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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3101-3107
Reduced NFAT1 Protein Expression in Human Umbilical Cord Blood T
Lymphocytes
By
Suzanne Kadereit,
Shaden F. Mohammad,
Robin E. Miller,
Kathleen
Daum Woods,
Chad D. Listrom,
Karen McKinnon,
Alborz Alali,
Linda S. Bos,
Michelle L. Iacobucci,
Michael R. Sramkoski,
James W. Jacobberger, and
Mary J. Laughlin
From the University Hospitals Ireland Comprehensive Cancer Center,
Case Western Reserve University, Cleveland, OH; and the Lineberger
Comprehensive Cancer Center, University of North Carolina at Chapel
Hill, Chapel Hill, NC.
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ABSTRACT |
Umbilical cord blood (UCB) stem cells from related and unrelated
allogeneic donors have emerged as novel treatment for patients with
hematologic malignancies. The incidence and severity of acute graft-versus-host disease (GVHD) after UCB transplantation compares favorably with that observed in recipients of matched unrelated donor
allogeneic grafts, but remains a major cause of morbidity and
mortality. It has been shown that stimulated lymphocytes from UCB have
reduced production of cytokines including interferon- (IFN-) and
tumor necrosis factor- (TNF-), which play a role in GVHD
pathophysiology. We investigated the molecular mechanisms underlying
this reduced cytokine production by analyzing expression of nuclear
factor of activated T cells-1 (NFAT1) in UCB T cells. We detected no
constitutive expression of NFAT1 protein in unstimulated UCB T cells
compared with adult T cells. Moreover, although NFAT1 expression in UCB
T cells was upregulated after prolonged (40 hours) T-cell stimulation,
it was only partially upregulated when compared with adult controls.
Our observation of minimal NFAT1 expression after stimulation
correlated with reduced cytoplasmic IFN- and TNF- production in
UCB T cells studied simultaneously. Reduced NFAT1 expression may blunt
amplification of donor UCB T-cell alloresponsiveness against recipient
antigens, thereby potentially limiting GVHD incidence and severity
after allogeneic UCB transplantation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ALLOGENEIC TRANSPLANTATION cures a
significant number of patients with hematologic malignancies via
immunologic mechanisms termed graft-versus-leukemia (GVL).1
The major obstacle to a successful outcome after transplantation is
graft-versus-host disease (GVHD), resulting from donor T-cell
alloreactivity against recipient major and minor histocompatibility
antigens presented within the context of the major histocompatibility
complex (MHC).2 Umbilical cord blood (UCB) from related and
unrelated donors has recently emerged as a novel source of stem cells
for patients requiring allogeneic transplantation.3-5 The
incidence and severity of acute GVHD in UCB recipients compares
favorably with that observed in recipients of matched unrelated donor
grafts, but remains a major cause of morbidity and mortality. Of 562 patients transplanted with partially HLA mismatched UCB grafts, the
majority of whom were less than age 18 years, 23% experienced severe
acute GVHD (grade III to IV).5 In comparison, of 88 children transplanted with HLA-matched unrelated donor grafts, 37%
experienced severe grade III to IV acute GVHD.6
T cells are known to play a central role in mediating GVHD. Allogeneic
grafts depleted of T cells elicit reduced GVHD
posttransplant.7,8 Donor T cells activated by recipient
alloantigen produce Th1 type cytokines, thereby recruiting and
activating other effector cells, which amplify donor T-cell
alloreactivity and provoke tissue damage. Cytokines implicated directly
in tissue damage during GVHD include tumor necrosis factor-
(TNF-) and interferon- (IFN-).9,10
T cells in UCB are predominantly of naive phenotype
(CD45RA+) with reduced to absent CD45RO+
expression when compared with adult peripheral blood T
cells.11 Upon primary stimulation, UCB T cells demonstrate
activation marker expression equivalent to adult controls, as measured
by CD45RA+ downregulation and upregulation of CD25, CD69,
T-cell receptor (TCR), and p56lck.12-14
Moreover, upon primary TCR stimulation, UCB T cells and adult T cells
proliferate to the same extent, as measured by thymidine incorporation.12 Importantly, however, certain effector
functions of UCB T cells necessary to amplify T-cell responses to
foreign antigens, are reduced. For example, T-cell proliferation after secondary stimulation has been shown to be significantly reduced, and
alloantigen-specific cytotoxicity is decreased.15,16
Moreover, production of the cytokines interleukin-2 (IL-2), IL-4,
IFN-, and TNF- by activated UCB T cells is significantly reduced,
when compared with adult T cells.11,17
The transcription of many cytokine genes and immunomodulatory molecules
is regulated by nuclear factor of activated T cells-1 (NFAT1 or NFATp),
a member of the NFAT family.18 NFAT1 is expressed constitutively at relatively high basal levels in unstimulated adult T
cells and resides in latent form in the cytoplasm.19,20 NFAT1 mRNA expression in published reports is not significantly upregulated with short-term (2 to 3 hours) T-cell
stimulation.20,21 However, a recent report describes
expression of NFAT1 protein above basal constitutive levels measured in
unstimulated T cells, after prolonged (10 to 30 days) T-cell
priming.22 After TCR stimulation and consequent calcium
influx, constitutively expressed NFAT1 protein is activated through
dephosphorylation by the phosphatase calcineurin.23
Dephosphorylation of NFAT1 correlates with its translocation to the
nucleus and increase in DNA-binding activity.24,25
Cyclosporin A (CsA) and FK506, effective prophylactic and
treatment agents against GVHD and powerful inhibitors of cytokine production, have been shown to inhibit calcineurin directly, as well as
dephosphorylation and translocation of NFAT1.26-28 In
addition, NFAT1 gene-deleted studies further establish the necessity of NFAT1 protein for the transcriptional upregulation of the IL-4, IFN-, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-13, TNF-, CD69, Fas Ligand (FasL), and CD40L
genes.29 These observations together suggest a central role
for NFAT1 in regulating cytokine production by activated T cells during GVHD.
Because little is known about the regulation of NFAT1 during GVHD or
its activity in UCB T cells, we investigated NFAT1 expression in
unstimulated and stimulated UCB T cells compared with adult T cells. We
show here that NFAT1 protein expression is significantly reduced in
unstimulated UCB T cells and is only partially expressed after
prolonged (40 hours) primary TCR stimulation when compared with adult
controls. These findings correlate with reduced IFN- and TNF-
production by the same cells during prolonged primary TCR stimulation.
Furthermore, we show that lack of NFAT1 expression in UCB T cells is
not due to naive, CD45RA+ phenotype. Reduced NFAT1 protein
expression in UCB T cells may prevent amplification of activated donor
UCB T-cell responses to recipient alloantigens posttransplant.
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MATERIALS AND METHODS |
Cells.
Human UCB was collected into collection bags containing citrate
dextrose (Allegiance, Deerfield, IL) after vaginal deliveries or
Cesarean sections of full-term pregnancies from healthy normal mothers,
as approved by the Institutional Review Board at University Hospitals
of Cleveland. Adult blood was collected into citrate dextrose, after
informed consent of normal healthy adults. Mononuclear cells (MNC) were
separated by gradient centrifugation on Histopaque-1077 (Sigma Chemical
Co, St Louis, MO). Red blood cell contamination of UCB was reduced by
gravity separation on 6% dextran (Sigma) before gradient
centrifugation. MNC were used immediately fresh.
T-cell purification.
T cells were purified by negative depletion using a cocktail of
monoclonal antibodies including: CD11b, CD16, CD19, and CD56 (PharMingen, San Diego, CA) followed by anti-IgG magnetic bead depletion, following manufacturer's instructions, with modifications (Dynal, Lake Success, NY). Briefly, antibodies were added at 5 µg/mL
each to 20 × 106 cells/mL of MNC resuspended in
RPMI/10% fetal bovine serum (FBS) and incubated for 30 minutes on ice.
Cells were washed twice and incubated in RPMI/10% FBS at 20 × 106 cells/mL with recommended amounts of magnetic beads.
After purification, T-cell purity was analyzed by flow cytometry and
CD3+ T-cell purity ranged between 90% to 97% of viable
CD45+ cells. In studies of CD45RA/RO subsets,
CD45RA+ T cells were further purified from T
cells by depleting with 10 µg/mL of anti-CD45RO monoclonal antibody
in the same conditions as above (PharMingen). The resulting depleted
population was single positive for CD45RA and purity ranged between
80% to 85% of viable T cells. The positively selected
CD45RO+ population contained the CD45RO single-positive
population, as well as the double CD45RO/CD45RA-positive population.
Western blot analysis.
Purified T cells were lysed in 20 mmol/L Tris pH 7.6, 50 mmol/L KCl,
400 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton-X, 20% glycerol, 1 mmol/L
dithiothreitol (DTT), 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL pepstatin, 2 µg/mL leupeptin, 2 µg/mL aprotinin, and 10 mmol/L Na2MoO4.
Protein extracts corresponding to 0.3 × 106 cells
were loaded per lane and separated on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to
polyvinylidene fluoride (PVDF) membrane (Millipore,
Bedford, MA) and immunodetected with a mix of monoclonal antibodies
directed against NFAT1 (Transduction Laboratories, Lexington, KY) and
 -actin (Sigma). NFAT1 antibody was used at a dilution of 1/2,500,
the actin antibody at a dilution of 1/5,000. The bands were visualized using enhanced chemiluminescence (Amersham, Buckinghamshire, UK) and
band intensities were quantified by densitometry scanning on Sci Scan
5000 (U.S. Biochemical, Cleveland, OH). Band intensity was
normalized for loading with the actin band, and NFAT1 band density was
expressed in relative percentage of the strongest NFAT1 band on each
gel. Purified single-positive CD45RA+ T cells were
extracted after selection as outlined above and analyzed by
Western blot. Positively selected CD45RO+ T cells
were extracted directly on magnetic beads and analyzed in the same
conditions as outlined above.
Primary T-cell stimulation.
Each experiment was performed concomitantly on MNC from 2 UCB units and
peripheral blood MNC from 2 adults. MNC were stimulated in bulk culture
at concentration 2 × 106 cells/mL with 2 µg/mL
concanavalin A (Con A) (Sigma) in RPMI medium (GIBCO-BRL,
Gaithersburg, MD) with 10% FBS (GIBCO-BRL), 1 mmol/L
sodium-pyruvate (GIBCO-BRL), 0.1 mmol/L nonessential amino acids
(GIBCO-BRL), 10 mmol/L HEPES (GIBCO-BRL), and 58 µmol/L 2-mercaptoethanol (Sigma) for 40 hours. T cells for Western blot analyses were isolated before stimulation and after stimulation and
were lysed immediately.
Cytokine expression.
Intracellular cytokine staining was performed concomitantly on the same
MNC preparations using identical stimulation conditions as the Western
blot analyses. Staining was performed using FastImmune (Becton
Dickinson, San Jose, CA) according to the manufacturer's instructions
with the following changes. MNC were stimulated at concentration 2 × 106 cells/mL for 18 and 40 hours with 2 µg/mL
concanavalin A (Con A) (Sigma) in the presence of 5 µg/mL of
Brefeldin A (BFA) (Becton Dickinson) for the final 16 hours of
stimulation. Red blood cells were lysed and MNC washed and
permeabilized. A total of 0.5 × 106 MNC were stained
for 30 minutes at room temperature (RT) with the following
antibodies: anti-IFN--fluorescein isothiocyanate (FITC) or
anti-TNF--FITC in the presence of anti-CD3-allophycocyanin (APC) and anti-CD69-phycoerythrin (PE)
(Becton Dickinson). Cells were washed and fluorescence was acquired
immediately on Elite ESP flow cytometer (Coulter, Miami, FL). Data were
analyzed with WinList (Verity Software House Inc, Topsham, MN) and are
plotted as T-cell-specific (CD3+)
anti-cytokine-reactivity (FITC-fluorescence) versus surface CD69 reactivity.
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RESULTS |
Undetectable NFAT1 protein expression in unstimulated UCB T cells.
NFAT1 protein is expressed constitutively in human peripheral T cells
and can readily be detected by Western blotting.20,30 We
investigated NFAT1 expression in UCB T cells and compared this with
adult controls. We prepared whole cell extracts from freshly purified
unstimulated T cells and analyzed NFAT1 protein expression by Western
blotting. While NFAT1 protein was readily detectable in 3 × 105 unstimulated adult T cells
(Fig 1A, lanes 7 to 9), we found no detectable NFAT1 expression in an equivalent number of unstimulated UCB
T cells (Fig 1A, lanes 1 to 6), indicating reduced constitutive NFAT1
expression in unstimulated UCB T cells.
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| Fig 1.
Undetectable NFAT1 protein expression in unstimulated UCB
T cells. (A) T cells were purified from fresh MNC of 6 UCB units (lanes
1 through 6) and from 3 adult PBMC (lanes 7 through 9). Protein
extracts equivalent to 3 × 105 T cells were loaded per
lane and analyzed by Western blotting with a combination of anti-NFAT1
and anti--actin antibodies. (B) T cells were further separated into
CD45RA+ and CD45RO+ expressing cells and
protein extracts analyzed as above.
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NFAT1 protein expression in CD45RA/RO T-cell subpopulations.
T cells in UCB are predominantly of naive phenotype
(CD45RA+) with reduced to absent CD45RO+
expressing T cells, when compared with adult peripheral blood T
cells.11 Reduced NFAT1 protein expression might therefore be attributable to the predominance of naive, CD45RA+
expressing T cells, rather than an intrinsic trait of UCB. NFAT1 protein expression was therefore measured by Western blot in
CD45RA+ and CD45RO+ T-cell subpopulations from
adult peripheral blood and UCB. In the adult, equivalent levels of
NFAT1 protein were measured both in the CD45RA+ and
CD45RO+ T-cell subsets (Fig 1B). In UCB T cells, no NFAT1
expression was detected in either CD45RA+ or
CD45RO+ T-cell subpopulations.
Reduced NFAT1 expression after prolonged UCB T-cell stimulation.
Because we observed reduced NFAT1 protein expression in unstimulated
UCB T cells, we investigated if we could upregulate NFAT1 protein
expression by prolonged primary T-cell stimulation. Therefore, we
stimulated MNC from freshly isolated UCB and adult peripheral blood for
up to 40 hours with Con A, purified the T cells at time points 18 hours
and 40 hours and assessed NFAT1 expression by Western blot analysis.
Interestingly, we observed that in adult T cells, NFAT1 protein
expression was upregulated 2-fold after 40 hours of primary T-cell
stimulation (Fig 2A and B). In UCB T cells,
however, NFAT1 expression remained deficient after 18 hours, with only
22% of the expression of NFAT1 observed in adult at that time point, and was only partially upregulated after 40 hours of stimulation, averaging 45% of expression measured in adult T cells.
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| Fig 2.
Reduced upregulation of NFAT1 protein in UCB T cells
during primary stimulation. (A) MNC from 2 UCB units and 2 adults were
stimulated in parallel with Con A for 40 hours. At 0 hour (unstim), 18 hours, and 40 hours of stimulation, T cells were purified and extracted
and NFAT1 protein expression analyzed by Western blotting. The results
shown are representative of 5 independent experiments, comparing in
each experiment 2 UCB and 2 adult controls analyzed together and run on
the same gel. (B) Graphic representation of the mean (±SE) of the
quantification of relative NFAT1 expression after stimulation with Con
A. Gel loading was normalized for each lane with the actin band and the
intensity of each NFAT1 band was calculated as relative percentage of
the most intense NFAT1 band on each gel. The most intensive band was
set arbitrarily at 100% and relative percentages were then averaged
and graphed.
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Reduced cytoplasmic IFN- and TNF- expression in activated UCB T
cells correlates with reduced NFAT1 expression.
The transcription of numerous cytokines, including IFN- and TNF-,
is regulated by NFAT1.18 In stimulated lymphocytes from NFAT1 gene-deleted mice, IFN- and TNF- expression has been shown to be reduced.29
Because we found deficient constitutive NFAT1 expression in
unstimulated UCB T cells and only partial NFAT1 protein upregulation with prolonged TCR stimulation, we measured cytoplasmic expression of
IFN- and TNF- in UCB T cells at time points 18 hours and 40 hours
of Con A stimulation using the same UCB and adult MNC as for our
Western blot analyses. While performing the Con A stimulation experiments for NFAT1 Western blotting described above, MNC from the
same UCB and adult peripheral bloods, stimulated concomitantly in
identical study conditions, were analyzed for intracellular cytokine
expression. Intracellular cytokine expression was analyzed using
3-color flow cytometric analysis with directly conjugated monoclonal
antibodies specific for CD3, the early activation marker CD69, and the
cytokines IFN- and TNF-. Cytoplasmic IFN- and TNF-
expression were then measured in gated activated, CD69 expressing CD3+ T cells.
In accordance with the reduced NFAT1 expression measured by Western
blot in UCB T cells, we found a reduced number of IFN- and TNF-
producing cells in UCB T cells at the time point 18 hours of Con A
stimulation (Fig 3,
Table 1). While on average 3.5% of
adult activated T cells expressed IFN- and 5.8% expressed TNF-
after 18 hours of prolonged T-cell stimulation, we found on average
only 1.5% of activated UCB T cells expressing IFN- and 2.7%
CD3+/CD69+ UCB T cells expressing cytoplasmic
TNF-. After 40 hours of Con A stimulation, at the time point when
NFAT1 protein became detectable by Western blotting in UCB T cells and
doubled in adult controls, TNF- expressing T cells in UCB increased
to 6.3% and almost doubled in adult controls to 10.8%. Further
increases in IFN- producing T cells in either UCB or adult controls
were not observed between 18 hours and 40 hours of Con A stimulation.
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| Fig 3.
Cytoplasmic expression of IFN- and TNF- by UCB and
adult T cells. Intracellular staining was performed in parallel and on
the same MNC during the primary Con A stimulation experiments for NFAT1
analysis by Western blotting. MNC from UCB or adult peripheral blood
were left unstimulated or stimulated using Con A for 40 hours in
identical conditions and analyzed for intracellular expression of
cytokines by flow cytometry. At 0 hour (unstim), 18 hours, and 40 hours
of stimulation, CD3+ cells were gated and intracellular
cytokine expression in gated cells was plotted versus CD69 expression.
Numbers in histograms indicate the percentage of
CD3+/CD69+ cells expressing the cytokine.
(A) Intracellular staining for IFN-. (B) Intracellular staining for
TNF-.
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DISCUSSION |
Transcription factors of the NFAT family play a major role in the
regulation of cytokine genes and other effector molecules modulated
during immune responses. While putative binding sites for NFAT1 have
been described on the regulatory elements of many genes, only recent in
vivo data from NFAT1 gene-deleted mice confirmed the crucial role of
NFAT1 in the transcription of the IL-4, IFN-, GM-CSF, IL-13,
TNF-, CD69, FasL, and CD40L genes.29
The surface phenotype and functional deficiencies described for murine
NFAT1 gene-deleted lymphocytes resembles that of human UCB lymphocytes.
Activated lymphocytes from UCB produce reduced amounts of IL-4,
IFN-, TNF-, GM-CSF, and have reduced surface expression of
CD40L.4,11,31,32 These properties suggested reduced NFAT1
function in UCB lymphocytes. We therefore analyzed NFAT1 protein
expression in UCB T cells and compared it with expression in adult T
cells. We show herein, that in freshly isolated unstimulated UCB T
cells, constitutive NFAT1 protein expression is undetectable, as
measured by Western blotting, in conditions where it is readily detectable in adult T cells.
As T cells in UCB are predominantly of naive phenotype
(CD45RA+) with reduced to absent CD45RO+
expression when compared with adult peripheral blood T
cells,11 one might hypothesize that reduced
NFAT1 protein expression is attributable to the naive,
CD45RA+ phenotype of T cells in UCB, rather than an
intrinsic trait of UCB T cells. However, our analysis of NFAT1 protein
expression by Western blot in CD45RA+ and
CD45RO+ T-cell subpopulations from adult and UCB does not
support this hypothesis. Rather, we measured similar levels of NFAT1
expression in both adult CD45RA+ and CD45RO+
subpopulations and could not detect NFAT1 in either subpopulation in
UCB. These findings strongly suggest that the reduced constitutive expression of NFAT1 we observe in unstimulated UCB T cells is an
intrinsic property of cord blood.
Upregulation of NFAT1 protein during primary stimulation of adult T
cells has not been described. However, a recent report described
upregulation of NFAT1 protein levels after 10 to 30 days of priming of
peripheral adult T cells.22 Our data demonstrates that in
adult T cells, NFAT1 is upregulated 2-fold after 40 hours of
stimulation. Importantly, however, our results show that after 18 hours
of primary T-cell stimulation, there is still deficient NFAT1
expression seen in UCB T cells. Furthermore, after 40 hours of
stimulation, NFAT1 expression remains significantly reduced in UCB T
cells, averaging only 45% of adult expression.
Interestingly, our studies using concomitant measurement of NFAT1 by
Western blotting with cytoplasmic expression of IFN- and TNF-
show that upregulation of NFAT1 in adult T cells after 40 hours of
stimulation correlates with the late upregulation of cytoplasmic
TNF- expression. Furthermore, in agreement with reduced NFAT1
protein levels expressed by UCB T cells, we detected reduced TNF-
producing T cells in UCB at 18 hours time point of primary T-cell
stimulation. After 40 hours of T-cell stimulation, when NFAT1
expression in UCB T cells is upregulated to 45% of that measured in
adult T cells, TNF- production is noted to increase to almost one
half that of adult controls. IFN- expression increased above
baseline expression after 18 hours of Con A stimulation, but did not
increase further after 40 hours of stimulation in UCB and adult T
cells, suggesting dependence on transcription factors other than NFAT1
at this later time point. It is interesting to note here that
upregulation of IFN- mRNA in NFAT1 gene-deleted lymphocytes has been
shown to be less deficient than upregulation of TNF-
mRNA.29
One might argue that reduced IFN- and TNF- production could be
attributable to excess Th2 type cytokine production by UCB T cells.
However, previous reports point to reduced rather than increased Th2
type cytokine production by UCB T cells.11,17 This
phenotype of reduced expression of Th2 cytokines is also observed in
murine NFAT1 gene-deleted lymphocytes.29 In this model,
NFAT1 protein expression has been clearly correlated with reduced Th1
and Th2 cytokine upregulation.
Low expression of the pivotal transcription factor NFAT1 and reduced
IFN- and TNF- production would be expected to blunt amplification
of alloreactive donor T-cell responses against recipient antigens,
thereby limiting clinical GVHD severity after UCB allogeneic transplantation. IFN- and TNF- are known to play a central role in mediating the pathophysiology of clinical GVHD. Accordingly, levels
of these cytokines are significantly increased in allogeneic recipients
during GVHD reactions.9,33,34 Moreover, clinical trials
show that administration of anti-TNF- antibodies is effective for
prophylaxis and attenuation of GVHD in allogeneic bone marrow transplant recipients.35,36 Likewise, it has been shown
that administration of a xanthine derivative pentoxifylline, capable of
downregulating TNF- production, reduced GVHD incidence and severity
in bone marrow transplant recipients.37 IFN- also synergizes with lipopolysaccaride (LPS), released upon tissue damage in
allogeneic recipients receiving ablative chemoradiotherapy conditioning
regimens, to augment TNF- production during the early posttransplant
time period.38,39
In addition to affording a possible new strategy for specific immune
modulation, our results suggest a potential molecular mechanism
underlying the reported reduced incidence and severity of GVHD observed
in allogeneic UCB transplant recipients. Whether there is a possible
link between NFAT1 protein expression and incidence and severity
of GVHD in recipients of allogeneic UCB is currently under active investigation.
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ACKNOWLEDGMENT |
The authors thank the Labor and Delivery staff of McDonald's Women
Hospital for their enthusiastic support.
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FOOTNOTES |
Submitted March 17, 1999; accepted July 5, 1999.
Supported by Grant No. 6230-98 from The Leukemia Society of America.
M.J.L. is a Stephen Birnbaum Translational Research Investigator of the
Leukemia Society of America and Leukemia Scholar in Clinical Research.
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 Mary J. Laughlin, MD, Director, Allogeneic
Transplant Program, Case Western Reserve University, University
Hospitals Ireland Comprehensive Cancer Center, 11100 Euclid Ave,
Cleveland, OH 44106-5065; e-mail: mjl13{at}po.cwru.edu.
| |
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