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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2523-2529
Stable Mixed Hematopoietic Chimerism in Dogs Given Donor Antigen,
CTLA4Ig, and 100 cGy Total Body Irradiation Before and Pharmacologic
Immunosuppression After Marrow Transplant
By
Rainer Storb,
Cong Yu,
J. Maciej Zaucha,
H. Joachim Deeg,
George Georges,
Hans-Peter Kiem,
Richard A. Nash,
Peter A. McSweeney, and
John L. Wagner
From the Clinical Research Division, Fred Hutchinson Cancer Research
Center; and the Department of Medicine, University of Washington,
Seattle, WA.
 |
ABSTRACT |
Stable mixed chimerism can be established in dogs given a sublethal
dose of 200 cGy total body irradiation (TBI) before and immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP) for 28 and 35 days, respectively, after dog leukocyte
antigen-identical marrow transplantation. Most likely, the role of
pretransplant TBI was to provide host immunosuppression, since stable
mixed chimerism was also achieved in MMF/CSP-treated dogs when 450 cGy irradiation, targeted to cervical, thoracic, and upper abdominal lymph
nodes, was substituted for TBI. When TBI was reduced from 200 to 100 cGy, all grafts were rejected within 3 to 12 weeks. Here, we asked
whether stable engraftment after 100 cGy TBI could be accomplished by
first reducing the intensity of host immune responsiveness with help of
the fusion peptide CTLA4Ig, which blocks T-cell costimulation through
the B7-CD28 signal pathway. Accordingly, recipient T cells were
activated with intravenous (IV) injections of 106 donor
peripheral blood mononuclear cells (PBMC)/kg per day on days  7 to
1 before 100 cGy TBI, with concurrent administration of CTLA4Ig 4 mg/kg/d IV. All 7 dogs so treated showed initial mixed chimerism. Two
rejected their allografts after 8 and 20 weeks, respectively, and
survived with autologous marrow recovery; 1 mixed chimera was
unevaluable because of death at 3 weeks from intussusception; and 4 showed persisting mixed chimerism, including unirradiated marrow and
lymph node spaces, for now more than 46 to 70 weeks after transplant.
Data support the hypothesis that stable marrow allografts can be
established by combining nonmyeloablative pretransplant host
immunosuppression with posttransplant host and donor cell
immunosuppression using MMF/CSP.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
WE RECENTLY PROPOSED a new concept for
performing marrow allografts which was founded on the knowledge that
both host-versus-graft (HVG) and graft-versus-host (GVH) reactions are
effected by T cells in the major histocompatibility complex
(MHC)-identical setting.1 Accordingly, we designed a
regimen of posttransplant immunosuppression that was capable of not
only preventing graft-versus-host disease (GVHD),2 but also
controlling HVG reactions.3 The regimen consisted of
administration of the antimetabolite mycophenolate mofetil (MMF)
combined with the T-cell activation blocker cyclosporine (CSP). Using
MMF/CSP postgrafting enabled us to decrease the dose of total body
irradiation (TBI) otherwise needed for successful engraftment of major
histocompatibility complex (MHC)-identical littermate marrow in a
canine model from a myeloablative and toxic dose of 920 cGy to a
nonlethal dose of 200 cGy.3 Most likely, the major role of
the residual pretransplant TBI was in providing host immunosuppression
and not in creating "marrow space," since stable donor
engraftment was also achieved in MMF/CSP-treated dogs when 450 cGy
local irradiation to cervical, thoracic, and upper abdominal lymph
nodes was substituted for 200 cGy TBI.4 This finding raised
the question whether pretransplant TBI in the model could, in part or
completely, be replaced by noncytotoxic immunosuppression.
A number of biologic reagents are potential candidates for targeting
host immune responses, including those that modify first and second
signals of T-cell activation. Some of these have been used successfully
both in murine and canine models of hematopoietic stem-cell
transplantation.5-12 Specifically, sustained T-cell activation is likely to require signal amplification. An important component of this amplification is provided by the costimulatory molecule CD28 on T cells. Wülfing and Davis13 suggest
that CD28 costimulation brings about an active accumulation of
molecules at the interface of the T cell and the antigen-presenting
cell, thereby building an immunologic synapse that increases the
overall amplitude and duration of T-cell signaling. Here, we sought to block the costimulatory signal using CTLA4Ig, a soluble,
immunosuppressive fusion peptide that consists of the extracellular
domain of CTLA4 and the Fc portion of the IgG1 heavy chain
and binds to B7.1 and 7.2 (CD80 and CD86) with high
avidity.14-17 Antigen recognition through the T-cell
receptor, while CD28 costimulation is blocked by CTLA4Ig, resulted in
T-cell hyporesponsiveness as tested in both in vitro and in vivo
studies.18-24 Accordingly, in the present study, the marrow
recipients' T cells were activated by intravenous (IV) injections of
donor antigen in the form of peripheral blood mononuclear cells (PBMC)
for 7 days before conditioning with 100 cGy TBI while concurrently
administering CTLA4Ig. While a previous study had shown only transient
allogeneic engraftment following 100 cGy TBI when no pretransplant
CTLA4Ig was administered,3 most current dogs achieved
sustained engraftment of dog leukocyte antigen (DLA)-identical
littermate marrow.
| |
MATERIALS AND METHODS |
Litters of beagles, harriers, pit bull-beagle crossbreeds, and other
mixed breeds were either raised at the Fred Hutchinson Cancer Research
Center (Seattle, WA) or purchased from commercial kennels. The dogs
weighed from 7.2 to 11.1 (median, 10.5) kg and were 7 to 14 (median, 8)
months old. They were observed for disease for at least 2 months before
study. All were immunized for papillomavirus, leptospirosis, distemper,
hepatitis, and parvovirus. The research protocols were approved by the
Institutional Animal Care and Use Committee of the Fred Hutchinson
Cancer Research Center. Research was conducted according to the
principles outlined in the Guide for Laboratory Animal Facilities and
Care prepared by the National Academy of Sciences, National Research
Council. The kennels were certified by the American Association for
Accreditation of Laboratory Animal Care.
DLA-identical littermate donor/recipient pairs were chosen based on the
identity for highly polymorphic MHC class I and class II microsatellite
marker polymorphisms.25 Specific DLA-DRB1 allelic identity
was determined by direct sequencing.26
The day of marrow grafting was designated as day 0. Recipients were
injected IV with 106 marrow donor PBMC/kg/d on days
7 to 1. Additionally, they received IV CTLA4Ig 4 mg/kg/d
on days 7 to 1. On day 0, they were given a single dose
of TBI 100 cGy delivered at 7 cGy/min from 2 opposing cobalt-60
sources.27 Marrow was harvested from the donors under general anesthesia through needles inserted into humeri and
femora,27 and infused IV into the recipients at doses of
3.8 to 4.7 (median, 4.0) × 108 nucleated cells/kg
within 4 hours of TBI. Recipients were given postgrafting
immunosuppression that consisted of CSP 15 mg/kg twice daily orally
from day 1 to day 35, and MMF 10 mg/kg twice daily
subcutaneously from day 0 to day 27.3 Standard postgrafting care27 included twice-daily oral nonabsorbable antibiotics
(neomycin sulfate and polymyxin sulfate) beginning on day 5
until day 14 after transplant. Dogs did not require transfusions. Their
clinical status was checked twice daily. Upon completion of the
studies, dogs were euthanized and underwent complete autopsies
including histopathologic examinations of autopsy specimens.
Hematopoietic engraftment was assessed by sustained recoveries of
peripheral blood granulocyte and platelet counts after the postirradiation nadirs, histologic features of the marrow from biopsy
or autopsy specimens, and documentation of donor microsatellite marker
polymorphisms in nucleated cells from blood and marrow. Conversely,
graft rejection was defined as complete repopulation of the
hematopoietic system with cells of host type. Posttransplant marrow
aspirates from the humoral head were done under general anesthesia.
Donor and host hematopoietic cells were distinguished by microsatellite
marker polymorphisms as assessed in a polymerase chain reaction
(PCR)-based assay.28 The technique detected between 2.5%
and 97.5% mixtures of donor and host cells. Mixed hematopoietic chimerism was quantified by estimating the proportion of donor-specific DNA among host DNA using the storage phosphorimaging
technique.29
Results in current dogs were compared with those in 6 previously
reported dogs given the same treatment except for omission of
pretransplant donor PBMC and CTLA4Ig injections.3
Additionally, peripheral blood count changes in transplanted dogs were
compared to those of 12 dogs given 100 cGy TBI and no subsequent marrow transplant (unpublished observations, 1999).
Mixed leukocyte cultures (MLC)30 were performed to assess
the immunosuppressive effectiveness of CTLA4Ig on dog cells in vitro.
To this purpose, PBMC from healthy dogs were separated from heparinized
whole blood using a Ficoll-Hypaque gradient (density = 1.074). Cells were washed and then resuspended in Waymoth's medium
supplemented with 1% nonessential amino acids, 1% sodium pyruvate,
1% L-glutamine, and 20% heat-inactivated pooled, normal dog
serum. A total of 105 responder cells/well and
105 irradiated (2,200 cGy in vitro irradiation from a
cesium source) stimulator cells/well obtained from DLA-nonidentical
unrelated donors were cultured together in the presence of increasing
concentrations of CTLA4Ig or a control peptide, L-6, in round-bottom,
96-well plates for 6 days at 37°C in a humidified 5%
CO2 air atmosphere. On day 6, cultures were pulsed with 1 µCi of 3H-thymidine (3H-Td) for 18 hours
before harvest. 3H-Td incorporation was determined using a
 -scintillation counter (Beckman Instruments, Fullerton, CA). Data
were analyzed as mean counts per minute (cpm) of 3 replicates.
Samples for serum levels of CTLA4Ig were collected from 3 dogs before
and at various time intervals after a single IV administration of
CTLA4Ig 4 mg/kg/d. Serum CTLA4Ig levels were measured using a sandwich
enzyme-linked immunoadsorbent assay (ELISA).31 All tests
were performed in triplicate.
| |
RESULTS |
Table 1 lists data on the immunosuppressive
effectiveness of CTLA4Ig in an in vitro MLC assay. At CTLA4Ig
concentrations ranging from 0.625 to 10 µg/mL, 90% to 95%
inhibition of MLC reactivities was seen. At 0.3 µg/mL, inhibition
ranged from 55% to 90%, and at 0.1 µg/mL, it ranged from 35% to
60%. No significant suppression of MLC reactivity was seen with
comparable concentrations of the control peptide, L-6.
CTLA4Ig serum levels in 3 dogs given 7 daily injections of CTLA4Ig 4 mg/kg IV each are shown in Fig 1. Within 1 minute of the first injection, CTLA4Ig levels up to 200 µg/mL were
observed, with subsequent declines over 2 hours to levels ranging from
60 to 70 µg/mL. Subsequent daily trough levels ranged from 25 to 80 µg/mL. These levels were well above the range at which virtually complete suppression of MLC reactivity was observed in vitro.
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| Fig 1.
Mean CTLA4Ig serum concentrations (ELISA)31
in 3 dogs given daily injections of 4 mg of CTLA4Ig/kg. Dots indicate
data points for individual dogs.
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Table 2 summarizes the results of the
transplant studies. The 6 previously reported dogs not given
pretransplant donor PBMC and CTLA4Ig showed initial allogeneic
engraftment that lasted for 3 to 12 weeks with subsequent graft
rejection, and they survived with complete autologous
recovery.3 The 7 dogs given pretransplant PBMC and CTLA4Ig
also engrafted. One of the 7 died from an intussusception, a
CSP-associated toxicity in dogs, at 3 weeks with mixed donor/host hematopoietic chimerism present. This dog's early death precluded final evaluation of the fate of the allograft. Two dogs rejected their
grafts after 8 and 20 weeks, respectively, and survived with autologous
marrow recovery. Four dogs have remained stable mixed donor/host
chimeras for now more than 46 to 70 weeks after transplant.
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Table 2.
Marrow Grafts From DLA-Identical Littermates After
Conditioning With 100 cGy TBI Delivered as a Single Dose at 7 cGy/min
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|
Figure 2 illustrates the hematologic
changes and the results of microsatellite marker studies in one of the
current dogs (E519). The granulocyte nadir occurred at approximately
day 10 with a count of 3,000/µL followed by rapid recovery. The
platelet count nadir occurred at 60,000/µL on day 14 followed by
recovery, while the lymphocytopenia was more prolonged, and recovery
did not occur until after week 7. The lowest lymphocyte counts were on
the order of 600/µL. The microsatellite marker studies performed 41 to 44 weeks after transplant demonstrated the presence of donor cells among all nucleated peripheral blood cells, mononuclear cells, and
granulocytes.
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| Fig 2.
(A) Peripheral blood granulocyte, platelet, and
lymphocyte changes in a dog conditioned with 100 cGy TBI before and
MMF/CSP after transplantation of DLA-identical littermate marrow (BMT).
In addition, the dog was pretreated with donor PBMC and IV CTLA4Ig. (B)
Results of testing for microsatellite markers of donor and recipient
cells before transplant (lanes 1 and 2) and recipient cells up to 59 weeks after transplantation (lanes 3 through 21). "Blood"
indicates all nucleated peripheral blood cells; MNC, mononuclear cells;
G, granulocytes.
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Figure 3 illustrates the median peripheral
blood granulocyte, lymphocyte and platelet changes in all current dogs.
During the first 2 weeks after TBI, no obvious differences were seen between their lymphocyte counts and those of the previously
transplanted dogs not given pretransplant CTLA4Ig and of dogs not given
marrow grafts after exposure to 100 cGy TBI. The speed of recovery of counts between days 15 and 35 was marginally but not significantly slower in CTLA4Ig-treated dogs compared with dogs of the 2 other groups.
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| Fig 3.
Median peripheral blood platelet, granulocyte, and
lymphocyte changes in dogs given either 100 cGy TBI and no marrow
grafts (n = 12); 100 cGy TBI, marrow grafts, and postgrafting MMF/CSP
(n = 6), or pretransplant PBMC plus CTLA4Ig, 100 cGy TBI, marrow
grafts, and postgrafting MMF/CSP (n = 7).
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The complete results of microsatellite marker studies in 5 of the
evaluable current dogs are shown in Fig 4.
Continued allogeneic engraftment was seen in 4 and graft rejection in 1 of the 5 dogs. The degree of stable donor chimerism ranged from 10% to
60%.
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| Fig 4.
Microsatellite marker studies of donor and recipient
cells before transplantation and recipient cells after marrow
transplantation in 4 dogs given pretransplant PBMC plus CTLA4Ig, 100 cGy TBI, marrow grafts from DLA-identical littermates on day 0, and
postgrafting MMF/CSP for 4 and 5 weeks, respectively. BM, bone marrow
cells.
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Comparing the duration of mixed chimerism in the 6 current evaluable
CTLA4Ig-treated dogs (not included was dog E736 that died from
intussusception) with that among previously reported controls not given
CTLA4Ig by a 2-sided Mann-Whitney U test showed a significant
prolongation in current dogs (P = .02).
| |
DISCUSSION |
The present study is based on the assumption that allografts can create
their own space in the host's marrow through GVH reactions and,
consequently, that intensive cytoreductive and toxic conditioning regimens conventionally used for hematopoietic stem-cell transplants can be replaced by nonmyelotoxic immunosuppression capable of promoting
acceptance of grafts.1 Specifically, immunosuppressive agents delivered before transplant would be applied to blunt host T-cell responses, while immunosuppression delivered after transplant would serve to modify both host and donor immune reactivities, with
resulting mutual graft/host tolerance in the form of stable mixed
donor/host hematopoietic chimerism. Ideally, agents administered before
transplant would suppress only host cells with the potential to react
to donor antigens while other, say anti-viral, immune responses would
be left intact. Experimental evidence in support of this novel
transplant approach was provided by a previous canine study in which
pretransplant TBI was replaced by 450 cGy irradiation targeted to
cervical, thoracic, and upper abdominal lymph nodes.4 When
combined with posttransplant immunosuppression by MMF/CSP, pretransplant lymph node radiation resulted in stable, long-term mixed
chimerism also in those marrow and lymph node spaces that were shielded
from irradiation. Further evidence has come from successful clinical
studies in 2 patients with inherited T-cell deficiencies other than
severe combined immunodeficiency disease (SCID), in whom pretransplant
immunosuppression was omitted and stable grafts were established solely
with posttransplant MMF/CSP.32
The current study was a first step toward the ideal transplant program
in patients with malignant and nonmalignant hematologic diseases that
would rely entirely on nonmyelotoxic immunosuppression. The dose of TBI
used here, 100 cGy delivered at 7 cGy/min, was small and resulted only
in moderate and transient declines of peripheral blood cell counts,
even in dogs not rescued by subsequent marrow transplants. Previous
studies had shown 100 cGy TBI alone to be insufficient to assure stable
allografts in this model.3 Adding pretransplant activation
of host T-cell receptors through daily injections of donor PBMC along
with blockade of the second T-cell activation signal through
B7 CD28 allowed stable engraftment in 4 of 6 evaluable dogs
conditioned with 100 cGy TBI. The success with this approach is all the
more impressive since extensive previous studies had shown exposure of
canine recipients to donor PBMC or whole blood transfusions before
high-dose TBI (920 cGy single dose) to result in a high incidence of
graft rejection in the absence of concurrently administered
CTLA4Ig.33
Current CTLA4Ig-treated marrow transplant dogs had slower peripheral
blood count recoveries than either radiation controls or marrow grafted
dogs not given CTLA4Ig. The marginal differences in blood count
recoveries could be explained as follows. Recoveries in radiation
controls and in previous marrow transplant recipients were largely or
entirely derived from the considerable numbers of autologous
progenitors which had survived 100 cGy TBI the donor contributions in
the transplanted dogs were not only transient, but also weak and barely
above the levels of detection by PCR (data not shown). In contrast, the
donor contribution in current CTLA4Ig-treated dogs was strong in at
least 4 of the 6 evaluable recipients. This would imply the elimination
of a proportion of host progenitors via the graft and, accordingly, a
shift in the dependence of blood count recoveries from the relatively
large autologous toward the smaller pool of transplanted allogeneic progenitors. Given the differences in stem-cell pool sizes, slower recoveries would be anticipated in the allogeneically engrafted dogs.
A study by Wekerle et al34 in mice conditioned with 300 cGy
TBI (dose rate not given) has explored blockade of costimulatory signals to establish mixed hematopoietic chimerism. They found that
monotherapy with either an injection of CTLA4Ig on day 2 or a
monoclonal antibody to CD40L on day 0 failed to induce stable mixed
chimerism. However, combining injections of CTLA4Ig and antibody to
CD40L and, thereby, blocking 2 costimulatory signals, resulted in
"high levels (>40%) of stable (>8 months) multilineage donor
hematopoiesis." Improved cardiac and skin xenograft survivals were
also observed when both CLTA4Ig and antibody to CD40L were combined in
murine hosts.18 The combination of the 2 agents also
prolonged kidney grafts in primates better than either agent alone.22
There are at least 2 reasons why pretransplant CTLA4Ig was not
uniformly successful in the current model. First, the fusion peptide
not only blocks the positive signal from B7CD28, but it also
interrupts the desirable negative signal from B7CTLA4Ig, which serves to turn off T-cell activation.35,36 Second, it is known from studies in mice lacking CD28 that they still could mount
immune responses, although mice required repeated stimulations with
high doses of antigen.37-39 Also, one study has shown the development of acute GVHD in TBI-treated mice given hematopoietic grafts from CD28 donors.40 Another
study, however, failed to confirm that result.41 These
contrasting results were explained by the differences in the mouse
strain combinations used.
Nevertheless, taken together, these studies suggest that either
selective blockade of the B7CD28 pathway with intact
signaling through CTLA4 or the additional blockade of other
costimulatory signals, may permit to lower the TBI dose further or even
completely eliminate TBI in the current canine model of stem-cell transplantation.
| |
ACKNOWLEDGMENT |
The authors are grateful to the technicians of the Shared Canine
Resource and the Hematology and Transplantation Biology Laboratories. Barbara Johnston, DVM, provided veterinary support. We thank Dr Peter
Linsley, formerly of Bristol Myers Squibb Research, Seattle, WA, and
now Rosetta Inphamatics, Kirkland, WA, for the gift of CTLA4IG; Dr Wen
Chyi Shyu, Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, NJ, for measuring CTLA4Ig serum levels; Sabine Hadulco,
Roche Bioscience-Nutley, Nutley, NJ, for the gift of MMF; and Dr
Elizabeth C. Squiers, Sangstat Medical Corp, Menlo Park, CA, for the
gift of CSP. We are very grateful to Bonnie Larson, Helen Crawford,
Lori Ausburn, and Sue Carbonneau for their outstanding secretarial support.
| |
FOOTNOTES |
Submitted April 9, 1999; accepted May 26, 1999.
Supported in part by Grants No. CA78902, CA15704, HL36444, HL03701, and
DK42716 from the National Institutes of Health (NIH), Department of
Health and Human Services, Bethesda, MD. P.M. and H.J.D. are also
supported by grants from the Gabriella Rich Leukemia Foundation. G.G.
received support from NIH Grant No. DK09718. J.L.W. and C.Y. received
support from NIH Grant No. RR12558. R.S. also received support through
a prize from the Josef Steiner Krebsstiftung, Bern, Switzerland, and
the Laura Landro Salomon Endowment Fund. H.P.K. is a Markey Molecular
Medicine Investigator.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Rainer Storb, MD, Fred Hutchinson Cancer
Research Center, 1100 Fairview Ave N, D1-100, PO Box 19024, Seattle, WA
98109-1024.
| |
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