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Prepublished online as a Blood First Edition Paper on October 17, 2002; DOI 10.1182/blood-2002-05-1529.
TRANSPLANTATION
From The Queensland Institute of Medical Research, and
the Department of Pathology, University of Queensland, Herston,
Australia; the Mater Medical Research Institute, South
Brisbane, Australia; the Centre for Immunology and Cancer
Research, Princess Alexandra Hospital, Woolloongabba,
Australia; the Department of Oncology, Pharmacia, St
Louis, MO; the Department of Internal Medicine and Pediatrics,
University of Michigan, Ann Arbor, MI; and the Department of Stem Cell
Transplantation, Royal Brisbane Hospital, Brisbane,
Australia.
The granulocyte colony-stimulating factor (G-CSF) and
Flt-3 receptor agonist progenipoietin-1 (ProGP-1) has potent
effects on dendritic cell (DC) expansion and may be an alternative to G-CSF for the mobilization of stem cells for allogeneic stem cell transplantation (SCT). We studied the ability of stem cell grafts mobilized with this agent to induce graft-versus-host disease (GVHD) to
minor and major histocompatibility antigens in the well-described B6 Allogeneic stem cell transplantation (SCT) is
currently indicated in the treatment of a number of malignant and
nonmalignant diseases. However, use of the procedure is limited by its
serious complications, the most common of which is graft-versus-host
disease (GVHD). Recently, the use of granulocyte colony-stimulating
factor (G-CSF)-mobilized stem cell grafts has improved rates of immune and hematopoetic reconstitution, reduced transplantation-related mortality, and improved leukemia eradication after
SCT.1 G-CSF has been shown to induce TH2
differentiation in donor T cells prior to SCT and this has been
suggested to be a major protective mechanism from GVHD in this
setting.2 In support of this, administration of other
cytokines that induce TH2 differentiation either in
vivo3 or in vitro4 also protect from GVHD.
However, G-CSF may also reduce GVHD through effects on
monocytes,5-7 natural killer (NK) cells,8 and
natural killer T (NKT) T cells.9 The mechanism of
TH2 differentiation induced by G-CSF is obscure. The lack
of surface expression of G-CSF receptors by T cells supports the notion
that this is an indirect effect and in vitro data have implicated
monocytes as the responsible immunomodulatory cell.6,7 Recently, a subset of dendritic cells (DCs), known as plasmacytoid DCs,
has been identified in humans that appears to preferentially induce
TH2 differentiation following activation. The proportion of
plasmacytoid DCs is increased in the peripheral blood of healthy donors
receiving G-CSF.10 Although G-CSF may induce
TH2 differentiation before SCT, similar T-cell effects have
not been demonstrated in patients undergoing SCT. The
suggestion that increased numbers of donor plasmacytoid DCs mediate
changes in T-cell function and reductions in GVHD10
following allogeneic SCT therefore remains highly speculative.
In the mouse, splenic DCs (characterized by high CD11c and class
II expression) have been shown to consist of CD8hi and
CD8lo subtypes.11 The high levels of
interferon alpha (IFN Flt-3 ligand (Flt-3L) is a hematopoietic cytokine that
increases DC numbers 20- to 30-fold in mice14 and
humans.15 In murine bone marrow transplantation (BMT)
models, Flt-3 ligand given days 25 to 40 after BMT enhances donor DC
and T-cell expansion with a resultant increase in both GVHD and
graft-versus-leukemia (GVL).16 In contrast,
treatment of the donor with Flt-3L alone prior to BMT has no apparent
effect on subsequent GVHD.16 Progenipoietin-1 (ProGP-1) is
a synthetic chimeric molecule that stimulates both G-CSF and Flt-3
receptors and appears to be significantly more potent in the expansion
of stem cells and DCs than the combination of both native cytokines due
to higher binding affinity.17,18 We reasoned that ProGP-1
may offer an attractive system to study the effect of DC subsets on
GVHD after allogeneic SCT.
We therefore compared the ability of stem cell grafts mobilized by
either ProGP-1 or G-CSF to induce GVHD. Our data confirm that ProGP-1
expands large numbers of DCs in donor animals. Recipient animals are
protected from GVHD in association with an altered T-cell adhesive
phenotype and a reduced response to alloantigen after SCT. This confers
a secondary inability to augment inflammatory cytokine production and
GVHD of the gastrointestinal (GI) tract after SCT, thereby preventing
the induction of GVHD.
Mice
Cytokine treatment
Bone marrow transplantation Mice underwent transplantation according to a standard protocol as has been described previously.2,20 Briefly, on day 1,
B6D2F1 mice received 1100 cGy total body irradiation (137Cs
source at 108 cGy/min), split into 2 doses separated by 3 hours to
minimize GI toxicity. Donor spleens were chopped, digested in
collagenase and DNAse, then whole unseparated spleen cells were
resuspended in 0.25 mL of Leibovitz L-15 media (Gibco BRL, Gaithersburg, MD) and injected intravenously into recipients. In most
experiments, Ly-5a (H-2b, Ly-5.1+)
animals were used as donors. Survival was monitored daily,
recipient's body weight and GVHD clinical score were measured weekly.
Donor cell engraftment was determined by examining the proportion of Ly-5.1+ per (Ly-5.2+ + Ly-5.1+) cells in peripheral blood or spleen after transplantation.
Assessment of GVHD The degree of systemic GVHD was assessed by a scoring system that sums changes in 5 clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity (maximum index = 10).3,21-23 Individual mice were ear-tagged and graded weekly from 0 to 2 for each criterion without knowledge of treatment group. Animals with severe clinical GVHD (scores > 6) were killed according to ethical guidelines and the day of death deemed to be the following day.Splenocyte and DC preparation DC purification was undertaken as previously described.24 Briefly, spleens were chopped and digested in collagenase and DNAse. Light-density cells were selected by nycodenz density (1.077 g/L) centrifugation. Non-DC-lineage cells were depleted by coating with rat immunoglobulin G (IgG) antibodies to B cells (CD19), T cells (CD3, Thy1), granulocytes (Gr-1), and erythroid cells (Ter-119). The coated cells were then removed by magnetic beads coupled to anti-rat IgG (Dynal ASA, Oslo, Norway). At the end of this procedure, 65% to 85% of these cell populations were DCs (class II/CD11chi). DCs were presorted to remove autofluorescent macrophages (prior to phenotypic analysis) and then purified by fluorescence-activated cell-sorting (FACS) (FACS Vantage, Becton Dickinson, San Jose, CA) to more than 98% purity using phycoerythrin (PE) CD11c and PE-Cy5 B220 staining.T-cell depletion Splenocytes were depleted of T cells by incubation for 40 minutes (4 degrees) with hybridoma supernatants containing anti-CD4 (GK1.5), CD8 (3.155), and Thy1.2 (HO-13-4) monoclonal antibodies (mAbs). Cell suspensions were then incubated with rabbit complement (Cederlane Laboratories, Ontario, ON, Canada) for 30 minutes at 37 degrees and the process repeated. Resulting cell suspensions had less than 1% contamination of viable CD3 T cells.FACS analysis Fluorescein isothiocyanate (FITC)-conjugated mAbs to mouse Ly 5.1 and Ly 5.2 antigens, CD4, CD8, CD11c, class II, CD3, GR-1, CD11b, and B220, and identical PE-conjugated mAbs were purchased from PharMingen (San Diego, CA). In DC analysis, CyChrome CD4 and CD8 antibodies were also used from Pharmingen. Cells were first blocked with mAb 2.4G2 for 15 minutes at 4°C, followed by the relevant conjugated mAb for 30 minutes at 4°C. Finally, cells were washed twice with PBS/0.2% bovine serum albumin (BSA), fixed with PBS/1% paraformaldehyde, and analyzed by FACS Calibur (Becton Dickinson). Propidium iodide was added in the final wash to label dead cells. DC staining was undertaken on presorted cell populations in which autofluorescent cells were removed by high speed presorting (FACS Vantage) and subsequent analysis was performed the same day on unfixed cells.Cell cultures Culture media additives were purchased from Gibco BRL and medium was purchased from Sigma (St Louis, MO). Peritoneal macrophages were lavaged and pooled from individual animals within a treatment group before culture at 1 × 105 cells per well in flat-bottom 96-well Falcon plates (Lincoln Park, NJ) with or without lipopolysaccharide (LPS). Cell culture was performed in 2% fetal calf serum (FCS)/Dulbecco modified Eagle medium (DMEM) (day 7 cultures) supplemented with 50 U/mL penicillin, 50 µg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, 0.02 mM -mercaptoethanol, and 10 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.75, at
37°C in a humidified incubator supplemented with 5% CO2.
Supernatants were collected at 5 hours for tumor necrosis factor alpha
(TNF ) analysis by enzyme-linked immunosorbent assay (ELISA).
Peritoneal macrophages lavaged from animals 7 days after transplantation were more than 95% donor as determined by Ly5.1 staining. All other cell cultures were performed in 10% FCS/DMEM. In
in vitro experiments, purified B6 T cells were cultured in round-bottom
96-well plates (Falcon) with 105 irradiated (2000 R) B6D2F1
peritoneal macrophages (primary mixed lymphocyte culture
[MLC]) and supernatants harvested at 72 hours. Cultures were
then pulsed with 3H-thymidine (1 µCi [0.037
MBq] per well) and proliferation was determined 16 hours later on a
1205 Betaplate reader (Wallac, Turku, Finland). In secondary MLC,
purified T cells were cultured in flat-bottom 24-well plates (Falcon)
with irradiated (2000 R) B6D2F1 splenocytes. 6 days later, cells were
removed and restimulated with irradiated (2000 R) B6D2F1 peritoneal
macrophages. Supernatants were removed 24 hours later and
3H-thymidine added as above. In experiments of T-cell
function ex vivo, splenocytes were removed from animals 7 to 10 days
after transplantation and 3 to 6 spleens combined from each group.
These cells were plated in 96-well flat-bottom plates with platebound anti-CD3 and -CD28 mAbs (both 10 µg/mL) or 105
irradiated (2000 R) peritoneal macrophages lavaged from naive B6D2F1
(allogeneic) animals. At 40 hours, cultures were pulsed with
3H-thymidine (1 µCi [0.037 MBq] per well) and
proliferation was determined 16 hours later. In separation experiments,
CD4+ cells were positively selected from splenocyte
populations using the mini-MACS system (Miltenyi Biotech, Bergisch
Gladbach, Germany) or FACS (FACS Vantage). Following selection,
positive and negative fractions were FACS stained and each fraction had
less than 1% contamination of opposing CD4+ or
CD8+ cells. Purified CD4+ or CD8+
populations were then plated and analyzed as above.
51Cr release assays P815 (H-2d) or EL4 (H-2b) tumor targets (2 × 106) were labeled with 100 µCi (3.7 MBq) of 51Cr for 2 hours. After washing 3 times, labeled targets were plated at 104 cells per well in U-bottom plates (Costar, Cambridge, MA). CD8+ splenocytes from allogeneic BM transplant recipients (prepared by magnetic selection as described in "Cell cultures") were added to triplicate wells at varying effector-to-target ratios and incubated for 5 hours in the presence of IL-2 (10 U/mL). Maximal and background release were determined by the addition of Triton-X (Sigma) or media alone to targets, respectively. 51Cr activity in supernatants taken 5 hours later was determined in a scintillation counter and lysis was expressed as a percentage of maximum. Lysis was expressed in lytic units (1000/effector-to-target ratio that induced 10% and 20% lysis).Cytokine ELISAs The antibodies used in the TNF, IFN , IL-10, TGF, and
IL-4 assays were purchased from PharMingen. All assays were performed according to the manufacturer's instructions. Supernatants were collected after 4 to 5 hours of culture for TNF, and 40 hours for
IL-4, IL-10, and IFN analysis. Serum was stored at 70°C until
analysis. Briefly, samples were thawed and diluted 1:3 to 1:24 and
TNF, IFN, IL-10, and IL-4 proteins were captured by the specific
primary mAb, and detected by biotin-labeled secondary mAb followed by
horseradish peroxidase (HRP)-conjugated streptavidin. The
biotin-labeled assays were developed with TMB substrate
(Kirkegaard and Perry Laboratories, Gaithersburg, MD). Plates were read
at 450 nm using a microplate reader (Bio-Rad, Hercules, CA).
Recombinant cytokines (PharMingen) were used as standards for ELISA
assays. Samples and standards were run in duplicate and the sensitivity of the assays was 16 pg/mL to 20 pg/mL for TNF, 0.063 U/mL for IFN, and 15 pg/mL for IL-10 and IL-4. TNF from LPS-stimulated peritoneal cells is expressed as picograms per 105
macrophages, as previously described.22
Histology Formalin-preserved distal small bowel was embedded in paraffin, and 5 µm-thick sections were stained with hematoxylin and eosin for histologic examination. Slides were coded and examined in a blinded fashion by one individual (A.D.C.) using a semiquantitative scoring system for abnormalities known to be associated with GVHD.3,22,25 Specifically, 7 parameters each were scored for small bowel (villous blunting, crypt regeneration, crypt epithelial cell apoptosis, crypt loss, luminal sloughing of cellular debris, lamina propria inflammatory cell infiltrate, and mucosal ulceration). The scoring system for each parameter denoted 0 as normal; 0.5 as focal and rare; 1 as focal and mild; 2 as diffuse and mild; 3 as diffuse and moderate; and 4 as diffuse and severe, as previously published in human26,27 and experimental3,22,25 GVHD histology. Scores were added to provide a total score of 28.Statistical analysis Survival curves were plotted using Kaplan-Meier estimates and compared by log-rank analysis. The Mann-Whitney U test was used for the statistical analysis of cytokine data and clinical scores. P < .05 was considered statistically significant.
ProGP-1 pretreatment results in a marked expansion of CD8hi DCs In these studies we compared the effect on GVHD of donor pretreatment with ProGP-1 and G-CSF, the latter being the current cytokine used for the mobilization of allogeneic stem cells in clinical practice. Injections of control diluent, ProGP-1 (20 µg/animal), or G-CSF (10 µg/animal) were administered daily for 10 days. This administration regimen for ProGP-1 was shown in preliminary studies to result in the greatest expansion of stem cells and DCs, consistent with recent reports.28 The G-CSF dose was half that of the chimeric G-CSF/FLT-3L molecule ProGP-1. At the end of this treatment period, absolute numbers of splenocytes increased by 65% in the G-CSF group and 700% in the ProGP-1 group. As shown in Figure 1, the percentages of DCs, CD4 and CD8 T cells, and B cells were similar in control- and G-CSF-treated animals, consistent with our previous findings.20 In contrast, ProGP-1 resulted in a 10-fold increase in the percentage of CD11chi and CD11cdim/B220hi DCs (Figure 1) and a 65% reduction in the percentage of T cells. Significant (30%) reductions in the overall proportion of B220+/CD19+ cells were also noted following ProGP-1 administration. As expected, the proportion of granulocytes was significantly increased in both G-CSF- and ProGP-1-treated animals. To examine the profound effect of ProGP-1 on DC expansion more closely, DCs (CD11chi) were purified and phenotyped according to the expression of CD8 and CD4, as previously published.24 The percentage of CD11chi/CD8hi DCs increased in animals treated with either ProGP-1 or G-CSF compared with those treated with control diluent (Figure 2). This was most dramatic following ProGP-1 administration, which resulted in a 14-fold increase in the proportion of splenic CD8 DCs and a 100-fold increase in the absolute number of these cells. The DCs in the ProGP-1-treated donors included a CD8dim subset in which all DCs were CD11blo (relative to CD4 DC CD11b expression) and a larger CD8hi subset, in which the majority of DCs were also CD11blo (75%). The remaining 25% were CD11bneg. Identical cellular proportions and expansion were seen in the peripheral blood of ProGP-1-treated animals, confirming that the spleen phenotype was representative of that in the blood.
Donor pretreatment with ProGP-1 is superior to G-CSF in reducing the severity of GVHD We next examined the effects of ProGP-1 mobilization in a well-established murine SCT model (B6 Ly5a B6D2F1) that
induces GVHD to major and minor histocompatibility antigens. Although
this model utilizes spleen as a stem cell source rather than peripheral
blood, its validity has been proven by the informative data indicating
the beneficial effects of G-CSF on both GVHD and GVL2,20
that has since been confirmed clinically.1 In preliminary
experiments, we confirmed that the prolonged 10-day course of G-CSF was
at least equivalent in preventing GVHD as the standard 6-day course
used in clinical practice. Survival at day 70 was 50% versus 30% in
recipients of splenocytes from donors treated with 10 days (n = 10)
versus 6 days (n = 10) of G-CSF (P = .49). In addition,
clinical scores were not statistically different in the first 50 days
after transplantation (P > .12) although there was a
trend to less GVHD in recipients of the 10-day course of G-CSF at later
time points. Pretreatment of donors with 10 days of G-CSF was therefore
used as the relevant control to ProGP-1 in subsequent experiments. In
these experiments, allogeneic donor B6 animals received daily
injections of either control diluent, G-CSF, or ProGP-1 and splenocytes
were harvested on day 11. B6D2F1 recipient mice were irradiated with
1100 cGy of total body irradiation (TBI) and received transplants of
107 splenocytes from respective donors. To compensate for
the reduced T-cell dose in the ProGP-1 recipients, a further cohort of
recipients received transplants of ProGP-1 splenocytes in which
additional purified ProGP-1 T cells were added, so as to equilibrate
T-cell dose (3 × 106 T cells) across groups. As
shown in Figure 3 and as previously described,20 GVHD induced in this model is severe with all
recipients of control splenocytes dying in 2 weeks with characteristic
features of GVHD (weight loss, hunching, fur ruffling, etc). In
contrast, 100% of non-GVHD controls that received transplants of
T-cell-depleted allogeneic splenocytes survived, confirming that this
splenocyte dose contained sufficient stem cells to rescue lethally
irradiated recipients and that GVHD is mediated by donor T cells. Mice
undergoing allogeneic SCT using G-CSF splenocytes had a
significantly improved survival at day 70 compared with recipients of
allogeneic control splenocytes (50% versus 0%,
P < .001). Recipients of ProGP-1 splenocytes had a
survival at day 70 in excess of 90% which was significantly better
(P < .05) than recipients of G-CSF splenocytes. The
addition of more T cells to ProGP-1 splenocytes did not increase GVHD
mortality (Figure 3), although clinical GVHD scores were significantly
increased (data not shown). In order to further establish the magnitude of protection afforded by ProGP-1 mobilization over that seen with
G-CSF, cohorts of animals received transplants of escalating doses of
splenocytes from either ProGP-1- or G-CSF-treated donors. As
demonstrated in Figure 4A, survival in
recipients of 106 splenocytes (1.2 × 106 T
cells) from ProGP-1-treated donors was superior to that in recipients
of splenocyte doses from G-CSF donors that ranged from 4 × 106 to 100 × 106
(1.2 × 106 to 30 × 106 T cells). As
expected, GVHD mortality was dependent on splenocyte dose in both
groups. As shown in Figure 4B, survival in recipients of
60 × 106 ProGP-1-treated splenocytes
(7.2 × 106 T cells) was superior to that seen in
recipients of 10 × 106 G-CSF splenocytes
(3 × 106 T cells; 75% versus 40%,
P < .03). Survival was similar, however, when a dose of
100 × 106 ProGP-1 splenocytes (12 × 106 T
cells) was compared with 10 × 106 G-CSF splenocytes
(3 × 106 T cells; P = .26). In addition,
GVHD clinical scores (Figure 4C) were similar in surviving recipients
of 10 × 106 G-CSF splenocytes (3 × 106 T
cells) and 60 × 106 ProGP-1-treated splenocytes
(7.2 × 106 T cells). Given the differences in T-cell
doses that this represents, these data suggest that donor pretreatment
with ProGP-1 allows a 2- to 4-fold escalation in T-cell dose over that
possible with G-CSF.
Donor T-cell engraftment in the spleen 7 days after SCT was 94.7% ± 1.4% in recipients of control splenocytes, 95.4% ± 0.7% in recipients of G-CSF splenocytes and 96.5% ± 0.1% in recipients of ProGP-1 splenocytes. The proportion of donor cells in the peripheral blood of recipients of G-CSF and ProGP-1 splenocytes at day 75 after SCT was 99.4% ± 0.6% and 99.2% ± 0.4%, respectively. In recipients of T-cell-depleted splenocytes, 81% ± 3.2% of peripheral blood cells were donor (P < .05 versus G-CSF and ProGP-1), confirming that the prolonged survival following allogeneic G-CSF- and ProGP-1-treated splenocytes was not due to the presence of stable mixed donor-host chimerism. Donor pretreatment with ProGP-1 results in a T-cell phenotype with reduced capacity to induce GVHD The GVHD induced in these models is dependent on T-cell function20,29 and we therefore examined the effect of G-CSF and ProGP-1 administration on T-cell phenotype and function. CD3+CD4+ and CD3+CD8+ T cells from ProGP-1-treated donors demonstrated an almost complete loss of L-selectin expression whereas T cells from G-CSF-treated animals demonstrated an intermediate pattern of L-selectin loss (Figure 5). A similar pattern of expression was demonstrated in T cells from the peripheral blood of G-CSF- and ProGP-1-treated donors (data not shown). The reduction in L-selectin expression did not coincide with an increase in the proportion of CD44hi T cells (Figure 5), suggesting that the loss of L-selectin was not due to an expansion of memory T cells. In this regard, ProGP-1 and G-CSF did not induce T-cell activation as assessed by CD25 (Figure 5) and CD69 expression (data not shown). L-selectin and CD44 expression on splenic T cells from recipients of control and ProGP-1 splenocytes 4 days after transplantation was equivalent (40% and 90%, respectively), indicating that the loss of expression of these molecules prior to transplantation was transient. In studies of T-cell function, CD3+CD4+ T cells were purified as described and stimulated in vitro with mitogen. As shown in Table 1, cytokine treatment did not alter proliferative responses although both ProGP-1 and G-CSF significantly increased the production of the type 2 cytokines IL-4 and IL-10 whereas IFN production was unchanged. To study T-cell responses to
alloantigen in vivo after SCT, animals received transplants of
splenocytes from control-, G-CSF-, or ProGP-1-treated donors as in
Figure 1. Donor CD4 and CD8 T cells were purified from the spleens of
animals 7 days later. As shown in Table
2, CD4 T cells isolated from mice
undergoing allogeneic SCT with ProGP-1-treated (and to a
lesser extent G-CSF-treated) splenocytes failed to proliferate to host
antigen. Cytokine generation (IFN, IL-4, and IL-10) was also
impaired. This impairment in proliferation was not corrected by the
addition of exogenous IL-2 (50 U/mL) to MLC (control, 123 963
cpm ± 11 289 cpm; G-CSF, 31 382 cpm ± 1991 cpm; ProGP-1,
28 832 cpm ± 2368 cpm). However, cytotoxicity to host antigens in the donor CD8 population was not significantly altered (Table 2). The
difference in T-cell responses before and after SCT suggests that
ProGP-1 and G-CSF modulate T-cell function predominantly in vivo,
perhaps due to impaired T-cell homing and/or the effects of
additionally expanded donor cellular fractions or the products thereof.
Neither CD11chi nor CD11cdim/B220hi donor DCs from ProGP-1-treated animals provide protection from GVHD Protection from GVHD has been associated with the expansion of host CD8+CD11chi DCs by Flt-3L administration.30 This DC subset has been defined as the antigen-presenting cells (APCs) primarily responsible for antigen cross presentation31 whereas the human CD11c CD123+ plasmacytoid DCs have been
associated with G-CSF-mediated transplant tolerance.10 We
therefore studied the ability of highly purified donor DCs to provide
protection from GVHD. Animals received transplants of control-treated
B6 spleen supplemented with FACS-sorted (> 98% pure)
CD11chi splenic DCs (predominantly CD8+) or
CD11cdim/B220hi DCs from ProGP-1-treated
donors in numbers that reflected the proportion present in whole
ProGP-1-treated spleen (Figure 1). As shown in Figure
6, all animals that received these cell
populations died at a similar rate to control animals, suggesting that
neither population provided protection from GVHD in isolation.
ProGP-1 augments IL-10 and TGF determined in culture
supernatants. As shown in Figure 7,
ProGP-1 spleens produced high amounts of IL-10 and TGF relative to
control and G-CSF spleens. After transplantation using these cell
populations, a major reduction in TNF was demonstrated in cultures
of macrophages from recipients of ProGP-1-treated splenocytes (Figure
7C). Macrophages from recipients of G-CSF-treated splenocytes produced
intermediate quantities of TNF. These data confirm that donor
pretreatment with ProGP-1 results in a graft composition favoring
anti-inflammatory cytokine production.
The inhibition of GVHD following donor pretreatment with ProGP-1 is mediated through effects on the T cell Since GVHD is a T-cell-dependent process, we next studied whether the ability of ProGP-1 to reduce GVHD was mediated through effects on the donor T cell. To compare the capacity of the T cells from treated animals to induce GVHD in isolation, all transplant recipients received T-cell-depleted control splenocytes together with equivalent numbers of purified splenic T cells from either control-, G-CSF-, or ProGP-1-treated donors. As shown in Figure 8A, 100% of recipients of control T-cell-depleted splenocytes survived whereas 100% of recipients of control T-cell-depleted splenocytes supplemented with purified control T cells died of GVHD by day 20. In contrast, the median survival was increased in recipients of control T-cell-depleted splenocytes supplemented with purified G-CSF-treated T cells to 40 days and survival at day 70 increased to 25% (P < .001 versus recipients of control T cells). Recipients of control T-cell-depleted splenocytes supplemented with purified ProGP-1-treated T cells had significantly less GVHD than either group, with 90% survival at day 70 (P < .0001 versus recipients of G-CSF and control T cells). GVHD severity in surviving animals, as determined by clinical score, was also significantly reduced in recipients of purified ProGP-1-treated T cells relative to purified G-CSF-treated T cells (Figure 8B). These data indicate that both G-CSF and ProGP-1 alter the capacity of donor T cells to induce GVHD and that the enhanced survival of recipients of ProGP-1-treated allografts relates to T-cell effects.
T cells from ProGP-1-treated donors fail to induce GI tract injury
and systemic TNF levels were
determined in the sera of animals 5 days after transplantation. IFN
levels were significantly reduced in recipients of both G-CSF- and
ProGP-1-treated T cells (63 ± 6.4 U/mL versus 46.4 ± 8.0 U/mL
and 44.4 ± 5.3 U/mL), consistent with the ex vivo data. GVHD
mortality in this transplantation model is TNF
dependent22 and IFN primes mononuclear cells to produce
high TNF levels following stimulation with bacterial-derived antigens that are primarily derived from the GI tract.33
As shown in Figure 9A, T cells from
ProGP-1- and G-CSF-treated donors failed to induce severe GVHD of the
GI tract relative to recipients of control-treated T cells. In vivo,
TNF levels in the sera of recipients of ProGP-1 T cells were 10-fold
lower than those in recipients of control T cells and were
indistinguishable from non-GVHD controls (Figure 9B). Recipients of
G-CSF-treated T cells had TNF levels intermediate between
recipients of control and ProGP-1 T cells, consistent with the
mortality seen in this group.
We have shown that donor pretreatment with ProGP-1 significantly
reduces GVHD in an experimental SCT model and that this reduction is
greater than that seen following donor G-CSF pretreatment. Our data
confirm that donor pretreatment with ProGP-1 results in a significantly
greater expansion of CD11chi DCs and
CD11cdim/B220hi DCs than G-CSF pretreatment,
although these DCs do not appear to protect from GVHD in isolation. We
demonstrate that T cells from ProGP-1-treated donor animals have an
altered adhesive phenotype and an impaired capacity to induce GVHD.
This occurs in association with impaired CD4 T-cell proliferation and
function although cytolytic capacity remains intact. Finally, ProGP-1
impairs TNF ProGP-1 pretreatment results in an anergic proliferative response to
alloantigen after SCT. In conjunction with this, both TH1
and TH2 cytokine responses (including IL-10) to mitogen
were reduced, suggesting this effect was not associated with changes to
T-cell differentiation or the induction of a regulatory cell population. Donor pretreatment with ProGP-1 results in the production of large amounts of IL-10 and TGF The ability of G-CSF to alter T-cell cytokine profiles has been
previously documented although the changes to T-cell differentiation have varied.2,5,20 In contrast to a previous
study,2,20 we found that G-CSF and ProGP-1 increased the
generation of TH2 cytokines, although there was not a clear
reduction in TH1 cytokine production. This study differed
from the former in that we used purified CD4+ T cells
rather than an enriched T-cell preparation that contained CD4+ and CD8+ T cells and nonadherent
contaminating cells. In addition, we studied T-cell effects after a
more prolonged schedule of G-CSF, to match that for ProGP-1. To date,
there have been no experimental or clinical studies demonstrating that
donor pretreatment with G-CSF induces TH2 differentiation
after SCT, which represents the most informative period for analysis.
TH1 cells produce cytokines (especially IFN Markedly reduced L-selectin expression was observed on both CD4 and CD8 T cells following ProGP-1 administration. Since L-selectin is an essential molecule in the homing of naive T cells to lymphoid organs, it is possible that reduced expression may play a role in delaying the initiation of immune responses during the induction of GVHD. Furthermore, the effect of ProGP-1 on L-selectin was far more dramatic than that of G-CSF (Figure 2) and this may represent an additional mechanism by which ProGP-1 results in greater amelioration of GVHD than G-CSF alone. Changes to L-selectin expression have been previously described following G-CSF administration, as was the case in this study, and L-selectinlo cells have a reduced capacity to respond to alloantigen in vitro.39 Furthermore, the blockade of L-selectin is known to prevent experimental GVHD.40 Since reciprocal increases were not seen in CD44 expression, we hypothesize that the loss of L-selectin is likely to represent enzymatic cleavage or conformational distortion of this molecule (rather than expansion of memory T cells), perhaps due to products released from the expanded myelomonocytic lineage in G-CSF- and ProGP-1-treated donors. The T-cell cytotoxic pathways (involving perforin, granzymes, Fas-FasL,
and other members of the TNF T cells from ProGP-1-treated donors clearly have a reduced capacity to
induce systemic TNF G-CSF remains the mainstay of donor pretreatment in current allogeneic stem cell protocols. ProGP-1 is an extremely active molecule for the purpose of stem cell mobilization and appears to be 8 to 10 times more potent than G-CSF and 2 to 3 times more potent than the combination of the native G-CSF and Flt-3L molecules.17 ProGP-1 has a higher affinity for the G-CSF receptor than the native G-CSF molecule and the differences observed in GVHD in this study may relate to downstream effects as a consequence of increased potency through the G-CSF receptor-ligand pair alone. In support of this, we have previously demonstrated improved protection from GVHD following a 10-fold increase in G-CSF dose in this system.2,20 Alternatively, the additional agonist activity through the Flt-3L receptor may confer increased protection, presumably via the expansion of donor APCs. Clearly, pretreatment of the donor with Flt-3L alone does not result in a reduction in GVHD16 as may have been expected if donor DCs were important in controlling GVHD. Furthermore, treatment of recipients with FLT-3L augments both GVHD and GVL.44 Conversely, the recent report of reduced GVHD following recipient treatment with FLT-3L prior to SCT is intriguing and may relate to a change in host DC phenotype, activation status, or both,30 and is consistent with the established paradigm that host APCs are important in the induction of GVHD.45 Thus, unlike host APCs, the relative role of donor APCs (and DCs in particular) in the induction of GVHD remains unclear at this time. Human studies have demonstrated that G-CSF expands "lymphoplasmacytoid" DCs, which can induce TH2 differentiation in vitro10 and this has been suggested as a mechanism by which G-CSF can prevent acute GVHD. We confirm that the counterpart murine CD11cdim/B220hi DC is also expanded after G-CSF and ProGP-1 treatment. However, we could not find any evidence that this cell provides protection from GVHD in isolation, nor was the T-cell phenotype after SCT consistent with this theory. This does not exclude the possibility that these cells may confer protection from acute GVHD if transferred in higher numbers or in conjunction with other cell populations modulated by G-CSF or ProGP-1. Recent studies suggest that increased DC2 doses in the BMT setting are associated with reduced chronic GVHD46 and so a causal relationship between high DC2 doses, TH2 differentiation, and the increased incidence of chronic GVHD in the clinical SCT setting also seems unlikely. G-CSF is known to expand monocytes capable of inhibiting T-cell responses, in part through IL-10 production.6,7 Although IL-10 is capable of ameliorating GVHD,47,48 the ability of a monocyte population or DC precursor to prevent GVHD has not been tested in experimental models and now deserves further study. Allogeneic stem cell transplantation with G-CSF-mobilized stem cells represents a major advance in the management of hematologic malignancies due to the increased separation of GVHD and GVL compared with that following traditional BMT. Despite this, GVHD remains a major limitation of allogeneic SCT and further advances are required to improve the safety of this procedure. The data presented here suggest that in addition to enhancing stem cell mobilization, the administration of progenipoietin rather than G-CSF to stem cell transplant donors is likely to further reduce GVHD and deserves study in carefully conducted clinical trials.
G.R.H. is a Wellcome Trust Senior Overseas Research Fellow.
Submitted May 24, 2002; accepted September 27, 2002.
Prepublished online as Blood First Edition Paper, October 17, 2002; DOI 10.1182/blood-2002-05-1529.
Supported in part by grants from the National Health and Medical Research Council (NHMRC) and Queensland Cancer Fund (QCF).
J.K.W. is employed by Pharmacia, whose (potential) product was studied in the present work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Geoffrey Hill, Immunoregulation Laboratory, The Queensland Institute of Medical Research, 300 Herston Rd, Herston, QLD 4006, Australia; e-mail: geoffh{at}qimr.edu.au.
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© 2003 by The American Society of Hematology.
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  K. A. Markey, T. Banovic, R. D. Kuns, S. D. Olver, A. L. J. Don, N. C. Raffelt, Y. A. Wilson, L. J. Raggatt, A. R. Pettit, J. S. Bromberg, et al. Conventional dendritic cells are the critical donor APC presenting alloantigen after experimental bone marrow transplantation Blood, May 28, 2009; 113(22): 5644 - 5649. [Abstract] [Full Text] [PDF]
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A. C. Burman, T. Banovic, R. D. Kuns, A. D. Clouston, A. C. Stanley, E. S. Morris, V. Rowe, H. Bofinger, R. Skoczylas, N. Raffelt, et al. IFN{gamma} differentially controls the development of idiopathic pneumonia syndrome and GVHD of the gastrointestinal tract Blood, August 1, 2007; 110(3): 1064 - 1072. [Abstract] [Full Text] [PDF]
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K. P. A. MacDonald, R. D. Kuns, V. Rowe, E. S. Morris, T. Banovic, H. Bofinger, B. O'Sullivan, K. A. Markey, A. L. Don, R. Thomas, et al. Effector and regulatory T-cell function is differentially regulated by RelB within antigen-presenting cells during GVHD Blood, June 1, 2007; 109(11): 5049 - 5057. [Abstract] [Full Text] [PDF]
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V. Rowe, T. Banovic, K. P. MacDonald, R. Kuns, A. L. Don, E. S. Morris, A. C. Burman, H. M. Bofinger, A. D. Clouston, and G. R. Hill Host B cells produce IL-10 following TBI and attenuate acute GVHD after allogeneic bone marrow transplantation Blood, October 1, 2006; 108(7): 2485 - 2492. [Abstract] [Full Text] [PDF]
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E. S. Morris, K. P. A. MacDonald, and G. R. Hill Stem cell mobilization with G-CSF analogs: a rational approach to separate GVHD and GVL? Blood, May 1, 2006; 107(9): 3430 - 3435. [Abstract] [Full Text] [PDF]
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S. Rutella, F. Zavala, S. Danese, H. Kared, and G. Leone Granulocyte Colony-Stimulating Factor: A Novel Mediator of T Cell Tolerance J. Immunol., December 1, 2005; 175(11): 7085 - 7091. [Abstract] [Full Text] [PDF]
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T. Banovic, K. P. A. MacDonald, E. S. Morris, V. Rowe, R. Kuns, A. Don, J. Kelly, S. Ledbetter, A. D. Clouston, and G. R. Hill TGF-{beta} in allogeneic stem cell transplantation: friend or foe? Blood, September 15, 2005; 106(6): 2206 - 2214. [Abstract] [Full Text] [PDF]
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K. P. A. MacDonald, V. Rowe, H. M. Bofinger, R. Thomas, T. Sasmono, D. A. Hume, and G. R. Hill The Colony-Stimulating Factor 1 Receptor Is Expressed on Dendritic Cells during Differentiation and Regulates Their Expansion J. Immunol., August 1, 2005; 175(3): 1399 - 1405. [Abstract] [Full Text] [PDF]
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K. P. A. MacDonald, V. Rowe, A. D. Clouston, J. K. Welply, R. D. Kuns, J. L. M. Ferrara, R. Thomas, and G. R. Hill Cytokine Expanded Myeloid Precursors Function as Regulatory Antigen-Presenting Cells and Promote Tolerance through IL-10-Producing Regulatory T Cells J. Immunol., February 15, 2005; 174(4): 1841 - 1850. [Abstract] [Full Text] [PDF]
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 B. J. Johnson, T. T. T. Le, C. A. Dobbin, T. Banovic, C. B. Howard, F. d. M. L. Flores, D. Vanags, D. J. Naylor, G. R. Hill, and A. Suhrbier Heat Shock Protein 10 Inhibits Lipopolysaccharide-induced Inflammatory Mediator Production J. Biol. Chem., February 11, 2005; 280(6): 4037 - 4047. [Abstract] [Full Text] [PDF]
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 K. E. Lawlor, I. K. Campbell, D. Metcalf, K. O'Donnell, A. van Nieuwenhuijze, A. W. Roberts, and I. P. Wicks Critical role for granulocyte colony-stimulating factor in inflammatory arthritis PNAS, August 3, 2004; 101(31): 11398 - 11403. [Abstract] [Full Text] [PDF]
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E. S. Morris, K. P. A. MacDonald, V. Rowe, D. H. Johnson, T. Banovic, A. D. Clouston, and G. R. Hill Donor treatment with pegylated G-CSF augments the generation of IL-10-producing regulatory T cells and promotes transplantation tolerance Blood, May 1, 2004; 103(9): 3573 - 3581. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), G-CSF (10 µg/animal per day for 10 days, 
), or ProGP-1 (20 µg/animal per
day for 10 days, 
). Spleens were harvested on day 11, chopped,
digested, and phenotyped. DCs were either
CD11cdim/B220hi or CD11chi. (A)
Proportion of lineage cells per spleen. (B) Absolute numbers of lineage
cells per spleen. *P < .05 compared with
controls.
,
control allogeneic, n = 15), G-CSF- (
, G-CSF allogeneic,
n = 20), and ProGP-1-treated (
, ProGP-1 allogeneic, n = 15)
donors were harvested on day 11 and transplanted into lethally
irradiated (1100 cGy) B6D2F1 recipient mice. Additional ProGP-1 T cells
were added to a ProGP-1 (
, TCD allogeneic, n = 8) spleens
were transplanted as non-GVHD controls. Survival,
P < .0001 for control allogeneic versus all others,
P = .05 for G-CSF allogeneic versus ProGP-1
allogeneic.
, n = 10) or
CD11cdim/B220hi DCs (
, n = 5)
were added to control allogeneic splenocytes in numbers equivalent to
those in unseparated ProGP-1 spleen (106
CD11chi and 2.5 × 105
CD11cdim/B220hi). Syngeneic spleen (
, n = 4). (B) At 10 days after
transplantation, TNF