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First published online December 1, 2005
Stem Cells Vol. 24 No. 4 April 2006, pp. 936 -948
doi:10.1634/stemcells.SC-05-0395; www.StemCells.com
© 2006 AlphaMed Press

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THE STEM CELL NICHE

Flt3-Ligand–Mobilized Peripheral Blood, but Not Flt3-Ligand–Expanded Bone Marrow, Facilitating Cells Promote Establishment of Chimerism and Tolerance

Yiming Huanga, Magda Kuciab, Francine Rezzouga, Janina Ratajczakb, Michael K. Tannera, Mariusz Z. Ratajczakb, Carrie L. Schaniea, Hong Xua, Isabelle Fugier-Viviera, Suzanne T. Ildstada

a Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky, USA;
b Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, USA

Key Words. Facilitating cells • Tolerance • Mobilization • CXCR4 • Stromal cell–derived factor-1

Correspondence: Suzanne T. Ildstad, M.D., Director, Institute for Cellular Therapeutics, University of Louisville, 570 S. Preston Street, Suite 404, Louisville, Kentucky 40202-1760, USA. Telephone: 502-852-2080; Fax: 502-852-2085; e-mail: suzanne.ildstad{at}louisville.edu

Received on August 16, 2005; accepted for publication on November 22, 2005.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 
Facilitating cells (CD8+/TCR–) (FCs) enhance engraftment of limiting numbers of hematopoietic stem cells (HSCs). The primary component of FCs is precursor-plasmacytoid dendritic cells (p-preDCs), a tolerogenic cell expanded by Flt3-ligand (FL). In this study, we evaluated the function and composition of FL-expanded FCs. FL treatment resulted in a significant increase of FCs in bone marrow (BM) and peripheral blood (PB). When FL-expanded FCs were transplanted with c-Kit+/Sca-1+/Lin– (KSL) cells into allogeneic recipients, BM-FCs exhibited significantly impaired function whereas PB-FCs were potently functional. A significant upregulation of P-selectin expression and downregulation of VCAM-1 (vascular cell adhesion molecule 1) were present on FL-expanded PB-FCs compared with FL BM-FCs. Stromal cell–derived factor-1 (SDF-1), and CXCR4 transcripts were significantly increased in FL PB-FCs and decreased in FL BM-FCs. Supernatant from FL PB-FCs primed HSC migration to SDF-1, confirming production of the protein product. The FL PB-FCs contained a predominance of p-preDCs and natural killer (NK)–FCs, and NK-FCs were lacking in FL BM-FCs. The impaired function for BM-FCs was restored within 5 days after cessation of treatment. Taken together, these data suggest that FCs may enhance HSC homing and migration via the SDF-1/CXCR4 axis and adhesion molecule modulation. These findings may have implications in development of strategies for retaining function of ex vivo manipulated FCs and HSCs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 
Bone marrow transplantation (BMT) offers great promise for the treatment of a number of chronic disease states, including autoimmunity, organ tolerance, and the hemoglobinopathies. However, the widespread clinical application of this approach is dependent upon the development of less toxic methods to establish chimerism and avoid graft-versus-host disease (GVHD). The search for cells with facilitative and tolerogenic potential has therefore been the focus of intense investigation. The facilitating cells (FCs) are a CD8+/TCR– bone marrow (BM) sub-population that enhance engraftment of purified hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD [1–3]. FCs also potently enhance engraftment of suboptimal numbers of HSCs in syngeneic recipients [4]. Notably, the predominant cell subpopulation in the FC gate resembles precursor-plasmacytoid dendritic cells (p-preDCs) in phenotype and function, and removal of p-preDC FCs completely abrogates the facilitating effect [5]. Approaches to expand FC numbers and enhance their function could have a significant impact upon the use of BMT in treating nonmalignant disorders, especially when HSC numbers are limiting, as in cord blood transplantation.

Hematopoietic growth factors have been used to significantly expand and mobilize BM cells. Flt3-ligand (FL) is a soluble growth factor that binds to the Flt3 receptor, which is restricted to cells of hematopoietic origin [6–8]. It plays an important role in the proliferation, differentiation, maintenance, and long-term reconstitution of HSCs [9–13]. When FL is administered to mice, HSCs and hematopoietic progenitors in BM and spleen are expanded and mobilized into the peripheral blood (PB) [14]. FL treatment of recipients also expands CD8-{alpha}+ p-preDCs and suppresses donor T-cell responses to host antigens, as evidenced by suppression of GVHD [15]. Mice lacking FL have a deficiency in hematopoiesis affecting hematopoietic progenitor cells, dendritic cells (DCs), and natural killer (NK) cells [16, 17]. The engraftment potential of BM cells from FL receptor–deficient mice is significantly impaired compared with wild-type marrow [17]. We previously reported that mice treated with FL alone or in combination with G-CSF show a significant expansion of FCs and HSCs in BM and PB [18]. Mobilized peripheral blood mononuclear cells engrafted more efficiently than untreated whole BM in limiting dilution studies [18]. Moreover, there was a striking disparity in engraftment ability of FL BM HSCs versus FL PB HSCs. Although phenotypically similar, FL-expanded BM-HSCs were significantly impaired in engraftment ability, whereas PB-HSCs were potently enhanced in function [19].

In the present studies, we evaluated FC function after FL mobilization. FL-expanded FCs from PB exhibited enhanced function, in striking contrast to BM-FCs, which were significantly impaired in function. Of note, the predominant cell types in FL PB-FCs were p-preDCs and NK-FCs, whereas NK-FCs were lacking in FL BM-FCs. Chimeras prepared with FL-expanded PB-FCs exhibited donor-specific transplantation tolerance to skin grafts and did not develop GVHD. In light of the fact that adhesion molecules play a major role in HSC homing and migration, especially after mobilization, we examined FL-expanded PB-FCs and BM-FCs for differences to explain the disparity in function between the two compartments. P-selectin expression was significantly upregulated on PB-FCs compared with BM-FCs, whereas cell surface expression of other adhesion molecules that influence homing and engraftment was decreased or unchanged. Strikingly, the enhanced engraftment potential of PB-FCs was associated with significant upregulation of transcripts for the chemokine stromal cell–derived factor-1 (SDF-1) and its receptor CXCR4 (nearly 10-fold) compared with normal FCs and FL-expanded BM-FCs. These data suggest a possible collaborative role for FCs in enhancing HSC homing and migration during mobilization and after transplantation. The findings herein will have important implications in development of novel cell-based strategies to immunomodulate the donor and recipient as well as in approaches for ex vivo manipulation of donor HSCs to potentiate engraftment and tolerance yet avoid GVHD.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosures
 References
 
Animals
Five- to 6-week-old male C57BL/10SnJ (B10; H-2b); B10.BR.SgSnJ (B10.BR; H-2k); C57BL/6 (B6; H-2b); C3H/HeJ (C3H; H-2k); and BALB/cJ (H-2d) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). Animals were housed in a barrier animal facility at the Institute for Cellular Therapeutics, University of Louisville, Louisville, KY, and cared for according to National Institutes of Health (NIH) animal care guidelines.

FL Treatment
Recombinant human FL (kindly provided by Amgen, Thousand Oaks, CA, http://www.amgen.com) was diluted in 0.1% mouse serum albumin in saline (Sigma, St. Louis, http://www.sigmaaldrich.com). Donor B10.BR mice were injected with 10 µg once daily subcutaneously from days 1 to 10. Control mice received saline only.

Monoclonal Antibodies
All monoclonal antibodies (mAbs) used in this study were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen). Stem cell sorting experiments used directly conjugated mAbs: stem cell antigen (Sca)-1-phycoerythrin (PE; E13–161.7), c-Kit allophycocyanin (APC; 2B8), CD8-{alpha} fluorescein isothiocyanate (fluorescein isothiocyanate [FITC]; 53–6.7), Mac-1 FITC (M1/70), B220 FITC (RA3–6B2), Gr-1 FITC (11–26c.2a), ß-TCR FITC (H57–597). FC sorting experiments used ß-TCR FITC (H57–597), {gamma}-{delta}-TCR FITC (GL3), and CD8-{alpha} PE (53–6.7). Analysis of the composition of subpopulations in sorted FCs use CD11c FITC (HL3) and B220 for DCs, CD11b FITC (M1/70) for myeloid/macrophages, NK1.1 FITC (PK136) plus Pan-NK cells FITC (DX5) for NK cells, and CD19 APC (1D3) for B cells. H-2Kk FITC (AF3–12.1) and H-2Kb PE (AF6–88.5) mAbs were used for assessment of chimerism.

c-Kit+/Sca-1+/lin– Cells and FC Sorting
c-Kit+/Sca-1+/lin– (KSL) HSCs were prepared as previously described [20]. Briefly, BM from the tibias and femurs of mice was reduced to a single-cell suspension. Cells were incubated with antibodies against lineage markers (CD8-{alpha} FITC, Mac-1 FITC, B220 FITC, Gr-1 FITC, ß-TCR FITC), anti-Sca-1 PE, and anti-c-Kit APC for 30 minutes and washed twice. Cells were resuspended in cell-sort media (1 x Hanks’ balanced saline solution, 25 mM HEPES, 50 µg/ml gentamicin, and 2% fetal bovine serum [FBS; Gibco, Grand Island, NY, http://www.invitrogen.com]) at 2.5 x 106 cells per ml, and HSCs were sorted by live sterile sorting (FACSVantage; Becton, Dickinson and Company, Mountain View, CA, http://www.bd.com). Sorts of less than 95% purity were not used.

CD8+/TCR– FCs were sorted as previously reported [20]. Mobilized PB cells were harvested by cardiac puncture from anesthetized live donors and collected into heparinized tubes. BM and PB were incubated with anti-CD8-{alpha} PE, anti-ß-TCR-FITC, anti-{gamma}-{delta}-TCR-FITC mAbs for 30 minutes and washed twice. Cells were resuspended in cell-sort media at 2.5 x 106 cells per ml.

Reconstitution of Allogeneic Recipients with KSL Cells Plus FCs Expanded from PB or BM
All KSL cells were sorted from untreated B10.BR or B6 mice. FCs were sorted from PB or BM of 10-day FL-treated mice or from the BM of untreated B10.BR or B6 mice as controls. The FCs and KSL cells were mixed prior to transplantation. Recipient B10 or C3H mice were conditioned with 950 cGy of total body irradiation (TBI) using a cesium source (Gamma-cell 40; MDS Nordion, Ottawa, Ontario, Canada, http://www.mds.nordion.com). Five thousand KSL cells was transplanted alone or in combination with 30,000 FCs by lateral tail vein injection at least 6 hours after conditioning.

Characterization of Chimeras by Flow Cytometry
Flow cytometry was performed monthly on PB to assess donor chimerism. Chimerism was determined by measuring the percentage of donor (B10.BR or B6)– or recipient (B10 or C3H)–derived major histocompatibility complex (MHC) class I+ cells. Briefly, whole blood from recipients was collected into heparinized tubes, and aliquots of 100 µl were stained with anti-H-2Kb-FITC and anti-H-2Kk-PE for 30 minutes on ice. RBCs were lysed with ammonium chloride lysing buffer for 5 minutes at room temperature, then washed twice in FACS (fluorescence-activated cell sorting) medium, and fixed in 1% paraformaldehyde. Multilineage chimerism was assessed by staining PB with anti-H-2Kk PE versus anti-CD4, CD8, ß-TCR, NK 1.1, anti-Gr-1, or Mac-1 FITC as previously described [21].

Skin Grafts
Skin grafts were performed by techniques previously published [22]. Briefly, full-thickness skin grafts from the tail of B10.BR, B10 and BALB/c mice were harvested. Graft beds were prepared on the lateral thoracic wall, carefully preserving the panniculus carnosum. Three skin grafts (syngeneic, donor, and third-party) were placed on each animal. Each graft was separated from the others by a skin bridge of at least 3 mm. Skin grafts were covered by a double layer of petroleum gauze and a cast. The cast was removed after 7 days. Grafts were scored daily for percentage of rejection. Rejection was defined as complete when no residual viable graft could be detected.

Flow Cytometric Analysis of Adhesion Molecule Expression
FCs sorted from FL-treated BM and PB were stained with anti-CD106 (vascular cell adhesion molecule 1 [VCAM-1]) FITC, anti-CD54 (intercellular adhesion molecule 1 [ICAM-1]) FITC, anti-CD102 (ICAM-2) FITC, anti-CD62E (E selectin) PE, anti-CD62L (L selectin) PE, anti-CD62P (P-selectin) PE, and anti-CD44 (Pgp-1) FITC mAbs. After staining, cells were analyzed on a FACSCalibur with CellQuest software (Becton, Dickinson and Company). Isotype-specific controls were analyzed on gated FCs.

Real-Time Reverse Transcription–Polymerase Chain Reaction Analysis for SDF-1 and CXCR4
To analyze SDF-1, CXCR4, and RANTES mRNA levels, total mRNA was isolated from FL-expanded BM-FCs, PB-FCs, or untreated BM-FCs with the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, http://www1.qiagen.com) and was reverse-transcribed with TaqMan Reverse Transcription Reagents (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com). Detection of SDF-1, CXCR4, RANTES, and ß2-microglobulin mRNA levels was performed by real-time reverse transcription–polymerase chain reaction (RT-PCR) assay using an ABI PRISM 7000 Sequence Detection System (Advanced Biotechnologies Inc., Columbia, MD). A 25-µl reaction mixture contains 12.5 µl of SYBR Green PCR Master Mix, 100 ng of cDNA template, 5'-CGT GAG GCC AGG GAA GAG T-3' forward and 5'-TGA TGA GCA TGG TGG GTT GA-3' reverse primers for SDF-1; 5'-GAC CGC CTT TAC CCC GAT AG-3' forward and 5'-GCA GGA CGA GAC CCA CCA T-3' reverse primers for CXCR4; 5'-GCA AGT GCT CCA ATC TTG CA-3' forward and 5'-CTT CTC TGG GTT GGC ACA CA-3' reverse primers for RANTES; and 5'-CAT ACG CCT GCA GAG TTA AGC A-3' forward and 5'-GAT CAC ATG TCT CGA TCC CAG TAG-3' reverse primers for ß2-microglobulin, and was compared with BM-FCs from untreated controls.

The threshold cycle (Ct) (i.e., the cycle number at which the amount of amplified gene of interest reached a fixed threshold) was determined subsequently. Relative quantitation of SDF-1, CXCR4, and RANTES mRNA expression was calculated with the comparative Ct method. The relative quantitation value of target, normalized to an endogenous control ß2-microglobulin gene and relative to a calibrator, is expressed as 2–{Delta}–{Delta}Ct (fold difference), where {Delta}Ct = Ct of target genes (SDF-1, CXCR4, and RANTES) – Ct of endogenous control gene (ß2-microglobulin), and {Delta}{Delta}Ct = {Delta}Ct of samples for target gene – {Delta}Ct of calibrator for the target gene.

SDF-1 Migration Assays
Migration of KSL cells to supernatant (SN) from FL-mobilized PB-FCs in the presence of an SDF-1 gradient was performed as previously described [23]. Briefly, KSL cells were loaded in the upper chamber, and SN that had been collected after overnight culture of sorted FCs was added to the upper chamber to detect priming by the SN.

Adhesion Assays
Confluent monolayers of BM stroma cells were established in 24-well plates and grown in Iscove’s modified Dulbecco’s medium (Gibco) supplemented with 12.5% horse serum and 12.5% FBS (Gibco) as previously described [24]. Sca-1+ BM cells were seeded on the stromal cell layers for 1 or 4 hours. Cells were harvested by trypsin digestion, washed, and resuspended in methylcellulose supplemented with murine interleukin (IL)-3 and GM-CSF (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). On day 7, the number of colony-forming unit–granuloctye-macrophage (CFU-GM) colonies was scored using an inverted microscope.

Colony-Forming Cell Assay
KSL cells were cultured at a 1:3 ratio with FCs from PB or BM from 10-day FL-treated animals in methylcellulose containing mouse growth factors (MethoCult GF M3434; StemCell Technologies) in duplicate at 37°C in 5% CO2 and humidified atmosphere [24]. After 14 days of culture, colonies containing more than 50 cells were scored.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosures
 References
 
FL Administration Expands FCs in PB and BM
We first evaluated the effect of FL administration on the expansion and mobilization of FCs in mice. B10.BR mice were treated with FL daily for 10 days, and the absolute number and proportion of FCs in BM and PB were evaluated. The maximum increase in FCs was observed after 10 days of FL treatment, as compared with control mice (Fig. 1Go). The absolute number, as well as relative proportion, of FCs in PB (Fig. 1A, 1BGo) and BM (Fig. 1C, 1DGo) was significantly increased on days 8 and 10 (p < .003), increasing by 100- and 8.5-fold, respectively, at the peak on day 10.


Figure 1
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  		Figure 1. Kinetics and composition of FL-expanded FCs in PB or BM. PB was obtained daily, and peripheral blood mononuclear cells (PBMCs) were counted. FCs (CD8+/TCR–) were analyzed by flow cytometry, and the absolute number of FCs per mouse after harvest from femur and tibia in BM was calculated based on the total number of BM cell counts (C) or FC cells per µl of PB; (A) was calculated based on PBMC counts, and percentages of FCs in BM and PB (D, B) were based on the lymphoid gate. The results represent two separate experiments (n = four animals per group). (E): Composition of FC total in FL-treated PB-FCs, FL-treated BM-FCs, and unmanipulated BM-FCs. FCs were sorted from unmanipulated BM (i), FL-treated PB (ii), and FL-treated BM (iii) and analyzed for the composition of subpopulations known to comprise the FC total population. Each experiment was repeated at least two times. This is one representative of six animals analyzed. Abbreviations: BM, bone marrow; FC, facilitating cell; FL, Flt3-ligand; PB, peripheral blood.

 
Composition of FC Total in FL-Expanded PB Versus BM
The CD8+/TCR– FC total is a heterogeneous subpopulation comprised predominantly of B220+/CD11c+/CD11b– cells that resemble p-preDCs in function and phenotype [5]. P-preDCs are a subpopulation of immature DCs shown to be tolerogenic under certain circumstances in vitro [25, 26]. We recently demonstrated that p-preDC FCs are the predominant functional facilitative subset in FC total, and removal of p-preDCs from the FC gate completely abrogates facilitation [5]. However, p-preDC FCs are not as effective at facilitation as FC total. P-preDCs are expanded and activated by FL, as are FCs. We therefore compared FL PB-FCs with FL BM-FCs to evaluate the FC total cellular composition (Fig. 1EGo). As expected, the number of p-preDC FCs was significantly increased in PB-FCs and, to a lesser extent, in BM-FCs after FL treatment. Notably, the number of NK FCs remained the same in PB-FC total and was nearly absent in the BM-FC population after FL treatment. In light of the fact that pre-DC FCs are not as potent as FC total, these data suggest that the NK-FC population may be an important collaborative cell in FC total function.

FL-Expanded PB-FCs, but not FL-Expanded BM-FCs, Enhance Engraftment of Purified Allogeneic KSL Cells
We previously reported that the engraftment potential of HSCs mobilized with FL plus G-CSF or FL alone was superior to HSCs obtained from animals treated with G-CSF alone and from normal BM [18]. The function of FL-expanded FCs has not previously been evaluated. We therefore evaluated the function of FL-expanded FCs in PB and BM. FCs were sorted from BM or PB of B10.BR mice after 10 days of treatment with FL. Recipient B10 mice were conditioned with 950 cGy of TBI and transplanted with 5,000 KSL cells from untreated B10.BR mice plus 30,000 FL-expanded FCs. Control B10 mice received 5,000 KSL cells alone or 5,000 KSL cells plus 30,000 BM-FCs from untreated B10.BR donors. FL-expanded PB-FCs significantly enhanced KSL cells engraftment (p = .005; Fig. 2AGo). In striking contrast, FL-expanded BM-FCs were significantly impaired in function, with only 14% engrafting. To exclude a strain-specific effect, similar transplants were carried out using the B6 and C3H strain combination, with similar outcomes (Fig. 2BGo). Therefore, although FL expands FCs in both BM and PB, the PB-FCs are significantly superior in function and the FL-expanded BM-FCs were impaired in facilitating KSL cells engraftment in allogeneic recipients (p = .005).


Figure 2
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  		Figure 2. Kaplan-Meier survival calculation of recipients of KSL cells and FL-expanded FCs from PB or BM. Donors were treated once daily with FL (10 µg/mouse per day) for 10 days. Recipients were conditioned with 950 cGy of total body irradiation and transplanted with 5,000 KSL cells and 30,000 FCs. Results are from a total of three experiments. The results from two different strain combinations are shown (B10.BR ->B10 [A]; B6->C3H [B]). There was a significant difference in survival between recipients of FL-expanded FCs obtained from PB versus BM for both data sets (p = .005). Abbreviations: BM, bone marrow; FC, facilitating cell; FL, Flt3-ligand; KSL, c-Kit+/Sca-1+/lin–; PB, peripheral blood.

 
Recipients of FL-Expanded PB-FCs Plus KSL Cells Exhibit Functional Tolerance to Donor-Specific Skin Allografts
To test whether the chimerism achieved with transplantation of KSL cells plus FL-expanded PB-FCs induced donor-specific tolerance, skin grafts from B10.BR (KSL cells donor), B10 (syngeneic), and BALB/c (MHC-disparate third-party) mice were performed. Donor-specific skin grafts were accepted by chimeras (median survival time [MST] ≥ 120 days), whereas third-party grafts were promptly rejected (MST = 15 days) (Fig. 3AGo). Recipients of FL-expanded PB-FCs were analyzed for multilineage production at 3 months. All recipients analyzed (n = 11) exhibited donor multilineage production (Fig. 3BGo).


Figure 3
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  		Figure 3. Facilitated chimeras exhibit tolerance and donor multilineage production. (A): Survival of skin grafts in mixed allogeneic chimeras (B10.BR->B10). Donor-specific (B10.BR), syngeneic (B10), and third-party (BALB/c) skin grafts were transplanted 3 months after c-Kit+/Sca-1+/lin– cell transplantation (n = 5). Grafts were followed daily. (B): Recipients were also analyzed for donor multilineage production at 3 months by flow cytometry. Staining was performed for CD4+, CD8+, and {alpha}-ß-TCR+ T cells, NK cells, B cells, macrophages, and granulocytes. This is one representative from a total of 11 animals analyzed. Abbreviations: NK, natural killer; TCR, T cell receptor.

 
FL Increases P-Selectin Expression and Downregulates VCAM-1 on PB-FCs
Adhesive interactions are critical to the regulation of hematopoiesis and the homing of stem cells after transplantation. A number of cell adhesion molecules have been identified that regulate homing and adhesion of HSCs to BM stroma [27–30], most notably, VCAM-1, ICAM-1, ICAM-2, E-selectin, P-selectin, and CD44. We therefore compared the difference in expression between the three sources of FCs: normal marrow FCs, FL-expanded BM-FCs, and FL-expanded PB-FCs (Fig. 4AGo) for VCAM-1, ICAM-1, ICAM-2, E-selectin, L-selectin, P-selectin, and CD44. CD8+/TCR– FCs were sorted and then stained for adhesion marker expansion. There was a significant (p = .015) increase in expression of P-selectin on FL-expanded PB-FCs compared with the other two groups. To a lesser extent, a larger proportion of FCs from PB expressed L-selectin compared with control BM-FCs and FL-expanded BM-FCs. The other molecules of interest were not significantly different between the groups except that VCAM-1 was decreased in FL-treated PB-FCs compared with BM-FCs and FL-treated BM-FCs.


Figure 4
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  		Figure 4. FL upregulates P-selectin expression on PB-FCs but not on BM-FCs. Representative histograms of PB- and BM-FCs sorted from FL-treated mice and stained with monoclonal antibody (mAb) anti-VCAM-1, ICAM-1, ICAM-2, E-selectin, L-selectin, P-selectin, and CD44. Expression of these adhesion molecules on gated PB-FCs and BM-FCs with corresponding isotype-specific control mAbs used as negative controls. Abbreviations: BM, bone marrow; FC, facilitating cell; FL, Flt3-ligand; ICAM, intercellular adhesion molecule; PB, peripheral blood; VCAM; vascular cell adhesion molecule.

 
FL-Expanded PB-FCs Exhibit Significantly Increased Levels of Transcripts for SDF-1 and CXCR4
SDF-1 plays an important role in HSC homing [31]. CXCR-4, the receptor for SDF-1, is expressed on HSCs and is believed to be the central mechanism by which HSCs migrate to the SDF-1–producing stroma after transplantation. To evaluate the mechanism for impaired FL-expanded BM-FC function and enhanced FL PB-FC function, we evaluated whether these molecules were present in the different FC populations in order to test the hypothesis that FC act as a collaborative cell and co-migrate with HSCs after transplantation and during mobilization. Strikingly, transcripts for SDF-1 and CXCR4 were significantly increased in FL-expanded PB-FCs compared with control FCs obtained from BM of untreated donors (p = .001; Fig. 5AGo). Moreover, the expression of these transcripts was significantly decreased in the less potently functional FL-expanded BM-FCs compared with control BM-FCs (p = .002) and FL-expanded PB-FCs.


Figure 5
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  		Figure 5. CXCR4 and SDF-1 are present in FCs. (A): Transcripts for SDF-1 and CXCR4 are upregulated in FL-expanded PB-FCs. Changes in expression of mRNA for SDF-1, CXCR4, and RANTES between FL-expanded PB-FCs or FL-expanded BM-FCs evaluated by real-time reverse transcription–polymerase chain reaction. The data are combined from two independent experiments. *, p = .001 or **, p = .002 compared with purified FCs from normal BM. (B): SDF-1 migration assays were performed to evaluate the effect of SN from FCs to enhance migration of KSL cells. FC-SN was added to the upper chamber with KSL cells, and migration to an SDF-1 gradient was performed. Data are the mean ± SD of three experiments. Abbreviations: BM, bone marrow; FC, facilitating cell; FL, Flt3-ligand; KSL, c-Kit+/Sca-1+/lin–; PB, peripheral blood; SDF-1, stromal cell–derived factor-1; SN, supernatant.

 
To examine whether FCs enhance or prime migration of HSCs to an SDF-1 gradient, SN from FCs collected after overnight culture was added to the upper chamber with KSL cells and migration assays were performed. Controls consisted of medium alone. Notably, FC SN significantly enhanced migration of KSL cells compared with controls, confirming the production of protein product of SDF-1 by FCs (Fig. 5BGo).

Effect of FL-Expanded FCs on HSC Adhesion
Another important step in the complex process of HSC engraftment after transplantation is adhesion to stroma in the hematopoietic microenvironment. Adhesion of HSCs to stroma cell monolayers is an in vitro correlate to this process [23, 32–34]. To evaluate whether the enhanced function of FL-expanded PB-FCs versus FL-expanded BM-FCs is due to a change in adhesive interactions between FCs and HSCs, sorted FCs were mixed with Sca-1+ BM cells and co-incubated overnight or placed immediately in adhesion assays. The collected cells were then placed in methylcellulose and CFU-GM colonies enumerated. There was no significant difference in CFU-GM between FL-expanded BM-FCs + Sca-1+ BM cells compared with FL-expanded PB-FCs + Sca-1+ BM cells when the cells were immediately subjected to the adhesion assay (Fig. 6AGo). Similarly, no difference was detected between the ability of the three sources of FCs to maintain clonogenicity after overnight culture followed by adhesion and CFU-GM colony enumeration (Fig. 6BGo). Taken together, these data suggest that the disparity in facilitative function between FL-expanded PB-FCs and FL-expanded BM-FCs is not due to a change in FC-mediated adhesion of HSCs to stroma.


Figure 6
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  		Figure 6. Adhesion and colony-forming cell (CFC) assays. (A): Sca-1+ BM cells were incubated for 1 or 4 hours on primary stroma. Adherent cells were then placed in methylcellulose assays and counted 7 days later. Data represent two experiments performed in triplicate (p > .01). Sca-1+ BM cells with or without FCs were immediately subjected to adhesion assay. Adherent cells were then placed in colony-forming unit–granuloctye-macrophageassays. (B): Sca-1+ BM cells were co-incubated with FCs overnight and then subjected to adhesion assay. Controls consisted of hematopoietic stem cells (HSCs) alone. (C): FCs were sorted from FL-treated donors from BM and PB and placed in CFCs with BM KSL cells from unmanipulated donors. Each experiment was performed in duplicate and repeated two times. All three FC populations enhanced HSC clonogenicity. Abbreviations: BM, bone marrow; FC, facilitating cell; FL, Flt3-ligand; KSL, c-Kit+/Sca-1+/lin–; PB, peripheral blood.

 
FL-Expanded PB-FCs and BM-FCs Increase HSC Clonogenicity
We recently observed that FCs increase HSC clonogenicity by exerting an anti-apoptotic effect on HSCs (Rezzoug, manuscript in preparation). Using the colony-forming cell (CFC) assay, we compared the impact of FL-expanded PB-FCs and FL-expanded BM-FCs on HSC colony formation. There was no significant difference between FL-expanded PB-FCs and FL-expanded BM-FCs compared with BM-FCs performed as controls, as cells from both compartments enhanced HSC clonogenicity (Fig. 6CGo).

The Function of BM-FCs Is Restored 5 Days After Cessation of FL Treatment
To evaluate the duration of impaired function of FL-expanded BM-FCs, B10.BR mice were treated with a 10-day course of FL. Five days after cessation of growth-factor treatment, B10 recipient mice were conditioned with 950 cGy of TBI and transplanted with 5,000 KSL cells from untreated B10.BR donors mixed with 30,000 FCs from BM from day 10 versus day 15. Significantly enhanced facilitation occurred in the recipients of FL-expanded BM-FCs harvested 5 days after cessation of FL treatment (p = .04) compared with FCs harvested on day 10 of FL treatment (Fig. 7Go).


Figure 7
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  		Figure 7. The function of BM-FCs is restored 5 days after cessation of treatment. (A): B10 recipient mice conditioned with 950 cGy of total body irradiation and given 5,000 allogeneic KSL cells from untreated B10.BR donors either mixed with 30,000 purified FCs from FL-treated BM or from BM 5 days after treatment ends. (B): A significant difference in survival was observed between recipients of FCs obtained from FL-expanded BM (n = 6) versus BM 5 days after treatment ends (n = 7; p = .04), suggesting that the changes that occur due to FL treatment are quite rapidly restored in the BM compartment. Abbreviations: BM, bone marrow; FC, facilitating cell; FL, Flt3-ligand; KSL, c-Kit+/Sca-1+/lin–.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosures
 References
 
BMT offers great therapeutic potential for the treatment of autoimmune disorders and the induction of tolerance to transplanted organs. However, the widespread clinical application of this approach is dependent upon the development of safe, non-toxic approaches to establish chimerism and avoid the complications of conventional BMT. Hematopoietic growth factors have been used to significantly expand HSCs and tolerogenic cells and induce their egress into the peripheral circulation. Notably, FL (and to a lesser extent G-CSF) enhance production of tolerogenic cells such as p-preDCs [35] and FCs [18], whereas GM-CSF does not [36, 37]. Additionally, these factors have been used for ex vivo expansion of HSCs when numbers are limiting. The strategic use of growth factors to expand HSCs and tolerogenic cells that facilitate engraftment may offer a novel approach to HSC-based therapies [38]. Although these mobilizing agents are increasingly used in the clinic, their mechanism of action remains to be fully defined. Moreover, the fact that after mobilization, HSCs must make their way back to the BM microenvironment suggests that release of HSCs and subsequent homing back to the hematopoietic microenvironment are sequential events with physiological roles. A clear definition of these mechanisms will allow the development of rationally designed approaches to activate and expand the cells of interest yet retain engraftment capability.

We previously demonstrated that treatment of mice with FL and G-CSF induced a synergistic expansion and mobilization of FCs and HSCs compared with either agent alone [18]). Strikingly, the function of HSCs was significantly influenced by the hematopoietic compartment from which they were obtained after FL treatment. We found that HSCs obtained from PB exhibited enhanced engraftment potential, whereas those from the BM were significantly compromised in function in vivo [19]. This change was associated with a change in cell cycle and phenotype of the HSCs in the BM compartment to committed progenitors as a result of the growth-factor treatment [19]. The upregulation of FL receptor expression on HSCs has been shown to be accompanied by loss of self-renewal capacity [39]. This finding therefore prompted us to address the function of mobilized FCs in the present studies.

Our data demonstrate that FCs can be significantly expanded in BM and mobilized into the PB using FL treatment, with a peak in production at 10 days. Notably, as we observed for HSCs, there was a significant disparity between phenotype and function when the FCs expanded in BM were compared with FL-expanded FCs from PB. Although phenotypically similar, FL-expanded BM-FCs were significantly impaired in function compared with unmanipulated BM-derived FCs. In striking contrast, FCs obtained from PB were functional as evidenced by excellent engraftment, long-term survival, and multilineage production. These findings are significant because they may have direct implications in design of clinical protocols using growth factors for ex vivo expansion of cells. To date, attempts at ex vivo expansion of primitive HSCs have been associated with loss of self-renewal and engraftment capability of HSCs in vivo for undefined mechanisms [40]. Our present findings suggest that approaches to enhance or preserve expression of chemokines and adhesion molecules critical to homing may overcome this limitation. Moreover, they suggest a cautionary approach for the timing for harvest of BM from the iliac crest in donors who do not mobilize well, because FCs as well as HSCs [19] that remain in the BM compartment are significantly impaired in function at the peak of mobilization. However, it is important to note that within 5 days of cessation of FL treatment, the BM-FCs were again functional. An understanding of the underlying mechanism for this disparity is important in the context of developing clinically applicable mobilization strategies.

Adhesive interactions are critical to the regulation of hematopoiesis [27, 28]. A number of adhesion molecules have been demonstrated to be involved in the mobilization process (reviewed in [41]). The localization of hematopoiesis to BM involves developmentally regulated adhesive interactions between primitive HSCs and the stromal cell–mediated hematopoietic microenvironment [29]. A wide variety of cell adhesion molecules representing several adhesion molecule superfamilies participate in the adhesion of hematopoietic cells to BM stromal cells and their associated extracellular matrix components [27]. Both P- and E-selectins are essential for the homing of hematopoietic cells to the BM [30]. Additionally, HSCs can acquire P-selectin from the microenvironment during mobilization. When we compared the expression of these adhesion molecules on FCs from PB versus BM after FL expansion, we found that the disparity in function of the FL-expanded PB-FCs compared with FL-expanded BM-FCs was associated with significant changes in expression of selected adhesion molecules. Notably, FL induced a significant increase in expression of P-selectin on expanded PB-FCs compared with BM-FCs. To a lesser extent, L-selectin was also increased. The levels of the other adhesion molecules known to play a role in HSC homing and migration, including ICAM-1, ICAM-2, E-selectin, and CD44, on PB-FCs were similar to the levels found in unmanipulated BM-FCs and FL-expanded BM-FCs. Notably, VCAM-1 was downregulated on FL-expanded PB-FCs. The downregulation of VCAM-1 on HSCs during mobilization is associated with release of HSCs from the microenvironment [42], and a similar decrease on FCs would be predicted. Therefore, one could hypothesize that the upregulation of P-selectin, and possibly L-selectin, combined with downregulation of VCAM-1 on FL PB-FCs may enhance mobilization and subsequent homing to the BM, suggesting that FCs serve as a chaperone cell for enhancing HSC migration. These findings may be clinically important because ex vivo manipulation of cell-based therapies to optimize outcome by enhancing expression of these molecules could allow enhanced potency as well as preserve in vivo function.

Trafficking of HSCs is regulated by chemokines as well as adhesion molecules [43–45]. The outcome of HSC transplantation is influenced by the ability of the cells to home and repopulate specialized BM niches. Crosstalk between HSCs and the microenvironment results in a series of highly regulated events involving interplay between chemokines, growth factors, proteolytic enzymes, and adhesion molecules [41, 46]. The chemokine SDF-1 and its receptor CXCR4 play central roles in HSC trafficking and repopulation, especially to vascularized niches [47, 48]. Downregulation of CXCR4 on HSCs is observed during mobilization [49]. Moreover, SDF-1 has also been shown to have an anti-apoptotic effect on HSCs [50, 51]. Notably, there was a significant (10-fold) upregulation of CXCR4 and SDF-1 transcripts in FL-expanded PB-FCs and downregulation of both in FL-expanded BM-FCs. In light of the fact that FCs themselves migrate to an SDF-1 gradient (manuscript in preparation) and of our present findings that SN from FCs enhances migration of HSCs to an SDF-1 gradient and that FCs enhance engraftment of limiting numbers of syngeneic HSCs [4], one could hypothesize that FCs act as a collaborative cell to chemoattract and chaperone HSCs to the hematopoietic microenvironment after transplantation. One could hypothesize a model in which the SDF-1 produced by the FCs would chemoattract the HSCs, and simultaneously exert an anti-apoptotic effect, and enhanced adhesion would increase the efficiency of the migration process after transplantation. These data complement our recent observation that enhanced green fluorescent protein–positive FCs persist in BM after allogeneic transplantation [20] and point to a possible direct mechanism for FC function on HSCs. Notably, FCs produce large quantities of inducible TNF-{alpha}, constitutively produce interferon-{gamma} and other factors such as IL-6 which maintain quiescence of primitive HSC [5] function via Bcl-3 to prevent apoptosis of HSCs in vitro, significantly enhance HSC clonogenicity in vitro (Rezzoug, manuscript in preparation), and enhance homing of HSCs in vivo in syngeneic recipients, all pointing to a collaborative role in enhancing migration and maintaining quiescence of HSCs during transplantation. The fact that adhesion as well as enhanced clonogenicity of FCs on HSCs is not significantly different whether the FCs are obtained from FL-treated PB versus BM would suggest that these properties of FCs are distinct and that additional mechanisms (i.e., the SDF-1/CXCR4 axis) are more important in this complex process of homing and migration after growth-factor treatment.

We have recently found that the predominant cell type in the FC total population resembles p-preDCs, a subset of DCs that can be tolerogenic in vitro via generation of regulatory T cells [52], in phenotype and function [5]. Importantly, removal of the p-preDC FCs from FC total completely abrogates facilitation in both the allogeneic and syngeneic models for facilitation. However, p-preDC FCs are significantly less potent biologically compared with FC total, suggesting that an additional FC cell or activation state is required for FC function [5]. Also contained within the BM-FC gate are NK cells, IgM+ CD19+ B cells, and monocytes. Notably, mobilized PB-FCs were composed almost exclusively of p-preDCs and NK-FCs. In contrast, NK-FCs were significantly decreased in the BM after FL treatment. It was recently reported that NK cells and immature DCs interact and reciprocally regulate each other via cellular "crosstalk" that requires direct cellular interactions in regulating innate and adaptive immune responses [53]. In light of the findings herein and the fact that p-preDCs do not produce IL-10 whereas FCs (and NK cells) do [5], we hypothesize the NK-FC subpopulation may similarly potentiate p-preDC FCs to contribute to the overall function of FC total. Studies are under way to test this hypothesis.

Chimeras prepared with FL-expanded PB-FCs exhibit donor-specific tolerance to skin grafts and do not develop GVHD. In fact, FCs were recently reported to themselves directly suppress GVHD via generation of foxp3-producing CD4+/CD25+ regulatory T cells [54]. The fact that FCs themselves suppress GVHD [54], as well as enhance engraftment of suboptimal numbers of syngeneic [4] and allogeneic HSCs [1], makes them a potentially attractive cell for establishing chimerism to induce tolerance. The strategic use of tolerogenic-promoting growth factors such as FL may allow a novel approach to induce tolerance with reduced toxicity. Moreover, FCs may be valuable as an approach to increase the success of ex vivo expansion of HSCs when numbers are limiting, given that maintenance of pluripotency after ex vivo expansion and maintenance of engraftment efficiency have been major limitations to date [40]. Our present findings underscore the importance of in vivo models in assessing function of growth-factor expanded cells. Although the enhanced clonogenicity of FCs on HSCs was similar for FL PB-FCs and FL BM-FCs, as was the adhesion, there was a significant disparity in facilitating function in vivo between the two populations. If we had used only in vitro assays, this disparity, and the other mechanistic differences we detected, would not have been identified.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosures
 References
 
FL treatment significantly expands FCs in PB and BM. However, although phenotypically similar, only the PB-FCs are functional. FL induces a significant upregulation of P-selectin expression and downregulation of VCAM-1 on PB-FCs, suggesting that these molecules may play an important role in the function of FCs to promote homing and engraftment of HSCs. FL mobilization is associated with a highly significant increase in CXCR4 and SDF-1 transcripts in FL-expanded PB-FCs. As we define which hematopoietic growth factors and critical collaborative cells enhance engraftment in vivo, novel strategies to immunomodulate the donor and/or recipient with these agents to reduce the risk of conventional conditioning and enhance HSC engraftment and tolerance will be possible.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 
We thank Drs. Christina L. Kaufman, Thomas C. Mitchell, and Zwi Berneman for helpful comments; Chris Perry, Barry Udis, Mary Jane Elliott, and Thomas Miller for technical assistance; Carolyn DeLautre for manuscript preparation; the staff of the animal facility of the University of Louisville for outstanding animal care; and Amgen for generously providing the growth factors. This research was supported in part by NIH RO1 HL63442, NIH R01 DK069766 [GenBank] -01A1, and NIH R01 HL076794-02, the Commonwealth of Kentucky Research Challenge Trust Fund, the Jewish Hospital Foundation, and the University of Louisville Hospital.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
References
 
The authors indicate no potential conflicts of interest.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 

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