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TRANSPLANTATION
From the Division of Hematology, Oncology Blood
and Marrow Transplantation, Department of Pediatrics; Transplant
Immunology Laboratory, Department of Surgery; Department of Medicine;
and Division of Animal Resources and Department of Pathology; all of
Emory University School of Medicine, Atlanta, GA.
The morbidity and mortality associated with sickle cell disease
(SCD) is caused by hemolytic anemia, vaso-occlusion, and progressive multiorgan damage. Bone marrow transplantation (BMT) is currently the
only curative therapy; however, toxic myeloablative preconditioning and
barriers to allotransplantation limit this therapy to children with
major SCD complications and HLA-matched donors. In trials of
myeloablative BMT designed to yield total marrow replacement with donor
stem cells, a subset of patients developed mixed chimerism. Importantly, these patients showed resolution of SCD complications. This implies that less toxic preparative regimens, purposefully yielding mixed chimerism after transplantation, may be sufficient to
cure SCD without the risks of myeloablation. To rigorously test this
hypothesis, we used a murine model for SCD to investigate whether
nonmyeloablative preconditioning coupled with tolerance induction
could intentionally create mixed chimerism and a clinical cure.
We applied a well-tolerated, nonirradiation-based, allogeneic transplantation protocol using nonmyeloablative preconditioning (low-dose busulfan) and costimulation blockade (CTLA4-Ig and
anti-CD40L) to produce mixed chimerism and transplantation tolerance to
fully major histocompatibility complex-mismatched donor marrow.
Chimeric mice were phenotypically cured of SCD and had normal RBC
morphology and hematologic indices (hemoglobin, hematocrit,
reticulocyte, and white blood cell counts) without evidence of graft
versus host disease. Importantly, they also showed normalization of
characteristic spleen and kidney pathology. These experiments
demonstrate the ability to produce a phenotypic cure for murine SCD
using a nonmyeloablative protocol with fully histocompatibility
complex-mismatched donors. They suggest a future treatment strategy
for human SCD patients that reduces the toxicity of conventional BMT
and expands the use of allotransplantation to non-HLA-matched donors.
(Blood. 2002;99:1840-1849) Patients with sickle cell disease (SCD) suffer from
both episodic acute complications and chronic, progressive, multisystem decline. Although medical treatments are life-extending, only stem cell
transplantation offers an effective cure. There are currently 2 major
barriers to stem cell transplantation for SCD: (1) the high morbidity
and mortality associated with conventional bone marrow transplantation
(BMT) and (2) the scarcity of acceptable stem cell
donors.1,2 (1) Conventional BMT can cure SCD but requires
toxic myeloablative preconditioning regimens to achieve donor cell
engraftment.3,4 These intensive preparative regimens have
many toxic adverse effects, including potential organ failure and a
long-term risk of malignancy. In heavily pretreated patient populations, the morbidity and mortality of transplantation can outweigh the morbidity and mortality of SCD.2 A dilemma is now developing between early treatment with stem cell transplantation (shown to increase survival and disease-free survival when compared with transplantation after more disease-related complications have
occurred) and a delayed approach, during which medical management ameliorates the symptoms of SCD until a later age when definitive therapy can be instituted.1,2 Unfortunately, this latter course may decrease the chance of successful stem cell transplantation. (2) The paucity of matched related donors has severely limited the
number of SCD patients eligible for transplantation. In fact, in the
Seattle consortium study, only 6.5% of potential SCD patients were
found to be eligible for stem cell transplantation based on disease
severity, and of these only 14% had an HLA-matched related
donor.1,3 The lack of matched donors compounds the problem
of transplantation-mediated toxicity due to the aggressive regimens
used to gain alloengraftment. Thus, for widespread transplantation to
be successful, it is essential to develop methods for the generation of
allochimerism that have low levels of morbidity and mortality.
The transplantation experience with SCD suggests that a cure can be
achieved even without total replacement of recipient stem cells.3-6 Walters et al reported that 4 of 50 patients
treated with conventional myeloablative preconditioning unintentionally developed mixed donor/recipient hematopoiesis.3
Importantly, those patients with stable mixed chimerism developed no
further sickle-related complications. Recently, Iannone et al achieved varying degrees of mixed normal/sickle hematopoiesis by titrating bone
marrow from a sickle mouse into a lethally irradiated normal mouse.7 Increasing levels of sickle red blood cells (RBCs) in the chimeric mice resulted in increasing hematopoietic and solid
organ pathology. Importantly, solid organ pathology was reported even
at sickle RBC percentages that normalized hematologic parameters, such
as the reticulocyte count and hemoglobin level. This implies that
relying on partial correction of hematologic indices may not predict
true systemic cure of SCD in the setting of moderate levels of mixed
RBC chimerism. Although the Iannone study was able to investigate
certain pathophysiologic sequelae of mixing normal and sickle red
cells, many questions about the effect of mixed chimerism could not be
addressed due to the recipient mice lacking the sickle genotype.
Given the results of recent studies, there is expanding interest in
protocols that are nonmyeloablative and that intentionally produce
stable mixed chimerism.8,9 The problem to overcome is one
of tolerance because there must be a coexistence of both host and donor
cells for stable mixed chimerism to be achieved. While the initial
protocols used relatively nonspecific immunosuppressive agents to
induce transplantation tolerance, recent murine studies have focused on
blocking T-cell activation pathways as a targeted approach for
developing donor-specific tolerance and long-term mixed
chimerism.10-15 These studies have shown that disruption of the T-cell costimulation signal mediated by the CD28/B7 or CD40/CD40L pathways at the time of BMT can lead to anergy of
donor-reactive host T cells and produce long-term tolerance to the
graft.10-15 Our group has developed a novel regimen for
murine BMT that employs nonmyeloablative preconditioning with low-dose
busulfan coupled with costimulation blockade of the CD28/B7 and CD40L
pathways.14 This regimen produces long-term, mixed
chimerism and robust tolerance to a fully major histocompatibility
complex (MHC)-mismatched allograft without the requirement of
myeloablation in a variety of mouse strains.
To rigorously test the ability for such a transplantation regimen to be
successful in SCD, we used a transgenic knock-out mouse that lacks all
murine hemoglobins and instead produces exclusively human Animals used in the study
BMT protocol
Flow cytometric analysis Peripheral blood was analyzed by staining with fluorochrome-conjugated antibodies (anti-CD3, anti-CD5, anti-CD11b, anti-GR1, anti-B220, anti-H-2Kd, anti-H-2Ks, anti-H-2Kb, anti-V 5.1/5.2, Pharmingen, San Diego, CA;
and anti-CD4 and anti-CD8, Caltag Laboratories, Burlingame, CA) or Ig
isotype controls (Pharmingen) followed by RBC lysis and washing with a
whole blood lysis kit (R&D Systems, Minneapolis, MN). Stained cells
were analyzed either using WinList (Verity Software House, Topsham, ME)
or CellQuest (Becton Dickinson, Mountain View, CA) software on either a
FACScan or FACSCalibur flow cytometer (Becton Dickinson). WBC chimerism was determined by staining with either donor (anti-H2Kd
[BALB/c] or anti-H2Ks [SJL]) or recipient
(anti-H2Kb) antibodies and specific lineage markers and
analysis by flow cytometry. Background staining with
anti-H2Kd or anti-H2Ks was typically less than
1%. V deletion was determined by staining with V5 antibodies and
specific lineage markers and analysis by flow cytometry.
Analysis of hematologic characteristics Complete blood counts were performed on a Hemavet 1500 blood analyzer (1500 R series, CDC Technologies, Oxford, CT). Reticulocyte counts were performed by flow cytometry of peripheral blood labeled with antibodies specific for RBCs (anti-Ter-119, Pharmingen) and WBCs (anti-CD45, Pharmingen) and a fluorescent label of RNA, thiazole (Sigma, St Louis, MO). Reticulocyte counts were defined as the percent of peripheral blood cells that were Ter-119+, thiazole+, and CD45 . "Stress"
reticulocytes18 were also analyzed by labeling with an
antibody against the transferrin receptor (CD71, Pharmingen).
Blood smears Smears of peripheral blood were made under ambient air (ie, oxygenated conditions) and then Wright-stained prior to microscopic analysis.Tissue histology Tissues were immersion-fixed in 4% paraformaldehyde prior to embedding and cutting. After hematoxylin and eosin staining, a veterinary pathologist examined the sections in a blinded manner.RBC studies RBC population half-life. Half-life was determined by a pulsed biotinylation experiment performed essentially as previously described.19 Briefly, 50 mg/kg N-hydroxysuccinimide biotin (Calbiochem, San Diego, CA) (initially dissolved at a concentration of 50 mg/mL in N,-N,-dimethylacetamide and diluted into 250 µL normal saline just prior to use) was injected intravenously into engrafted or naive sickle animals. This produced a biotin pulse label to the peripheral blood. Blood was obtained either from the retro-orbital venous plexus or through a tail nick at regular intervals after biotinylation. The percentage of peripheral RBCs that were biotinylated was determined by flow cytometry using fluorescent streptavidin-cychrome (Pharmingen) to identify biotinylated cells and a fluorescent Ter-119-phycoerythrin antibody (Pharmingen) to identify RBCs. The decay of biotinylation is directly related to the clearance of the biotinylated RBCs from the peripheral circulation and thus can be used to determine the half-life of the RBC population. Plasma-membrane phosphatidylserine exposure. Exposure was measured by the percentage of cells that were positive in annexin V (Pharmingen) binding assays. Annexin V binding assays were performed by incubating 1 × 106 peripheral blood cells with 5 µL annexin V and appropriate lineage-specific antibodies in annexin binding buffer (Pharmingen) for 30 minutes at room temperature. Cells were then washed once with annexin binding buffer and analyzed by flow cytometry to determine the percentage of annexin V+ cells. Scramblase assays. RBC scramblase enzyme assays were performed essentially as previously described.20,21 Briefly, 2 × 106 peripheral blood cells were incubated with 3 mM of the fluorescent phosphatidylcholine analog palmitoyl-C6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylcholine (NBD-PC; Avanti Polar Lipids, Birmingham, AL) in phosphate-buffered saline containing 1 mM CaCl2 for 30 minutes at 37°C. Cells were then cooled on ice and washed into buffer containing 10 mg/mL defatted bovine serum albumin (Sigma) to back-exchange noninternalized phospholipid. Cells were then incubated with appropriate lineage-specific antibodies (Pharmingen) for 20 minutes at 4°C prior to analysis by flow cytometry. Analysis of RBC chimerism RBC chimerism was determined by differential hemoglobin electrophoresis of donor and recipient hemoglobin. Donor-globin consists of murine "major" and "minor" -globin isomers,
which have different electrophoretic mobilities than recipient human sickle -globin. Hemoglobin electrophoresis was performed on the Helena Titan III electrophoresis system (Helena Laboratories, Beaumont,
TX). Gels were scanned, and percent donor or recipient hemoglobin was
determined by densitometry using Kodak 1-D Image Analysis software
(Kodak, Rochester, NY).
Determining tolerance to donor antigen by CFSE assays Splenic and mesenteric lymph node cells were harvested from experimental mice. After RBC lysis and nylon wool passage, cells were incubated in 10 µM of the fluorescent dye, CFSE (Molecular Probes, Eugene, OR). Irradiated (1.8 Gy [1800 rads]) sickle, BALB/c, or C3H mice then received 1 × 107 to 1 × 109 CFSE-labeled cells intravenously. After 66 to 72 hours, splenocytes were harvested from the recipients, the RBCs lysed, and the remaining cells stained with anti-CD4 and anti-CD8 or isotype controls and analyzed by flow cytometry as described above.Determining hematopoietic balance in recipient hematopoietic organs Mice were killed, and their splenocytes and bone marrow were harvested with conventional techniques. Hematopoietic balance was specified by determining the percent of bone marrow and spleen cells that were either RBCs (Ter-119+, CD45),
reticulocytes (Ter-119+, CD45,
thiazole+), or WBCs (Ter-119,
CD45+).
Creation of stable chimeras using busulfan and costimulation blockade Sickle mice16 were generously provided by Paszty and colleagues and treated with a regimen that included busulfan (20 mg/kg) pretreatment on day 1, BMT with TDBM from BALB/c mice on day 0, and
costimulation blockade with 500 µg each of anti-CD40L and CTLA4-Ig on
days 0, 2, 4, and 6. The T-cell content of transplanted cells was
determined by flow cytometry and represented less than 0.4% of donor
cells (data not shown). Additional animals received control protocols,
including either (1) 20 mg/kg of busulfan without any bone marrow
rescue, (2) TDBM alone, (3) TDBM and busulfan but no costimulation
blockade, or (4) TDBM and costimulation blockade but no busulfan.
Figure 1 shows the transient
myelosuppressive effect of 20 mg/kg busulfan given to 10 sickle mice.
All recipients survived the busulfan treatment and recovered
hematopoiesis without the need for stem cell rescue (absolute
neutrophil count nadir = 1600 at 14 days, platelet nadir = 170 000
at 14 days). Thus, pretreatment with 20 mg/kg busulfan is
nonmyeloablative in the sickle mice.
Mice who received control protocols, including BMT alone (5 mice), BMT
after pretreatment with 20 mg/kg busulfan (no costimulation blockade, 5 mice), or BMT and costimulation blockade (no busulfan, 9 mice) showed
low to undetectable levels of peripheral WBC chimerism (< 2.5%)
throughout the experimental period (Figure
2A). In contrast, 10 of 13 mice who
received the full protocol of BMT, 20 mg/kg busulfan, and costimulation
blockade achieved multilineage WBC mixed chimerism (Figure 2A). Two of
these engrafted mice died during anesthesia at 3 months after
transplantation (with no signs of graft versus host [GVH] disease
prior to their death), and 1 mouse was killed at 2 months for
histologic analysis. Chimerism for the remaining 7 mice (mean,
43% ± 10%) peaked 3 months after transplantation and was stable
for more than 150 days (Figure 2A). Three mice rejected the graft.
These mice displayed transient levels of WBC chimerism at 1 to 2 months
after transplantation but fully reverted to the recipient WBC and RBC
phenotype by 3 to 4 months after transplantation (data not shown).
Figure 2B shows the engraftment kinetics of individual cell lineages in the peripheral blood of chimeric mice. As expected, donor granulocytes (GR1) and macrophages (CD11b) appear first in the periphery, followed by donor B cells (B220) and T cells (CD3). Figure 2C shows representative flow cytometric data from which the values shown in Figure 2A,B were drawn. The peripheral chimerism in individual engrafted animals was reflected in their hematopoietic organs. Mean chimerism for bone marrow (50%), spleen (45%), and thymus (25%) is shown in Figure 2D. The chimerism noted in the animals that received the full protocol of BMT, 20 mg/kg busulfan, and costimulation blockade was not donor strain-specific, because 5 sickle mice undergoing transplantation with busulfan, costimulation blockade, and TDBM from SJL mice (which are of the H-2Ks rather than H-2Kd genotype) showed similar levels of WBC and RBC chimerism (data not shown). Engrafted mice showed no signs of GVH disease: Their individual body weights remained stable (mean pretransplantation weight, 32 ± 1 g; mean posttransplantation weight, 36 ± 4 g) and posttransplantation necropsies, in which organs (including the skin, heart, lungs, spleen, liver, kidney, and intestinal tract) were specifically evaluated for signs of GVH disease, showed normal histology. Peripheral white cell chimerism was comparable to previous results
using busulfan and costimulation blockade in allogeneic wild-type
mice,14 whereas peripheral RBC chimerism was strikingly higher in the sickle transplantation recipients. Quantification of
donor (normal BALB/c
Mice receiving TDBM alone or TDBM plus busulfan (no costimulation
blockade) showed no donor RBC (hemoglobin electrophoresis) or WBC (flow
cytometry) engraftment. Thus, in the absence of costimulation blockade,
the sickle mice were able to totally reject the donor graft. However,
in the setting of BMT plus costimulation blockade (but no busulfan), 5 of 9 control mice developed significant red cell chimerism (10%-54%,
Figure 4) despite minimal WBC chimerism (< 2.5%, Figure 2A). This probably reflects very low levels of stem
cell engraftment and massive expansion of healthy red cell precursors,
leading to significant peripheral read-out of donor hemoglobin.
Chimeric mice are specifically tolerant to donor and recipient antigen BALB/c mice express I-E and therefore delete V5-bearing T
cells, whereas sickle mice do not express I-E and specifically utilize
V5.1/2 on about 2% to 3% of CD4+ T
cells.22,23 As anticipated, the 9 animals that received BMT and costimulation blockade but no busulfan (and who showed low to
undetectable WBC engraftment, Figure 2A) failed to delete donor-reactive V5+CD4+ T cells (Figure
5A). In contrast, engrafted animals that
received TDBM along with busulfan and costimulation blockade developed near complete deletion of CD4+V5+ T cells by
day 60. Part of this decrease in total
V5+CD4+ T cells could have been due to the
mixture of donor (V5) and recipient
(V5+) CD4+ T cells even without deletion.
However, given that 82% of CD4+ T cells showed V5
deletion at 150 days (Figure 5A), while CD4+-T-cell
chimerism was only 33% ± 8% at this same time point (data not
shown), most of the reduction of V5+CD4+ T
cells is likely due to specific V5+-T-cell deletion.
The percentage of V8-bearing CD4+ T cells, normally
expressed on 15% to 25% of BALB/c and sickle CD4+ T
cells, was similar in all groups, indicating that the T-cell deletion
was donor-specific in nature (data not shown). These results suggest
that the bone marrow-derived I-E-bearing donor cells influence the
selection of the T-cell repertoire in engrafted mice, ultimately
conferring robust long-term donor-specific tolerance.
Coincident with the long-term chimerism seen in the busulfan-treated animals was the development of specific tolerance to the allogeneic BALB/c bone marrow graft without GVH disease (Figure 5B). A rigorous in vivo alloproliferation assay using CFSE dye was performed to test for the presence of alloreactivity.24 T cells were harvested from spleens and mesenteric lymph nodes of animals that were at least 100 days after transplantation. After labeling with 10 µM CFSE, T cells were transferred into recipient mice (BALB/c, sickle, or C3H) previously supralethally irradiated. Splenocytes were harvested 72 hours later and analyzed via flow cytometry. While CD4+ and CD8+ T cells from nonengrafted groups underwent extensive cell division in response to both BALB/c and third-party (C3H) hosts but not to sickle hosts (data not shown), T cells from the engrafted mice generated no proliferative response to either donor (BALB/c) or recipient (sickle) hosts (Figure 5B). As expected, clear proliferative responses were present when naive BALB/c T cells were transferred into sickle hosts (data not shown) or when chimeric T cells were transferred to a third party (C3H, Figure 5B). These results confirm the specific absence of donor and recipient alloreactive CD4+ or CD8+ T cells capable of cell division in chimeric animals. Chimeric mice are cured of sickle cell disease Engrafted sickle mice demonstrated a phenotypic cure of their SCD by a variety of parameters. As seen in Figure 6A,B, a striking absence of irreversibly sickled cells in peripheral blood smears occurred after transplantation in mice that were conditioned with busulfan and costimulation blockade. Engrafted mice also demonstrated normalization of their hematologic abnormalities (Figure 6C), including hemoglobin (45 g/L corrected to 100 g/L [4.5 g/dL corrected to 10 g/dL]), hematocrit (0.16 corrected to 0.40 [16% corrected to 40%]), and peripheral thiazole+ reticulocyte percentage (49% corrected to 3.5%), consistent with a reversal of their hemolytic anemia. Furthermore, the abnormally elevated WBC count seen in naive sickle mice was corrected in engrafted mice (20 × 109/L to 5.1/ × 109/L [20 000/µL to 5100/µL]).
The health of the newly emerging chimeric red cells was assessed by 3 physiologic markers. First, RBC population half-life was determined through a pulsed biotinylation experiment19 (Figure 6D). Untreated and engrafted sickle mice were injected intravenously with N-hydroxysuccinimide biotin to label the peripheral blood with biotin as previously described.19 RBC half-life was determined by the decay of the biotinylated RBCs over time by flow cytometry. Consistent with previous work from this laboratory,25 RBCs from the naive sickle animals had exceedingly short peripheral half-lives (0.8 days) compared with normal control C57BL/6 mice (half-life 18 days). Engrafted animals had a RBC half-life indistinguishable from that of normal mice, consistent with replacement of the diseased red cell compartment with normal RBCs. Second, we measured the production of transferrin+ "stress" reticulocytes in engrafted and nonengrafted mice. These cells are an indication of overactive erythropoiesis and are thought to contribute to the increased adhesion of sickle reticulocytes in the microvasculature.18,26,27 Figure 6E shows that the percent of these cells decreases from 27% to 3% in engrafted mice, consistent with normalization of red cell turnover in these animals. Third, we examined plasma membrane phosphatidylserine (PS) exposure, which is known to be increased in sickle red cells.28,29 PS is thought to contribute to increased clearance of these cells by macrophages and monocytes and may also contribute to abnormal endothelial adhesion.30 Two assays were used: annexin V binding, which measures exposed PS residues directly,31 and NBD-PC internalization, which measures the scramblase enzyme that leads to PS exposure on the plasma membrane.20,21,32 Figure 6E shows that sickle mice consistently show a high PS exposure prior to transplantation (measured by annexin V binding), but engrafted mice demonstrate a significant decrease in this PS exposure. Figure 6E also shows that a dramatic decrease in the number of RBCs with active scramblase occurs after engraftment, consistent with the decline in PS exposure described above. Although the focus of this study is the effect of full RBC replacement
in the face of partial WBC engraftment, as noted above, a subset of
mice (5 of 9) receiving BMT and costimulation blockade (but not
busulfan) did develop partial RBC chimerism. Of those 5 mice, 2 died
unexpectedly (1 at 3 months, 1 at 4 months of age, neither with signs
of GVH disease) and thus were not fully analyzed. The remaining mice
were analyzed for the effects of mixed RBC chimerism on murine SCD. As
shown in Figure 7, there was a
progressive normalization of both hematologic (hemoglobin and
hematocrit) and physiologic (reticulocyte count and spleen size)
parameters in mice with increasing RBC engraftment. Further analysis
also showed partial correction of NBD-PC internalization, annexin V binding, and splenic hematopoeitic balance in mice with mixed donor and
host RBCs (data not shown). A separate study designed to rigorously
determine the effect of progressively increasing amounts of normal RBCs
in the sickle background is currently underway (L.S.K. et al, in
preparation).
The spleens in the engrafted mice exhibit signs of reversal of characteristic sickle pathology One of the hallmarks of murine sickle cell pathophysiology is the dramatic increase in spleen size compared with that of normal animals.16,17,25 This is related to the immense requirement for erythropoiesis in order to replenish the rapid destruction of peripheral sickle RBCs.16,17,25 As shown in Figure 8A, the spleen undergoes a significant decrease in size in engrafted mice (from 7.3% total body weight in age-matched naive sickle mice to 0.7% total body weight in engrafted mice measured 5 months after transplantation). Figure 8B shows that while the spleen functions as a largely erythropoietic organ in untreated sickle mice, it undergoes a reprogramming in the engrafted cohort and resumes a more normal balance between white and red cell hematopoiesis. Figure 9A,B shows a histologic comparison of the spleens from naive and engrafted mice. In engrafted mice there is a striking resolution of the characteristic hyperactive hematopoiesis and red cell pooling characteristic of SCD and the acquisition of normal splenic architecture.
Renal histology is normal in engrafted mice In addition to the defects observed in both the peripheral blood and the hematopoietic organs, sickle mice also demonstrate solid organ pathology similar to that seen in patients with SCD. 16,17 As in the original description of this murine SCD model,16,17 we have noted pathologic changes in many organs, including the kidney, liver, lung, and heart in untreated sickle animals. To determine the effect of BMT on organ structure and histology, necropsies were performed on age-matched naive and engrafted animals and tissues were prepared for histologic analysis. We found that engrafted animals had normal histology of all organs tested, including the kidney, liver, heart, and lungs. Representative of the histologic normalization that occurred in these animals, Figure 9C,D shows a comparison of renal histology in age-matched untreated and engrafted mice. Figure 9C shows the membranoproliferative glomerulonephritis consistently observed in untreated sickle mice. Figure 9D shows that engrafted animals had normal renal histology, including normalization of glomerular capsular space and glomerular membrane thickness.
Stem cell transplantation is the only current treatment that offers an effective cure for SCD.3,4,33,34 However, the availability of this treatment is limited by the lack of HLA-matched related donors and the toxicity of the myeloablative preconditioning regimens.1,2 While alternative stem cell sources are being explored, such as unrelated cord blood stem cells and partially matched relatives, the use of these types of donor cells has required strong, toxic, preparative regimens to myeloablate all residual host cells. High induction-related morbidity/mortality is not acceptable in SCD, because good medical support can extend life to 40 to 60 years without transplantation.2 Indeed, the first 2 adults undergoing transplantation by the Seattle consortium died of toxicity-related complications, which halted the adult clinical trial. Moreover, Vermylen et al showed that survival and disease-free survival after BMT were consistently worse in those children who met the Seattle criteria for severe disease than in those who were relatively asymptomatic.4 BMT will be available to most SCD patients only when a minimally toxic regimen can be established. Furthermore, given the scarcity of matched-related donors, a truly successful approach will need to cross allogeneic barriers. Walters et al reported that a subset of patients who were treated with
a myeloablative preparative regimen and BMT (designed to produce total
replacement with donor cells) developed stable mixed
chimerism.3 These mixed-chimeric patients had no further sickle cell-related clinical events. This clinical experience leads to
2 major lines of experimental inquiry: (1) Can a regimen be developed
to intentionally produce tolerance and mixed WBC chimerism, and will
this result in a reliable cure of SCD? (2) What is the minimal level of
RBC chimerism that can lead to both a hematologic and systemic cure of
SCD? The first step toward the ultimate goal of answering these
questions in a clinical setting is to perform controlled studies in a
bona fide animal model of SCD. The recent creation of transgenic mice
in which human sickle In this study we have focused on the first question posed above and have intentionally induced mixed WBC chimerism in the transgenic sickle mice with a well-tolerated, nonmyeloablative preconditioning regimen employing low-dose busulfan. Furthermore, we have added costimulation blockade of the CD40/CD40L and CD28/B7 pathways to induce stable donor-specific tolerance to fully MHC-mismatched bone marrow without GVH disease. This protocol resulted in WBC mixed chimerism and complete replacement of the peripheral RBC compartment in 10 of 13 animals within 1 month of transplantation that was stable throughout the observation period (> 150 days). The fact that engrafted mice demonstrated a significantly higher level of donor RBCs in the peripheral blood compared with donor WBCs (100% vs 43%, respectively) is likely due to the dramatically increased clearance of sickle RBCs from the circulation, with RBC read-out reflecting the lineage-specific survival of donor RBCs. Thus, even with partial stem cell engraftment and mixed WBC chimerism, complete normalization of the peripheral RBC defect occurred in engrafted sickle mice. The replacement of the RBC compartment with normal cells resulted in a clinical cure of murine SCD by all parameters tested, including reversal of hemolytic anemia, disappearance of sickled forms on peripheral blood smears, reduction of RBC PS exposure, and normalization of the hematopoietic balance in the spleen. Furthermore, engrafted mice showed a reduction in peripheral WBC count, which is elevated in the untreated sickle cohort. This reduction in peripheral WBC count is particularly interesting given that several lines of evidence indicate that leukocytes play a major role in sickle cell pathophysiology.36,37 Indeed, elevated WBC count is an epidemiologic risk factor for increased morbidity and mortality in SCD,38 including pulmonary complications,39 stroke,40 and silent brain infarction.41 Moreover, decline in WBC count was the hematologic parameter most strongly correlated with clinical response to hydroxyurea in reducing the severity of SCD.42 Importantly, in addition to the correction of the sickle hematologic abnormalities noted above, engrafted mice also showed normalization of solid organ structure and histology. Again, this is a strong indication that whole-body cure of murine SCD was accomplished with our transplantation protocol. All sickle mice treated with TDBM alone or TDBM plus busulfan (no costimulation blockade) fully rejected their bone marrow graft and developed no WBC or RBC chimerism. In contrast, significant peripheral RBC chimerism (10%-54%) was obtained in a subset of mice (5 of 9 animals) that did not receive busulfan, but that did receive TDBM and costimulation blockade, despite minimal WBC chimerism (< 2.5% by flow cytometry). The higher level of donor RBCs compared with WBCs in the peripheral blood again likely reflects their survival advantage over the rapidly cleared sickle cells. Given the recent discovery of common lymphoid and myeloid precursors,43,44 the intriguing possibility also exists of a common erythroid/megakaryocyte precursor and that enhanced engraftment of such a cell may occur in sickle mice. Clinically, the ability to engraft RBCs without any busulfan treatment may be very important. Given the extreme fragility of adult SCD patients subjected to conventional BMT, a regimen that would produce RBC chimerism without any chemotherapy may allow even the sickest patients an option of long-term disease amelioration. This development of mixed RBC chimerism in a subset of mice given BMT and costimulation blockade without busulfan pretreatment applies to the second question posed above: What is the minimal level of RBC chimerism that can lead to both a hematologic and systemic cure of SCD? Although beyond the scope of the present study, the small number of animals who serendipitously developed mixed RBC chimerism (Figure 7) does gi |
Top
Abstract
Introduction
-globin,
-globin, and sickle 
). Three mice became
transiently engrafted but rejected their grafts on or before 60 days.
These mice completely reverted to the host WBC and RBC phenotype by 3 to 4 months after transplantation (data not shown). Control animals
received 1 of 3 treatments: TDBM alone (n = 5, 
), TDBM plus
busulfan (no costimulation blockade, n = 5, 
), or TDBM plus
costimulation blockade but no busulfan (n = 9, 
) Animals in each
of the control groups showed less than 2.5% peripheral WBC chimerism.
(B) Lineage-specific chimerism over time. The percent of each lineage
(granulocytes [GR1], macrophages [CD11b], B cells [B220], and T
cells [CD3]) that was donor type (H2-Kd) was identified
by flow cytometry over time. Values shown are the mean for each time
point from 7 engrafted animals ± SEM. 
,
GR1; 
, CD11b; 
, CD3. (C) Representative flow cytometric analysis.
At left is a histogram showing H2-Kd staining of a chimeric
animal with both host (H2-Kd-negative) and donor
(H2-Kd-positive) WBCs. At right is a dot plot showing a
representative quadrant analysis of CD3 cells versus H2-Kd.
(D) WBC chimerism in the bone marrow (
), and thymus
(
). Engrafted sickle cell mice (n = 4) showed 45% ± 15%
chimerism (H2-Kd-positive cells by flow cytometry) in the
splenic compartment, 50% ± 10% chimerism in the bone marrow, and
25% ± 15% chimerism in the thymus.
