboxyfluorescein succinimidyl ester dilution, Sca-1 and c-kit expression. Naı¨ve and bone marrow–homed cells were challenged for apoptosis ex vivo. Results. The Fas receptor and ligand were markedly upregulated to 40% to 60% (p ! 0.001 vs 5–10% in naı¨ve cells) within 2 days after syngeneic transplantation, while residual host cells displayed modest and delayed upregulation of these molecules (w10%). All linLSca+c-kit+ cells were Fas+FasL+, including 95% of Sca-1+ and 30% of c-kit+ cells. Fas and FasL expression varied in donor cells that homed to bone marrow, spleen, liver and lung, and was induced by interaction with the stroma, irradiation, cell cycling, and differentiation. Bone marrow– homed donor cells challenged with supralethal doses of FasL were insensitive to apoptosis (3.2% ± 1% vs 38% ± 5% in naı¨ve bone marrow cells), and engraftment was not affected by pretransplantation exposure of donor cells to an apoptotic challenge with FasL. Conclusion. There was no evidence of Fas-mediated suppression of donor and host cell activity after transplantation. Resistance to Fas-mediated apoptosis evolves as a functional characteristic of hematopoietic reconstituting stem and progenitor cells, providing them competitive engraftment advantage over committed progenitors. Ó 2007 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Although hematopoietic stem cell transplantations have been performed successfully for several decades, the mechanisms of stem cell engraftment are not fully understood. In the process of preconditioning for transplantation, recipients often receive treatments that induce massive injury to the bone marrow, including both its cellular and stromal elements. Endogenous hematopoietic cells rarely survive myeloablative radiation. Donor hematopoietic stem and progenitor cells (HSPC) find their way to the host bone marrow, where they seed and engraft to reconstitute the Offprint requests to: 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; E-mail: anadir@012. net.il

hematopoietic system. In the early stages of engraftment, donor cells operate within a skewed cytokine environment that includes multiple apoptotic signals. Under physiological conditions, bone marrow cells express Fas at low levels [1–4], and are protected from apoptosis by a series of antiapoptotic factors, including FLIP, Bcl-2, survivin, and caspase-8L (an antiapoptotic splice variant of caspase-8) [4–8]. Stem and progenitor cells endowed with these protective factors guarantee maintenance of the important element in this developmental system. Along hematopoietic cell differentiation the receptor is expressed in proliferating and differentiating progenitors, serving as a negative regulator of distal differentiation in all lineages [9–14]. Such enhanced expression of Fas has been observed in cultured hematopoietic progenitors, and

0301-472X/07 $–see front matter. Copyright Ó 2007 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2007.07.010

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was associated with impaired viability and reduced clonogenesis [1–3,15–20]. It was assumed that excessive expression of Fas in HSPC exposed to injury signals after transplantation promotes execution of apoptosis in donor cells [21–24], and is involved in suppression of their activity [18,19]. For example, ex vivo incubated murine HSPC [18] and human CD34þ HSPC showed deficient homing to, and engraftment in, the murine bone marrow, in parallel with expression of the Fas receptor [20]. All these detrimental effects were efficiently induced by activating antiFas antibodies and were reversed by blocking anti-Fas antibodies and soluble Fas-ligand (FasL) [3,18–20,25,26]. Importantly, under selected conditions, soluble FasL acts as an antiapoptotic factor that competitively binds Fas without its activation [25–27]. The Fas receptor has been also attributed a role in suppression of host cell activity, and marrow hypoplasia caused by graft-vs-host disease was ameliorated by injection of FasL-defective cells [28]. In variance, a series of studies have suggested that the Fas–FasL interaction is not associated with impaired donor cell engraftment. Infection of hematopoietic progenitors with viral vectors encoding FasL resulted in improved short-term engraftment [29], suggesting that the infected cells were insensitive to this proapoptotic ligand. Similar results were recently observed after expression of a FasLchimeric protein on the surface of grafted cells, aiming to induce donor antigen-specific immune nonresponsiveness [30]. We recently showed that expression of ectopic FasL protein in donor lineage-negative bone marrow cells (lin BMC) alleviated the need for large numbers of cells (megadose) to overcome allogeneic barriers [31,32]. These immunogenic activities of FasL-expressing cells were similar to those obtained by expression of FasL in donor antigen-presenting cells [29,33] and splenocytes [34–36]. Insensitivity of hematopoietic progenitors to FasL-induced apoptosis was primarily attributed to low levels of Fas expression and high levels of antiapoptotic factors, such as FLIP [7]. Nevertheless, other studies demonstrated that the subset of engrafting human HSPC in severe combined immunodeficient mice was Fasþ [19], and donor cells upegulated FasL in human allogeneic transplants [17]. A quite unexpected result was the FasL-mediated improved engraftment of syngeneic donor cells, where antigen disparity is not expected to affect the outcome of transplantations [30]. The donor cell-FasL apparently targeted Fasþ stromal cells, because the engraftment deficit was not restored in lpr chimeras with Fasþ BMC and Fas-defective stroma. These data motivated further evaluation of the role of Fas and FasL in the transplant setting. In this study, we focused on the patterns of Fas and FasL expression in donor cells, and the physiological significance of this molecular interaction in the process of engraftment. The specific questions addressed here include: 1) do bone marrow–derived cells succumb to apoptosis upon encountering a hostile environment? 2) does the Fas

receptor mediate suppression of donor cell activity?, and c) do host and donor cells physically eliminate or inhibit each other? To answer these questions, we monitored the patterns of Fas receptor and ligand in grafted and host cells in a murine model of HSPC transplantation, and assessed their susceptibility to Fas-mediated apoptosis. A streptavidin-FasL chimeric protein that simulates the activity of membranous FasL due to streptavidin aggregation served as an apoptotic challenge [30,34–36]. We found that although bone marrow–homed donor cells acutely express Fas, activation of this receptor does not lead to suppression of donor and host cell activity. The grafted cells display a remarkable resistance to Fas-mediated apoptosis, a functional characteristic that is dissociated from Fas expression.

Materials and methods 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-Faslpr/J (lpr, H2Kb, CD45.2), B6Smn.C3-Tnfsf6gld/J (gld, H2kb, CD45.2) and C57BL/6-TgN(ACTbEGFP)1Osb (GFP, H2kb), purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed in a barrier facility, and all procedures were approved by the Institutional Animal Care Committee. Recipients were conditioned by sublethal (850 rad) and lethal (950 rad) total body irradiation using an x-ray irradiator (RadSource 2000, Alpharetta, GA, USA) at a rate of 106 rad/minute. Mice were routinely conditioned 18 to 24 hours before transplantation. Notably, x-ray irradiation is different from g-irradiation in myeloablative dose and toxicity [30]. For transplantation, cells suspended in 0.2 mL phosphate-buffered saline (PBS) were infused into the lateral tail vein.

Cell isolation, characterization, and staining Whole BMCs were harvested from femurs and tibia in PBS (Beit Haemek, Israel) in aseptic conditions. For immunomagnetic separation of lineage-negative (lin) BMC, cells were incubated for 45 minutes at 4 C with saturating amounts of rat 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, USA). Antibody-coated cells were washed twice with PBS containing 1% fetal calf serum (Biological Industries, Kibutz Haemek, Israel) and 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, USA). Unconjugated lin BMC were collected by exposure to a magnetic field, and the efficiency of separation was reassessed by flow cytometry using a cocktail of fluorescein isothiocyanate–labeled mAb against the lineage markers (eBioscience and BD Pharmingen, Erembodegem, Belgium). To achieve a higher degree of purity (O95%), the immunomagnetic separation was repeated in some cases. For staining with an intracellular dye, the cells were incubated for 20 minutes with 2.5 mM of 5-(and-6-)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR, USA), washed and resuspended.

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Apoptotic challenge using FasL protein The streptavidin-FasL chimeric protein was previously shown to transduce potent apoptotic signals to Fasþ cells [30,34–36]. Cells were incubated (5  106 cells/mL) for 24 hours in a-minimum essential medium supplemented with StemPro Nutrient Supplement (Stem Cell Technologies, Vancouver, BC), 2 mM L-glutamine, 50 mM 2b-ME. In some cases, the medium was supplemented with 10 ng/mL stem cell factor and 100 ng/mL thrombopoietin. All supplements were purchased from PeproTech (Rocky Hill, NJ, USA). Cells were challenged by addition of 75 to 250 ng/mL streptavidin-FasL chimeric protein for 18 to 24 hours, followed by flow cytometric analysis of apoptosis and death.

Semi-quantitative reverse transcription polymerase chain reaction Total RNA will be extracted from the cells using either EZ-RNA II extraction reagent or RNeasy mini columns (Qiagen, Hilden, Germany). RNA was used in the reverse transcription reaction along with pd(T)12-18 primers (30 cycles). The polymerase chain reaction step was performed using the following set of primer pairs: mouse FASdForward 50 GCCTTGGTTGTTGACCA, Reverse 50 GTACCAGCACAGGAGCA, generating a 300 bp fragment; mouse FAS-ligand – Forward 50 ACCGCCATCACAACCA, Reverse 50 TCAACCTCTTCTCCTCCA, generating a 500-bp fragment. Primers for b-actin were used as an internal control and normalization of expression.

Organ harvesting Spleen, lung, and liver were harvested after intracardiac perfusion of 30 mL cold PBS containing 100 U heparin. Tissues were sectioned into pieces and processed: the lung was digested in 380 U/mL collagenase type V (Sigma, St. Louis, MO, USA) for 60 minutes at 37 C, the liver was digested in 1500 U/mL collagenase for 20 minutes at 37 C. All tissues, including spleen, were filtered over a 40-mm mesh, and cell suspensions were washed twice with PBS.

Statistical analysis Data are presented as mean 6 standard deviations 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 ! 0.05.

Results Adsorption of FasL protein on the surface of cells Cells were suspended in 5 mM freshly prepared EZ-Link SulfoNHS-LC-Biotin (Pierce, Rockford, IL, USA) in PBS for 30 minutes at room temperature [30]. After two washes with PBS, cells were incubated with streptavidin-FasL chimeric protein (100 ng protein/106 cells) in PBS. Efficiency of adsorption was evaluated by flow cytometry using primary goat anti-streptavidin mAb (Zymed, San Francisco, CA, USA) counterstained with secondary porcine anti-goat IgG (R&D Systems, Minneapolis, MN, USA), and anti-FasL antibodies (clone MFL-4, BD Pharmingen).

Flow cytometry Measurements were performed with a Vantage SE flow cytometer (Becton Dickinson). Nucleated peripheral blood and BMC were isolated by centrifugation over a Ficoll gradient, according to the manufacturer’s instructions (Cedarlane, Ontario, Canada). Cells were washed in PBS, incubated for 45 minutes at 4 C with labeled primary mAb or counterstained with a fluorochrome-labeled secondary mAb. Donor chimerism in syngeneic transplants was determined from the percentage of donor and host peripheral blood lymphocytes using mAbs against minor antigens CD45.1 (clone A20, eBioscience) and CD45.2 (clone 104, eBioscience). Cell death and apoptosis was determined in cells incubated with 5 mg/mL 7-aminoactinomycin-D (7-AAD, Sigma) and Annexin-V (IQ products, Groningen, The Netherlands). The Fas receptor and FasL were identified with a primary labeled mAb, clone 15A7 (eBioscience), and clone MFL4 (BD Pharmingen), respectively. Cell surface markers of putative stem cells were identified as Sca-1 (clone D7, eBioscience) and c-kit (clone 2B8, eBioscience). Biotinylated antibodies were counterstained with streptavidin conjugated to fluorescein isothiocyanate, phycoerythrin, allophycocyanin and peridinin chlorophyll a protein (BD Pharmingen). Positive staining was determined on a log scale, normalized with control cells stained with isotype control antibodies.

Dynamic expression of Fas and FasL in grafted cells Changes in expression of the Fas receptor and its ligand in hematopoietic cells occur in response to multiple environmental factors and differentiation events [1–3,15–23]. The bone marrow stroma is the only microenvironment that provides a site for definitive engraftment of HSPC. We recently reported upregulation of the Fas receptor and its cognate ligand in transplanted cells that homed to the bone marrow of allogeneic recipients [30]. To test whether upregulation of Fas and FasL expression characterized only bone marrow–homed cells, we analyzed donor cells that homed to the various organs of syngeneic hosts (CD45.1/ CD45.2). Figure 1A presents measurements of Fas/FasL expression in bone marrow–homed cells at 24 hours after transplantation of lin BMC. The levels and time course of expression of these molecules varied in the different organs. Both the receptor and its ligand were upregulated in donor cells that homed to the bone marrow (Fig. 1A and B) and lung (Fig. 1C). In variance, the grafted cells that homed to the spleen (Fig. 1D) and liver (Fig. 1E) primarily upregulated their FasL expression. These molecules were transiently expressed in donor cells that homed to the lung and liver (to peak levels at 24 hours posttransplantation), whereas they were progressively expressed in bone marrow and spleen-homed cells. In parallel, radiation injury induced expression of these molecules in the parenchymal cells of these organs (Fig. 1B, C, D, and E). The concomitant upregulation in parenchymal and grafted cells in the various organs suggested that the expression of Fas and FasL was partially induced by factors (chemokines and cytokines) released as a result of radiation injury.

Figure 1. Patterns of Fas and FasL expression. Expression of Fas and FasL was assessed by flow cytometry after syngeneic transplants (CD45.1/CD45.2) of lin bone marrow cells (BMC) into irradiated mice (850 rad total body irradiation). (A, B) Using a gate for donor cells labeled with anti-CD45.1- phycoerythrin (PE) monoclonal antibodies (mAb) (filled pattern), Fas was measured with peridinin chlorophyll (PerCP)–labeled mAb and FasL with fluoresceinisothiocyanate (FITC)–labeled mAb. Residual host BMCs were similarly identified by anti-CD45.2 allophycocyanin mAb (bold line). Parenchymal cells in other tissues were identified as CD45.1-negative: (C) lung, (D) spleen, and (E) liver (n 5 5). Data represent mean 6 SD. *p ! 0.05. (F) Naı¨ve lin (96% pure) and whole BMC (wBMC) were assayed by reverse transcription polymerase chain reaction for presence of mRNA encoding Fas and FasL before transplantation. Data are representative of three independent experiments showing similar results.

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Nevertheless, other inductive stimuli of Fas and FasL expression varied in the different organs. Transplantation into nonirradiated hosts resulted in expression of these molecules in bone marrow–homed cells at levels of approximately 55% of those observed in irradiated hosts. To determine whether the increased expression of Fas and FasL was regulated at the level of RNA transcription, we performed a reverse transcription polymerase chain reaction analysis of the grafted cells (before transplantation). Nucleated whole BMCs expressed significant levels of mRNA encoding Fas and trace amounts of mRNA encoding FasL (Fig. 1F). mRNA transcripts for both molecules were undetectable in the lin BMC used for the transplants. Thus, the appearance of the Fas and FasL proteins on the grafted cells was a result of induced transcription and translation. Bone marrow–homed donor cells are not submitted to Fas-mediated suppression A series of previous studies demonstrated increased mortality of hematopoietic progenitors through Fas ligation under various conditions [3,16–18]. The remarkable upregulation in Fas-receptor expression suggested that donor cells became sensitive to apoptosis. To test this possibility, radiation-conditioned mice (850 rad) were transplanted with syngeneic lin BMC (CD45.1/CD45.2), and the bone marrow–homed cells were harvested from the femoral marrow after 2 days. These cells were exposed to an apoptotic challenge with the FasL protein in vitro in the absence of supporting chemokines and serum to enhance cell susceptibility to apoptosis (Fig. 2A). The FasL challenge revealed a remarkable resistance of the bone marrow resident cells to apoptosis (Fig. 2B). Among the residual host BMC that survived radiation, the resistance to FasL-induced apoptosis was expected, due to low levels of Fas expression. In variance, a significant fraction of the bone marrow–homed donor cells were positive for Fas at 48 hours posttransplantation. Measurements of apoptosis in reference to Fas and lineage marker expression (Fig. 2A) demonstrated that the majority of Fasþ bone marrow–homed donor cells were insensitive to FasL-induced apoptosis. To determine whether the expression of Fas and FasL and the resistance to Fas-mediated apoptosis were characteristics of the particular subset of bone marrow–homed cells, the same challenge was applied to naı¨ve BMC used for transplantation. Incubation of whole BMC with FasL protein resulted in apoptotic death of approximately 40% of the cells (Fig. 2C). The most sensitive subset was within the linþ cells, with relative low fractional death of the lin BMC. The high rates of FasL-induced apoptosis in whole BMC, of which only 5% were Fasþ at the onset of incubation, suggested that the receptor was upregulated in these cells during the incubation period. Measurements of Fas expression and annexin incorporation revealed that upregulation of the receptor in both lin and linþ subsets was

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enhanced by the presence of FasL in the medium (Fig. 2D). However, apoptosis occurred primarily in the linþ subset, and a significant fraction of the linFasþ BMC remained viable (p ! 0.001). Taken together, these data pointed to dissociation between Fas-receptor expression and sensitivity to apoptosis, which was significantly accentuated by the process of homing to the bone marrow. Depletion of FasL-sensitive cells does not abolish the hematopoietic reconstituting potential Next, we questioned whether the stem and progenitor cells responsible for durable and short-term hematopoietic reconstitution of myeloablated mice reside within the apoptosis-sensitive or apoptosis-resistant subsets of BMC. Nucleated BMC were preincubated with the FasL protein for 24 hours, and the 40% to 50% dead cells were eliminated by Ficoll centrifugation. Cell death was recorded primarily in granulocytes (GR-1þ) and erythroid progenitors (Ter-199þ). Transplantation of equal numbers of FasLincubated and fresh BMC (5  105) into sublethally conditioned (850 rad) syngeneic recipients (CD45.1/CD45.2) resulted in equivalent levels of donor chimerism at 3 weeks (26% 6 3% and 24% 6 4%, respectively). All recipients proceeded to develop full donor chimerism at 14 weeks posttransplantation. BMCs of the chimeras were used as donors to secondary myeloablated (950 rad) recipients (CD45.2). All secondary recipients (n 5 7) displayed full donor chimerism after 12 to 16 weeks. Taken together, these data indicated that both the short and long-term hematopoietic reconstituting cells were unaffected by brief exposure to the proapoptotic FasL protein. Assuming that the progenitors resided in the lin fraction, the same experiment was performed by exposing lin BMC to FasL before transplantation. Similar levels of chimerism (n 5 6) attained after transplantation of lin BMC incubated for 24 hours with medium (66% 6 5.8%) and with FasL protein (65% 6 4.1%) provided direct evidence of the insensitivity of progenitors to apoptosis. Physiological Fas activation in donor cells and host In the context of bone marrow transplantation there are several Fas/FasL–interactions: 1) autocrine donor Fas activation by FasL, 2) donor FasL with host Fas, 3) donor Fas with host FasL (Fig. 3A). To isolate these possible interactions we used Fas-defective (lpr) and FasL-defective (gld) donors and recipients. Syngeneic transplants (CD45.1/ CD45.2) were performed with 5  105 lin BMC and sublethal host irradiation (850 rad) to attain mixed chimerism (Fig. 3). The first general observation was the deficient short-term engraftment of both lpr and gld cells in wildtype (wt) recipients, and reciprocally of wt cells in both lpr and gld hosts (p ! 0.05). Expression of FasL protein on the surface of donor cells, previously shown to improve the engraftment of wt cells [30], restored the engraftment deficit (p ! 0.001) of gld cells in wt recipients (Fig. 3C).

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Figure 2. Resistance of hematopoietic reconstituting cells to Fas-mediated apoptosis. (A) Bone marrow (BM) cells of mice were harvested 2 days after syngeneic transplants (CD45.1/CD45.2), donor cells were identified with anti-CD45.1 phycoerythrin (PE) and were stained with a cocktail of allophycocyanin-labeled monoclonal antibody against lineage markers (GR-1, Mac-1, Ter119, CD5, B220). Cells were then submitted to an apoptotic challenge with FasL protein (250 ng/mL) for 18 hours (n 5 5). Cells incubated in (FasL-free) medium served as controls. Fas expression (anti-Fas–peridinin chlorophyll [PerCP]) and annexin-V incorporation (fluorescein isothiocyanate [FITC]) were measured by gating on the linþ and lin subsets of donor and host cells. (B) Summary of apoptotic death (annexin V) in BM resident cells at 48 hours after transplantation and ex vivo incubation with FasL protein (n 5 6). (C) Naı¨ve nucleated whole BM cells (wBMC) (5  106 cells/mL) were incubated for 18 hours in culture medium with and without 250 ng/mL FasL protein (n 5 5), to determine death and apoptosis in reference to lineage marker expression. (D) Apoptosis was measured in reference to Fas-receptor expression in naı¨ve nucleated wBMC after incubation with and without 250 ng/mL FasL protein (n 5 5).

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Figure 3. Engraftment of cells deficient in Fas and FasL. All recipients were conditioned with a sublethal dose of 850 rad and infused with 5  105 naı¨ve and FasL-coated syngeneic lin bone marrow cells (BMC) (CD45.1/CD45.2). Chimerism was determined in peripheral blood at 3 weeks posttransplantation. (A) Deficient engraftment of lpr cells and in gld recipients (n 5 8). (B) Deficient engraftment of lpr cells in wild-type (wt) recipients (n 5 8) and of wt cells in lpr recipients (n 5 10). (C) Deficient engraftment of gld cells in wt recipients (n 5 8) and of wt cells in gld recipients (n 5 11) was efficiently reversed by expression of FasL protein on the surface of donor cells (n 5 9).

A similar effect was observed when FasL-expressing wt cells were transplanted into gld recipients (p ! 0.001), suggesting donor FasL–host Fas interaction and apparent autocrine Fas/FasL activity in the engrafting cells. In all experimental groups, mice proceeded to develop full donor chimerism at 16 weeks posttransplantation. The integrated interpretation of these data pointed to a 20% to 25% deficient engraftment of cells lacking the Fas receptor and ligand, evidence of trophic activities of this molecular interaction in the proximal stages of hematopoietic cell engraftment.

Cellular factors that affect Fas-receptor expression Expression of the death receptors might be induced by division and early differentiation, as some donor cells engage in cycling early upon homing to the bone marrow [37–40]. Therefore, the expression of Fas/FasL was monitored in donor cells during the proximal seeding process in reference to lineage marker expression and CFSE dilution. The bone marrow was harvested at 48 hours after transplantation of O95% pure lin BMC, and donor cells were identified in reference to lineage marker expression (Fig. 4A). The expression of Fas and FasL was determined by gating

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Figure 4. Fas and FasL expression in cycling cells. Lin bone marrow cells (BMC) (O90% pure) were transplanted into irradiated syngeneic recipients (850 rad total body irradiation), and the bone marrow–homed cells were analyzed after 48 hours for lineage marker expression and carboxyfluorescein succinimidyl ester (CFSE) dilution. (A) Donor cells positive for phycoerythrin (PE)-labeled monoclonal antibodies (mAb) were analyzed for lineage marker expression using a cocktail of allophycocyanin (APC)-labeled antibodies against granulocytes (GR-1), macrophages (Mac-1), B lymphocytes (B220), T lymphocytes (CD5), erythroid precursors (Ter119), and natural killer cells (NK1.1). Fas and FasL were determined using peridinin chlorophyll (PerCP)-labeled mAb by gating on the donor lin (bold line) and linþ subsets (n 5 6). (B) The fraction of linþ BMC of donor origin increased early after transplantation. (C) Fas and FasL were upregulated in both lin and linþ subsets. (D) CFSE dilution was evident in all bone marrow–homed donor cells, and a fraction divided at fast rates (n 5 7). (E) Upregulation of Fas and FasL was evident in slow and fast cycling cells, most prominent in the latter. Tx 5 transplantation.

on the bone marrow–homed donor lin and linþ subsets. At this time point, approximately 15% to 20% of the donor cells expressed lineage markers (Fig. 4B). Expression of

both Fas and FasL was more accentuated in those bone marrow–homed cells that expressed lineage markers (p ! 0.001), indicating that one of the inductive factors was early

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differentiation of hematopoietic progenitors (Fig. 4C). Notably, the homing and seeding efficacy of lin BMC is w10-fold higher than that of linþ BMC [41]. Similarly, approximately one fourth of cells displayed significant CFSE dilution, indicative of cell division after homing (Fig. 4D). Both the Fas receptor and ligand were markedly upregulated primarily in dividing cells (p ! 0.01, Fig. 4E). Taken together, these data indicated that expression of the receptors was associated primarily with early cell cycling and differentiation upon homing to and seeding in the host bone marrow. Murine stem and progenitor cells fall largely within the subset phenotypically defined as linSca-1þc-kitþ [42,43]. To determine Fas and FasL expression in reference to the presence of these markers on the surface of bone marrow–homed cells, syngeneic transplants were performed with CFSEþlin BMC. Virtually all CFSEþlinSca-1þckitþ cells, which best correspond to the repopulating HSPC, were positive for Fas (Fig. 5A). Likewise, all phenotypically characterized HSPC expressed FasL at 48 hours posttransplantation (Fig. 5B).

Discussion In this study, we demonstrate that Fas-receptor expression is dissociated from the sensitivity to apoptosis in engrafting HSPC. Soon after transplantation, a fraction of donor cells that homed successfully to the host bone marrow displayed acute upregulation of Fas, and virtually all the putative HSPC characterized by linSca-1þc-kitþ were positive for this receptor. However, these cells were resistant to FasL-induced apoptosis, as could be expected for a subset of cells coexpressing FasL. The answers to the initial questions posed in this study are: 1) Fas-mediated apoptosis is infrequent in donor cells that seed in the marrow of irradiated hosts, 2) Fas receptor does not mediate suppression of donor cell activity, 3) donor and host cells that reside in the bone marrow do not physically eliminate each other. The parameter of physiological significance was resistance of the hematopoietic progenitors to Fas-mediated apoptosis. In the case of host cells, resistance to Fas-mediated apoptosis was due to the low levels of receptor expression. In the bone marrow–homed donor cells, the clear dissociation between Fas expression and sensitivity to apoptosis challenges the long-held concept that this receptor is involved in suppression of donor cell activity after transplantation [3,16–18,44,45]. We found no evidence of reciprocal physical repression between host and donor cells, both of which are insensitive to this apoptotic trigger. Our findings are consistent with the competitive model of hematopoietic reconstitution, where the progeny is equally distributed among the residual host and donor cells that co-reside in the bone marrow [46–49]. Instead of serving as a mechanism of donor–host competition, early upregulation of Fas may offer an engraftment advantage to the most primitive

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progenitors in the graft, which are insensitive to apoptosis. It is possible that the Fas/FasL axis is involved in prioritizing the engraftment of primitive precursors over cells positioned in distal stages of differentiation, which are sensitive to Fas-mediated apoptosis [9–14,25,26]. Not only that there was no evidence of Fas-mediated donor cell suppression, its expression was rather beneficial to the outcome of stem cell transplantations. The apparent normal hematopoiesis in Fas-defective (lpr) and FasLdefective (gld) mice, and the minor disturbances observed under stress conditions, indicate that absence of a functional Fas–FasL interaction is well-compensated throughout development [50]. However, mice deficient in these molecules, used either as donors or recipients, showed diminished engraftment as compared to the wild-type counterparts. The differences were not of magnitudes that suggest this molecular pair as a critical mechanism, but the contribution to engraftment may be significant under borderline conditions. Transplants using lpr and gld cells provide insights into several features of Fas–FasL involvement. Deficient engraftment was more pronounced in Fas-defective grafts (lpr/wt) than in Fas-defective recipients (wt/lpr). We have recently reported that ectopic expression of FasL protein on the surface of donor HSPC improved engraftment substantially, not only in allogeneic transplants through abrogation of alloimmune responses, but also in syngeneic transplants [30]. The apparent target of donor cell FasL was the stromal Fas rather than the residual host cells, which were negative for this receptor. However, ectopic expression of FasL protein obviated the role of donor cell FasL (gld/wt) and also of host FasL (wt/gld), suggesting that a trophic event in donor cells underlies differential engraftment. These data reinforce recent observations of stimulated clonogenicity of murine HSPC in culture by a proapoptotic isoform of FasL protein (unpublished data). Fas is a death receptor that integrates multiple environmental and intrinsic parameters in hematopoietic cells [1– 3,9–23]. Data presented here include information on some of the triggers and mechanisms that drive the upregulated expression of Fas and FasL. It is difficult to dissociate between intrinsic cellular and environmental factors that affect expression, because these signals are closely associated. The lin BMC used for transplantation lacked mRNA encoding for the Fas receptor and its ligand. Considering the relatively short lifetime of these molecules on the surface of cells in vivo, in particular FasL [27,51], sustained expression must have involved activation at the transcriptional and translational levels. An intrinsic mechanism that might be partially responsible for expression of both molecules is the transition in cell-cycle phase [1,15,18–21]. The marrow-seeded cellular grafts engage in cell cycle at variable rates, with a majority of cells remaining mitotically quiescent or slow cycling, and a minority of the cells commencing rapid proliferation and

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Figure 5. Expression of Fas and FasL in putative stem and progenitor cells. Lin carboxyfluorescein succinimidyl ester (CFSE)þ bone marrow cells were transplanted into irradiated syngeneic recipients (850 rad total body irradiation), and the bone marrow–homed donor cells were analyzed after 48 hours for lineage markers, Sca-1 and c-kit by gating on the CFSEþ cells (n 5 5). (A) All linSca-1þ and linSca-1þc-kitþ cells were positive for Fas, whereas only a fraction of linc-kitþ cells expressed the receptor (bold line). The control histogram presents Fas expression (B) Distribution of Fas and FasL expression before transplantation in cells expressing the candidate hematopoietic stem and progenitor cells markers Sca-1 and c-kit. Approximately 30% of the c-kitþ cells expressed Fas.

differentiation [37–40,48,49]. CFSE dilution and early expression of lineage markers were observed in 20% to 25% of the bone marrow–homed cells, and both events were associated with upregulation of Fas and FasL expression [52,53]. Differential cycling may be one of the reasons for the striking differences in the tempo and amount of Fas and FasL expression in the residual host cells and in the grafted cells. The residual host cells showed modest levels of Fas expression only 2 to 3 days after irradiation, much

less pronounced than the expression in donor cells. The modest and delayed upregulation of Fas in residual host BMC could be due to limited ability of the irradiated cells to perform transcriptional and translational tasks. Differences in the inductive stimuli delivered by various stromal microenvironments to the grafted cells were apparent from the gradual induction in the bone marrow and spleen, and a more acute and transient expression in the lung and liver. The differential stimulatory effects of these

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irradiated organs were also evident from the joint induction of Fas and FasL expression in the bone marrow and lung, and the preferential induction of FasL in the spleen and the liver. Tumor necrosis factor-a and interferon-g are candidate injury–associated factors that stimulate Fas expression in hematopoietic cells [15–18,21–24]. Thus, expression of Fas and FasL is an integrated consequence of a multitude of intrinsic and environmental triggers that jointly induced these molecules. If upregulation of FasL was meant to help the grafted cells to survive in their hostile environment, the liver is of particular interest because hepatocytes are generally sensitive to Fas-mediated apoptosis, while Fas engagement is beneficial to liver growth [54]. Based on the data presented here, we propose the following scenario for involvement of the Fas–FasL signaling pathway in the early stages of hematopoietic cell engraftment. Upon donor cell homing to the bone marrow, the inflammatory environment and the stroma induce expression of the receptor and ligand. This may be a physiological response of immune-hematopoietic cells upon encountering a hostile environment, or exposure to stress conditions. Expression of Fas converts the donor cells responsive to environmental factors that modulate their activity, influences that are rather supportive than detrimental to hematopoietic cell engraftment. Elimination of the more differentiated progenitors by apoptosis increases the engraftment chances of more primitive stem and progenitor cells. This may be considered as a mechanism of HSPC enrichment within the process of seeding and early engraftment, in continuation of an earlier enrichment process attributed to superior homing of HSPC as compared to committed progenitors [41,49]. Resistance to Fas-mediated apoptotic death evolves as a functional characteristic of those donor cells that engraft successfully. Several potential applications can be envisioned, based on insensitivity of hematopoietic reconstituting progenitors to Fas-mediated apoptosis. For example, targeted therapy can be used against apoptosis-sensitive hematopoietic malignancies to reduce tumor burden. The high homing efficiency of hematopoietic progenitors to the bone marrow [41] can be used to target Fas-sensitive intramarrow malignant cells using hematopoietic progenitors armed with FasL [31]. Due to the high toxicity of FasL, it should be preferentially used ex vivo to eliminate residual cancer cells in autologous transplants. A recent study showed the feasibility to eliminate graft-vs-host T-cell effectors by pretransplantation presentation of alloantigens in conjunction with FasL [55]. Our current data suggest that T cells need not be isolated before this procedure, and the entire cellular graft may be safely exposed to FasL. Acknowledgments 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

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(to J.S., I.Y.), American Heart Association Postdoctoral Fellowship (0120396B to E.S.Y.), NIH (R21 DK61333, R01 AI47864 to H.S.), and JDRF innovative grant #5-2005-1102 (to N.A.).

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