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Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3968-3975
Graft-Versus-Host Disease and Graft-Versus-Leukemia Effect in Mice
Grafted With Peripheral Newborn Blood
By
Véronique de La Selle,
Eliane Gluckman, and
Martine Bruley-Rosset
From INSERM Unité 267, Immunogénétique des
Allogreffes, Villejuif, France; and Unité de transplantation de
moelle osseuse, Hôpital Saint-Louis, Paris, France.
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ABSTRACT |
We previously used peripheral newborn blood (NBB) as a possible in
vivo experimental model for cord blood (CB) transplantation and showed
that B10.D2 NBB cells successfully reconstituted adult (DBA/2 × B10.D2)F1 mice without causing graft-versus-host disease (GVHD),
probably because of their phenotypic and functional immaturity. Here we
investigated the influence of T-cell maturation occurring in NBB cells
during the early postbirth period on the degree of engraftment, the
incidence of GVHD, and the graft-versus-leukemia (GVL) potential. These
parameters were compared in recipients grafted with bone marrow (BM)
cells. We observed an increased percentage of CD4+ mature
T cells accompanied by the acquisition of proliferative responses to
phytohemagglutinin (PHA) and to allogeneic cells of day-5 NBB cells.
The capacity of day-2 NBB to engraft was moderately reduced and
recipients developing GVHD were occasionally observed after the graft
of day-5 NBB cells. No GVL effect was evidenced regardless of the time
of postbirth blood collection. However, the GVL effect can be obtained
by the delayed infusion of donor mature T cells to recipients grafted
with day-0 NBB, without causing GVHD. In contrast, the same protocol
applied to mice grafted with BM cells induced GVHD mortality of all
recipients. Interleukin (IL)-10 but not IL-2 messenger RNA was
expressed in NBB cells as opposed to BM cells. These findings suggest
that, in terms of GVHD incidence, delayed infusion of mature T cells as
post-transplant tumor immunotherapy would be more effective when
applied after CB than after BM transplantation.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
IN RECENT YEARS, THE number of
transplantations using human umbilical cord blood (CB) instead of bone
marrow (BM) has increased regularly, yielding promising
results.1,2 Indeed, CB may represent a particularly
desirable source of transplantable cells because previous studies
indicate that it contains a large number of hematopoietic
progenitors.3 In addition, it has been reported that these
cells possess a naive phenotype,4 a reduced alloreactivity,5-8 and a limited capacity to cause
graft-versus-host disease (GVHD).1,2,9 Because many CB
transplantations concern children with malignant
diseases,1,2,9 it is important to preserve an intact
antileukemic effect. Yet the low potential of CB cells to induce a GVHD
might be associated with a lack of graft-versus-leukemia (GVL) effect
because effector T cells responsible for GVHD contribute, at least
partially, to the GVL effect.10 On the other hand, natural
killer (NK) and lymphokine-activated killer (LAK) cells
were shown to mediate this effect.5,11,12 If NK lytic cell
activity is minimal in CB, increased LAK activity can be enhanced in
vitro by incubation with interleukin-2 (IL-2).5,11,12 All
these data are encouraging but are not conclusively indicative of the
real in vivo capacity of CB cells to mediate a GVL effect in the
absence of GVHD.
In a previous study, we explored an experimental murine model for CB
transplantation and showed the powerful capacity of peripheral blood
(PB) cells from B10.D2 newborn mice (NBB cells) to engraft fully in
adult (DBA/2 × B10.D2)F1 mice without inducing a GVHD across the
minor histocompatibility antigens (mHAgs) barrier.13
The purpose of this report was to examine whether, in our experimental
murine model, we could show that a GVL effect exists in the absence of
a GVHD across the mHAgs barrier. Yet our previous data clearly showed
that murine NBB cells displayed more immature phenotypic and functional
characteristics than human CB cells.13 Therefore in
attempts to mimic the human situation, we investigated the
immunological reactivity and the GVHD and GVL capacity of NBB cells
collected from B10.D2 mice either on the day of birth or 2 and 5 days
later. It is known that phenotypic maturation and acquisition of
immunocompetence progressively develop during the early postbirth
period at rates that depend on the cell compartment and the function
studied.14-17 However, most of the studies were done in
thymus or peripheral lymphoid organs and no data are available for PB.
Here we report a significant increase in the percentage of
CD4+ mature T cells in day-5 NBB accompanied by the
acquisition of proliferative responses to phytohemagglutinin (PHA) and
to allogeneic cells. Yet the capacity of NBB cells to engraft
irradiated F1 recipients was significantly reduced when NBB was
collected 2 days after birth and mice developing GVHD were only
occasionally observed after the graft of day-5 NBB cells. However, no
GVL effect was observed regardless of the time of postbirth blood
collection. Additional experiments indicated that a GVL effect can be
obtained, without causing GVHD, by the delayed infusion of donor mature T cells to recipients grafted with NBB collected on the day of birth.
In contrast, the same protocol applied to mice grafted with BM cells
induced GVHD mortality of all recipients. We hypothesize that the
protection against GVHD by NBB cells involves the intervention of
regulatory mechanisms.
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MATERIALS AND METHODS |
Mice.
B10.D2 (H-2d, Mls-1b2b), DBA/2
(H-2d, Mls-1a2a), (DBA/2 × B10.D2)F1 (H-2d/d,
Mls-1a/b2a/b), C57BL/6 (H-2b,
Mls-1b2b) mice were bred in our own animal
facility.
PB collection.
The PB from individual adult or newborn B10.D2 mice was collected on
Calciparin.13 The volume of blood obtained from one newborn
mouse ranged from 40 to 50 µL. For in vitro experiments, PB
from six to eight individual newborn mice from the same litter was
pooled and mononuclear cells were prepared using Ficoll/Hypaque gradients (1090; Pharmacia, Uppsala, Sweden). PB from one adult B10.D2
mouse (500 to 800 µL) was similarly separated on Ficoll/Hypaque gradient (1077; Pharmacia). For in vivo experiments, the blood of two
newborn mice was pooled before injection into recipients.
Graft procedure.
(DBA/2 × B10.D2)F1 mice were exposed to a 137Cs
radiation source (RX 30/55 irradiator; Gravatom Industries Ltd,
Gosport, UK) at a dose rate of 0.75 Gy/minute. Lethally
irradiated (9.5 Gy) recipients were infused intravenously with
107 BM cells and 8 × 106 lymph node
B10.D2 cells, or with 6 × 105 adult PB B10.D2 cells,
or with 106 PB cells (two B10.D2 newborn mice) collected on
the day of birth or 2 and 5 days later.
Phenotype characterization.
Mononuclear cells from B10.D2 adult or newborn PB and from adult BM
were directly labeled with fluorescein isothiocyanate (FITC)- or
phycoerythrin (PE)-conjugated anti-CD4, anti-CD8, anti-TCR  / monoclonal antibodies (MoAb) (Pharmingen Clinisciences,
Montrouge, France) and anti-Thy-1 MoAb (Coulter,
Margency, France). Unconjugated rat MoAb against stem-cell
antigen (SCA-1, clone Fall-3; Pharmingen) and mouse MoAb against
Lyt-1.1 allele (Coulter) were shown by FITC-conjugated goat antirat
immunoglobulin (Ig) (GAR, Coulter) or by FITC-conjugated goat antimouse
Ig (GAM, Coulter), respectively.13 Briefly, 5 × 105 cells were incubated for 30 minutes at 4°C with the
corresponding MoAb. After washing, the cells were stained with the
specific second reagent. PE-conjugated anti-CD4 and FITC-conjugated
anti-CD8 MoAbs were mixed before incubation. Samples of 10,000 cells
were analyzed in a flow cytometer (Profile II, Coulter) and the
percentage of single- or double-positive cells was calculated.
Mixed lymphocyte reaction (MLR) and mitogen stimulation.
Cells (2 × 105 per well) from B10.D2 adult or newborn
PB and from B10.D2 adult BM and spleen were cultured with PHA or
concanavalin A (Con A) mitogens and with irradiated splenocytes (4 × 105 per well) from C57BL/6 mice. Proliferation was
evaluated 2 days (cultures with mitogens) or 3 days (cultures with
irradiated spleen cells) later by 3H-thymidine
incorporation, added during the last 18 hours of culture.
Evaluation of GVL effect.
To assess the GVL effect, two protocols were used (1) F1 recipients
were injected intravenously with 105 P815 tumor cells
(mastocytoma from DBA/2 mice) 1 day before lethal irradiation and
grafting with B10.D2 BM cells or NBB cells collected on different days
after birth and (2) 103 P815 cells were injected
intravenously into F1 mice 2 to 3 months after the graft of BM or NBB
cells collected on different days after birth.
RNA extraction and cDNA synthesis.
2 × 106 Cells from adult spleen, BM, and NBB were
stimulated by Con A and pelleted after 24 hours of culture. Total RNA
was extracted with RNABle (Eurobio, les Ulis, France) and ethanol precipitated with the addition of 5 µg of glycogen before
resuspension in 20 µL of water. Single-strand complementary DNA
(cDNA) was synthesized using the First-Strand synthesis kit (Pharmacia
Biotech, Orsay, France) and then diluted in 30 µL of water.
Oligonucleotides.
C, IL-2, interferon- (IFN), IL-4, and IL-10 primers were
obtained from Life Technologies (GIBCO BRL, France).
Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR).
One µL of cDNA was added to 50 µL of amplification mixture (2.5 mmol/L MgCl2, 10 mmol/L dNTPs, 12.5 pmol/L of IL 3
and 5 primers with 1.5 u/reaction Taq polymerase) and overlaid
with mineral oil (Sigma Chemical Co, St Louis, MO). The mixtures were submitted to 45 cycles, each consisting of 45 seconds at 94°C, 1 minute at 60°C, and 1 minute at 72°C. Ten µL aliquots of the reaction mixture were withdrawn through oil at 35, 40, and 45 cycles
and analyzed by ethidium bromide staining of 2% agarose gel. For
quantitative PCR, 5 µL of the reaction mixture was sampled through
oil at different cycle numbers and transferred into avidin-coated wells
containing 95 µL of TRIS-EDTA buffer for quantitation of the amplified products in a liquid-hybridization-enzyme-linked immunosorbent assay (ELISA) with luminometry readings.18
Statistical Analysis.
Statistical analysis for in vitro experiments was performed using
Student's t test. Survival data were analyzed using the  2 test that compared the percentage of surviving mice
presented in Figs 1, 3, and 4. The
Mann-Whitney nonparametric test was used to compare the rate of
mortality (Fig 2).
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| Fig 1.
In vivo survival of lethally irradiated (DBA/2 × B10.D2)F1 mice either receiving no cells (control,  ) or
grafted with B10.D2 cells from adult PB (···), TCD BM
(-··-), BM, and lymph nodes (-·-) or NBB collected on days 0 (--), 2 (- -), and 5 ( ) after birth. Mortality was recorded
daily and mice were examined for the presence of clinical GVHD (number
of live mice/total number of mice).
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| Fig 2.
In vivo survival of lethally irradiated (DBA/2 × B10.D2) F1 mice grafted with B10.D2 cells from adult TCD BM
(-·-) or from NBB collected on days 0 (--), 2(- -), and 5 (···) after birth. F1 mice grafted with BM and spleen cells from
F1 mice (ISO, -··-) served as controls. All recipient mice
received 105 P815 cells one day before irradiation and
grafting. Mortality was recorded daily and mice were examined for the
presence of tumor metastasis (number of live mice/total number of
mice).
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RESULTS |
Phenotypic maturation of NBB cells after birth.
PB was collected from B10.D2 mice either on the day of birth, 2 or 5 days later, or from adult (2- to 3-month-old) mice. After purification
on a Ficoll/Hypaque gradient, mononuclear cells were stained with
various labeled MoAbs. The changes in composition of NBB during the
early postnatal period are illustrated in
Table 1 and compared with that
of adult PB and BM. As already reported in our preceding
paper,13 the majority of T cells of newborn mice expressed
a CD4+CD8+ Thy-1+ immature
phenotype with few CD4+ or CD8+ cells with a
high level of TCR /. The cell composition of NBB collected on the
day of birth did not differ significantly from that of adult BM except
for the presence of CD4+CD8+ cells. Between day
0 and day 5, the percentage of CD4+CD8+ and of
Thy-1+T cells decreased significantly (P = .05 and
P = .01, respectively) and that of CD4+ but not of
CD8+ T cells gradually rose above that of adult BM cells
(P = .003). The percentage of T cells expressing TCR
high increased in parallel (P = .01).
Surprisingly, the mean percentage of SCA-1+ progenitor
cells did not decrease significantly after birth until day 5, although
it varied greatly between different blood samples.
Acquisition of immunological competence.
Previous data showed that exposure to T-cell mitogens or allogeneic
cells induces only a minor proliferative response of NBB cells
collected on the day of birth.13 The results presented in
Table 2 show a significant increase in the response to
PHA (P = .027) but not to Con A (P = .08) of NBB cells
collected on day 5 in comparison with cells collected from NBB on day
0. The low reactivity to allogeneic cells observed in day-0 NBB cells increased (P = .04) in day-5 NBB cells. The proliferative
responses of peripheral blood cells remain significantly weaker in
newborn mice than in adult mice. These data clearly show that NBB T
cells progressively acquire immunological reactivity but that they are not yet fully competent by day 5 after birth.
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Table 2.
Proliferative Response of Cells From Newborn Blood or
Adult Spleen, Peripheral Blood and BM to T-cell Mitogens and Allogeneic
Cells
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Engraftment GVHD incidence in F1 mice receiving NBB cells collected
on different days after birth.
In our transplantation model, all F1 recipients grafted with BM mixed
with lymph node cells died because of acute GVHD against incompatible
host DBA/2 mHAgs (Fig 1). Conversely, F1 mice receiving only BM cells
depleted of mature Thy-1+ T cells (TCD BM) survived and
became fully chimeric. F1 recipients grafted with adult PB died,
because of the absence of engraftment due to the lack of
SCA-1+ progenitor cells (Table 1), with a rate of mortality
similar to that of irradiated F1 mice receiving no cells (control). PB was collected from newborn mice either on the day of birth or 2 and 5 days later. The blood of two newborn mice, containing around
106 mononuclear cells, was pooled before injection into
lethally irradiated (DBA/2 × B10.D2)F1 mice. The percentage
survival was significantly reduced (2 test; P = .052) when
recipients were grafted with day-2 NBB cells (47%) compared with day-0
NBB cells (78%). In these two groups, early mortalities (before day
10) were due to a lack of engraftment attested by the microscopic size
of the spleen. Mice grafted with day-5 NBB cells reacted differently; 5 of the 12 mice (42%) died after day 30 with signs of GVHD. None of the
mice surviving over 100 days developed signs of GVHD.
Immunohematopoietic reconstitution of NBB-engrafted F1 recipients.
We evaluated the degree of chimerism of recipients grafted 2- to
3-months earlier by measuring the residual percentage of lymph node T
cells expressing the Lyt-1.1 marker specific to recipient DBA/2
haplotype. The content of T cells of donor origin was high (82.3% to
88.9%) in all groups of mice regardless of the source of cells used
for transplantation (Table 3). The percentages of
CD4+ T cells as well as of Ig+ B cells were not
significantly different from those found in normal B10.D2 mice and in
BM-engrafted F1 mice, showing that the recovery was complete for these
subpopulations 2 to 3 months after the graft. However, the percentages
of CD8+ T cells remained significantly reduced (P < .05) in all grafted groups.
Absence of a GVL effect in NBB-engrafted F1 recipients.
To investigate whether a GVL effect exists or not in F1 mice grafted
with newborn blood, 105 P815 tumor cells were injected
intravenously into recipients 1 day before irradiation and
reconstitution with NBB collected on days 0, 2, and 5 after birth. The
incidence of tumor relapse was compared with that of F1 recipients
grafted with TCD BM cells of B10.D2 origin that did not develop lethal
GVHD (Fig 1) and with that of F1 recipients grafted with F1 BM plus
spleen cells (ISO). Previous data19 and Fig 2 showed that
seven of eight TCD BM-grafted mice receiving 105 P815 cells
died of leukemia with a median survival time (MST) of 30 days. This
mortality rate is not significantly different (P = .13) from
that of ISO-grafted mice (MST = 25 days; Fig 2). All mice grafted with
day-2 and day-5 NBB cells died quickly (MST = 12 and 11 days,
respectively) and only 2 of 14 mice grafted with day-0 NBB cells
survived (MST = 18 days). The survival of day-2 and day-5 NBB-grafted
mice but not of day-0 NBB-grafted mice was significantly shorter than
that of TCD BM-grafted mice (P < .001). All recipients dying
before day 12 had a highly atrophic spleen, indicating the absence of
engraftment. From 2 weeks after P815 injection, irradiation, and
grafting, most recipients, regardless of the origin of the grafted
cells, died with evidence of tumor metastasis in the spleen and liver,
thus showing the absence of a GVL effect in NBB-grafted mice as in TCD
BM- and ISO-grafted mice.
Effect on GVHD development of delayed infusion of donor spleen cells
to grafted recipients.
Several studies have reported a beneficial antitumoral effect after
injection of donor lymphocytes several days or weeks after TCD BM
transplantation, but GVHD was frequently described.20-22.
We first examined whether infusion of 106 B10.D2 donor
spleen cells 7 and 14 days after transplantation of day-0 NBB, or of
adult TCD BM, induced lethal GVHD. Because engraftment was better with
day-0 than with day-2 NBB cells, and because their phenotype and
functional characteristics do not differ significantly, we used day-0
NBB as the source of transplantable cells for the subsequent
experiments. We eliminated day-5 NBB cells because they induced some
degree of GVHD. The survival of F1 recipients grafted with NBB alone
(78%) was not significantly affected by the two additional injections
of donor splenocytes (73%) (Fig 3). Conversely, the
injection of splenocytes into F1 recipients grafted with TCD BM
significantly (P < .001) increased the percentage
of mortality (95% with an MST = 48 days) by inducing GVHD.
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| Fig 3.
In vivo survival of lethally irradiated (DBA/2 × B10.D2)F1 mice grafted with B10.D2 cells from adult TCD BM (- -) or from NBB (--) collected on the day of birth. 106
B10.D2 donor splenocytes (Spl) were injected or not 7 and 14 days after
the graft of NBB (-·-) or of TCD BM (···) cells. Mortality
was recorded daily and mice were examined for the presence of clinical
GVHD (number of live mice/total number of mice).
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Influence of delayed infusion of NBB-grafted recipients with donor
spleen cells on GVL effect.
P815 tumor cells (103) were administered intravenously to
F1 recipients reconstituted 3 months earlier with NBB alone, NBB plus
splenocytes, or TCD BM cells. Relapse rate was recorded and compared
with that of naive F1 mice injected with the same number of P815 cells
(Fig 4). Addition of splenocytes conferred significant protection against tumor growth in 10 out 16 mice (62%) grafted with
NBB cells when compared with recipients grafted with NBB (P = .016) or with TCD BM (P = .004) cells
alone.
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| Fig 4.
Percent mortality by tumor relapse of lethally irradiated
(DBA/2 × B10.D2)F1 mice grafted with B10.D2 cells from day-0 NBB
either alone (--) or mixed with splenocytes (NBB+Spl, - -) or
from TCD BM (-··-) alone. Naive F1 mice served as controls
(-·-). All recipient mice were injected intravenously with
103 P815 tumor cells 2 to 3 months after grafting.
Mortality was recorded daily and mice were examined for the presence of
tumor metastasis (number of live mice/total number of mice).
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Comparison of lymphokine messenger RNA (mRNA) expression by Con
A-stimulated adult BM, spleen, and NBB cells.
To understand why no GVHD developed after injection of splenocytes into
mice grafted with NBB, as opposed to mice grafted with BM, we
postulated that some cells present in NBB possess regulatory properties
expressed through secreted soluble interleukins. It was found that on
anti-CD3 stimulation early postnatal T cells produce abundant IL-4 that
inhibited IL-2 secretion in response to primary stimulation in
vitro.15 We therefore studied the lymphokine mRNA
expression profile of BM and NBB cells on Con A stimulation and
compared it with that of adult spleen cells. mRNA was extracted from
NBB collected on the day of birth and from
adult BM cells and spleen cells after stimulation with Con A for 24 hours. Lymphokine mRNA expression level was first measured by semiquantitative RT-PCR amplification by using IL-2, IFN, IL-4,
IL-10, and C-specific primers. PCR products were collected after 35, 40, and 45 cycles of amplification and the results of mRNA expression
are summarized in Fig 5. Approximately
comparable levels of C mRNA were obtained with mRNA derived from
spleen and NBB cells and slightly less for mRNA derived from BM cells. IL-2 and IFN mRNA were detectable in stimulated spleen cells after
35 cycles whereas IL-4 and IL-10 mRNA appeared after 40 cycles but at
very low levels in the case of IL-10. Stimulated BM cells expressed
similar but low levels of mRNA transcripts for IL-2, IFN, and IL-4
(detectable after 40 cycles) but not at all for IL-10. NBB cells
expressed IFN and IL-4 mRNA after 40 cycles and very low levels of
IL-2 mRNA transcripts at 45 cycles. In contrast, IL-10 transcripts were
detectable after 40 cycles at higher levels than in spleen cells.
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| Fig 5.
IL gene expression in adult spleen, BM, and in NBB. Cells
from spleen, BM, and NBB were stimulated with Con A for 24 hours and
mRNA extracted. Semiquantitative RT-PCR amplification was performed on
cDNA using C-, IL-2-, IFN-, IL-4-, and IL-10-specific primers.
PCR products were analyzed on agarose gel after 35, 40, and 45 cycles
of amplification.
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A quantitative RT-PCR assay was further developed19 to
estimate the relative levels of mRNA for IL-2 and IL-10 and compare the
number of amplification cycles necessary to generate similar amounts of
cDNA PCR products. For a similar level of C mRNA, NBB cells
expressed more IL-10 mRNA (two-cycle difference corresponding to a
fourfold increase) than spleen cells, and BM cells did not reach
detectable levels of mRNA (Fig 6). IL-2
mRNA was not detected in NBB cells whereas spleen cells expressed
approximately four times more mRNA than BM cells.
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| Fig 6.
Quantitation of C, IL-2, and IL-10 mRNA by RT-PCR
amplification. At sequential cycle numbers, cDNA PCR samples
synthesized from mRNA, prepared from adult spleen ( ), adult BM
( ), and NBB ( ). Con A-stimulated cells were collected and
transferred onto avidin-coated microtiter plates. Quantitation of the
amplified products was performed in a liquid-hybridization-ELISA assay
with luminometry readings.
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DISCUSSION |
Our experimental model was designed to evaluate the GVHD incidence and
the antileukemic potential associated with the transplantation of PB
from newborn mice in comparison with the transplantation of
T-cell-depleted adult BM. We previously showed that the blood of one
newborn mouse contained a sufficient number of progenitors to
reconstitute lethally irradiated F1 recipients with about 80% engraftment.13 In the genetic combination used, donor and
recipient differed in multiple mHAgs and the injection of BM mixed with mature lymphoid cells caused 100% GVHD mortality, whereas injection of
TCD BM or NBB cells generated no GVHD signs. The phenotypic and
functional immaturity of T cells from NBB explained these results13 and prompted us to investigate the influence of
T-cell maturation during the neonatal period and of the acquisition of immune reactivity on the degree of engraftment, the incidence of GVHD,
and the antileukemic potential.
There was a progressive increase in NBB of CD4+ T cells
expressing TCR / and a concomitant decrease in
CD4+CD8+ T cells. These observations are in
accordance with studies of T-cell maturation that identified
CD4+CD8+ immature T cells in lymph nodes but
not in the spleens of newborn mice.16 These
CD4+CD8+ cells are thymus-derived and exported
during the first few days of life. They had dropped in number by day 8 and disappeared by day 10.16 The percentage of
SCA-1+ progenitor cells fluctuated but differences were not
statistically significant. Simultaneously, T cells acquired the
capacity to proliferate on stimulation with PHA and allogeneic cells,
although less so than adult PB. The degree of engraftment of F1 mice
grafted with day-2 NBB was significantly reduced, probably because of graft failure shown on autopsy by the absence of cells in lymphoid organs. In contrast, F1 recipients grafted with day-5 NBB reconstituted but some mice died with clinical signs of GVHD. We interpreted these
data by postulating that the presence of 13% of mature T cells in the
blood at that time favors the engraftment but induces a partial
antihost response.
The antileukemic potential was tested by intravenously injecting
105 P815 cells into F1 recipients 1 day before lethal
irradiation (which eliminated 90% to 99% of tumor cells) and cell
reconstitution. This protocol was first used to mimic the human
situation in which tumor cells pre-exist in the patient before
conditioning and transplantation. At this P815 dose, 87% of recipients
grafted with TCD BM cells and 93% grafted with F1 isogeneic cells
relapsed with evidence of spleen and liver metastasis because of the
absence of antihost T-cell responses. Nearly all the recipients grafted
with day-2 and day-5 NBB cells died of graft failure before day 15. To
explain this observation, we postulate that tumor cell growth prevented the cell reconstitution of lymphoid organs by NBB progenitors that are
more immature and therefore require more time than BM progenitors to
engraft. Mice grafted with day-0 NBB cells that died after day 18 exhibited evidence of tumor metastasis. In this case, the functional
unresponsiveness of T cells was responsible for the lack of GVL effect
for ISO- and TCD BM-engrafted mice.
It has been reported in experimental and human BMT, that a GVL effect
can be generated by the delayed infusion of donor T cells,20-22 but a substantial incidence of GVHD is
associated with this therapy. In our model, injection of
106 donor B10.D2 spleen cells 7 and 14 days after the graft
of adult TCD BM cells induced marked GVHD that was lethal for 17 of 18 recipients. Interestingly, the delayed splenocytes treatment applied to
F1 recipients engrafted with day-0 NBB cells did not cause GVHD. To
explain this observation, we hypothesized that an active suppression
mediated by neonatal cells or factors is responsible for the
tolerization of mature T cells specific for host mHAgs. It was shown
that CD4+ T cells from neonatal lymph nodes secrete large
amounts of IL-4 on stimulation with anti-CD3 that inhibit IL-2
production by adult T cells.15 In addition, susceptibility
of newborns to transplantation tolerance has been attributed to their
high level of IL-4 production.23 We therefore compared the
pattern of cytokines produced by NBB and BM cells. We found that BM
cells expressed a moderate amount of IL-2, IFN, and IL-4 mRNA but no
IL-10 mRNA, whereas NBB cells expressed mRNA transcripts for IFN,
IL-4, and IL-10 but very little for IL-2. Studies in mice showed that
in newborn spleen or lymph node cells, the secretion of
interleukins15 or the expression of interleukin
genes17 on stimulation was low for IL-2, IFN, and IL-10,
and high for IL-4 and thus differed from that of peripheral NBB cells.
Interestingly, single-positive CD4+ thymocytes differ from
their progeny, naive peripheral CD4+ cells, because they
produce mainly IL-4, IL-10, and IFN, but little
IL-2.17,24 In view of the presence of
CD4+CD8+ cells and of the lymphokine pattern
that we found, it appears that NBB cells resemble thymocytes more than
peripheral cells. This hypothesis is consistent with results showing
that the functional phenotype of recent thymic emigrants represents the
exclusive T-cell type in the periphery during the first week of life
both in mice15,17 and in humans.4
Comparison between BM and NBB cells showed no difference in IL-4 mRNA
expression and thus excludes the implication of this cytokine in the
GVHD inhibition. In contrast, IL-2 mRNA is expressed in BM cells and
not in NBB cells and, conversely, IL-10 mRNA is expressed in NBB cells
and not in BM cells. Although lymphokine protein was not measured in
the assays, it is reasonable to assume that mRNA expression for a
particular lymphokine indicates that the corresponding protein was
secreted. IL-10 is known to be synthesized by Th2 cells or by other
types of cells such as B cells, macrophages, and mast
cells.25 It immunosuppresses cytokine production and antigen-specific proliferation of Th1 cells26 and also
induces the differentiation of a new regulatory CD4+ T-cell
subset.27 Recent data showed that chronic activation of
both human and murine CD4+ T cells in the presence of IL-10
gives rise to CD4+ T-cell clones of low proliferative
capacity, producing low levels of IL-2 and no IL-4.27 These
antigen-specific T-cell clones suppress the proliferation of
CD4+ T cells in response to antigen and prevent a
pathogenic T-cell response. In our model, IL-10 produced by NBB cells
when stimulated by incompatible host DBA/2 mHAgs might inhibit the
activation of mature T splenocytes injected after the graft
of NBB cells, and thus prevent induction of GVHD. Conversely, the
delayed infused splenic T cells were able to induce GVHD when combined
with adult BM cells that are unable to synthesize IL-10. This finding
does not exclude the participation of other immunosuppressive cytokines such as transforming growth factor (TGF) not measured
in this work.
We further showed that the delayed infusion of splenocytes induced
resistance to P815 tumor growth in 62% F1 mice grafted 2 to 3 months
earlier with NBB cells, whereas injection of the same dose of tumor
cells induced relapse in 83% mice grafted with NBB cells alone. Most
importantly, these experiments point to a dissociation between the
capacity of mature T cells to achieve GVHD and GVL reactions.
The restricted tissue expression of some mHAgs10,28,29
raises the possibility that mHAgs present on leukemic cells are not
expressed on normal cell targets of GVHD and hence that effector T
cells implicated in GVHD differ from those implicated in GVL.
To explain why this adoptive treatment induced a GVL effect but no GVHD
in NBB-engrafted mice, we hypothesized that donor T cells that became
tolerant to host mHAgs implicated in GVHD,13 further
tolerized (infectious tolerance) delayed infused splenic mature T cells
specific of host mHAgs but not of T cells specific of tissue
antigens expressed on P815 mastocytoma cells. Those T cells were not
tolerized because of the absence of tumor cells at the time of mature
T-cell infusion, thus explaining the preserved GVL effect.
Despite phenotypic and functional differences, murine NBB and human CB
cells share common immunological features such as a low capacity to
induce a GVHD,1,2,9 alterations in the cytokine production
pattern,7,12,30-32 and presence of suppressor T cells or
factors.33,34 Although recorded with a model that does not
exactly reflect the human situation, the present results suggest that,
in terms of GVHD incidence, the delayed infusion of mature T cells as
post-transplant adoptive immunotherapy would be more effective in
eliminating residual disease or in treating leukemia relapse when
applied after CB transplantation than after BMT. A recent clinical
report35 supports this assumption because complete
remission was achieved in a child with acute lymphoblastic leukemia
who, following CB transplantation, was administered adoptive immunotherapy consisting of several infusions of donor PB leukocytes.
| |
ACKNOWLEDGMENT |
We thank N. Riché and L. Majbruch for technical assistance and J. Roué for typing the manuscript.
| |
FOOTNOTES |
Submitted March 12, 1998;
accepted July 13, 1998.
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 Martine Bruley-Rosset, MD, INSERM Unité
267, Immunogénétique des Allogreffes 12, Avenue Paul
Vaillant-Couturier 94807, Villejuif cedex, FRANCE.
| |
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Abstract
Introduction