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Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3949-3959
Engraftment of Severe Combined Immune Deficient Mice Receiving
Allogeneic Bone Marrow Via In Utero or Postnatal Transfer
By
Bruce R. Blazar,
Patricia A. Taylor,
Ron McElmurry,
Lina Tian,
Angela Panoskaltsis-Mortari,
Sylvia Lam,
Chris Lees,
Thomas Waldschmidt, and
Daniel A. Vallera
From the University of Minnesota Cancer Center and Department of
Pediatrics, Division of Bone Marrow Transplantation and Pediatric
Oncology, Therapeutic Radiology, University of Minnesota, Minneapolis,
MN; and the Department of Pathology, University of Iowa, Iowa City, IA.
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ABSTRACT |
Although in utero transplantation (IUT) has been shown to be
effective in treating human severe combined immune deficiency (SCID), the relative merit of IUT as compared with
postnatal bone marrow transplantation (BMT) for SCID is unknown.
Therefore, comparative studies were undertaken in mice to determine the
engraftment outcome in these two settings. Because T-cell depletion
(TCD) reduces graft-versus-host disease (GVHD) severity but compromises
alloengraftment, studies were performed with TCD or non-TCD BM and GVHD
risk was assessed using a tissue scoring system and by the adoptive
transfer of splenocytes from engrafted mice into secondary recipients. Non-SCID recipients received pre-BMT irradiation to simulate those circumstances in which conditioning is required for alloengraftment. IUT recipients of non-TCD and especially TCD BM cells in general had
higher levels of donor T-cell and myeloid peripheral blood (PB)
engraftment than nonconditioned SCID recipients. Increased TCD or
non-TCD BM cell numbers in adult SCID recipients resulted in similar
levels of PB engraftment as IUT recipients. However, under these
conditions, mean GVHD scores were higher than in IUT recipients. The
majority of adoptive transfer recipients of splenocytes from IUT
recipients were GVHD-free, consistent with the in vitro evidence of
tolerance to host alloantigens. Total body irradiation (TBI)-treated
mice that had the highest engraftment had evidence of thymic damage as
denoted by a higher proportion of thymic and splenic T cells with a
memory phenotype as compared with IUT recipients. IUT mice had vigorous
thymic reconstitution by 3 weeks of age. Our data indicate that IUT has
a number of advantages as compared with postnatal BMT. Future studies
examining the fine specificity of immunoreconstitution in IUT versus
postnatal BMT are indicated.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
IMMUNOLOGICAL correction of human severe
combined immune deficiency (SCID) has recently been accomplished by in
utero transplantation (IUT) with HLA-disparate bone marrow
(BM).1 At birth, T-cell engraftment and function were
readily demonstrated, whereas B-cell engraftment was low. The
alternative to IUT for SCID is postnatal bone marrow transplantation
(BMT). HLA-disparate donor BM administered postnatally to SCID patients
can engraft and restore immune function in some
patients.2-16 However, uniform engraftment is not observed
in this setting. When engraftment does occur, T cells typically engraft
far better than B cells or myeloid cells. This split (donor/host)
chimerism can leave the recipient with humoral immune deficiency if the
recipient has few B cells or if the recipient's B-cell dysfunction is
not corrected by normal donor T cells. Pre-BMT conditioning regimens will improve the likelihood of B-cell and myeloid cell engraftment but
are associated with damage to organs such as the lung, liver, developing central nervous system (CNS), and the endocrine
system.16-19 In addition, conditioning regimens may damage
the lymphoid microenvironment, which further hampers immune system
development.
IUT offers the possibility of avoiding some of the problems associated
with haploidentical BMT for SCID. Theoretically, IUT would have the
benefit of allowing the immune system development to occur in utero.
Alloengraftment might be easier than postnatal BMT, because the fetal
immune system is naive and, at least in terms of immunological
rejection mechanism(s), the fetus is preimmune. Immune restoration may
be easier after IUT than after postnatal BMT with myeloablation,
because conditioning regimens may damage the microenvironment necessary
for optimal immune development. There is an opportunity for immune
system recovery in utero so that, by birth, as shown in the X-SCID
case, elements of the immune system may have been
restored.1 Graft-versus-host (GVH) risk may be reduced
after IUT if the fetal microenvironment is not permissive for the
expansion of adult T-cell effectors.
Although many theoretical reasons have been offered suggesting that IUT
is preferable to postnatal BMT for SCID, there are not sufficient data
upon which to make a definitive conclusion. We have established a
murine IUT model for SCID. Allogeneic adult BM transferred into fetal
SCID recipients results in multilineage progenitor cell engraftment. We
have focused on the engraftment and graft-versus-host disease (GVHD)
effects of a large series of SCID recipients of IUT. Although T-cell
depletion (TCD) is most often used to reduce the GVHD risk in SCID
recipients of HLA-disparate donor BM, TCD BM may be more difficult to
engraft in some recipients than non-TCD (NTCD) BM. Thus,
IUT SCID recipients received either TCD or NTCD BM to determine the
effect of donor T cells on alloengraftment and GVHD risk.
Because postnatal BMT is an alternative to IUT, in many instances
comparative data to IUT recipients were obtained for adult SCID
recipients of TCD or NTCD BM. An additional option in postnatal recipients is the use of conditioning therapy pre-BMT. Some SCID patients are conditioned to promote initial BM engraftment or in
instances in which a second BM graft is needed. Therefore, mice that
received total body irradiation (TBI) and postnatal BMT also were
examined. The overall goal of this study was to determine the
comparative engraftment, GVHD risk, and rapidity of
reconstitution in IUT as compared with postnatal BMT recipients. We
present data that show that IUT compares favorably with postnatal BMT
for the treatment of SCID recipients with respect to lymphoid engraftment and GVHD risk.
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MATERIALS AND METHODS |
Mouse strains.
C57BL/6 (B6:H2b) and BALB/c (H-2d) were
purchased from the National Institutes of Health (Bethesda, MD).
BALB/c-SCID (H-2d, BALB/c background genes) were purchased
from Taconic (Germantown, NY). B10.BR (H2k) mice were
purchased from The Jackson Laboratory (Bar Harbor, ME). Donors and
postnatal BMT recipients were 6 to 12 weeks of age. All mice were
housed in microisolator cages under specific pathogen-free conditions
and were fed ad libitum according to University of Minnesota Research
Animal Resources guidelines.
In utero intraperitoneal microinjections.
Our procedure has been previously described in detail.20,21
In brief, BM cells were harvested from 6- to 12-week-old B6 (allogeneic) or BALB/c (congenic) donors by flushing cells from the
hind leg bones with phosphate-buffered saline (PBS). Cells were
filtered through nylon mesh (Nitex HC3-41; Tetko, Chicago, IL) to
remove clumps before injection. Where indicated, BM was TCD by
treatment with anti-Thy1.2 (clone 30-H-12; provided by Dr David Sachs,
Cambridge, MA) + complement, as previously described. TCD or NTCD BM
cells (1 to 4 × 106) were suspended in 5 to 10 µL
for injection into each BALB/c-scid/scid fetus. Pregnant females at
days 15 and 16 of a 21-day gestation period were anesthetized with
sodium pentobarbital injected intraperitoneally. The abdomen was
entered with a midline ventral approach, and the uterine horns were
exteriorized with jeweller's forceps. Cells were injected
intraperitoneally into each fetus through hand-drawn pipettes. The
muscle layers were closed with nonabsorbable synthetic 4-0 sutures, and
metal surgical clips were used to close the skin.
Postnatal BMT.
Nonconditioned BALB/c-SCID recipients were infused intravenously with
the indicated number of TCD or NTCD (range, 5 to 100 × 106) BM. For TBI studies, BALB/c mice were conditioned with
600 cGy TBI from an x-ray source (dose rate, 41 cGy/min) on day
 1 and then infused with 5 to 100 × 106 TCD BM
cells intravenously on day 0.
Flow cytometry.
Whole PB cells or single-cell suspensions of BM, spleen, lymph node
(LN), or thymus were prepared in buffer (PBS + 5% colostrum-free bovine serum + 0.015% sodium azide). Pelleted cells were incubated for
15 minutes at 4°C with 0.4 µg of an anti-Fc receptor monoclonal antibody (MoAb; clone 2.4G2, rat IgG2b) to prevent Fc binding. Two-color or three-color flow cytometry using directly conjugated (fluorescein isothiocyanate, phycoerythrin) MoAbs or biotinylated MoAb
with pridinin chlorophyll protein (PerCP) was performed to assess
chimerism and lineage content of lymphohematopoietic cells. Optimal
concentrations of directly conjugated MoAbs were added to a total
volume of 100 to 130 µL and incubated for 1 hour at 4°C. The
following MoAbs were included: anti-H2b specific MoAb
(clone EH-144, mouse IgG) and anti-H2d specific MoAb (clone
34-5-8S, mouse IgG2a). Additional MoAbs obtained from PharMingen (San
Diego, CA) included lineage-specific markers: anti-CD4 (clone GK1.5,
rat IgG2b), anti-CD8 (clone 53-6.7, rat IgG2a), pan-B cell (B220: clone
Ra3-6B2, rat IgG2a or CD19:clone 1D3, rat IgG2a), and CD44 as an
indicator of memory cell phenotype. All samples were analyzed on a
FACScalibur (Becton Dickinson, Palo Alto, CA) using Cell Quest
software. Forward and 90° side-scatter were used to identify and
gate PB cells with lymphocyte, monocyte, and granulocyte
characteristics. A minimum of 10,000 events was examined. Background
subtraction using a directly conjugated irrelevant antibody control was
performed for each sample.
GVHD assessment by tissue scoring and adoptive transfer into
secondary recipients.
Recipients were monitored for the occurrence of GVHD symptomatology,
including ruffled fur, diarrhea, hunched posture, and lethargy,22 and by twice weekly quantitation of body
weights (for postnatal BMT mice). Liver, lung, colon, skin, and spleen were obtained for histological assessment using a semiquantitative scoring system (0.5 to 4.0 grades) as shown below (with each grade followed by its histological features).
Grade 0: normal.
Grade 0.5: minimal perivascular cuffing (liver, lung); occasional
necrotic cells (spleen); occasional necrotic crypt cell, minimal
infiltration in lamina propria and submucosa (colon).
Grade 1: perivascular cuffing, 1 to 2 cells in thickness, involving up
to 15% of vessels (liver, lung); necrotic/apoptotic cells, up to 10 cells/mm2 of tissue (spleen); necrotic cells in up to 15%
of crypts, minor infiltration of up to 20% of lamina propria (1 to 2 cell thickness in intermucosal areas and submucosa) (colon).
Grade 1.5: perivascular cuffing, 1 to 2 cells in thickness, involving
up to 15% of vessels and infiltration into parenchyma proper (liver,
lung); Necrotic/apoptotic cells, up to 10 cells/mm2 of
tissue and occasional hemolysis (spleen); necrotic cells in up to 15%
of crypts, minor infiltration of less than or equal to one third of the
lamina propria (1 to 2 cell thickness in intermucosal areas and
submucosa) (colon).
Grade 2: perivascular cuffing, 2 to 3 cells in thickness, involving up
to 25% of vessels and infiltration into parenchyma proper (liver,
lung); necrotic/apoptotic cells,  20 cells/mm2 of tissue,
and occasional hemolysis with abnormal architecture (spleen); necrotic
cells in 25% of crypts, infiltration of less than or equal to one
third of the lamina propria (3 cell thickness in intermucosal areas and
submucosa) (colon).
Grade 2.5: perivascular cuffing, 2 to 3 cells in thickness, involving
25% to 50% of vessels and infiltration into parenchyma proper (liver,
lung); necrotic/apoptotic cells, 20 cells/mm2 of tissue,
and hemolysis in 25% of the tissue with abnormal architecture
(spleen); necrotic cells in 25% to 50% of crypts, infiltration of
less than or equal to one third of lamina propria (3 to 4 cell
thickness in intermucosal areas and submucosa) (colon).
Grade 3: perivascular cuffing, 4 to 5 cells in thickness, involving
25% to 50% of vessels, peribronchiolar cuffing (2 to 3 cells) and
infiltration into parenchyma proper (liver, lung); necrotic/apoptotic
cells, 40 cells/mm2 of tissue, hemolysis in 25% to 50%
of tissue with abnormal architecture and areas of leukopenia involving
25% of tissue, formation of fibrous bands (spleen); necrotic cells
in greater than 50% of crypts, infiltration of lamina propria (5 to 6 cell thickness in intermucosal areas and submucosa) with loss of 25%
of goblet cells (colon).
Grade 3.5: perivascular cuffing, 6 to 7 cells in thickness, involving
greater than 50% of vessels, peribronchiolar cuffing (4 to 5 cells,
lung), necrotic foci (liver) and infiltration into parenchyma proper
with severe disruption of structure (liver, lung); necrotic/apoptotic
cells, up to 40 cells/mm2 of tissue, hemolysis evident in
greater than 50% of tissue with abnormal architecture and areas of
leukopenia involving 25% to 50% of tissue, formation of fibrous bands
(spleen); necrotic cells in greater than 50% of crypts, infiltration
of lamina propria resulting in displacement of 50% of mucosa with
loss of 50% of goblet cells (colon).
Grade 4: perivascular cuffing, greater than 8 cells in thickness,
involving greater than 50% of vessels, peribronchiolar cuffing (>6
cells, lung), large necrotic foci (liver), and infiltration into
parenchyma proper with necrotic lesions (liver, lung); large areas of
necrosis and hemolysis evident in greater than 50% of tissue with
abnormal architecture and large areas of leukopenia involving greater
than 50% of tissue (spleen); necrotic cells in greater than 50% of
crypts, infiltration of lamina propria resulting in displacement of
greater than 50% of mucosa with loss of 75% to 100% of goblet cells
(colon).
Mice were monitored daily for survival. GVHD risk also was analyzed in
vivo by secondary transfer experiments. Splenocytes (107
per recipient) obtained from long-term chimeras that were GVHD-free by
clinical assessment were infused into BALB/c-SCID secondary recipients
pretreated with anti-asialo-GM1 antisera (Wako Chemicals, Richmond, VA)
at a dose of 25 µL on days 4 and 2 to deplete host NK
cells that might participate in graft resistance.
In vitro assessment of anti-host responsiveness.
T-cell function was measured using an in vitro mixed lymphocyte
reaction (MLR) culture of splenocytes obtained from long-term chimeras.
Single-cell suspensions of splenocytes were washed and mixed with
irradiated (30 Gy) splenocyte stimulators from B6 (donor strain),
BALB/c (host strain), or B10.BR (third-party strain) mice. The mixture
was resuspended in Dulbecco's Minimal Essential Medium (Bio Whittaker,
Walkersville, MD), 10% fetal calf serum (Hyclone, Logan, UT), 2 mercaptoethanol (5 × 10 5 mol/L;
Sigma, St Louis, MO), 10 mmol/L HEPES buffer, 1 mmol/L sodium pyruvate
(GIBCO BRL, Grand Island, NY), and amino acid supplements (1.5 mmol/L
L-glutamine, L-arginine, and L-asparagine; Sigma), antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin; Sigma). For MLR analysis,
2 × 105 cells responders and 5 × 105 stimulators were plated into 96-well round-bottom
(Costar, Acton, MA) plates and placed at 37°C and 10%
CO2 for 2 to 6 days. Tritiated thymidine (1 µCi) was
added 16 hours before harvesting and counting in the absence of
scintillant on a beta counter. For assessment of cytokine levels
produced during an MLR reaction, supernatants were harvested from bulk
cultures established in 24-well plates and analyzed for interleukin-2
(IL-2), IL-4, IL-10, and interferon  (IFN) protein concentrations
by enzyme-linked immunosorbent assay (ELISA; R & D Systems,
Minneapolis, MN).
Statistical analysis.
The Kaplan-Meier product-limit method was used to assess the survival
of mice. The log-rank statistic was used to test differences between
groups.
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RESULTS |
PB engraftment in recipients treated via IUT or postnatal BMT: Role of
donor BM-derived T cells and postnatal TBI on donor T-, B-, and myeloid
cell engraftment.
A large series of experiments was performed in which the same
donor-recipient allogeneic strain combination (B6  BALB/c
background) was used for IUT and for postnatal BMT. IUT recipients were
treated at a fixed dose (4 × 106) of BM. To determine
the role of donor BM-derived T cells in alloengraftment, in utero or
postnatal recipients received either TCD or NTCD BM. Because some human
SCID recipients undergoing first BMT with TCD HLA-disparate donor BM
and many recipients undergoing second BMT receive pre-BMT conditioning
therapy, a group of non-SCID murine recipients received TBI to simulate
the effect of TBI conditioning therapy on engraftment and outcome in
humans. An additional group of IUT treated mice received syngeneic donor NTCD BM as an indication of the extent of lymphoid reconstitution achievable by IUT in the absence of an allogeneic effect.
H2 phenotyping of whole PB performed 6 to 9 weeks after BM transfer in
IUT recipients demonstrated high levels of PB donor cells
(Fig 1A). Approximately one third of
injected mice are live-born (typical range, 15% to 68%). Control
noninjected pregnant mice in our colony give rise to a day-7 postnatal
survival rate of 50% to 75%, which is higher than that of
IUT-injected recipients. The proportion of donor PB cells in adult SCID
recipients of TCD or NTCD BM was higher in recipients receiving a
larger dose of BM cells. At the lowest BM cell dose (5 × 106), adult SCID recipients of NTCD BM had a higher mean
percentage of PB donor cells than those that received TCD BM. At a BM
cell dose of 25 × 106, the mean percentage of PB
donor cells in adult SCID recipients of either TCD or NTCD BM was
comparable to that of IUT recipients. Engraftment, as defined by the
presence of  1% H2b+ PB cells, was 100% in IUT
recipients and 98% to 100% of postnatal SCID recipients. The overall
incidence of low level (1% to 10% PB cells of donor origin) chimeras
was 5% (7/153) or 4% (4/107) for IUT recipients of NTCD or TCD BM,
respectively. For nonconditioned adult SCID recipients of low numbers
of NTCD or TCD BM cells (5 × 106), the incidence of
low level chimeras was 13% or 21%, respectively. The infusion of
higher numbers of BM cells was associated with a reduced incidence of
low-level chimeras in nonconditioned adult SCID recipients of NTCD BM
(0/34) or TCD (1/20). TBI conditioning of BALB/c recipients provides
the highest percentage of donor PB cells and none of the TBI-treated
recipients was a low-level chimera. Because engraftment of IUT and
postnatal SCID recipients was found to be stable at time periods
greater than 5 months posttransfer, only the initial age 6- to 9-week
PB typing data, which are available on all mice, are shown.
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| Fig 1.
Comparative PB engraftment analysis in IUT and postnatal
BMT recipients. BALB/c-SCID recipients were treated via IUT or
postnatal BMT, with the indicated B6 BM cell dose shown in parentheses.
Recipients received TCD or NTCD BM as indicated. Additional control
groups consisted of BALB/c-SCID recipients of BALB/c BM (congenic
[cong.]) and BALB/c recipients were treated with TBI and received B6
TCD BM. Where indicated, controls are non-BMT B6 mice. The mean
proportion of PB-expressing H2b (A), CD4 (C), CD8 (D), and
B220 (B cells) (E) antigens are listed on the y-axis. The mean
percentage of donor myeloid engraftment (B) was calculated by dividing
the percentage of H2b+ Mac1+ cell
population by the total Mac1+ cells. A composite graph
(F) of the relative mean proportion of donor CD4+,
CD8+, B220+, Mac1+, and host
Mac1+ cells in individual mice in each group is shown.
The number of recipients studied (no.) in each group is listed at the
bottom of (F). Symbols for (F): ( ) CD4; ( ) CD8; ( ) B220; ( )
H2b Mac1; ( ) H2d Mac1. One standard error of the mean values ranged
0% to 4% for all values shown, except for a value of 9% for
H2b expression in SCID TCD25 recipients.
*Significant differences between IUT TCD recipients and other groups.
For the assessment of the proportion of PB expressing donor lymphoid
markers, the (#) indicates significant differences between the
indicated groups and the non-BMT B6 control mice.
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Because myeloid lineage cells are short-lived and there is not a
competitive advantage for donor over host myeloid cells in SCID
recipients, assessment of donor myeloid engraftment provides a more
reliable indicator of pluripotent hematopoietic progenitor cell
engraftment than the lymphoid compartment. Because recipients have
myeloid (but not lymphoid) cells, the degree of myeloid engraftment can
be quantified by comparing the proportion of myeloid cells derived from
donor (H2b) BM as opposed to the recipient
(H2d). The beneficial effect of donor T cells on myeloid
engraftment in IUT and postnatal BMT SCID recipients is readily
demonstrable (Fig 1B). Although there is a titratable effect of BM cell
dose on the percentage of of myeloid cells derived from donor rather than host BM in adult SCID recipients of either TCD or NTCD BM, adult
SCID recipients of very high doses TCD BM (100 × 106)
were unable to achieve the level of myeloid cell engraftment observed
in IUT recipients of 4 × 106 NTCD BM. Adult SCID
recipients of 25 × 106 but not 5 × 106 TCD BM cells had comparable levels of myeloid
engraftment as IUT recipients of TCD BM, albeit significantly lower
than IUT recipients of NTCD BM. Although TBI-treated mice achieved full donor myeloid engraftment, these recipients had significant morbidity (27% loss in pre-BMT body weight in the first week post-BMT) and mortality (30% by 6 weeks post-BMT) from the conditioning process. Collectively, these data indicate that a high level of myeloid engraftment requires either donor T cells, high numbers of BM cells, or
TBI conditioning (for postnatal BMT recipients).
The goal of SCID therapy by BMT is to reconstitute the immune system.
Donor T- and B-cell engraftment is necessary for this purpose in SCID
patients with combined T- and B-cell defects. The proportion of PB
composed of CD4+ or CD8+ T cells or B cells was
examined as an indication of lymphoid engraftment, because recipients
do not have lymphoid cells and therefore all lymphoid cells are of
donor origin. As compared with non-BMT B6 controls, IUT recipients of
either TCD allogeneic or NTCD congenic or allogeneic BM had similar
proportions of PB CD4+ T cells (Fig 1C). Similar findings
were observed in adult SCID recipients that received higher BM cell
doses (25 to 100 × 106). IUT recipients of allogeneic
donor grafts had a mean percentage of PB CD8+ T cells that
most closely approximated values observed in non-BMT controls (Fig 1D).
Adult SCID recipients of NTCD BM or small doses of TCD BM had lower
values and TBI-treated mice had the lowest values for donor PB
CD8+ T cells. Because the proportion of the PB that is of
myeloid origin is low (average approximately 20% of PB), it is not
surprising that the mean percentage of PB B cells in the various groups
was inversely related to the degree of T-cell engraftment, because the
PB is primarily composed of T or B cells (Fig 1E and F). In all groups,
10% of the PB was composed of B cells.
Multiorgan system engraftment in IUT or postnatal BMT recipients.
To obtain a quantitative assessment of donor cell engraftment, BM,
spleen, and thymus were examined in representative IUT and postnatal
BMT recipients at 3 to 5 months of age. Adult SCID recipients of low BM
cell doses had significantly lower levels of donor engraftment than
those receiving higher doses. Some of the adult SCID recipients of the
highest doses of NTCD had clinical GVHD. Therefore, to obtain the
comparative data of IUT versus postnatal BMT for SCID, we elected to
focus on the comparison of adult SCID recipients of 25 × 106 TCD or NTCD BM to IUT recipients.
BM (Fig 2A) and thymus (Fig 2B) were
examined as the primary sites for the generation of T cells, B cells,
and myeloid cells. In the BM, donor myeloid lineage engraftment was
highest in TBI-treated mice and adult SCID recipients of NTCD BM, with
progressively lower levels seen in IUT recipients of NTCD BM, adult
SCID recipients of TCD BM, and then IUT recipients of TCD BM. Thus,
pre-BMT conditioning or the inclusion of donor BM T cells provided the
highest levels of donor myeloid engraftment. In general, IUT recipients
had lower levels of myeloid engraftment than adult SCID recipients.
Donor CD19+ B cells were found in both IUT and postnatal
BMT recpients, with higher levels observed in the latter groups. Thymic
reconstitution was vigorous in all groups except for postnatal mice
that received TBI. TBI-treated mice were noted to have few immature
CD48 cells and a low number of
CD4+8+ thymocytes, although more mature
CD4+8 and
CD48+ cells were comparable in number to
the other groups.
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| Fig 2.
Assessment of alloengraftment in central (BM, thymus) and
peripheral (spleen) lymphohematopoietic compartments. BM (A), thymus
(B), and spleen (C) was obtained from 4- to 6-month-old chimeras (n = 5 to 6 mice/group) and analyzed for the presence of donor cells. In BM
and spleen, the number of donor CD4+, CD8+,
CD19+ B cells, donor Mac1+
(Mac1b), and host Mac1+ (Mac1d)
cells were quantified. The thymus was analyzed for T-cell
differentiation consisting of immature
CD48, intermediate
CD4+8+, and mature
CD4+8 or
CD48+ cells. The absolute number of cells
is shown on the y-axis. One standard error of the mean values for
absolute cell number are designated by bars. *Significant differences
between IUT TCD recipients and other groups. #Significant differences
between the indicated groups and the non-BMT B6 control mice.
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The spleen (Fig 2C) was examined as a primary site for quantifying
T-cell, B-cell, and myeloid peripheralization. In the spleen, donor
myeloid and B-cell numbers were both highest in IUT recipients of NTCD
BM or TBI-conditioned postnatal BMT recipients, with the lowest values
observed in adult SCID recipients of NTCD BM. Comparable values for
CD4+ and CD8+ T-cell reconstitution were
observed in all groups except for the adult SCID recipients of NTCD BM.
These data are consistent with an ongoing GVH process in the latter
group. To determine whether IUT and postnatal BMT recipients were
equally capable of generating naive T cells, T cells localized to the
spleen and LN of clinically healthy mice were phenotyped for the
expression of cell surface determinants associated with a naive or
memory cell (CD44hi) phenotype. CD4+ and
CD8+ T cells in the spleen and LN from TBI-treated
postnatal BMT recipients had a higher density of CD44 antigen as
compared with IUT recipients of NTCD BM or normal controls
(Table 1). CD44 antigen density on
CD4+ or CD8+ T cells in spleen or LN of IUT
mice was comparable to controls.
A potential advantage of IUT versus postnatal BMT is the possibility of
achieving immunological reconstitution during the time period before
birth. We therefore sought to determine how quickly thymic and
peripheral (splenic) reconstitution occurs after IUT. Thymic
reconstitution in normal and SCID controls was compared with IUT
recipients at 1, 3, and 6 weeks of age. By 3 weeks of age
(Fig 3A), IUT recipients had significantly
higher numbers of thymocytes than SCID controls, and by 6 weeks of age, IUT recipients had comparable numbers of thymocytes as normal controls.
Splenocyte numbers were significantly higher in IUT than in SCID
recipients at all time periods (Fig 3B). These data are consistent with
rapid lymphoid engraftment that is evident by 3 to 6 weeks of age.
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| Fig 3.
Rapidity of thymic and splenic reconstitution in SCID
recipients treated via IUT. BALB/c-SCID fetal recipients received
106 B6 TCD BM cells on day 15 to 16 of gestation. Controls
consisted of noninjected BALB/c-SCID and noninjected BALB/c wild-type
mice. At the indicated times postnatally, 4 to 8 mice were studied for
thymic (A) and splenic (B) cellularity. *Significant differences in
cellularity as compared with normal age-matched B6 controls. The
absolute cell number is listed on the y-axis. The bars designate one
standard error of the mean.
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Anti-host responses of IUT and post-BMT recipients.
IUT reconstituted mice were uniformly healthy appearing at all time
periods of observation. To search for subclinical GVH responses, coded
tissue (liver, lung, colon, and spleen) samples were examined for
histological evidence of GVHD. Groups of mice selected for analysis
were chosen according to the criteria used for multiorgan phenotyping
(Fig 2). IUT recipients of TCD or NTCD or TBI recipients of TCD BM had
minimal evidence of GVHD (Table 2). In
contrast, SCID recipients of either high doses of TCD or NTCD BM had
GVHD of moderate severity. Although IUT mice were not weighed during
the time period of weaning, these mice appeared healthy without obvious
weight loss. TBI-treated mice had an early loss in body weight that was
regained in less than 1 month post-BMT. SCID recipients of TCD BM had a
transient 6% decrease in mean weights at 1 month post-BMT. SCID
recipients of NTCD BM had a 10% decrease in mean body weights
beginning 1 month post-BMT that never recovered to their peak post-BMT
body weights. These data are consistent with the mean GVHD scores of
liver, lung, and spleen, which were significantly higher in the adult
SCID recipients as compared with the IUT recipients of congenic BM. For
each tissue analyzed, there was either a significant difference or a
statistical trend (.1 = P > .05) toward more severe GVH in
adult SCID recipients as compared with IUT recipients of TCD BM.
To more rigorously test whether IUT and TBI-treated recipients were
tolerant to host alloantigens in vivo, splenocytes (107)
from representative high-level chimeric mice from these groups were
adoptively transferred into SCID recipients
(Fig 4). Phenotyping of donor splenocytes
used for adoptive transfer did not show any significant differences in
the proportions of CD4+ or CD8+ T cells in the
various groups, including the non-BMT controls. Splenocytes from IUT
recipients of TCD BM were unable to cause lethal GVHD in secondary
recipients, in contrast to the 78% lethality observed after the
infusion of splenocytes from non-BMT controls. Splenocytes from IUT
recipients of NTCD and TBI-treated recipients had a 30% and 10%
lethality rate, respectively. Secondary recipients of splenocytes from
IUT or postnatal BMT had mean weights that exceeded pre-BMT values in
all groups except the recipients of control splenocytes. Adoptive
transfer experiments were not performed with adult SCID, because these
recipients already had evidence of GVHD clinically and by histological
evaluation.
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| Fig 4.
The adoptive transfer of splenocytes from high-level
chimeras as an assessment of alloreactivity in vivo. Splenocytes
(107) from B6 controls, IUT recipients of TCD or NTCD, or
TBI-conditioned BALB/c recipients were transferred into NK-depleted
BALB/c-SCID recipients. The number of mice per group and the P
value comparison with B6 controls are indicated. On the x-axis is days
posttransfer and on the y-axis is the proportion of mice surviving.
|
|
Because splenocytes obtained from IUT recipients of NTCD BM resulted in
the highest incidence of GVHD in secondary recipients, we proceeded to
determine whether splenocytes from this group of donors had in vitro
evidence of anti-host alloresponsiveness. For comparison purposes,
non-BMT controls and TBI-treated recipients of TCD BM were concurrently
analyzed. Day 4, the time of peak responses, is represented
(Fig 5), although similar trends were present at all time points analyzed (data not shown). As compared with
normal controls, bulk MLR cultures established from IUT recipients and
from TBI-treated recipients showed anti-host responses to be of a
similar magnitude to anti-donor responses. Anti-third party responses
were intact, although modestly lower in the IUT NTCD as compared with
the TBI-treated group.
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| Fig 5.
Ex vivo assessment of anti-host and anti-third-party
alloreactivity. MLR cultures were established using splenocytes
obtained from nonmanipulated B6 controls (A) or 4- to 6-month-old
BALB/c-SCID recipients receiving B6 NTCD via IUT (B) or TBI-conditioned
BALB/c recipients of B6 TCD BM (C). Day-4 peak MLR responses to B6
(donor strain), BALB/c (host strain), or B10.BR (third-party strain)
alloantigen-bearing stimulator cells were quantified. Mean values are
shown. In all instances, one standard deviation of the mean was less
than 84 cpm. Similar findings were observed on days 3 and 5 (not
shown). The individual recipient animal number is presented as
indicated on the x-axis. On the y-axis are the cpm obtained in the
absence of scintillant amplification.
|
|
Consistent with the anti-host hyporesponsiveness seen in the IUT NTCD
group, as compared with control cultures, supernatants from these bulk
cultures had markedly lower levels of IL-2 and IFN in both the IUT
NTCD BM and TBI TCD BM recipients on days 2 and 3 of culture
(Fig 6A and B). Anti-host responses in IUT recipients peaked later than controls (data not shown), consistent with
a lower precursor frequency of anti-host reactive cells. Therefore, it
is not surprising that IL-2 levels in the supernatant reached control
levels later in culture (day 4), past the time of peak proliferative
response of controls. IL-2 and IFN responses to third-party
alloantigens in both groups peaked later than non-BMT controls, with
responses in TBI recipients being of a modestly higher magnitude at the
time of peak proliferative response (Fig 6C and D). The consistently
low IFN levels in response to host (Fig 6B) and third-party (Fig 6D)
alloantigens in the IUT and TBI groups could be related to an intrinsic
immune dysfunction in these two groups. Mean values for IL-4 and IL-10
protein concentration were less than 15 pg/mL in all three groups in
response to donor, host, or third-party stimulators, except for an
IL-10 value of 27 pg/mL obtained from day-5 MLR cultures containing
splenocytes from TBI-treated recipients and third-party stimulators.
The modest differences in IL-2 and IFN cytokine levels cannot be
readily explained by the splenic T-cell content in these cultures,
because control and IUT NTCD recipients had comparable percentages of CD4+ and CD8+ T cells that were both higher
than in the splenocytes from the TBI-treated group. Because the mean
CD44 antigen density on CD4+ and especially
CD8+ T cells was higher in splenocytes obtained from the
TBI-treated group as compared with non-BMT controls or IUT recipients,
it is possible that the modest differences in proliferative and
cytokine responses observed using splenocytes obtained from TBI or IUT recipients could be due to a higher proportion of T cells with a memory
cell phenotype in the latter group.
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| Fig 6.
IL-2 and IFN protein concentrations in supernatants
obtained from bulk MLR cultures. Splenocytes obtained from recipients
analyzed in Fig 5 also were studied for the production of IL-2 or
IFN in bulk MLR cultures. Supernatants obtained at the indicated
time periods were analyzed by ELISA for the concentration of IL-2 (A
and C) or IFN protein (B and D) released in response to host (A and
B) or third-party (C and D) alloantigen-bearing stimulator cells. On
the x-axis is the days of MLR culture. On the y-axis is the protein
concentration in picograms per milliliter. *Significant differences
between the indicated groups and nonmanipulated B6 controls.
|
|
The high level of engraftment observed after IUT with either TCD or
NTCD is associated with low anti-host alloresponses in vivo and in
vitro. IUT recipients of TCD BM appear to be at a lower risk for GVHD
than comparably engrafted adult SCID recipients of NTCD BM, whereas IUT
recipients of NTCD BM have a relatively low but not absent risk of
GVHD.
| |
DISCUSSION |
We have provided the first extensive comparative analysis of the
alloengraftment and GVHD side effects of adult B6 NTCD or TCD BM
infused in utero or postnatally into BALB/c-SCID recipients. In this
study, we have shown that IUT can lead to high levels of PB, thymic,
and splenic lymphoid alloengraftment in SCID recipients, with minimal
histological evidence of GVHD. The presence of low numbers of
BM-derived donor T cells facilitated alloengraftment in IUT recipients
without significantly increasing GVHD-induced tissue destruction. At
comparable levels of PB lymphoid alloengraftment, nonconditioned
postnatal SCID recipients had more GVH-induced tissue destruction than
IUT recipients. TBI conditioning of nonimmune deficient postnatal
recipients that provided the highest level of alloengraftment damaged
the thymus and compromised the generation of naive T cells that
repopulate the lymph node and spleen of recipients. IUT reconstituted
the thymus and spleen between 3 and 6 weeks of age. Thus, IUT provides
a rapid means of achieving lymphoid reconstitution at an early age of
life and suggests that SCID recipients treated by IUT have certain
advantages over adult SCID recipients treated by postnatal BMT.
It is possible that the high levels of PB alloengraftment seen in IUT
recipients are due to the relatively large BM dose infused. The
infusion of higher numbers of BM cells (25 × 106)
was required in nonconditioned SCID recipients to prevent the development of low-level (1% to 10% PB of donor origin) chimeras that
would be an insufficient immune reconstitution setting in human SCID
recipients to provide immune surveillance against infectious agents.
IUT recipients had a smaller incidence of low level chimeras than adult
nonconditioned SCID recipients, regardless of whether these mice
received NTCD or TCD BM. Additional studies indicated that none of 31 (0%) IUT recipients of NTCD BM at a dose of 106 cells
failed to engraft or was a low-level chimera. At a dose of
106 and a weight of 1 to 2 g (day-15 fetus), the estimated
NTCD BM dose is 0.5 to 1 × 109/kg. For adult
nonconditioned SCID recipients of 5 × 106 NTCD BM
cells, which did not preclude graft rejection, the estimated NTCD BM
cell dose is 0.2 × 109/kg; for recipients of 25 × 106, which did preclude graft rejection, the
estimated NTCD BM dose is 1 × 109/kg. Because very
young SCID mice would have lower NK activity, which does not fully
develop until 4 to 5 weeks of age, it is possible that engraftment
rates would be improved if very young SCID mice were used. Although the
high BM cell dose alone could be responsible for the high level of
alloengraftment observed in IUT recipients, as discussed below,
nonconditioned adult SCID recipients of comparable NTCD BM cell doses
have more GVHD-induced tissue destruction than IUT recipients.
Alloengraftment of SCID recipients via IUT was not entirely due to a
selective expansion of mature lymphoid cells or their progenitors but
rather involved the engraftment of multilineage progenitor and stem
cell populations. In a different type of severe combined immune
deficiency (Janus-3 kinase [Jak3K]), small numbers of
Jak3K-expressing lymphoid cells will repopulate the recipient's lymphoid system, whereas there is no competitive advantage of donor
myeloid cells in Jak3K deficient mice.23 Because myeloid cells have a short half-life and SCID mice do not have a known defect
in myeloid progenitor cells, evaluation of the myeloid compartment
provides a better indicator of the requirement of donor T cells in
facilitating donor stem cell alloengraftment. Our data show that
myeloid engraftment, a reflection of multilineage progenitor and stem
cell engraftment, is achievable in IUT or postnatal SCID recipients of
sufficient numbers of BM cells. A clear effect of the inclusion of
donor BM-derived T cells was observed in PB myeloid engraftment in both
the IUT and nonconditioned adult SCID recipients. Adult SCID recipients
of high numbers of BM cells (25 × 106) had a greater
degree of BM myeloid engraftment than IUT recipients, although IUT
recipients of NTCD BM had higher myeloid engraftment and less GVHD than
adult SCID recipients of TCD BM.
The inclusion of donor BM-derived T cells for transfer into murine IUT
recipients facilitated alloengraftment without increasing GVHD risk.
The role of donor T cells in facilitating alloengraftment also has been
examined in fetal sheep (reviewed in Zanjani et al24). The
transfer of T-cell-containing cord blood cells, newborn BM, or adult
BM permitted a higher degree of alloengraftment. However, GVHD was
increased in each instance. In baboons, the infusion of T-replete BM
was associated with GVHD. The T-cell content of the progenitor cells
infused into sheep or baboons would have exceeded the proportion of T
cells present in murine BM (2% to 4%). In the sheep model, TCD BM
avoided GVHD but led to diminished alloengraftment. In a recent report
of a human SCID recipient successfully treated by IUT, the repetitive
infusion of rigorously TCD, stem cell-enriched haploidentical BM
permitted high levels of T cells with low levels of myeloid engraftment and no GVHD.1 Murine IUT with BM may represent a situation in which there are sufficient BM-derived T cells to promote
alloengraftment but insufficient numbers to cause significant GVHD. In
that regard, Archer et al25 showed that 44% to 66% of
NOD/SCID mice, deficient in NK and macrophage activity, had a mean of
30% donor PB at 4 weeks of age after intraperitoneal injection on day
13.5 with purified lineage-depleted BM (0.8 × 106
cells). Although direct comparisons cannot be made between our study
and that of Archer et al25 due to potential differences in
day of injection and technical aspects of the injection process, lineage-depleted BM would contain fewer T cells than TCD BM. Because we
have found that NTCD BM engrafts better than TCD BM in IUT recipients,
the higher PB alloengraftment reported in our series may be due to the
presence of higher proportions of donor BM-derived T cells infused in
our experiments.
The high engraftment and low GVHD of the murine IUT studies reported
here may be due to the fact that the microenvironment of the murine
fetus may be more conducive to the alloengraftment of mouse BM than the
generation of GVHD. Higher doses of NTCD or TCD BM administered to
nonconditioned SCID recipients that resulted in comparable
alloengraftment were associated with more GVHD-induced tissue
destruction. Consistent with the greater histological evidence of GVHD,
both thymic and splenic cellularity were reduced in adult SCID
recipients of these high BM cell doses, presumably due to GVHD-induced
involution. The use of TBI to condition postnatal recipients provided
the highest level of PB and BM myeloid alloengraftment. At the TBI dose
administered, TCD BM readily engrafted. TBI damaged the central
(thymus) and peripheral (spleen, lymph node) lymphoid compartments. The
generation of naive T cells was compromised. Significant morbidity and
mortality was observed in TBI-treated recipients. Thus, TBI
conditioning, which offers the highest likelihood of successful
alloengraftment, is associated with substantial side effects and leads
to the impaired naive T-cell repopulation of the lymph node and splenic
compartments.
An important aspect of the present studies was the demonstration that
IUT SCID recipients had rapid recovery of the thymus and spleen by 3 to
6 weeks of age; IUT provides an advantage of beginning the
alloengraftment process before birth. The extent to which the pregnancy
and fetal risks associated with IUT outweigh waiting to perform BMT in
newborn SCID recipients is unknown and will require further
investigation. However, it is noteworthy that the human SCID recipient
treated with TCD haploidentical BM had normal numbers of
CD8+ T cells and B cells at birth.1
CD4+ T cells, which were low at birth, progressively
increased in number, reaching normal numbers at 5 months of age. T
cells were of donor origin and B cells of host origin, an important
distinction to the results obtained in murine SCID recipients treated
by IUT. It is not known whether the high levels of donor B-cell
engraftment seen in IUT-treated murine SCID recipients is due to an
earlier defect in B-cell development than in the human
IL-2Rc-deficient SCID recipient and/or an overall higher
alloengraftment seen in murine as compared with human recipients.
Because myeloid cells in the human SCID recipient were of host origin
and, in the mouse, a mean of 20% to 40% of myeloid cells were of
donor origin in the PB of murine SCID recipients, at least part of the
explanation would appear to include a more ready engraftment of the
mouse than the human fetus. Despite the high engraftment results
reported in murine SCID recipients, it is worth emphasizing that the
engraftment of non-SCID, non-stem cell defective IUT recipients with
allogeneic BM has been problematic in murine series reported to date.
In our previous studies with transplacental injections, alloengraftment was transient in this setting.26 Carrier et
al27 reported alloengraftment in 50% of day 12 to 13 gestational recipients injected ip with day 11 to 13 fetal liver cells,
although the levels ranged from 0.0001% to 0.6%. IUT treatment of
nonimmunodeficient sheep typically provides alloengraftment levels of
approximately 15% donor cells, although repetitive infusions can
increase mean levels to 30%.24 A challenge in the field
will be to identify strategies that are sufficient to routinely
repopulate a fetal microenvironment in which there is not a competitive
advantage to a particular lineage of progenitor cell, as is the case
for murine SCID recipients.
In our murine studies as well as the human SCID patient,1
donor T cells have a markedly blunted response to host alloantigens in
MLR culture while retaining third-party responses. The adoptive transfer of splenocytes from murine IUT recipients into host-strain SCID recipients was not able to uncover an alloresponse in recipients of TCD BM, although some recipients of NTCD did experience GVHD. Although we have previously observed that IUT NTCD reconstituted SCID
mice have circulating levels of IgM at least as high as control B6
mice,20 additional studies will be required to determine the fine specificity of T-cell and B-cell responses to antigenic challenge and to determine whether there are unique mechanism(s) responsible for tolerance induction after IUT versus postnatal BMT.
In summary, we have shown that, at comparable levels of T-cell, B-cell,
and myeloid PB alloengraftment, IUT recipients had less GVHD tissue
destruction than nonconditioned adult SCID recipients. TCD of donor BM
did not preclude alloengraftment of IUT recipients. TBI, which resulted
in the highest alloengraftment when used to condition non-SCID
recipients, impaired thymic T-cell production and the generation of
naive T cells. IUT recipients of TCD or NTCD BM were tolerant to host
alloantigen-bearing cells as assessed in vitro and in vivo. Because
SCID recipients treated by IUT had rapid reconstitution of the thymic
and splenic compartments, our data would indicate that IUT treatment of
fetal SCID recipients has a number of potential advantages as compared
with BMT of nonconditioned adult SCID recipients. Because the major
limitation for the more widespread use of IUT is alloengraftment,
approaches to improve alloengraftment can be readily tested in SCID
recipients that are permissive for lymphoid but less permissive for
myeloid and stem cell engraftment.
| |
FOOTNOTES |
Submitted May 25, 1998;
accepted July 7, 1998.
Supported in part by National Institutes of Health Grants No.
R01-HL49997 and R01-HL52952.
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 reprint requests to Bruce Blazar, Box 109 Mayo Bldg, University
of Minnesota Hospital, Minneapolis, MN 55455.
| |
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P. A. Taylor, M. J. Ehrhardt, C. J. Lees, J. Tolar, B. J. Weigel, A. Panoskaltsis-Mortari, J. S. Serody, V. Brinkmann, and B. R. Blazar
Insights into the mechanism of FTY720 and compatibility with regulatory T cells for the inhibition of graft-versus-host disease (GVHD)
Blood,
November 1, 2007;
110(9):
3480 - 3488.
[Abstract]
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A. Panoskaltsis-Mortari, K. V. Tram, A. P. Price, C. H. Wendt, and B. R. Blazar
A New Murine Model for Bronchiolitis Obliterans Post Bone Marrow Transplant
Am. J. Respir. Crit. Care Med.,
October 1, 2007;
176(7):
713 - 723.
[Abstract]
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W. H. Peranteau, M. Endo, O. O. Adibe, A. Merchant, P. W. Zoltick, and A. W. Flake
CD26 inhibition enhances allogeneic donor-cell homing and engraftment after in utero hematopoietic-cell transplantation
Blood,
December 15, 2006;
108(13):
4268 - 4274.
[Abstract]
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P. A. Taylor, A. Panoskaltsis-Mortari, G. J. Freeman, A. H. Sharpe, R. J. Noelle, A. Y. Rudensky, T. W. Mak, J. S. Serody, and B. R. Blazar
Targeting of inducible costimulator (ICOS) expressed on alloreactive T cells down-regulates graft-versus-host disease (GVHD) and facilitates engraftment of allogeneic bone marrow (BM)
Blood,
April 15, 2005;
105(8):
3372 - 3380.
[Abstract]
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C Veltkamp, R B Sartor, T Giese, F Autschbach, I Kaden, R Veltkamp, D Rost, B Kallinowski, and W Stremmel
Regulatory CD4+CD25+ cells reverse imbalances in the T cell pool of bone marrow transplanted TG{varepsilon}26 mice leading to the prevention of colitis
Gut,
February 1, 2005;
54(2):
207 - 214.
[Abstract]
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P. A. Taylor, A. Panoskaltsis-Mortari, J. M. Swedin, P. J. Lucas, R. E. Gress, B. L. Levine, C. H. June, J. S. Serody, and B. R. Blazar
L-Selectinhi but not the L-selectinlo CD4+25+ T-regulatory cells are potent inhibitors of GVHD and BM graft rejection
Blood,
December 1, 2004;
104(12):
3804 - 3812.
[Abstract]
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C. A. Wysocki, S. B. Burkett, A. Panoskaltsis-Mortari, S. L. Kirby, A. D. Luster, K. McKinnon, B. R. Blazar, and J. S. Serody
Differential Roles for CCR5 Expression on Donor T Cells during Graft-versus-Host Disease Based on Pretransplant Conditioning
J. Immunol.,
July 15, 2004;
173(2):
845 - 854.
[Abstract]
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A. Panoskaltsis-Mortari, J. R. Hermanson, E. Taras, O. D. Wangensteen, I. F. Charo, B. J. Rollins, and B. R. Blazar
Post-BMT lung injury occurs independently of the expression of CCL2 or its receptor, CCR2, on host cells
Am J Physiol Lung Cell Mol Physiol,
February 1, 2004;
286(2):
L284 - L292.
[Abstract]
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A. R. Rao, M. P. Quinones, E. Garavito, Y. Kalkonde, F. Jimenez, C. Gibbons, J. Perez, P. Melby, W. Kuziel, R. L. Reddick, et al.
CC Chemokine Receptor 2 Expression in Donor Cells Serves an Essential Role in Graft-versus-Host-Disease
J. Immunol.,
November 1, 2003;
171(9):
4875 - 4885.
[Abstract]
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J. C. Segovia, G. Guenechea, J. M. Gallego, J. M. Almendral, and J. A. Bueren
Parvovirus Infection Suppresses Long-Term Repopulating Hematopoietic Stem Cells
J. Virol.,
August 1, 2003;
77(15):
8495 - 8503.
[Abstract]
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B. R. Blazar, B. M. Carreno, A. Panoskaltsis-Mortari, L. Carter, Y. Iwai, H. Yagita, H. Nishimura, and P. A. Taylor
Blockade of Programmed Death-1 Engagement Accelerates Graft-Versus-Host Disease Lethality by an IFN-{gamma}-Dependent Mechanism
J. Immunol.,
August 1, 2003;
171(3):
1272 - 1277.
[Abstract]
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A. Panoskaltsis-Mortari, J. R. Hermanson, E. Taras, O. D. Wangensteen, J. S. Serody, and B. R. Blazar
Acceleration of idiopathic pneumonia syndrome (IPS) in the absence of donor MIP-1alpha (CCL3) after allogeneic BMT in mice
Blood,
May 1, 2003;
101(9):
3714 - 3721.
[Abstract]
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T. J. Waldschmidt, A. Panoskaltsis-Mortari, R. T. McElmurry, L. T. Tygrett, P. A. Taylor, and B. R. Blazar
Abnormal T cell-dependent B-cell responses in SCID mice receiving allogeneic bone marrow in utero
Blood,
December 15, 2002;
100(13):
4557 - 4564.
[Abstract]
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M. Gonzalez, S. A. Quezada, B. R. Blazar, A. Panoskaltsis-Mortari, A. Y. Rudensky, and R. J. Noelle
The Balance Between Donor T Cell Anergy and Suppression Versus Lethal Graft-Versus-Host Disease Is Determined by Host Conditioning
J. Immunol.,
November 15, 2002;
169(10):
5581 - 5589.
[Abstract]
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W. H. Peranteau, S. Hayashi, M. Hsieh, A. F. Shaaban, and A. W. Flake
High-level allogeneic chimerism achieved by prenatal tolerance induction and postnatal nonmyeloablative bone marrow transplantation
Blood,
August 28, 2002;
100(6):
2225 - 2234.
[Abstract]
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J. S. Serody, S. E. Burkett, A. Panoskaltsis-Mortari, J. Ng-Cashin, E. McMahon, G. K. Matsushima, S. A. Lira, D. N. Cook, and B. R. Blazar
T-lymphocyte production of macrophage inflammatory protein-1alpha is critical to the recruitment of CD8+ T cells to the liver, lung, and spleen during graft-versus-host disease
Blood,
November 1, 2000;
96(9):
2973 - 2980.
[Abstract]
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J. C. Zeller, A. Panoskaltsis-Mortari, W. J. Murphy, F. W. Ruscetti, S. Narula, M. G. Roncarolo, and B. R. Blazar
Induction of CD4+ T Cell Alloantigen-Specific Hyporesponsiveness by IL-10 and TGF-{beta}
J. Immunol.,
October 1, 1999;
163(7):
3684 - 3691.
[Abstract]
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A. W. Flake and E. D. Zanjani
In Utero Hematopoietic Stem Cell Transplantation: Ontogenic Opportunities and Biologic Barriers
Blood,
October 1, 1999;
94(7):
2179 - 2191.
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Top
Abstract
Introduction