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Stem Cells 2003;21:200-207 www.StemCells.com
© 2003 AlphaMed Press

Cardiac Allograft Acceptance after Localized Bone Marrow Transplantation by Isolated Limb Perfusion in Nonmyeloablated Recipients

Nadir Askenasya, Esma S. Yoleukb, Haval Shirwanb, Zhiliang Wangc, Daniel L. Farkasd

a Frankel Laboratory of Bone Marrow Transplantation, Schneider Children’s Medical Center of Israel, Petach Tikva, Israel;
b Institute for Cellular Therapeutics, and Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA;
c The Starzl E. Thomas Transplantation Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA;
d Minimally Invasive Surgical Technologies Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA

Key Words. Bone marrow transplantation • Isolated limb perfusion • Hematopoietic chimerism • Heart grafts • Tolerance

Nadir Askenasy, M.D., Frankel Laboratory of Bone Marrow Transplantation, Center for Stem Cell Research, National Center of Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, 14 Kaplan Street, Petach Tikva 49202 Israel. Telephone: 972-3-641-1475; Fax: 972-3-641-1475; e-mail: anadir{at}012.net.il


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Donor-specific tolerance to cardiac grafts may be induced by hematopoietic chimerism. This study evaluates the potential of localized bone marrow transplantation (BMT) performed by isolated limb (IL) perfusion to induce tolerance to secondary cardiac grafts without myeloablative conditioning. BALB/c recipients (H2d) preconditioned with lethal and sublethal doses of busulfan were injected i.v. and IL with 107 whole bone marrow cells (wBMCs) from B10 donors (H2b). Two hours after IL infusion of PKH-labeled wBMCs into myeloablated hosts, there were few labeled cells in the host peripheral blood (p < 0.001 versus i.v.) and femurs of the infused limb contained 57% ± 7% PKH-labeled blasts (p < 0.001 versus 8% ± 0.6% after i.v.). Femurs of the noninfused limbs contained 60-70 PKH-labeled blasts (p < 0.001 versus i.v.-BMT) after 2 days and 47% ± 5% of 0.32 x 107 donor cells (p < 0.001 versus 78% ± 4% of 1.2 x 107 donor cells in infused femurs) after 4 weeks. The survival rates of myeloablated hosts were 90% and 80% after i.v. and IL infusion, respectively, and the chimeras had 78%-84% donor peripheral blood cells. In recipients conditioned with 35 mg/g busulfan, the levels of donor chimerism in peripheral blood were 33% ± 4% and 21% ± 4% at 3 weeks after i.v.- and IL-BMT, respectively. Transplantation of donor-matched (H2b) secondary vascularized hearts in these chimeras after 3 weeks resulted in graft survival for periods exceeding 8 weeks, while third-party (H2k) allografts were acutely rejected (p < 0.001 versus H2b). These data indicate that IL perfusion is a reliable alternative procedure for establishment of hematopoietic chimerism and donor-specific tolerance without myeloablative conditioning.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mixed hematopoietic chimerism is an efficient strategy for induction of donor-specific tolerance to secondary solid organs [1]. The use of bone marrow transplantation (BMT) in clinics is limited by the high morbidity and mortality rates associated with recipient preconditioning, graft-versus-host disease (GVHD), and failure of engraftment [2]. These aspects have been extensively studied in an attempt to minimize the toxicity and maximize the success of engraftment by design of nonlethal conditioning modalities and engineering of the donor inoculum [3–8]. Recent approaches of nonmyeloablative conditioning include a low dose of irradiation in conjunction with selective immunosuppression, such as T-cell depletion and inhibition of stimulatory and costimulatory pathways [5–9]. These strategies are particularly adequate for induction of tolerance in the clinical setting because, after simultaneous transplantation of organs and bone marrow cells (BMCs), the solid graft needs only transient protection prior to establishment of hematopoietic chimerism. Furthermore, mixed hematopoietic chimerism lowers the incidence of GVHD [3] and prevents chronic rejection [10], a major cause of graft failure that is refractory to immunosuppression [11].

Optimization of the site of hematopoietic cell injection is one of the variables recently considered within the effort to improve the outcome of BMT [12–14]. The conceptual advantages of BMC injection into the bone marrow (BM) or an isolated limb (IL) preparation include: A) direct injection of the cells to their destination eliminates the homing process, B) depletion of the antigen-disparate donor cells by the host immune system during systemic circulation is avoided [15], and C) introduction of the cells into their natural site of engraftment improves their competitiveness for vacant niches in the host BM [16]. In fact, injection of the cells into a smaller hematopoietic space creates a local megadose effect, shown to improve the efficiency of T-cell-depleted BMC (TCD-BMC) and stem cell engraftment across antigen barriers without myeloablative conditioning [17, 18]. Experimentally, there is evidence that infusion of cells into the BM and into the IL is associated with a lower incidence of GVHD and superior engraftment of TCD-BMCs [13, 14]. Assessment of the early fate of donor cells after intra-BM injection revealed systemic dissipation of the cells, suggesting that this procedure was very similar to infusion into a peripheral vein [14]. In contrast, vascular isolation of the limb and infusion of cells into the femoral artery resulted in initial localization of the cells in the perfused extremity. This study aims to determine whether localized BMT using the IL preparation induces hematopoietic chimerism and tolerance to cardiac allografts.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Preparation
B10 (C57Bl/10Sn, H2b), B10.BR (C57Bl/SgSn, H2k), and BALB/c (H2d) mice purchased from Jackson Laboratories (Bar Harbor, ME; http://www.jax.org) were housed in a pathogen-free facility. Recipients aged 8-10 weeks were anesthetized with Avertin (12-17 µg/g, i.p.; Sigma; St. Louis, MO; http://www.sigmaaldrich.com) and conditioned 36 hours before transplantation with busulfan (i.p.), an agent with a predominant cytoreductive activity [19–21]. Busulfan was dissolved in dimethyl sulfoxide at a concentration of 24 mg/ml and was diluted fivefold in water at 40°C before injection. Donor BMCs suspended in: A) 0.2 ml phosphate-buffered saline (PBS) were injected into the lateral tail vein (i.v.), and B) 2 ml Krebs-Henseleit physiological solution (KH) were infused into the femoral artery (IL). The Institutional Animal Care Committee of the University of Louisville approved all the procedures.

IL Perfusion
Blood flow to the limb was occluded with a tourniquet, and the femoral artery and vein were clamped and cannulated with 24-gauge catheters using a Leica GZ6 surgical stereoscope (Leica; Northvale, NJ; http://www.leicacamera.com), as previously described [14]. The femoral artery was perfused with KH [22] using a miniperistaltic pump (Instech; Plymouth Meeting, PA; http://www.instechlabs.com) at a rate of 0.2 ml/min in three stages: 5 minutes KH, 10 minutes donor cells in KH (2.5 x 107 cells/ml), and 10 minutes KH. Outflow from the femoral vein was collected into a container. The low infusion volume aimed to minimize extravasation and accumulation of cells in the soft tissues of the limb. Perfusion was performed within the minimum possible duration, usually 1 hour, to avoid endothelial permeabilization and injury to BM stroma due to ischemia. The two ends of the femoral artery were secured to a short segment of the 24-gauge catheter. The femoral vein was occluded after perfusion, diverting blood drainage from the limb to the deep veins. The limb was reperfused, usually after 60 minutes of vascular occlusion, by gradual release of the arterial clamp. Injury was assessed by measurements of creatine kinase (CK) activity in the peripheral blood of animals 30 minutes after surgery. CK activities were measured spectrophotometrically using a coupled hexokinase/glucose 6-phosphate dehydrogenase enzymatic system (procedure 45-UV, Sigma Diagnostics; St. Louis, MO; http://www.sigmaaldrich.com), as previously described [23].

Isolation of BMCs and PKH Staining
BMCs were harvested from femurs and tibiae crushed in Hank’s balanced salt solution (HBSS), suspended using an 18-gauge needle, filtered through a 30-µm sterile nylon mesh, collected by centrifugation (400 g for 10 minutes at 4°C), and resuspended in HBSS containing 2% fetal calf serum (FCS) (GIBCO; Grand Island, NY; http://www.invitrogen.com). RBC lysis was performed by incubation with ammonium chloride for 4 minutes at room temperature. For cellular labeling, 1 ml of 4 µM freshly prepared PKH67 was added to 2 x 107 cells/ml Diluent C. Samples were agitated at room temperature for 5 minutes, and staining was terminated by addition of 4 volumes HBSS containing 10% FCS. Cells were collected by centrifugation (400 g for 10 minutes at 4°C) and washed twice with HBSS. The average recovery of the staining procedure was 90%, with a viability of 95% as determined with the trypan blue exclusion test.

Tracing of PKH-Labeled BMCs
Samples of peripheral blood were collected by tail bleeding into heparinized serum vials in 200 µl HBSS, and after RBC lysis, the nucleated cells were collected by centrifugation (400 g for 10 minutes at 4°C). The fraction of PKH-labeled cells in peripheral blood was determined by flow cytometry (Coulter Elite; Miami, FL; http://www.beckman.com) on the blast gate. In some samples, low-density cells were collected by centrifugation over a gradient of lymphocyte separation medium (vide infra).

Characterization of Donor Chimerism
Percent donor cells in peripheral blood and femurs of the hosts were determined using monoclonal antibodies (mAbs): phycoerythrin-(PE)-labeled anti-H2b mAb for B10 donors and fluorescein isothiocyanate-(FITC)-anti-H2d mAb for BALB/c recipients (Pharmingen; San Diego, CA; http://www.pharmingen.com). Donor cells in the peripheral blood were assayed in blood samples collected in heparinized vials. Low-density cells were collected by centrifugation (1,000 g for 20 minutes at 4°C) over lymphocyte separation medium (1.087 g/ml; CedarLane; Hornby, ON, Canada; http://www.cedarlanelabs.com), washed twice with HBSS, and fixed with 0.5% paraformaldehyde. Analysis of donor cells was performed by flow cytometry on the lymphocyte and blast gates. In some cases, cells were labeled with FITC-anti-CD3 and FITC-anti-B220 mAbs to determine the percentage of donor T and B lymphocytes, respectively. For determination of Sca-1+ c-kit+lineage– stem cells in the host femurs, cells were isolated from harvested bones (as described above). Then, the cells were incubated with biotinylated anti-H2b mAb, washed twice with PBS containing 1% bovine serum albumin (BSA), and incubated with excess streptavidin conjugated to M-450 magnetic beads (Biotin Binder Kit; Dynal Inc.; Lake Success, NY; http://www.dynal.no). Rosetted donor cells were precipitated by exposure to a magnetic field, and the supernatant containing non-H2b cells was removed. Donor BMCs were resuspended and treated with DNase according to the manufacturer’s instructions (Dynal). These cells were incubated with PE-anti-Sca-1, allophycocyanin-(APC)-anti-c-kit, and a cocktail of FITC-labeled mAbs against lineage markers: CD5, GR-1, CD45R, Ly-76, T-cell receptor-(TCR)-{alpha}ß, and TCR-{gamma}{delta} (Pharmingen). Analysis of donor cells was performed by flow cytometry.

Heterotopic Heart Transplantation
Hearts were transplanted as primary vascularized grafts into naïve recipients and chimeras using a modified procedure for the mouse [24]. Briefly, hearts were harvested from donors euthanized by CO2 asphyxiation and cooled on ice throughout the surgical procedure. Recipient intra-abdominal aortae and inferior vena cavae were exposed and end-to-side anastomoses secured graft aortae and pulmonary arteries, respectively, using 10-0 Novafil sutures (Davis & Geck, Kendall; Mansfield, MA; http://www.kendallhq.com). Spontaneous contractile activity was initiated by reperfusion and warming of the graft. Cardiac grafts were assessed by daily palpation, and rejection was defined as cessation of spontaneous contractile activity.

Statistical Analyses
Data are presented as mean ± standard deviation (SD) for each experimental protocol. Donor hematopoietic chimerism was evaluated for reproducibility by linear regression of duplicate measurements in each experimental group. Differences between the experimental protocols were estimated with a post-hoc Scheffe t-test, and significance was considered at p < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Allogeneic BMC Engraftment after i.v. and IL Infusion
All BALB/c recipients (n = 10 in each group) conditioned with 145 µg/g busulfan died within 19 days after i.v. or IL injection of cell-free medium. The 30-day survival rates after i.v. and IL injection of 107 allogeneic (B10->BALB/c) wBMCs were 90% and 80%, respectively (n = 10 in each group). At 4 weeks, the levels of H2b donor chimerism in the peripheral blood were 84% ± 5% and 78% ± 4% in the i.v. and IL protocols, respectively. In i.v.-injected hosts, 44% ± 5% and 35% ± 5% of the H2b donor cells were CD3+ and B220+, respectively. In IL-injected hosts, 37% ± 5% and 38% ± 5% of the H2b cells were CD3+ and B220+, respectively.

The Fate of BMCs Transplanted into the IL
IL perfusion caused an increase in CK activity in the peripheral blood of recipients from 29 ± 4 (n = 5) to 115 ± 13 µmol/min/ml (n = 15). Release of muscle enzymes was caused by the surgical procedure itself and was observed in sham-operated mice with and without arteriovenous perfusion. To assess whether the IL preparation was efficient in localizing the transplantation, donor cells were labeled with PKH membrane linkers. The fate of infused cells was assayed in peripheral blood samples and femurs of the recipients. Briefly, 107 PKH-labeled wBMCs from B10 donors were injected i.v. or IL into myeloablated BALB/c recipients (5 mice in each group). Blood sampling after i.v. injection showed a decrease in PKH-labeled blasts from 4.7%-5.8% at 5-30 minutes to 1.3% ± 0.2% at 2 hours (Fig. 1AGo). In contrast, there were very few PKH-labeled blasts in the peripheral blood after IL-BMT (p < 0.001 versus i.v.-BMT).



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  		Figure 1. Distribution of PKH-labeled cells 2 hours after injection into a peripheral vein (i.v.) and infusion into the femoral artery (IL) of an isolated limb. Myeloablated B10.BR recipients were injected with 107 allogeneic wBMCs labeled with PKH67 (n = 5). A) Blood levels of circulating PKH-labeled blasts. B) Contents of PKH-labeled blasts in the femurs after i.v. injection (represents the mean of the two femurs), the femur of the injected limb (IL), and the contralateral limb. Data are shown as mean ± SD. *p < 0.05, IL versus i.v.

 
Two hours after injection, femurs of recipients injected i.v. (n = 5) contained 0.6% ± 0.1% PKH-labeled cells and 8% ± 0.6% PKH-labeled blasts (Fig. 1BGo). Analysis of the contents of PKH-labeled cells in the femurs of the infused and contralateral limbs was performed 2, 6, and 48 hours after the arterial clamp was removed (n = 5 mice in each group). In femurs from the infused limbs, 42% ± 5% of the BMCs and 57% ± 7% of the cells in the blast gate (p < 0.05 versus total BMCs) were PKH+ after 2 hours (Fig. 1BGo). After 2 days, the femurs of perfused limbs contained 29% ± 6% PKH+ BMCs and 51% ± 6% PKH+ cells in the blast gate (p < 0.005 versus total BMCs). The number of cells in the contralateral femurs was at the edge of the detection threshold of flow cytometry (Fig. 2Go). Detailed examination of the cellularity of the contralateral femurs by fluorescence microscopy revealed 12 ± 3 (range 6-17) PKH-labeled cells 2 hours after IL-BMT, which was greater by 2.5- and sixfold at 6 and 48 hours, respectively.



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  		Figure 2. Femoral contents of PKH-labeled BMCs. Myeloablated BALB/c recipients were injected with 107 wBMCs labeled with PKH67 into the tail vein (i.v.) and using the isolated limb (IL) preparation. Femurs were harvested after 2 hours to determine the number of PKH-labeled cells by flow cytometry on the blast gate (RI). Analysis of PKH-labeled cells in mice injected i.v. represents the mean of the two femurs. Femurs contralateral to the infused limb contained few cells, compared with the retention of a large number of cells by the femur of the injected limb. The differences between the readouts represent various cell counts and do not reflect quantitative differences in the cellularity of the femurs.

 
Differences in the cellular contents of femurs from the infused and contralateral limbs persisted for several weeks. At 4 weeks, the femurs of the injected limbs contained 1.2 x 107 cells, which were 78% ± 4% H2b donor (n = 5). The contralateral femurs contained 0.32 x 107 cells in which only 47% ± 5% were H2b donor (n = 5, p < 0.001 versus injected limb). To assess whether hypocellularity of the noninfused femurs of the busulfan-ablated recipients offered favorable conditions for proliferation of a smaller number of progenitors that migrated to those bones, donor BMCs from both femurs (n = 4 mice) were stained for Sca-1 and c-kit [25, 26]. In the injected femurs, 0.22% ± 0.04% of the cells were Sca-1+ lineage– (Fig. 3AGo) and 78% ± 9% of those cells were c-kit+ (Fig. 3BGo). In the contralateral femurs, 0.31% ± 0.04% of the cells were Sca-1+ lineage– (Fig. 3C pGo, < 0.001 versus injected limb) and 44% ± 5% of those cells were c-kit+ (Fig. 3D pGo, < 0.001 versus injected limb). Thus, the fraction of Sca-1+c-kit+lineage– cells in the contralateral femurs (0.07% ± 0.01% of total cells) was significantly lower (p < 0.01) than the fraction in the infused femurs (0.17% ± 0.03% of total cells).



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  		Figure 3. Differences in distributions of hematopoietic cells in femurs of infused (A and B) and contralateral (C and D) limbs 4 weeks after transplantation. Donor cells were immunomagnetically isolated, and analysis was performed using FITC-labeled mAbs against lineage markers, PE-anti-Sca-1, and APC-anti-c-kit mAbs. Percent values are given in reference to the total number of donor cells (H2b) in the host femoral BM.

 
Acceptance of Cardiac Grafts
For induction of tolerance to secondary heart grafts, BALB/c mice (H2d) were conditioned with 35 µg/g busulfan and were injected i.v. (n = 7) and IL (n = 16) with 107 wBMCs from B10 donors (H2b). All the BALB/c recipients survived conditioning with 35 µg/g busulfan and i.v. or IL infusion of cell-free medium (n = 5). Two other groups of BALB/c mice conditioned with 35 µg/g busulfan were injected i.v. (n = 7) and IL (n = 16) with 107 wBMCs from B10 donors. Three weeks after i.v. and IL injection, the levels of H2b donor cells in the peripheral blood of the hosts were 33% ± 4% and 21% ± 4% (n = 6, p < 0.005), respectively (Fig. 4AGo). After 11 weeks post-transplantation, the levels of H2b donor chimerism were lower in mice injected both i.v. and IL (p < 0.05 versus 3 weeks). The chimeras presented lower levels of CD3+ cells after IL infusion, both at 3 and 11 weeks post-transplantation (p < 0.05 versus i.v. injection, Fig. 4BGo).



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  		Figure 4. Donor chimerism 4 and 11 weeks after injection of 107 H2b cells into H2d recipients conditioned with 35 µg/g busulfan. A) Percent donor H2b cells in the peripheral blood of recipients injected into a peripheral vein (i.v.) and into the femoral artery (IL). B) Percent CD3+ and B220+ cells in the H2b donor fraction. Error bars represent standard deviations. *p < 0.05, IL versus i.v. #p < 0.05, 11 weeks versus 3 weeks.

 
Hearts from H2b donors were acutely rejected by untreated H2d recipients with a mean time of 9.5 ± 1 days (Table 1Go). In contrast, transplantation of BMC-matched secondary hearts (H2b) 3 weeks after i.v.- and IL-BMT resulted in extended survival of the allografts for periods exceeding 8 weeks (n = 5). Hearts from third-party B10.BR donors (H2k) transplanted 3 weeks after transplantation of B10 BMCs (H2b) were acutely rejected, at a tempo comparable with that observed in allogeneic donor-recipient pairs without BMT (Table 1Go).


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  		Table 1. Survival of cardiac grafts after systemic and IL-BMT
 

   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Donor-specific tolerance to secondary grafts may be induced by hematopoietic chimerism, while preserving immunoreactivity against third-party antigens [3–8]. The novel finding in this study is that localized transplantation of hematopoietic cells in one limb had the same tolerogenic effect as systemic transplantation. Considering the relative safety of the IL perfusion procedure [27, 28], IL-BMT may have significant clinical applications for induction of donor-specific immune nonresponsiveness to solid organs via hematopoietic chimerism without sustained immunosuppressive therapy.

During the first 2 hours after IL-BMT, there were small numbers of PKH-labeled cells in the peripheral blood of recipients (Fig. 1AGo), suggesting efficient seeding of donor cells in the marrow space of the perfused limb. Release of a small number of cells into systemic circulation after restitution of blood flow to the isolated limb was a consistent observation in this preparation [14], which may reflect the reciprocity of the homing and mobilization processes [29]. The number of circulating cells was expected to be small, considering that perfusion of isolated femurs in situ is efficient in removing nonadherent cells from the marrow space [30]. In that same study, we demonstrated that the femur has the capacity to host seeding of large numbers of BMCs, which explains the retention of a significant number of donor cells in femurs of perfused limbs.

Within the first 2 days post-BMT, there was a nonsignificant decrease in the number of cells in femurs of the infused limbs (from 57% ± 7% to 51% ± 6%), and there was a slow increase in the number of PKH-labeled cells in the contralateral femurs. Cell counts in the contralateral femurs indicated a mean redistribution of ~30 cells per day/femur during the first 2 days after IL-BMT. This estimate is comparable with the reported low flow rates of cells from femurs to the humera of mice [31]. After 4 weeks, the femurs of injected limbs contained 38-fold higher numbers of cells than the contralateral limbs of busulfan-myeloablated recipients. The asymmetry between bones of the infused and contralateral hind limbs suggests that hematopoiesis was predominantly localized within the first days to weeks after IL-BMT. However, retention of a large number of donor cells in bones of the infused limbs served as a source of secondary systemic engraftment over time. The levels of donor chimerism at 3-4 weeks exceeded the contribution of one femur and tibia (8%-9%) to the total hematopoietic space in the mouse [32], which would be expected if engraftment remained localized over a long period. Redistribution of cells in the host bones via a hematogenic route and secondary engraftment [33] is a process that proceeds at a relatively slow pace, and its rate is proportional to the number of cells infused [34]. At 4 weeks, the noninjected femurs of myeloablated hosts contained ~15% of the donor cells found in femurs of injected limbs, and the fraction of stem cells (defined as Sca-1+ c-kit+lineage–) was 2.5-fold lower. These data suggest that hematopoietic stem cells that migrated to the hypocellular environment of the noninfused bones proliferated more.

Recipient conditioning with busulfan most likely increased the rate of hematopoietic cell mobilization from the injected limb [31], and systemic preconditioning augmented the rate of secondary seeding of circulating cells in hematopoietic bones [35]. We have recently reported changes in the levels of donor chimerism proportional to the dose of busulfan and the size of donor inoculum used for IL-BMT [14]. This predominantly cytoreductive agent, which affects mainly slow-cycling and quiescent primitive stem cells in G0 [19–21], was chosen for evaluation of the localized BMT approach because its cytoreductive effect is more pronounced than its immunosuppressive activity. We assumed that localized BMT significantly reduced the need for systemic immunosuppression at the time of transplantation. Comparison of the success of engraftment with various conditioning modalities suggests that a dose of busulfan of 15-25 µg/g is equivalent to 100-200 rad of total body irradiation [36–38]. Thus, the dose of 35 µg/g busulfan used for partial conditioning in this study was extrapolated to the minimal dose of total body irradiation (~400 rad) required for allogeneic stem cell engraftment [31, 39–41]. Recipients with levels of 16%-33% donor chimerism (in the peripheral blood) accepted donor-matched secondary heart allografts, while preserving immunoreactivity against third-party antigens.

The IL preparation was initially designed for local treatment of malignancies, in particular, malignant melanoma in the lower extremities [28, 42]. The complications associated with this procedure are mainly related to the toxic effects of the various chemotherapeutic agents to the blood vessels of the extremity [27]. However, injection of hematopoietic cells into the femoral artery of a limb that was isolated from the systemic circulation can provide several advantages. In principle, direct inoculation of the limb eliminates the homing process, prevents the depletion of antigen-disparate cells during systemic circulation, and creates a local megadose effect [13–18]. Thus, instead of injection of a large number of cells into a peripheral vein, a megadose effect may be achieved by injection of fewer cells into a smaller hematopoietic space.

Recent evidence indicates that injection of donor cells into the BM reduces the incidence of GVHD [13], and we showed a lower incidence of the reaction after IL perfusion [14]. Interestingly, we observed a lower fraction of donor CD3+ cells in mice injected IL than in mice injected i.v. Donor T-cell-mediated GVHD is a severe complication in humans, however, mice are not particularly prone to develop this reaction. Thus, assessment of this reaction and the way it is affected by the route of donor cell administration should be performed in GVHD-prone species [43]. GVHD may be prevented by injection of TCD-BMCs. In fact, engraftment of TCD-BMCs was found to be superior when cells were directly injected into the BM and into an isolated limb compared with the i.v. route [13, 14]. This effect may be attributed to the megadose effect, where i.v. injection of large numbers of TCD-BMCs overcomes antigenic barriers and yields superior efficiencies of engraftment [8, 17, 18]. In addition to a larger number of hematopoietic stem and progenitor cells, transplant of TCD-BMCs requires more aggressive conditioning [44, 45]. Because isolated limb perfusion improves the efficiency of TCD-BMC engraftment and also reduces the incidence of GVHD, localized conditioning to the transplanted limb may be a significant advantage of IL-BMT. The usefulness of the IL preparation should be further assessed experimentally to determine the efficiency of engraftment of purified stem cells, the possible use of localized preconditioning, and the tolerogenic effect when solid organs are simultaneously transplanted.

In summary, this study demonstrates that localized BMT performed by IL perfusion is an efficient route of transplantation to: A) reconstitute hematopoiesis in myeloablated allogeneic recipients; B) achieve mixed hematopoietic chimerism in partially conditioned recipients, and C) induce donor-specific immune nonresponsiveness to secondary cardiac grafts. The safety of IL perfusion makes IL-BMT a potential clinically relevant modality for induction of donor-specific immune nonresponsiveness without myeloablative conditioning.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Dr. Suzanne T. Ildstad, Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky, for the outstanding support of this study, Dr. Tatiana Zorina, Division of Immunogenetics, Children’s Hospital, University of Pittsburgh, for the stimulating discussion, and Mrs. Judy Montibeller and Mrs. Lisa McGow for the excellent technical assistance. This study was supported in part by AHA grant 9960386V and the Edward and Leah Frankel Trust for Bone Marrow Transplantation, Schneider Children’s Medical Center of Israel.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received May 10, 2002; accepted for publication October 3, 2002.



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