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Departments of
*
Surgery,
Pediatrics, and
Medicine, The Carlos and Marguerite Mason Transplantation Biology Research Center, Emory University School of Medicine, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiation, depletion of the peripheral immune system) or
administration of "mega" doses of bone marrow to facilitate
reliable engraftment. Although encouraging, many issues remain that may
restrict or prevent clinical application of such strategies. We
describe an alternative, nonirradiation based strategy using a single
dose of busulfan, costimulation blockade, and T cell-depleted donor
bone marrow, which promotes titratable macrochimerism and a reshaping
of the T cell repertoire. Chimeras exhibit robust donor-specific
tolerance, evidenced by acceptance of fully allogeneic skin grafts and
failure to generate donor-specific proliferative responses in an in
vivo graft-versus-host disease model of alloreactivity. In this model,
donor cell infusion and costimulation blockade without busulfan were
insufficient for tolerance induction as donor-specific
IFN--producing T cells re-emerged and skin grafts were rejected at
100 days. When applied to a murine 
-thalassemia model, this
approach allows for the normalization of hemologic parameters and
replacement of the diseased red cell compartment. Such a protocol may
allow for clinical application of mixed chimerism strategies in
patients with end-stage organ disease or
hemoglobinopathies. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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irradiation and/or depletion of the
peripheral immune system (3, 4, 5, 6, 7, 8). Even though traditional
conditioning methods may be acceptable in the setting of hemopoietic
malignancy, their application to patients awaiting organ
transplantation or suffering from genetic hemoglobinopathies has been
prohibitive due to concerns over toxicity and the risk of graft-vs-host
disease (GVHD). More recently, it has been reported that simultaneous blockade of costimulatory signals and administration of supraphysiological doses of non-T cell-depleted donor BM (non-TDBM) obviate the need for pretransplant conditioning (9, 10). In these protocols, large numbers of donor BM cells (6.7 x 109 BM cells/kg), given under the protection of costimulation blockade, engraft and produce low-level (4–12%) macrochimerism and transplant tolerance. Though appealing, these protocols require quantities of non-TDBM that are, at present, clinically unfeasible in the setting of cadaveric organ transplantation, and the degree of donor chimerism that they achieve may be too low to effectively treat hemoglobinopathies. Consequently, we sought to develop a regimen that would allow for titratable degrees of hemopoietic chimerism dependent on the intended application—lower levels for the induction of organ transplant tolerance and higher levels for the treatment of hemoglobinopathies, such as sickle cell anemia or the various thalassemias.
To minimize peripheral toxicities while promoting engraftment with
clinically relevant numbers of donor BM cells, we explored the use of
busulfan in combination with costimulatory blockade. Busulfan is an
alkylating agent that produces preferential depletion of early
hemopoietic stem cells (HSCs) (11, 12). It is commonly used in a
multidose fashion and always in conjunction with other chemotherapeutic
agents for recipient conditioning in many clinical BM transplant
regimens (13). As such, busulfan presented an attractive
alternative, nonirradiation-based conditioning approach for
investigation. Here we show that in conjunction with simultaneous
blockade of the CD40 (anti-CD40 ligand (anti-CD40L)) and CD28
(CTLA4-Ig)-costimulatory pathways, minimally myelosuppressive doses of
busulfan promote titratable, high-level chimerism using low numbers of
allogeneic TDBM in murine models of organ transplantation and
-thalassemia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult male 6- to 8-wk-old C57BL/6 (H-2b), BALB/c (H-2d), C3H/HeJ (H-2k), and C57BL/6 SCID (H-2b) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6JHbbd3th male mice (H-2b) were provided by D. Archer (Emory School of Medicine, Atlanta, GA). All mice were housed in specific pathogen-free conditions and in accordance with institutional guidelines.
BM preparation and treatment regimens
BM was flushed from tibiae, femurs, and humeri. Ferromagnetic T cell depletion with anti-CD3 (PharMingen, San Diego, CA) or anti-CD90 and a MACS separation column system (Miltenyi Biotec, Auburn, CA) was performed and confirmed by flow cytometry (anti-CD3, anti-CD4, anti-CD8, and anti-CD5 Abs; PharMingen). Red cell lysis was performed using a Trizma base ammonium chloride solution. The BM cells were resuspended at 2 x 107 cells/500 µl sterile saline and injected i.v. Hamster anti-mouse CD40L mAb (MR1; Bioexpress, Lebanon, NH) and CTLA4-Ig (Bristol-Myers Squibb, Princeton, NJ) were administered on days 0, 2, 4, 6, 14, 28 (500 µg/dose i.p.). In vivo depletion of CD4+ T cells was accomplished by using 100 µg anti-CD4 mAb (GK1.5) i.p. on days -3, -2, -1, 0, and weekly thereafter.
Skin grafting
Full thickness skin grafts (1 cm2) were
transplanted on the dorsal thorax of recipient mice and secured with a
Band-Aid for 7 days. Rejection was defined as the complete loss of
viable epidermal graft tissue. Statistical significance was determined
using the Mann-Whitney U test.
Flow cytometric analysis
Peripheral blood was analyzed by staining with
fluorochrome-conjugated Abs (anti-CD3, anti-CD5,
anti-CD11b, anti-CD11c, anti-GR1, anti-B220,
anti-H-2Kd,
anti-I-Ad,
anti-H-2Kb, anti-V11,
anti-V5.1/5.2, anti-V8.1/8.2 (PharMingen), anti-CD4,
anti-CD8 (Caltag, Burlingame, CA), or immunoglobulin isotype
controls (PharMingen, Caltag)) followed by RBC lysis and washing with a
whole blood lysis kit (R&D Systems, Minneapolis, MN). Stained cells
were analyzed using CellQuest software on a FACSCalibur flow cytometer
(BD Biosciences, Mountain View, CA). Percentage of donor chimerism was
determined as follows: (H-2Kd+ cells/total
cells) x 100.
Cytotoxicity assays
BALB/c CL.7 cells (a fibroblast cell line) were used as targets
and were suspended at 1 x 107/ml with
750 µCi 51Cr (NEN Life Science Products,
Boston, MA) for 90 min at 37°C. Target cells were washed three times
and plated at 1 x 104 targets/well.
Effectors were prepared as nylon wool-passaged splenocytes and plated
at the appropriate ratios in quadruplicate. Total lysis was measured by
the addition of 2% Triton-X to targets, and spontaneous lysis by the
addition of RPMI 1640 supplemented with 10% FCS without
effector cells. After 5 h, the supernatant was harvested and
analyzed by -counting. Percentage of specific lysis was determined
by the use of the following formula: 100 x (cpm unknown -
cpm spontaneous)/(cpm total - cpm spontaneous).
IFN- ELISPOT assays
Allospecific T cell responses were measured by IFN- ELISPOT
assays using nylon wool-passed splenocytes from experimental C57BL/6
mice. The capture Ab, rat anti-mouse IFN- (clone R4-6A2;
PharMingen), was incubated at 4 µg/ml in PBS (100 µl/well) at 4°C
overnight in ester-cellulose-bottom plates (Millipore, Bedford, MA).
After washing, various dilutions of responder cells were added.
Irradiated (2000 rad) transiently (16 h) adherent splenocytes (60–70%
CD11c+) were used as stimulators at a 1:10
stimulator-to-responder ratio. Effector cells were incubated for 14–16
h at 37°C with or without stimulators. After the culture period,
biotinylated anti-mouse IFN- (clone XMG1.2; PharMingen) was
added at 4 µg/ml (100 µl/well). After 2–3 h at 4°C, unbound Ab
was removed, and HRP-avidin D (Sigma, St. Louis, MO) was added. Spots
were developed with the substrate 3-amino-9-ethyl-carbazole (Sigma)
with 0.015% H2O2. Each
spot represents an IFN--secreting cell, and the frequency was
determined by dividing the number of spots counted in each well by the
total number of cells plated at that dilution.
CFSE assay
Splenic and mesenteric lymph node cells were harvested from experimental mice. After RBC lysis and nylon wool passage, cells were incubated in 10 µM CFSE (Molecular Probes, Eugene, OR). Irradiated (1800 rad) BALB/c, C57BL/6, or C3H mice then received 1 x 107 to 1 x 109 CFSE-labeled cells i.v. After 66–72 h, splenocytes were harvested from the recipients, the RBCs lysed, and the remaining cells stained with anti-CD4 (PharMingen) and anti-CD8 (Caltag) and analyzed by flow cytometry as above.
Hemologic monitoring
Hemavet series multiple species hemology instrument (1500 R series; CDC Technologies, Oxford, CT) was used to determine the complete blood counts.
Hemoglobin electrophoresis
Hemoglobin electrophoresis was performed using a cystamine hemoglobin cellulose acetate gel electrophoresis procedure (14). Briefly, 2 µl of whole blood was mixed with 7 µl of a solution containing 83 mM cystamine, 0.25% ammonium hydroxide, and 0.01 M DTT. The mixture was incubated at room temperature for 15 min before applying to cellulose acetate gels (Helena Laboratories, Beaumont, TX) and electrophoresed for 45 min at 350 V in SupraHeme buffer (Helena Laboratories). Gels were poststained using Ponceau S (Sigma) for hemoglobin visualization.
Reticulocyte counts
Reticulocytes were quantified by staining whole blood with the RNA-specific label Thiazole Orange (Sigma), anti-CD45, and Ter-119 Abs (PharMingen). Reticulocytes are defined as Ter-119 positive, Thiazole Orange-positive, CD45-negative.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initially we examined the ability of a single dose of busulfan to
promote hemopoietic chimerism in the absence of an immunologic barrier
using congenic mice. C57BL/6 (B6) recipients
(H-2b, CD45.2) were treated with a single
busulfan dose (0, 10, 20, or 30 mg/kg i.p. (below the
LD50 dose of 136 mg/kg, with marrow rescue; Ref.
15) 1 day before i.v. infusion of 2 x
107 B6.SJL (H-2b, CD45.1)
TDBM cells (6.6 x 108 BM cells/kg). As
expected, levels of donor hemopoietic chimerism, measured by peripheral
blood cell flow cytometry, were directly proportional to the busulfan
dose administered (Fig. 1
A).
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B). As in the congenic experiment, the
level of chimerism was directly proportional to the dose of busulfan.
Animals in control groups failed to demonstrate hemopoietic chimerism
at any time point (Fig. 1, B and C and data not
shown). Furthermore, the levels of hemopoietic chimerism seen in
allogeneic transplants were similar to the levels seen in mice
receiving congenic TDBM, indicating that the addition of donor cells
and costimulation blockade had effectively eliminated the immunological
barrier to allogeneic TDBM transplantation.
Although our initial experiment achieved high-level chimerism using an
engrafting dose of TDBM that was only one-tenth the quantity used in
recent reports without recipient conditioning (9, 10), we
wished to determine whether lower doses of TDBM could induce stable
mixed chimerism. The engrafting dose of TDBM (day 6) was titrated from
2 x 107 to 0. At >120 days posttransplant,
peripheral donor cells correlated directly with the engrafting dose of
TDBM (2 x 107 (65%), 1 x
107 (57%), 5 x 106
(37%), 2 x 106 (26%); Fig. 1D). Remarkably, stable macrochimerism was achieved by using
an engrafting dose of as few as 2 x 106
TDBM cells (6.7 x 107 BM cells/kg). These
results indicate that the level of chimerism attained is titratable
either by altering the engrafting dose of marrow or by modifying the
dose of busulfan. In other experiments we have found that either
irradiated BM or splenocytes could be substituted for the tolerizing
dose of BM (data not shown).
Tomita et al. (19) reported that 3 Gy whole body
irradiation (WBI) was the minimal dose required to produce reliable
long-term engraftment of syngeneic pluripotent HSCs. In addition, they
evaluated the toxicity profile associated with WBI-based BM transplant
protocols and concluded that 3 Gy was essentially nonmyelosuppressive.
Furthermore, a similar protocol involving 3 Gy WBI and costimulatory
blockade has been proven sufficient to produce reliable levels of
chimerism in an allogeneic model (8). For comparison, we
assessed the toxicity of our busulfan-based protocol (2 x
107 BALB/c TDBM (6.7 x
108 BM cells/kg), 500 µg costimulation blockade
0, 2, 4, 6, and 20 mg/kg busulfan day -1; n = 5) and
an irradiation-based protocol (2 x 107 donor BM
cells, 450 µg anti-CD40L at day 0, 500 µg CTLA4-Ig at day 2,
and 3 Gy irradiation at day 0; n = 5). As shown in Fig. 2, both protocols are minimally myelosuppressive (white cell count nadir:
irradiation-based protocol, day 13, 2.86 x
103/mm3; busulfan-based
protocol, day 13, 4.04 x
103/mm3). Furthermore,
greater than 200 animals have been treated with our busulfan-based
regimen with only one death (resulting from anesthesia). These results
demonstrate that titratable, high-level chimerism can be achieved
safely in the absence of irradiation.
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Next, we tested the effects of the tolerance protocol in
experimental hemoglobinopathy and skin transplantation models. Although
previous protocols have achieved transplant tolerance and mixed
chimerism in the absence of recipient conditioning using
supraphysiologic doses of T cell-replete BM (5.3–6.6 x
109 BM cells/kg), the levels of chimerism were
modest (4–12%) (9, 10). In the setting of
hemoglobinopathies, red cell chimerism in this range may be
insufficient to correct the pathophysiology of disease. Therefore, we
assessed the degree to which our chimerism induction protocol could
promote replacement of the red cell compartment in the
Hbbth2 murine model of thalassemia
(20). This thalassemia model, created by an
insertional disruption of the mouse adult major globin gene,
results in perinatal death of homozygotes, whereas heterozygotes
survive but display a phenotype similar to human thalassemia
intermedia, characterized by shortened RBC survival, anemia, and
reticulocytosis. For this experiment, thalassemic heterozygote
recipients (H-2b) were treated with a tolerizing
dose of BALB/c TDBM (day 0), costimulation blockade (days 0, 2, 4, and
6), busulfan (20 mg/kg, day 5), and an engrafting dose (6.6 x
108 BM cells/kg) of BALB/c TDBM (day 6). Control
recipients received costimulation blockade and TDBM without busulfan.
Assessments of leukocyte and red cell chimerism, hemoglobin levels
(Hb), and reticulocyte counts were performed before protocol induction,
and at 2 wk, 4 wk, and monthly following transplantation. As in the
previous experiments using B6 recipients, leukocyte chimerism (24.2%
± 3) developed only in recipients treated with costimulation blockade,
and TDBM and busulfan, not in recipients receiving costimulation
blockade and TDBM alone (data not shown). Furthermore, near complete
replacement of the pathologic Hb band by the functional BALB/c major
Hb allele was observed in the chimeric recipients, but not in the
control group (Fig. 3A). Importantly, reticulocyte counts (Fig. 3B) and
Hb (data not shown) in the chimeric, thalassemic mice normalized,
indicating that the pathologic phenotype of the disorder had been
eliminated.
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A). Recipients receiving TDBM and costimulation blockade
without busulfan showed greatly prolonged survival (Fig. 4A), but ultimately rejected their allografts. In contrast,
animals receiving busulfan, TDBM and costimulation blockade accepted
their skin grafts for >460 days without evidence of rejection (Fig. 4A, p < 0.008). Similar results were
obtained in the reciprocal strain combination (data not shown).
Importantly, the concomitant placement of donor tissue did not prevent
the development of hemopoietic chimerism. In addition to the skin graft
model, we have also achieved similar results in pancreatic islet and
heterotopic heart allograft models (N. Shirasugi, A. B. Adams, M. M.
Durham, T. C. Pearson, and C. P. Larsen, manuscript in
preparation).
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100 days after the
original transplant with donor (BALB/c) and third-party (C3H/HeJ) skin
grafts. Control animals promptly rejected both BALB/c and C3H/HeJ skin
grafts median survival time (MST) = 10 and 12 days, respectively, data
not shown). Administration of TDBM and blockade of costimulatory
pathways without the induction of mixed chimerism (i.e., the group
receiving anti-CD40L mAb, CTLA4-Ig, and TDBM but no busulfan)
significantly prolonged primary allograft survival but did not promote
lasting tolerance (original graft MST = 107 days; donor-specific
regraft MST = 8 days, Fig. 4A). In contrast, mice that
received busulfan, TDBM, and costimulation blockade became high-level
chimeras, uniformly accepted the second donor-specific BALB/c skin
grafts (MST > 350 days, Fig. 4B), and promptly
rejected C3H/HeJ grafts (MST = 10 days, Fig. 4B).
Importantly, the original BALB/c skin grafts and the chimeric state
were unperturbed following placement of a second skin graft (Fig. 4A). In addition, we have achieved robust tolerance and
stable chimerism using a single dose of 2 x
107 TDBM cells on the day of skin transplantation
with a single dose of busulfan 24, 12, or 6 h before TDBM infusion
(data not shown). Donor BM and costimulation blockade transiently eliminates anti-donor T cell responses but mixed chimerism is required for sustained unresponsiveness
To examine the tolerant state, we explored the ability of treated
and control mice to generate anti-donor T cell cytolytic (CTL) and
IFN- (ELISPOT) responses after challenge with a donor skin graft
both at early (day 10) and late (>100 days) time points. Splenic T
cells were prepared from B6 recipients of BALB/c skin graft that
received TDBM and costimulation blockade, TDBM and busulfan, TDBM and
costimulation blockade with busulfan, no treatment, or from naive B6
animals. Untreated B6 mice generated both large numbers of
IFN--producing cells (Fig. 5A) and strong CTL responses (Fig. 5B) 10 days
after skin grafting. During the induction period (at day 10) both the
generation of IFN--producing cells and CTL responses were inhibited
in all groups receiving costimulation blockade and essentially
abrogated in animals receiving costimulation blockade and BM (with or
without busulfan; Fig. 5, A and B). However, at
later time points (>100 days), animals treated with TDBM and
costimulation blockade without busulfan generated significant numbers
of donor-reactive IFN--producing cells and anti-donor CTL
activity after rechallenge with a second donor skin graft; in contrast,
those treated with TDBM, costimulation blockade, and busulfan failed to mount any anti-donor
CTL activity or IFN- response (Fig. 5, A and
C). Both groups mounted similar anti-third party (C3H,
H-2k) responses (data not shown). These results
indicate that the initial, transient hyporesponsiveness to donor Ag
established by TDBM in the presence of costimulation blockade wanes
over time, possibly due to the emergence of new thymic emigrants or to
the decay of regulatory T cell function. In support of the latter
hypothesis, animals treated with costimulation blockade, TDBM, and
busulfan demonstrate significant levels of donor-derived class
II-bearing cells (CD11c+,
I-Ad+) in the thymus, whereas animals receiving
only donor cells and costimulation blockade failed to show thymic
engraftment (data not shown).
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Previous reports have indicated that long-term survival induced by
CD40/CD40L blockade and donor-specific transfusion requires the
participation of CD4+ T cells (18).
However, it not is known whether protocols that induce tolerance via
the establishment of mixed chimerism also require
CD4+ T cells. To explore this question, we
depleted recipients of CD4+ T cells in vivo with
an anti-CD4 mAb, before and during tolerance induction. In the
absence of CD4+ cells, animals treated with donor
BM, 20 mg/kg busulfan, and costimulation blockade (as above) uniformly
failed to become chimeric, implying an essential role for
CD4+ cells during chimerism induction (skin graft
MST = 29 days, n = 7, data not shown). To
investigate whether CD4+ cells were also
necessary for tolerance/chimerism maintenance we depleted long-term
chimeras (>300 days post transplant) of CD4+
cells as above. In contrast to the induction phase, where
CD4+ cells play a pivotal role, depletion of
CD4+ T cells during the maintenance phase did not
perturb either skin graft survival or the chimeric state (data not
shown). Because there is strong evidence that dominant regulatory
mechanisms may play a crucial role in tolerance maintenance in other
models, we also performed adoptive transfer experiments to test for
evidence of regulation (21). We adoptively transferred T
cells from tolerant-chimeric mice, naive B6 mice, or mixtures of
tolerant and naive T cells into C57BL/6 SCID mice (B6 SCID) receiving
both BALB/c and C3H skin grafts. At 150 days after therapy
institution (last BM on day 6 and last costimulation blockade on day
28), T cells were prepared from the spleens of mice that had been
rendered specifically tolerant to BALB/c skin grafts (but rejected
third party) with our protocol. Next, B6 SCID mice received 5 x
106 transferred T cells from chimeric-tolerant
animals (TDBM, costimulation blockade, busulfan), cells from tolerant
animals mixed with 5 x 106 T cells from
naive B6 mice, or only cells from naive B6 mice. T cells from naive
animals quickly rejected donor and third party grafts (MST = 10
and 12 days, respectively; Fig. 5D). In contrast, 100% of
animals receiving T cells from tolerant animals accepted BALB/c skin
grafts (>300 days) while rejecting third party allografts (MST =
12 days; Fig. 5D). However, when naive T cells were mixed
with the tolerant T cells, prompt rejection of the BALB/c skin grafts
was observed (MST = 12 days). These data confirm that T cells from
animals receiving our protocol of TDBM, busulfan, and costimulation
blockade are robustly and specifically tolerant to the marrow donor and
suggest that although regulatory mechanisms may play an important role
during tolerance induction, they are unlikely to be the major mechanism
by which tolerance is maintained in this model. However, given the
requirement for CD4+ T cells during the tolerance
induction period, additional experiments focusing on the role of
CD4+ CD25+ regulatory cells
are clearly warranted.
Clonal deletion of alloreactive T cells is the primary mechanism for tolerance maintenance
BALB/c mice delete V11- and V5-bearing T cells, whereas B6
mice do not express I-E and use V11 on 4–5% of
CD4+ T cells and V5.1/2 on 2–3% of
CD4+ T cells (22, 23). As
anticipated, control groups (costimulation blockade or TDBM or busulfan
alone) failed to delete donor-reactive V11+ or
V5+CD4+ T cells (Fig. 6). In contrast, recipients of BALB/c TDBM, busulfan, and costimulation
blockade therapy developed near complete deletion of
CD4+V11+ and
CD4+V5+ T cells by day
60. The percentage of V8-bearing CD4+T cells,
which are expressed on 15–20% of BALB/c and B6
CD4+T cells, was similar in all groups,
indicating that the T cell deletion was donor specific in nature
(Fig. 6).
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D). However, strong proliferative responses
to third party (C3H) were similar in all groups (Fig. 5D).
Taken together with repertoire analysis, the absence of
CD4+ or CD8+ T cells
capable of cellular division in this GVHD model provides further
evidence that the tolerant state achieved with this protocol results in
near complete elimination of the donor-specific T cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Within the past year, it has been reported that administration of supraphysiological doses of non-TDBM obviates the need for pretransplant conditioning (9, 10). In these protocols, administration of very large numbers of donor BM cells (6.7 x 109 BM cells/kg), under the protection of costimulation blockade, produces durable hemopoietic chimerism and induces a robust state of deletional donor-specific transplantation tolerance. Although these "mega"-dose BM approaches are appealing in that they avoid the associated toxicity of preconditioning, there are also issues that must be addressed and/or overcome to allow for their clinical application. For example, these protocols rely upon the use of unseparated BM cells. Specifically, T cells were not removed from the preparations. Although leaving T cells in the preparation may enhance HSC engraftment, the risk of potentially lethal GVHD is proportional to the T cell mass in the BM inoculum. Although the percentage of T cells in the BM is relatively low, the mega-doses of BM required for these protocols transfers vastly more T cells than the current methods used in clinical BM transplantation. A second concern centers on the level of chimerism achieved with these methods. Even though the initial application of hemopoietic chimerism may focus on the induction of transplantation tolerance, the impact of a safe, reliable chimerism induction strategy might have a greater impact on the treatment of genetic hemoglobinopathies (e.g., sickle cell anemia and the thalassemias). Unfortunately, the levels of chimerism achieved using these mega-dose BM strategies were relatively low (4–12%) and may prove to be insufficient not only to provide functional RBCs but also to reduce the number of pathogenic defective red cells. Finally, the absolute numbers of BM cells required to achieve the relatively modest levels of chimerism observed in these studies may be clinically impractical in many settings. In these experiments, BM from multiple (2, 3) donor mice was required to transplant a single recipient. Although peripheral stem cell mobilization in living donors may circumvent this problem, the time constraints and complexity of cadaveric donation would, at present, appear to preclude the application of this approach for the transplantation of multiple organs from a single cadaveric donor.
Although there are many practical issues that must be addressed for tolerance induction to become a clinical reality, we believe there are three critical conceptual features that must be considered in the design of any tolerance induction strategy. First, the strategy must provide a means to control the existing population of donor-specific T cells in the periphery. Second, it must provide a method to control donor-specific T cells that may be generated in the future. Finally, the regimen must protect the allograft from irreversible immunologic injury during tolerance induction and maintenance.
We have shown that the combination of busulfan, TDBM, and
costimulation blockade promotes hemopoietic chimerism and robust
transplantation tolerance in mouse models of transplantation and
genetic hemoglobinopathies. Furthermore, the doses of busulfan required
are well tolerated, minimally myelosuppressive, and permit titratable,
high-level chimerism. Like other successful macrochimerism induction
protocols, we have observed a robust state of donor-specific tolerance
to secondary skin grafts and specific deletion of superantigen-specific
(CD4+V11+ and
V5+) T cells that are normally deleted in the
donor strain. Using new technology, we have provided evidence of robust
tolerance of both CD4+ and
CD8+ alloreactive T cells by showing a complete
lack of donor-specific responsiveness in vivo using the CSFE model and
cytokine ELISPOT assays after rechallenge with skin grafts. In
particular with the ELISPOT assay, we show that although administration
of BM and costimulation blockade produces prolonged hyporesponsiveness
in the absence of chimerism, the donor-reactive IFN--producing T
cells re-emerge by day 100. We have also shown that this minimally
myelosuppressive mixed chimerism protocol is able to restore normal red
cell parameters in a murine model of thalassemia. These data
suggest that this method for induction of hemopoietic chimerism and
transplantation tolerance may potentially be useful in several clinical
disease states where traditional BM transplantation is currently
precluded including solid organ transplantation and hemologic
diseases.
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Footnotes |
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2 A.B.A. and M.M.D. contributed equally to this work.
3 Current address: Department of Surgery, Seoul National University College of Medicine, Seoul, Korea.
4 E.K.W. and C.P.L. share senior authorship.
5 Address correspondence and reprint requests to Dr. Christian P. Larsen, Emory University, Woodruff Memorial Building, 1639 Pierce Drive, Atlanta, GA 30322. E-mail address: clarsen{at}emory.org
6 Abbreviations used in this paper: BM, bone marrow; GVHD, graft-vs-host disease; HSCs, hemopoietic stem cells; CD40L, CD40 ligand; WBI, whole body irradiation; TDBM, T cell-depleted donor BM; Hb, hemoglobin level; MST, median survival time.
Received for publication February 14, 2001. Accepted for publication May 7, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and CTLA4. J. Clin. Invest. 101:2446.[Medline]
-globin gene. Proc. Natl. Acad. Sci. USA 90:3177. 11-bearing T cells. J. Exp. Med. 169:1405.This article has been cited by other articles:
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  J. Kurtz, F. Raval, C. Vallot, J. Der, and M. Sykes CTLA-4 on alloreactive CD4 T cells interacts with recipient CD80/86 to promote tolerance Blood, April 9, 2009; 113(15): 3475 - 3484. [Abstract] [Full Text] [PDF]
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 B. H. Koehn, M. L. Ford, I. R. Ferrer, K. Borom, S. Gangappa, A. D. Kirk, and C. P. Larsen PD-1-Dependent Mechanisms Maintain Peripheral Tolerance of Donor-Reactive CD8+ T Cells to Transplanted Tissue J. Immunol., October 15, 2008; 181(8): 5313 - 5322. [Abstract] [Full Text] [PDF]
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T. Fehr, S. Wang, F. Haspot, J. Kurtz, P. Blaha, T. Hogan, M. Chittenden, T. Wekerle, and M. Sykes Rapid Deletional Peripheral CD8 T Cell Tolerance Induced by Allogeneic Bone Marrow: Role of Donor Class II MHC and B Cells J. Immunol., September 15, 2008; 181(6): 4371 - 4380. [Abstract] [Full Text] [PDF]
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 A. A. Wilson, L. W. Kwok, A.-H. Hovav, S. J. Ohle, F. F. Little, A. Fine, and D. N. Kotton Sustained Expression of {alpha}1-Antitrypsin after Transplantation of Manipulated Hematopoietic Stem Cells Am. J. Respir. Cell Mol. Biol., August 1, 2008; 39(2): 133 - 141. [Abstract] [Full Text] [PDF]
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 B. Metzler, P. Gfeller, G. Wieczorek, J. Li, B. Nuesslein-Hildesheim, A. Katopodis, M. Mueller, and V. Brinkmann Modulation of T cell homeostasis and alloreactivity under continuous FTY720 exposure Int. Immunol., May 1, 2008; 20(5): 633 - 644. [Abstract] [Full Text] [PDF]
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D. Stapler, E. D. Lee, S. A. Selvaraj, A. G. Evans, L. S. Kean, S. H. Speck, C. P. Larsen, and S. Gangappa Expansion of Effector Memory TCR V{beta}4+CD8+ T Cells Is Associated with Latent Infection-Mediated Resistance to Transplantation Tolerance J. Immunol., March 1, 2008; 180(5): 3190 - 3200. [Abstract] [Full Text] [PDF]
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L. M. Ide, B. Gangadharan, K.-Y. Chiang, C. B. Doering, and H. T. Spencer Hematopoietic stem-cell gene therapy of hemophilia A incorporating a porcine factor VIII transgene and nonmyeloablative conditioning regimens Blood, October 15, 2007; 110(8): 2855 - 2863. [Abstract] [Full Text] [PDF]
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B. H. Koehn, M. A. Williams, K. Borom, S. Gangappa, T. C. Pearson, R. Ahmed, and C. P. Larsen Fully MHC-Disparate Mixed Hemopoietic Chimeras Show Specific Defects in the Control of Chronic Viral Infections J. Immunol., August 15, 2007; 179(4): 2616 - 2626. [Abstract] [Full Text] [PDF]
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 N. Najafian, M. J. Albin, and K. A. Newell How Can We Measure Immunologic Tolerance in Humans? J. Am. Soc. Nephrol., October 1, 2006; 17(10): 2652 - 2663. [Abstract] [Full Text] [PDF]
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A. Nakao, H. Toyokawa, K. Kimizuka, M. A. Nalesnik, I. Nozaki, R. J. Bailey, A. J. Demetris, T. E. Starzl, and N. Murase Simultaneous bone marrow and intestine transplantation promotes marrow-derived hematopoietic stem cell engraftment and chimerism Blood, August 15, 2006; 108(4): 1413 - 1420. [Abstract] [Full Text] [PDF]
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 S. J Knechtle Development of tolerogenic strategies in the clinic Phil Trans R Soc B, September 29, 2005; 360(1461): 1739 - 1746. [Abstract] [Full Text] [PDF]
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C. C. Kemball, E. D. H. Lee, V. Vezys, T. C. Pearson, C. P. Larsen, and A. E. Lukacher Late Priming and Variability of Epitope-Specific CD8+ T Cell Responses during a Persistent Virus Infection J. Immunol., June 15, 2005; 174(12): 7950 - 7960. [Abstract] [Full Text] [PDF]
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A. Bushell, E. Jones, A. Gallimore, and K. Wood The Generation of CD25+CD4+ Regulatory T Cells That Prevent Allograft Rejection Does Not Compromise Immunity to a Viral Pathogen J. Immunol., March 15, 2005; 174(6): 3290 - 3297. [Abstract] [Full Text] [PDF]
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P. Zhou, S. J. Balin, M. Mashayekhi, K. W. Hwang, D. A. Palucki, and M.-L. Alegre Transplantation Tolerance in NF-{kappa}B-Impaired Mice Is Not Due to Regulation but Is Prevented by Transgenic Expression of Bcl-xL J. Immunol., March 15, 2005; 174(6): 3447 - 3453. [Abstract] [Full Text] [PDF]
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B. Metzler, P. Gfeller, M. Bigaud, J. Li, G. Wieczorek, C. Heusser, P. Lake, and A. Katopodis Combinations of Anti-LFA-1, Everolimus, Anti-CD40 Ligand, and Allogeneic Bone Marrow Induce Central Transplantation Tolerance through Hemopoietic Chimerism, Including Protection from Chronic Heart Allograft Rejection J. Immunol., December 1, 2004; 173(11): 7025 - 7036. [Abstract] [Full Text] [PDF]
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H. Ito, Y. Takeuchi, J. Shaffer, and M. Sykes Local irradiation enhances congenic donor pluripotent hematopoietic stem cell engraftment similarly in irradiated and nonirradiated sites Blood, March 1, 2004; 103(5): 1949 - 1954. [Abstract] [Full Text] [PDF]
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L. S. Kean, E. A. Manci, J. Perry, C. Balkan, S. Coley, D. Holtzclaw, A. B. Adams, C. P. Larsen, L. L. Hsu, and D. R. Archer Chimerism and cure: hematologic and pathologic correction of murine sickle cell disease Blood, December 15, 2003; 102(13): 4582 - 4593. [Abstract] [Full Text] [PDF]
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G. Andersson, B. M. W. Illigens, K. W. Johnson, D. Calderhead, C. LeGuern, G. Benichou, M. E. White-Scharf, and J. D. Down Nonmyeloablative conditioning is sufficient to allow engraftment of EGFP-expressing bone marrow and subsequent acceptance of EGFP-transgenic skin grafts in mice Blood, June 1, 2003; 101(11): 4305 - 4312. [Abstract] [Full Text] [PDF]
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P. Blaha, S. Bigenzahn, Z. Koporc, M. Schmid, F. Langer, E. Selzer, H. Bergmeister, F. Wrba, J. Kurtz, C. Kiss, et al. The influence of immunosuppressive drugs on tolerance induction through bone marrow transplantation with costimulation blockade Blood, April 1, 2003; 101(7): 2886 - 2893. [Abstract] [Full Text] [PDF]
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N. E. Phillips, T. G. Markees, J. P. Mordes, D. L. Greiner, and A. A. Rossini Blockade of CD40-Mediated Signaling Is Sufficient for Inducing Islet But Not Skin Transplantation Tolerance J. Immunol., March 15, 2003; 170(6): 3015 - 3023. [Abstract] [Full Text] [PDF]
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M. A. Williams, A. B. Adams, M. B. Walsh, N. Shirasugi, T. M. Onami, T. C. Pearson, R. Ahmed, and C. P. Larsen Primary and Secondary Immunocompetence in Mixed Allogeneic Chimeras J. Immunol., March 1, 2003; 170(5): 2382 - 2389. [Abstract] [Full Text] [PDF]
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 J. D. Down and M. E. White-Scharf Reprogramming Immune Responses: Enabling Cellular Therapies and Regenerative Medicine Stem Cells, January 1, 2003; 21(1): 21 - 32. [Abstract] [Full Text] [PDF]
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M. A. Williams, T. M. Onami, A. B. Adams, M. M. Durham, T. C. Pearson, R. Ahmed, and C. P. Larsen Cutting Edge: Persistent Viral Infection Prevents Tolerance Induction and Escapes Immune Control Following CD28/CD40 Blockade-Based Regimen J. Immunol., November 15, 2002; 169(10): 5387 - 5391. [Abstract] [Full Text] [PDF]
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M. Eto, H. Hackstein, K. Kaneko, K. Nomoto, and A. W. Thomson Promotion of Skin Graft Tolerance Across MHC Barriers by Mobilization of Dendritic Cells in Donor Hemopoietic Cell Infusions J. Immunol., September 1, 2002; 169(5): 2390 - 2396. [Abstract] [Full Text] [PDF]
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N. Shirasugi, A. B. Adams, M. M. Durham, A. E. Lukacher, H. Xu, P. Rees, S. R. Cowan, M. A. Williams, T. C. Pearson, and C. P. Larsen Prevention of Chronic Rejection in Murine Cardiac Allografts: A Comparison of Chimerism- and Nonchimerism-Inducing Costimulation Blockade-Based Tolerance Induction Regimens J. Immunol., September 1, 2002; 169(5): 2677 - 2684. [Abstract] [Full Text] [PDF]
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D. Forman, R. M. Welsh, T. G. Markees, B. A. Woda, J. P. Mordes, A. A. Rossini, and D. L. Greiner Viral Abrogation of Stem Cell Transplantation Tolerance Causes Graft Rejection and Host Death by Different Mechanisms J. Immunol., June 15, 2002; 168(12): 6047 - 6056. [Abstract] [Full Text] [PDF]
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L. S. Kean, M. M. Durham, A. B. Adams, L. L. Hsu, J. R. Perry, D. Dillehay, T. C. Pearson, E. K. Waller, C. P. Larsen, and D. R. Archer A cure for murine sickle cell disease through stable mixed chimerism and tolerance induction after nonmyeloablative conditioning and major histocompatibility complex-mismatched bone marrow transplantation Blood, March 1, 2002; 99(5): 1840 - 1849. [Abstract] [Full Text] [PDF]
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M. A. Williams, J. T. Tan, A. B. Adams, M. M. Durham, N. Shirasugi, J. K. Whitmire, L. E. Harrington, R. Ahmed, T. C. Pearson, and C. P. Larsen Characterization of Virus-Mediated Inhibition of Mixed Chimerism and Allospecific Tolerance J. Immunol., November 1, 2001; 167(9): 4987 - 4995. [Abstract] [Full Text] [PDF]
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; 20 mg/kg, 
; 10 mg/kg, 
; 0 mg/kg,
; n = 5/group). Similar results were obtained in
two experiments. B, Allogeneic BM transplant with
busulfan. B6 (H-2b) recipients were treated with 500
µg CTLA4-Ig and anti-CD40L (costimulation blockade, CB) i.p. on
days 0, 2, 4, 6, 14, and 28, varying doses of busulfan were given on
day 5, and recipients were transplanted with allogeneic BALB/c TDBM
(2 x 107 cells i.v. on days 0 and 6) (30 mg/kg, 
; 1 x 107, 
, n = 3). All animals (B6)
received a fully allogeneic skin graft (BALB/c, H-2d) on
day 0. Control groups (no treatment; BM, busulfan; or costimulation
blockade, busulfan) promptly rejected the BALB/c skin graft. One
hundred days after protocol initiation animals were rechallenged with a
second donor (BALB/c, H-2d) and third party (C3H,
H-2k) skin grafts. At 100 days, primary skin graft survival
in BM, costimulation blockade group (
, MST = 10
days), whereas the secondary donor grafts went on to 100% survival for
over 350 days (•, p = 0.001 compared with
control). Similar results have been achieved in three additional
experiments.
, n = 3) and >100 days (