International Journal of
Molecular Sciences
Article
Immunomodulation Induced by Stem Cell
Mobilization and Harvesting in Healthy Donors:
Increased Systemic Osteopontin Levels after
Treatment with Granulocyte
Colony-Stimulating Factor
Guro Kristin Melve 1,2 , Elisabeth Ersvaer 3 , Çiğdem Akalın Akkök 4 , Aymen Bushra Ahmed 5 ,
Einar K. Kristoffersen 1,2 , Tor Hervig 1,2 and Øystein Bruserud 2,5, *
1
2
3
4
5
*
Department of Immunology and Transfusion Medicine, Haukeland University Hospital, N-5021 Bergen,
Norway; guro.kristin.melve@helse-bergen.no (G.K.M.); einar.kristoffersen@uib.no (E.K.K.);
tor.audun.hervig@helse-bergen.no (T.H.)
Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway
Department of Biomedical Laboratory Sciences and Chemical Engineering, Faculty of Engineering and
Business Administration, Bergen University College, N-5020 Bergen, Norway; elisabeth.ersver@hib.no
Department of Immunology and Transfusion Medicine, Oslo University Hospital, Ullevål, N-0424 Oslo,
Norway; uxciak@ous-hf.no
Division for Hematology, Department of Medicine, Haukeland University Hospital, N-5021 Bergen,
Norway; aymen.bushra.ahmed@helse-bergen.no
Correspondence: oystein.bruserud@haukeland.no; Tel.: +47-55-97-50-00
Academic Editor: Maurizio Muraca
Received: 28 April 2016; Accepted: 11 July 2016; Published: 19 July 2016
Abstract: Peripheral blood stem cells from healthy donors mobilized by granulocyte
colony-stimulating factor (G-CSF) and harvested by leukapheresis are commonly used for allogeneic
stem cell transplantation. The frequency of severe graft versus host disease is similar for patients
receiving peripheral blood and bone marrow allografts, even though the blood grafts contain more
T cells, indicating mobilization-related immunoregulatory effects. The regulatory phosphoprotein
osteopontin was quantified in plasma samples from healthy donors before G-CSF treatment, after
four days of treatment immediately before and after leukapheresis, and 18–24 h after apheresis.
Myeloma patients received chemotherapy, combined with G-CSF, for stem cell mobilization and
plasma samples were prepared immediately before, immediately after, and 18–24 h after leukapheresis.
G-CSF treatment of healthy stem cell donors increased plasma osteopontin levels, and a further
increase was seen immediately after leukapheresis. The pre-apheresis levels were also increased in
myeloma patients compared to healthy individuals. Finally, in vivo G-CSF exposure did not alter
T cell expression of osteopontin ligand CD44, and in vitro osteopontin exposure induced only small
increases in anti-CD3- and anti-CD28-stimulated T cell proliferation. G-CSF treatment, followed
by leukapheresis, can increase systemic osteopontin levels, and this effect may contribute to the
immunomodulatory effects of G-CSF treatment.
Keywords: allogeneic transplantation; hematopoietic stem cell mobilization; granulocyte
colony-stimulating factor; osteopontin; apheresis
1. Introduction
Osteopontin is a glycosylated phosphoprotein synthesized and secreted by various cells [1].
The ability to interact with several cell surface receptors, including certain integrins and CD44, makes
Int. J. Mol. Sci. 2016, 17, 1158; doi:10.3390/ijms17071158
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Int. J. Mol. Sci. 2016, 17, 1158
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osteopontin a functional regulator of cell adhesion, migration, and survival for a wide range of
cells [1]. Binding of osteopontin to the intracellular part of CD44 is important for cytoskeletal
functions [2,3], transcriptional regulation, and anti-apoptotic signaling in normal and malignant
cells [1,4–6]. Finally, osteopontin is important for normal hematopoiesis and is a component of the
hematopoietic stem cell niche, where it regulates the location and cycling of normal stem cells [7,8].
Osteopontin is widely expressed by immunocompetent cells and upregulated both during
inflammation and in various tumors [1,9–15]. It has pro-inflammatory effects by stimulating
chemotaxis of various immunocompetent cells and by increasing pro-inflammatory cytokine release
from macrophages [9] and expression of antigen-presenting and costimulatory molecules by dendritic
cells [16]. It is also important for B cell proliferation and immunoglobulin production and is released by
activated B cells and T cells as a Th1-associated cytokine [17–19]. However, osteopontin may also have
anti-inflammatory effects [1], as observed both in animal models [19,20] and human disease [20,21].
Osteopontin is also important for growth regulation of acute lymphoblastic, and probably also
acute myeloid leukemia, cells located at the endosteal stem cell niche [22,23]. Studies in humans
have demonstrated that plasma osteopontin levels can reflect local inflammation [24] as well as tumor
hypoxia and, thereby, chemo-sensitivity [25].
Systemic administration of granulocyte colony-stimulating factor (G-CSF) is commonly applied
to mobilize hematopoietic stem cells for collection by leukapheresis [26–28]. Several apheresis systems
have been developed for efficient harvesting of mononuclear cells [29–31]. Peripheral blood stem
cell grafts are widely used for allogeneic and autologous hematopoietic stem cell transplantation
(allo- and auto-HSCT) in hematological diseases, solid tumors and immune disorders [26,32–36], and
increasingly in autoimmune and non-malignant gastrointestinal diseases [37–39]. Additionally, G-CSF
mobilized progenitor cells are applicable in regenerative medicine and immunotherapy, and have, e.g.,
been tried in coronary and limb ischemia, as a possible source for differentiation of dendritic cells and
for isolation of mesenchymal stromal cells [40–44].
One important complication associated with allo-HSCT is acute graft versus host disease (acute
GVHD). The risk of acute GVHD seems to be similar for peripheral blood and bone marrow
allografts [45], suggesting that the potentially adverse effect of the larger number of donor T cells in
peripheral blood allografts is counteracted by immunomodulation of graft T cells during mobilization
or harvesting.
Animal models suggest that osteopontin stimulates CD8+ T cell-mediated GVHD [46]. This effect
may be caused either by pre-transplant modulation of immunocompetent cells in the allogeneic
stem cell grafts, or by post-transplant modulation caused by osteopontin in the graft supernatant or
osteopontin released in the recipient. Osteopontin has several immunomodulatory effects, and in
this context we investigated the levels of osteopontin in autologous and allogeneic stem cell donors
and stem cell grafts during mobilization/harvesting and in allogeneic stem cell recipients following
graft infusion.
2. Results
2.1. Plasma Osteopontin Levels of Healthy Stem Cell Donors Increase during Granulocyte Colony-Stimulating
Factor (G-CSF) Treatment and Reach a Maximal Level Immediately Following Stem Cell Harvesting
by Leukapheresis
The median plasma osteopontin levels in healthy allogeneic stem cell donors prior to G-CSF
therapy was 45 ng/mL (variation range: 27–62 ng/mL), see Table 1 and Figure 1. During G-CSF
treatment, and immediately prior to leukapheresis, the osteopontin concentration in the stem cell
donors was increased to a median level of 50 ng/mL (range: 19–75 ng/mL, p = 0.008). The healthy
allogeneic stem cell donors were compared to a group of 15 healthy platelet donors who did not receive
any kind of treatment prior to the apheresis. These healthy platelet donors showed no significant
differences compared to the healthy stem cell donors with respect to age, gender distribution, or
baseline white blood cell counts (Table 2). The pre-apheresis osteopontin concentrations of the platelet
Int. J. Mol. Sci. 2016, 17, 1158
Int. J. Mol. Sci. 2016, 17, 1158
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3 of 17
donors
(medianof44the
ng/mL;
28–60
ng/mL)
did notrange:
differ28–60
from ng/mL)
the pre-treatment
levels
of the
concentrations
plateletrange:
donors
(median
44 ng/mL;
did not differ
from
allogeneic
stem
cell
donors
either
(Table
1).
pre-treatment levels of the allogeneic stem cell donors either (Table 1).
Allogeneic stem cell donors
Figure 1.1.Plasma
Plasma
osteopontin
levels
in healthy
allogeneic
cell
donors
stem cell
Figure
osteopontin
levels
in healthy
allogeneic
stem cellstem
donors
during
stemduring
cell mobilization
mobilization
and
harvesting.
Peripheral
blood
plasma
osteopontin
concentrations
were
determined
and harvesting. Peripheral blood plasma osteopontin concentrations were determined prior to
prior to stimulation
with granulocyte
colony-stimulating
(G-CSF)
(A),mobilization
after stem and
cell
stimulation
with granulocyte
colony-stimulating
factor (G-CSF)factor
(A), after
stem cell
mobilization prior
and toimmediately
to apheresis
(B), immediately
after apheresis
(C) start
and
immediately
apheresis (B),prior
immediately
after apheresis
(C) and approximately
24 h after
approximately
of
apheresis (D).24 h after start of apheresis (D).
Table 1.
1. The
The effect
effect of
of granulocyte
granulocyte colony-stimulating
colony-stimulating factor
factor (G-CSF)
(G-CSF) treatment,
treatment, apheresis
apheresis procedures
procedures
Table
and
allogeneic
stem
cell
transplantation
on
plasma
osteopontin
(OPN;
Upper
part)
and
G-CSF
and allogeneic stem cell transplantation on plasma osteopontin (OPN; Upper part) and G-CSF
(Lower part)
part) concentration.
concentration. (Upper
(Upper part)
part) From
From the
the top,
top, the
the plasma
plasma OPN
OPN levels
levels are
are presented
presented for
for the
the
(Lower
four study
study groups:
groups: (i)
(i) prior
treatment of
of allogeneic
allogeneic stem
stem cell
cell donors;
donors; (ii)
(ii) immediately
four
prior to
to and
and after
after G-CSF
G-CSF treatment
immediately
before and
and after
after apheresis
apheresis and
in the
the apheresis
apheresis product
product for
for each
each study
study group
group undergoing
undergoing apheresis;
before
and in
apheresis;
and
(iii)
in
allotransplanted
patients
8–12
h
prior
to
start
of
stem
cell
infusion
andh after
12–16infusion;
h after
and (iii) in allotransplanted patients 8–12 h prior to start of stem cell infusion and 12–16
infusion;
(Lower
part)
Plasma
G-CSF concentrations
given for allogeneic
stem cell
donors
prior
to
(Lower
part)
Plasma
G-CSF
concentrations
are given are
for allogeneic
stem cell donors
prior
to and
after
and after
G-CSF and
treatment
and for stem
autologous
stemonly
cell after
donors
after
the G-CSF
therapy. All
G-CSF
treatment
for autologous
cell donors
theonly
G-CSF
therapy.
All concentrations
concentrations
are given
mediansranges
with variation
ranges in parentheses.
are
given as medians
withasvariation
in parentheses.
Patients/Donors
Patients/Donors
Allogeneic
stem
Allogeneic
stemcell
celldonors
donors
Autologous
stemcell
celldonors
donors
Autologous
stem
Healthy platelet donors
Healthy
platelet donors
Allogeneic HSC recipients
Allogeneic HSC recipients
Patients/Donors
Procedure
Procedure
G-CSF stimulation
G-CSF stimulation
Stemcell
cellapheresis
apheresis
Stem
Stem
Stemcell
cellapheresis
apheresis
Platelet apheresis
Platelet apheresis
Allogeneic stem cell
Allogeneic stem
transplantation
cell transplantation
Procedure
Pre-Procedure
Pre-Procedure
OPN (ng/mL)
OPN (ng/mL)
45 (27–62)
Post-Procedure
Post-Procedure
OPN (ng/mL)
OPN (ng/mL)
50 (19–75)
45 (27–62)
(19–75)
5050
(19–75)
8989
(41–356)
(41–356)
44 (28–60)
50 (19–75)
(31–87)
5656
(31–87)
109
(55–473)
109
(55–473)
46 (33–56)
126 (80–438)
103 (72–260)
44 (28–60)
126 (80–438)
Pre-Procedure
G-CSF
(pg/mL)
Pre-Procedure
46 (33–56)
103 (72–260)
Post-Procedure
G-CSF
(pg/mL)
Post-Procedure
p Value
p Value
0.008
0.008
0.006
0.006
0.008
0.008
NS
NS
NS
Apheresis Product
Apheresis Product
OPN (ng/mL)
OPN (ng/mL)
-
53 (29–73)
53 (29–73)
86 (7–328)
86 (7–328)
48 (25–75) 1
1
48 (25–75)
Not applicable
NS
p Value
Not applicable
Apheresis Product
G-CSF
(pg/mL)Product
Apheresis
Patients/Donors
Procedure
p Value
G-CSF
(pg/mL) 10,780
G-CSF
(pg/mL)
G-CSF (pg/mL)
Allogeneic stem cell donors
G-CSF stimulation
50 (22–241)
(3687–31,947)
0.0003
6673 (1704–21,152)
Autologous
stem
celldonors
donors
G-CSF
18,366
(9861–46,314)
12,906
(8863–41,139)
Allogeneic
stem
cell
G-CSFstimulation
stimulation Not determined
50 (22–241)
10,780
(3687–31,947) Not determined
0.0003
6673
(1704–21,152)
1 The
Autologous
stem
cell donors values
G-CSF
stimulationin platelet
Not determined
(9861–46,314)
determined
12,906of(8863–41,139)
osteopontin
measured
concentrate18,366
supernatants
were Not
adjusted
for dilution
the
products
with platelet
additive
solution
plasma,
63% T-sol).supernatants
NS, not significant.
The
osteopontin
values
measured
in(37%
platelet
concentrate
were adjusted for dilution of
the products with platelet additive solution (37% plasma, 63% T-sol). NS, not significant.
1
Int. J. Mol. Sci. 2016, 17, 1158
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Table 2. Clinical and biological characteristics of healthy stem cell donors, autotransplanted myeloma
patients, healthy platelet donors, and allotransplant recipients. Number of individuals, age, and
gender (M: male, F: female) are presented for each study group. Median basal white blood cell
counts (WBC ˆ 109 /L) are given for the study groups undergoing apheresis. White blood cell counts
and peripheral blood (PB) concentrations of CD34+ stem cells before start of apheresis and yield of
CD34+ stem cells are given for G-CSF stimulated allogeneic and autologous donors (multiple myeloma
patients). All values are presented as medians with the variation ranges given in parentheses.
Total White Blood Cell Count in
the Grafts
Group
Age
Gender (M/F)
Allogeneic stem cell
donors (n = 22)
51 (25–77)
Autologous stem
cell donors (n = 15)
CD34+ Cells after
G-CSF Treatment
Baseline Level
(ˆ109 /L)
After G-CSF
(ˆ109 /L)
PB Level
(ˆ103 /mL)
Yield
(ˆ106 /kg)
14/8
5.9 (3.1–13.4)
46.0 (30.1–76.3)
44.1 (16.7–147.8)
5.4 (0.8–22.4)
57 (44–67)
9/6
5.4 (2.5–9.0)
10.8 (2.7–43.7)
39.9 (9.7–175.0)
5.3 (1.1–27.9)
Platelet donors
(n = 15)
47 (26–62)
8/7
6.0 (4.7–13.5)
-
-
-
Allogeneic HSCT
recipients (n = 16)
47 (35–63)
7/9
-
-
-
-
HSCT, hematopoietic stem cell transplantation.
The G-CSF-treated allogeneic stem cell donors showed a further increase of the median
osteopontin concentration to 56 ng/mL (range: 31–87 ng/mL, p = 0.008, Table 1) immediately after
leukapheresis, but 18–24 h after start of apheresis the median level had declined to 54 ng/mL (range:
29–76 ng/mL, p = 0.014, Figure 1). In contrast, the control group of healthy platelet donors showed
stable osteopontin levels throughout the observation period without significant altered concentrations
immediately after apheresis or 18–24 h after start of apheresis (Table 1).
Plasma G-CSF concentrations in allogeneic stem cell donors prior to and after mobilization were
also investigated. The median pre-treatment G-CSF level was 50 pg/mL (range: 22–241 pg/mL) and
after four days of G-CSF it was 10,780 pg/mL (range: 3687–31,947 pg/mL); see lower part of Table 1.
G-CSF and osteopontin levels then showed no significant correlation.
There were no significant associations between osteopontin plasma levels and apheresis time
(median: 305 min; range: 231–377 min) the absolute number of total blood volumes processed during
apheresis (median: 3.6; range: 1.6–6.6), or apheresis device applied.
2.2. Plasma Osteopontin Levels Show an Inverse Correlation with Peripheral Blood Neutrophil Levels during
G-CSF Therapy but No Association with Peripheral Blood Levels or Yields of CD34+ Cells
We used simple linear regression analyses with one way analysis of variance (ANOVA) to
study the correlation between healthy stem cell donor osteopontin levels (all donors included in the
analysis) and the corresponding peripheral blood levels of total leukocytes (Table 2) and leukocyte
subsets. Plasma osteopontin levels immediately prior to leukapheresis showed significant inverse
correlations with the corresponding peripheral blood neutrophil counts (median: 38.5 ˆ 109 /L;
range: 24.3–66.4 ˆ 109 /L; R2 = 0.381; p = 0.002) and total peripheral blood leukocyte counts (median:
46.0 ˆ 109 /L; range: 30.1´76.3 ˆ 109 /L; R2 = 0.366; p = 0.003). With this exception, there were no
significant associations between osteopontin levels and the total leukocyte counts or the levels of
neutrophils, monocytes, total lymphocytes, CD3+ lymphocytes, or CD34+ cells in peripheral blood or
in the stem cell graft at any other time point.
2.3. Myeloma Patients (Autologous Stem Cell Donors) Show Increased Plasma Osteopontin Levels after G-CSF
Therapy Compared with Healthy Allogeneic Stem Cell Donors
Plasma samples from myeloma patients receiving G-CSF therapy for mobilization of autologous
stem cells were available only immediately before leukapheresis (after five days of G-CSF treatment);
the plasma osteopontin levels then showed a wide variation and were significantly increased for
Int. J. Mol. Sci. 2016, 17, 1158
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Int. J. Mol. Sci. 2016, 17, 1158
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the myeloma patients (median 89 ng/mL; range 41–356 ng/mL) compared with the pre-apheresis
levels of the
healthy
stem cell
donors
(Mann-Whitney
U test,
p =were
0.001).
presented higher
in Table
presented
in Table
1 (lower
part),
the pre-harvesting
G-CSF
levels
alsoAs
significantly
for1
(lower part),
the pre-harvesting
levels
were
also significantly
higher
patients
myeloma
patients
(median 18,366G-CSF
pg/mL;
range
9861–46,314
pg/mL) than
forfor
themyeloma
healthy stem
cell
(median
18,366
pg/mL;
range
9861–46,314
pg/mL)
than
for
the
healthy
stem
cell
donors
(median:
donors (median: 10,780 pg/mL; range: 3687–31,947 pg/mL; p = 0.005). There was no significant
10,780 pg/mL;
range:
3687–31,947 pg/mL;
p = 0.005).
Thereplasma
was no levels
significant
between
correlation
between
pre-harvesting
G-CSF and
osteopontin
in thecorrelation
myeloma patients.
pre-harvesting
osteopontin
plasma
levelspatients
in the myeloma
As shown
in Table
1 and
As
shown in G-CSF
Table and
1 and
Figure 2,
myeloma
had a patients.
significant
increase
in plasma
Figure
2,
myeloma
patients
had
a
significant
increase
in
plasma
osteopontin
level
during
apheresis,
osteopontin level during apheresis, but the increase in median osteopontin level 24 h after apheresis
but not
the increase
in median
osteopontin level 24 h after apheresis did not reach statistical significance.
did
reach statistical
significance.
0.008
600
400
400
200
200
0
B
NS
600
C
Apheresis
0
C
D
Post-apheresis
Figure 2. Plasma osteopontin levels in autologous stem cell donors (myeloma patients) after stem cell
Figure 2. Plasma osteopontin levels in autologous stem cell donors (myeloma patients) after stem cell
mobilization
andimmediately
immediately
to apheresis
(B), immediately
after(C)apheresis
(C) and
mobilization and
priorprior
to apheresis
(B), immediately
after apheresis
and approximately
approximately
24
h
after
start
of
apheresis
(D).
24 h after start of apheresis (D).
2.4. Osteopontin Levels Are Higher in Autografts from Myeloma Patients than in Allografts from Healthy
2.4. Osteopontin Levels Are Higher in Autografts from Myeloma Patients than in Allografts from Healthy Stem
Stem
Cell Donors
Cell Donors
We
then compared
compared osteopontin
osteopontin concentrations
We then
concentrations in
in the
the apheresis
apheresis products
products from
from autologous
autologous and
and
allogeneic
stem
cell
donors
and
healthy
platelet
donors.
Autologous
stem
cell
grafts
allogeneic stem cell donors and healthy platelet donors. Autologous stem cell grafts from
from myeloma
myeloma
patients
showed significantly
significantlyhigher
highersupernatant
supernatant
osteopontin
levels
than
allografts
= 0.002)
patients showed
osteopontin
levels
than
thethe
allografts
(p =(p
0.002)
and
and
the
platelet
concentrates
(p
=
0.005);
the
results
are
summarized
in
Table
1
and
presented
the platelet concentrates (p = 0.005); the results are summarized in Table 1 and presented in detail in
in
detail
Figure
3. The osteopontin
levels
auto- and
were
higher than
unstimulated
Figure in
3. The
osteopontin
levels in autoand in
allografts
wereallografts
higher than
unstimulated
plasma
levels in
plasma
levels
autologous
and
donors,
but didfrom
notthe
differ
significantly
fromlevels
the
autologous
and in
allogenic
donors,
but allogenic
did not differ
significantly
corresponding
plasma
corresponding
plasma Due
levels
therapy.
Due tosolution
dilutionaswith
plateletinadditive
solution
during G-CSF therapy.
toduring
dilutionG-CSF
with platelet
additive
described
the experimental
as
described
in
the
experimental
section,
the
osteopontin
levels
in
platelet
concentrates
were
lower
section, the osteopontin levels in platelet concentrates were lower than the corresponding plasma
than
corresponding
plasma
in the platelet
donors,
and
low compared
to allogeneic
and
levelsthe
in the
platelet donors,
and levels
low compared
to allogeneic
and
autologous
stem cell
grafts (median:
autologous
stem
cell
grafts
(median:
18
ng/mL;
range:
10–28
ng/mL).
The
patients
treated
with
the
18 ng/mL; range: 10–28 ng/mL). The patients treated with the platelet concentrates thus received
platelet
concentrates
thus
received
relatively
low
amounts
of
osteopontin
during
platelet
infusion.
relatively low amounts of osteopontin during platelet infusion. However, after correction for the
However,
afterthere
correction
the dilution
factor,
there osteopontin
was no significant
difference
between
dilution factor,
was no for
significant
difference
between
levels in platelet
concentrates
osteopontin
levels
in
platelet
concentrates
and
stem
cell
grafts
from
healthy
donors
or
and stem cell grafts from healthy donors or between platelet concentrates and peripheral bloodbetween
samples
platelet
and(Table
peripheral
blood
from theconcentrates
platelet donors
1, Figure
3).samples from the platelet donors (Table 1, Figure 3).
Int. J. Mol. Sci. 2016, 17, 1158
Int. J. Mol. Sci. 2016, 17, 1158
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Figure 3.
levels
in apheresis
products,
i.e., peripheral
bloodblood
stem cell
grafts
platelet
Figure
3. Osteopontin
Osteopontin
levels
in apheresis
products,
i.e., peripheral
stem
celland
grafts
and
concentrates.
The
osteopontin
levels
were
determined
in
allogeneic
stem
cell
products
from
platelet concentrates. The osteopontin levels were determined in allogeneic stem cell products from
G-CSF-mobilized healthy
cell donors
donors (n
(n =
= 22),
22), autologous
autologous stem
stem cell
cell products
products derived
derived from
from
G-CSF-mobilized
healthy stem
stem cell
myeloma
patients
mobilized
by
chemotherapy
plus
G-CSF
(n
=
15),
and
platelet
concentrates
from
myeloma patients mobilized by chemotherapy plus G-CSF (n = 15), and platelet concentrates from
unstimulated healthy
healthyplatelet
platelet
donors
15).osteopontin
The osteopontin
levels measured
in platelet
unstimulated
donors
(n = (n
15).=The
levels measured
in platelet concentrate
concentrate
supernatants
were
adjusted
for
dilution
of
the
products
with
platelet
additive
supernatants were adjusted for dilution of the products with platelet additive solution (37% solution
plasma,
(37%solution).
plasma, 63% solution).
63%
2.5. Pretransplant
Osteopontin Levels
Levels of
of Allotransplant
Allotransplant Recipients
Recipients Are
Are Increased
Increased and
and the
the High
High Levels
Levels Are
Are Not
Not
2.5.
Pretransplant Osteopontin
Altered Following
Following the
the Infusion
Infusion of
of Osteopontin-Containing
Osteopontin-Containing Stem
Stem Cell
Cell Grafts
Grafts
Altered
The pre-transplant
pre-transplant osteopontin
osteopontin levels
levels in
in allotransplant
allotransplant recipients
recipients were
were high
high (median:
(median: 126
126 ng/mL;
ng/mL;
The
range:
80–438
ng/mL)
and
were
significantly
higher
than
the
levels
in
healthy
individuals
range: 80–438 ng/mL) and were significantly higher than the levels in healthy individuals (p < 0.001;
(p < Table
0.001; see
1), and
even than
higher
the myeloma
patients
= 0.02).
Theinfusion
infusionof
of the
the
see
1), Table
and even
higher
forthan
thefor
myeloma
patients
(p = (p
0.02).
The
osteopontin-containing allograft
alter the
the plasma
plasma levels
levels significantly;
significantly; the
the levels
levels remained
remained
osteopontin-containing
allograft did
did not
not alter
high
in
the
allotransplant
recipients
both
when
tested
one
day
post-transplant
and
for
eight
patients
high in the allotransplant recipients both when tested one day post-transplant and for eight patients
also
tested
later
after
the
transplantation
(median:
six
days
after
infusion;
range:
4–13
days).
also tested later after the transplantation (median: six days after infusion; range: 4–13 days).
Additional analyses
analysesshowed
showed
association
between
recipient
osteopontin
plasma
levels
Additional
no no
association
between
recipient
osteopontin
plasma levels
(Table
1)
+ stem
+
(Table
1)
and
(i)
patient
age
and
gender;
(Table
2)
(ii)
allograft
content
of
leukocytes,
CD34
and (i) patient age and gender; (Table 2) (ii) allograft content of leukocytes, CD34 stem cells, CD3+
+ T cells, neutrophils, monocytes, lymphocytes or platelets measured as absolute numbers
cells,
CD3
T
cells,
neutrophils,
monocytes, lymphocytes or platelets measured as absolute numbers or as the
or
as
the
number
of
cells
per kg
patient
body
weight
number of cells per kg patient
body
weight
(Table
3). (Table 3).
As
presented
in
Table
3,
the
median
time
until
neutrophil reconstitution
reconstitution with
with peripheral
peripheral blood
blood
As presented in Table 3, the median time until neutrophil
9
neutrophil counts
countsabove
above0.5
0.5ˆ× 10
109/L
days was
was day
day +17
+17 (range:
(range: day
day +13
+13
neutrophil
/Lon
onthe
thefirst
first of
of three
three consecutive
consecutive days
9/L for the first of three
9
to
+28).
Furthermore,
the
median
time
of
platelet
counts
above
50
×
10
to +28). Furthermore, the median time of platelet counts above 50 ˆ 10 /L for the first of three
consecutive days
days was
was day
day +15
+15 (range:
(range: day
day +11
+11 to
to +39).
+39). There
association between
between
consecutive
There was
was no
no significant
significant association
osteopontin levels
levelsand
and
hematopoietic
reconstitution.
Finally,
the investigated
16 patients
osteopontin
timetime
untiluntil
hematopoietic
reconstitution.
Finally, for
the 16 for
patients
investigated
acute
GVHD
grade
II–IV
was
seen
in
two
patients,
early
death
before
day
+100
in four
acute GVHD grade II–IV was seen in two patients, early death before day +100 in four patients, chronic
patients,inchronic
GVHD and
in nine
patients,
and leukemia
relapse These
in fourobservations
patients. These
observations
GVHD
nine patients,
leukemia
relapse
in four patients.
suggest
that our
suggest
that
our
16
patients
are
representative
for
allotransplanted
patients.
16 patients are representative for allotransplanted patients.
Int. J. Mol. Sci. 2016, 17, 1158
7 of 17
Int. J. Mol. Sci. 2016, 17, 1158
7 of 17
Table
3. Allogeneic
stemstem
cell cell
grafts
derived
from
healthy
donors;
thethe
levels
of of
various
cells
in in
thethe
grafts
Table
3. Allogeneic
grafts
derived
from
healthy
donors;
levels
various
cells
and grafts
the post-transplant
clinical
course
of
the
allotransplant
recipients.
The
cell
content
of
the
stem
and the post-transplant clinical course of the allotransplant recipients. The cell content of the cell
grafts
infused
to 16infused
allotransplant
recipients is
presented
as the absolute
in the graft
(graft
stem cell grafts
to 16 allotransplant
recipients
is presented
as the numbers
absolute numbers
in the
content)
and
as
the
infused
cell
doses
per
kg
(infused
cells).
graft (graft content) and as the infused cell doses per kg (infused cells).
Cell Type
Total WBC
Total+ WBC
CD34
stem cells
CD34+ stem
cells
CD3+ T cells
CD3+ T cells
Neutrophils
Neutrophils
Monocytes
Monocytes
Lymphocytes
Lymphocytes
Platelets
Platelets
Cell Type
Graft Content (×108) 8
Graft Content (ˆ10 )
791 (342–2495)
791(2.4–6.7)
(342–2495)
4.6
(2.4–6.7)
2784.6
(71–490)
278 (71–490)
285285
(112–1048)
(112–1048)
127127
(18–563)
(18–563)
(105–759)
346346
(105–759)
7068
(3176–11,449)
7068
(3176–11,449)
Infused Cells (×106/kg)
Infused Cells (ˆ106 /kg)
109 (376–3054)
109(3.3–6.8)
(376–3054)
5.5
(3.3–6.8)
395.5
(10–61)
39 (10–61)
45 45
(15–133)
(15–133)
16 16
(3–69)
(3–69)
(14–96)
50 50
(14–96)
9607
(3655–14,260)
9607
(3655–14,260)
Post-Transplant Course 1 1
Post-Transplant Course
Neutrophil reconstitution
17 (13–28)
Neutrophil
reconstitution
17 (13–28)
Platelet reconstitution
15 (11–39)
Platelet reconstitution
15 (11–39)
aGVHD
2/16
aGVHD
2/16
cGVHD
9/16 9/16
cGVHD
Early
death
4/16 4/16
Early
death
Relapse
Relapse
4/16 4/16
- -
1
1 Neutrophil
Neutrophil
andand
platelet
reconstitution
is given
the first
of three
days after the
transplantation
platelet
reconstitution
is as
given
as the
firstconsecutive
of three consecutive
days
after the
with neutrophil counts above 0.5 ˆ 109 /L and platelet transfusion
independence with platelet counts above
9/L and platelet
transfusion
independence
with
transplantation
with
neutrophil
counts
above
0.5
×
10
50 ˆ 109 /L. aGVHD: acute graft versus host disease grade II–IV, cGVHD: chronic graft versus host disease,
9/L. aGVHD: acute graft versus host disease grade II–IV, cGVHD:
platelet
counts
above
50
×
10
early death: defined as death before day +100 after transplantation, WBC: white blood cell count. All values
arechronic
presented
as versus
medians
with
the variation
rangesdefined
given inasparentheses
or day
as fractions
of the
total number of
graft
host
disease,
early death:
death before
+100 after
transplantation,
16 WBC:
patients.
white blood cell count. All values are presented as medians with the variation ranges given in
parentheses or as fractions of the total number of 16 patients.
2.6. T and B Lymphocytes Show High Expression of the CD44 Osteopontin Receptor and these High Levels Are
Maintained
during
Stem CellShow
Mobilization
and Harvesting
2.6. T and
B Lymphocytes
High Expression
of the CD44 Osteopontin Receptor and these High Levels Are
Maintained during Stem Cell Mobilization and Harvesting
Interaction between osteopontin and the CD44 receptor mediates chemotaxis of lymphocytes and
Interaction
between
osteopontin
and the CD44
receptor
chemotaxis
of lymphocytes
macrophages
[47]. We
investigated
the expression
of CD44
by mediates
viable donor
lymphocytes
during stem
macrophages
We investigated
the expression
of CD44highly
by viable
donor lymphocytes
during
cell and
mobilization
and[47].
harvesting;
the receptor
was generally
expressed
and all comparisons
+
stem
cell
mobilization
and
harvesting;
the
receptor
was
generally
highly
expressed
and
allMFI
are therefore based on the mean fluorescence intensity (MFI), see Figure 4. In CD19 B cells
+ B
comparisons
are
therefore
based
on
the
mean
fluorescence
intensity
(MFI),
see
Figure
4.
In
CD19
was reduced from 31,869 to 25,519 (mean values, n = 15) during G-CSF stimulation (p = 0.022).
cells MFI was reduced from 31,869 to 25,519 (mean values, n = 15) during G-CSF stimulation
No significant G-CSF induced change in CD44 expression was detected in CD3+ T cell populations;
(p = 0.022). No significant G-CSF induced change in CD44 expression was detected in CD3+ T cell
neither was there any significant effect of apheresis on CD44 expression in T and B cells. T cell and
populations; neither was there any significant effect of apheresis on CD44 expression in T and B
B cell
CD44-APC MFI did not show any significant correlation to plasma levels of osteopontin or
cells. T cell and B cell CD44-APC MFI did not show any significant correlation to plasma levels of
G-CSF
at any sampling
osteopontin
or G-CSFpoint.
at any sampling point.
B and T cells
100
**
**
80
60
*
40
20
+
+
+
+
8
D
8
+
+
na
ï
m ve
em
or
y
Tr
1
C
m
+
4
D
4
C
C
D
C
D
na
ïv
e
em
or
y
+
+
3
+
C
D
8
+
D
4
3
C
D
C
D
+
C
D
3
C
C
D
19
+
0
Figure 4. Expression of CD44 in unstimulated (grey-colored bars) and in vivo G-CSF stimulated
Figure 4. Expression of CD44 in unstimulated (grey-colored bars) and in vivo G-CSF stimulated
(black-colored bars) peripheral blood leukocytes from healthy allogeneic stem cell donors. The
(black-colored bars) peripheral blood leukocytes from healthy allogeneic stem cell donors. The results
results are presented as the mean fluorescence intensity (MFI) given as mean values ± standard error
are presented as the mean fluorescence intensity
(MFI) given as mean values ˘ standard error
of the mean (SEM). (Left): The results for CD19+ B cells and CD3+ T cells with CD4+ and CD8+ main
of the
mean (SEM). (Left): The results for CD19+ B cells and CD3+ T cells with CD4+ and
subsets are shown; (Middle): CD4+ and CD8+ naïve (CD45RA+) T cell subsets are compared with the
+
+ T cell subsets are
CD8corresponding
main subsets
are memory
shown; (CD45RA
(Middle):
CD4+ and
CD8+T naïve
(CD45RA
−) subsets
T cell
and with
regulatory
type 1 )(Tr1)
cells (CD4+
´
compared
with
the
corresponding
T
cell
memory
(CD45RA
)
subsets
and
with
T
regulatory
−
+
+
+
hi
hi
CD45RA CD49b LAG-3 ); (Right): Transitional B cells (CD19 CD24 CD38 ) together with maturetype
+ CD45RA
´ CD49b+ LAG-3
+ (Right):
+ CD24
hi CD38
hi )
+CD24
+CD38
+) and memory
−) B-cells
+CD24
lowCD38
hi)
1 (Tr1)
cells
(CD4
Transitional
B cells
(CD19
(CD19+CD24);hi38
and plasmablasts
(CD19
(CD19
+ ) and memory (CD19+ CD24hi 38´ ) B-cells and plasmablasts
together
with mature
(CD19+significant
CD24+ CD38
are presented.
Statistically
differences
are indicated (** p = 0.001, * p = 0.05).
+
low
hi
(CD19 CD24 CD38 ) are presented. Statistically significant differences are indicated (** p = 0.001,
* p = 0.05).
Int. J. Mol. Sci. 2016, 17, 1158
8 of 17
CD44 expression was consistently higher for CD3+ T cells than for CD19+ B cells; as expected,
8 of 17
both CD4+ and CD8+ CD45RA´ memory T cells showed significantly higher CD44 expression than
+ naïve T cells (Figure 4). Particularly high
+ B cells;
CD45RA
expression
was
found
the subset of
CD44
expression was consistently higher for CD3+ TCD44
cells than
for CD19
as in
expected,
+
+
+
+
−
both CD4
and Tr1
CD8cells
CD45RA
memoryactivation
T cells showed
significantly
CD44type
expression
CD49b
LAG-3
(lymphocyte
gene-3
positive Thigher
regulatory
1 cells)than
[48].
+ naïve T cells (Figure 4). Particularly high CD44 expression
CD45RA
was found
subset of
We also
compared CD44 expression in the main CD19+ B cell subsets
[49],ininthe
unstimulated
and
+ LAG-3+ Tr1 cells (lymphocyte activation gene-3 positive T regulatory type 1 cells) [48].
+
CD49b
G-CSF stimulated peripheral blood mononuclear cells (PBMC) samples. Compared to the CD24 CD38+
also compared CD44 expression
the main CD19+ B cell subsets [49], in unstimulated
matureWe
subset,
transitional CD24hi CD38hiincells
showed significantly lower and CD24hi 38´ memory
and G-CSF stimulated peripheral blood mononuclear
cells (PBMC) samples. Compared to the
B cells significantly
higher CD44 expression. CD19+ CD24low CD38hi plasmablasts showed high CD44
CD24+CD38+ mature subset, transitional CD24hiCD38hi cells showed significantly lower and
expression
similar to B memory cells [50].
CD24hi38− memory B cells significantly higher CD44 expression. CD19+CD24lowCD38hi plasmablasts
To
summarize,
vivo G-CSF
therapy
resultedcells
in a [50].
modest reduction in CD44 expression in B cells
showed high CD44inexpression
similar
to B memory
exclusively,
and apheresis
procedures
did not
alter in
T aand
B cellreduction
CD44 expression
significantly.
To summarize,
in vivo
G-CSF therapy
resulted
modest
in CD44 expression
in B
Int. J. Mol. Sci. 2016, 17, 1158
cells exclusively, and apheresis procedures did not alter T and B cell CD44 expression significantly.
2.7. Osteopontin Causes a Minor Increase of in Vitro Proliferative T Cell Responses
2.7. Osteopontin Causes a Minor Increase of in Vitro Proliferative T Cell Responses
The effect of exogenous osteopontin on T cell proliferative responses was investigated for eight
effect of exogenous
oncultured
T cell proliferative
was investigated
healthyThe
individuals
(Figure 5).osteopontin
PBMC were
in vitro inresponses
the presence
of anti-CD3 for
andeight
anti-CD28.
healthy
individuals
(Figure
5).
PBMC
were
cultured
in
vitro
in
the
presence
of
anti-CD3
and with
We compared the proliferative responses for cultures prepared in medium alone and cultures
anti-CD28.50We
compared
the osteopontin
proliferative level
responses
for culturestoprepared
in medium
alone and
osteopontin
ng/mL,
i.e., the
corresponding
the plasma
level in healthy
stem cell
cultures with osteopontin 50 ng/mL, i.e., the osteopontin level corresponding to the plasma level in
donors (see Table 1). Osteopontin increased T cell proliferation, but this increase usually corresponded
healthy stem cell donors (see Table 1). Osteopontin increased T cell proliferation, but this increase
to less than 20% of the corresponding control cultures both when osteopontin was tested in culture
usually corresponded to less than 20% of the corresponding control cultures both when osteopontin
medium
without
G-CSF
and medium
supplemented
withsupplemented
G-CSF.
was tested
in culture
medium
without G-CSF
and medium
with G-CSF.
Figure 5. Peripheral blood mononuclear cells (PBMC) from eight healthy unstimulated donors were
Figure 5. Peripheral blood mononuclear cells (PBMC) from eight healthy unstimulated donors
cultured in serum-free medium and stimulated with anti-CD3 and anti-CD28. The effect of
were
cultured50
in ng/mL
serum-free
medium
and stimulated
with 10
anti-CD3
and anti-CD28.
osteopontin
without
G-CSF (left)
and with G-CSF
pg/mL (right)
on in vitro The
T celleffect of
osteopontin
50
ng/mL
without
G-CSF
(left)
and
with
G-CSF
10
pg/mL
(right)
on
in
vitro T cell
3
proliferation was assayed as H-thymidine incorporation expressed as median counts per minute
3 H-thymidine incorporation expressed as median counts per minute
proliferation
was
assayed
as
(cpm). The proliferation of normal PBMC in control cultures containing isotypic control antibodies
(cpm).
The
of normal
PBMC
in control cultures
containing isotypic control antibodies
instead
of proliferation
anti-CD3/anti-CD28
antibodies
corresponded
to <1000 cpm.
instead of anti-CD3/anti-CD28 antibodies corresponded to <1000 cpm.
Int. J. Mol. Sci. 2016, 17, 1158
9 of 17
3. Discussion
Osteopontin can mediate both pro- and anti-inflammatory effects through its binding to
specific receptors expressed by various immunocompetent cells [20,21]. In the present study we
describe that systemic osteopontin levels are altered during stem cell mobilization and harvesting.
Elevated osteopontin levels are detected in the stem cell grafts, and we hypothesize that osteopontin
may thereby affect the immunocompetent cells in the grafts.
Some of the statistically significant differences in osteopontin plasma levels described in our
present study were relatively small. However, the biological day-to-day variation, time of day variation,
and week-to-week variation in osteopontin level in healthy blood donors has been shown to be low [51].
Furthermore, several previous studies have demonstrated that differences corresponding to 15%–25%
of control levels reflect differences of biological and clinical significance, e.g., in cancer patients
and cardiovascular disease patients [52–54]. These observations suggest that even relatively small
variations in plasma osteopontin levels may have a clinical/biological relevance. Our own observations
are also in agreement with these previous observations, e.g., we had similar results in base-line samples
for our two independent groups of healthy individuals.
Our present study compared plasma osteopontin levels in two independent groups of healthy
individuals (G-CSF treated stem cell donors, untreated platelet donors) undergoing apheresis with
or without G-CSF stimulation. Osteopontin concentrations increased during G-CSF treatment, and
the levels showed a further increase after leukapheresis/stem cell harvesting. This was a transient
effect and osteopontin levels decreased during the 24 h period post harvesting. On the other hand, the
control group of healthy untreated platelet donors showed stable osteopontin levels with no detectable
effect of the apheresis.
We also compared the healthy allogeneic stem cell donors with a group of myeloma patients
receiving G-CSF treatment for mobilization of autologous stem cells; the myeloma patients then
showed higher pre-harvesting osteopontin levels and a similar increase as the healthy donors following
leukapheresis. The higher pre-harvesting osteopontin concentrations in myeloma patients may be due
to the combination of G-CSF and chemotherapy for autologous stem cell mobilization in these patients
and five days of treatment with G-CSF in contrast to four days of treatment in the allogeneic donors.
Alternatively, the difference could be disease dependent; increased levels in myeloma patients are
associated with disease burden and decrease when patients respond to anti-myeloma treatment [55,56].
It should be emphasized that only a minority of our patients achieved a complete response prior to the
autologous stem cell harvesting.
Samples drawn prior to G-CSF therapy were not available from our myeloma patients. In a
recent study of myeloma patients mobilized for stem cell harvest, no significant effect of G-CSF on
osteopontin levels could be detected [57]. However, as the regulation of the osteopontin concentration
during stem cell mobilizing in these patients is complex and influenced by both disease stage and
chemotherapy [55], possible effects of G-CSF might be difficult to detect.
Thus, the effect of apheresis (and possibly the effect of G-CSF treatment) on osteopontin levels is
not only seen in healthy donors, but also in myeloma patients. However, the levels were not altered in
healthy blood donors undergoing unstimulated thrombapheresis, which suggests that this is probably
an effect induced by the G-CSF therapy and not a general effect of all kinds of apheresis procedures.
This is further supported by reports of a relatively high degree of product manipulation and activation
in the apheresis device used for platelet collection [58,59]. In contrast to our findings, an eventual
effect of apheresis procedures on osteopontin levels would, therefore, be expected to be stronger
during platelet collection compared to stem cell apheresis. However, it is not possible to exclude that
differences in apheresis techniques between stem cell harvesting and platelet collection (e.g., processed
blood volume, separation techniques, anti-coagulation) contributed to the different effects of apheresis
on osteopontin levels.
G-CSF treatment both in healthy individuals and myeloma patients caused increased levels of
circulating neutrophils that express the osteopontin receptor CD44 [60]. One would, therefore, expect
Int. J. Mol. Sci. 2016, 17, 1158
10 of 17
increased binding of osteopontin to neutrophils during G-CSF treatment, but despite this increased
binding we could still detect increased osteopontin plasma levels during the treatment.
A recent study of patients with hematological malignancies described an association between
genetic CD44 polymorphisms and the efficiency of CD34+ cell mobilization [61], suggesting that
CD44-osteopontin are important regulators of stem cell retention to the bone marrow during G-CSF
mobilization, at least in myeloma patients. However, we did not observe any association between
osteopontin levels and CD34+ cell mobilization/yield, neither in the myeloma patients, nor in the
healthy stem cell donors.
We investigated the osteopontin levels in the graft supernatants. The high pre-harvesting plasma
levels and the difference between healthy stem cell donors, myeloma patients, and platelet donors
were also reflected in the osteopontin levels in the supernatants. The stem cell transplantation thereby
also includes an infusion of osteopontin.
The osteopontin receptor CD44 is widely expressed by immunocompetent cells; the T cell
expression was not altered by in vivo G-CSF exposure whereas B cell expression was moderately
decreased. Exposure of T cells to osteopontin during in vitro activation caused a slight increase in
anti-CD3 + anti-CD28 initiated T cell proliferation. These experiments show that osteopontin can alter
T cell responses when tested at concentrations corresponding to the in vivo levels. However, additional
studies are required to clarify whether this is a direct stimulatory effect on the proliferating cells, a
reduced effect of T regulatory cells or an indirect effect mediated by the accessory cells.
The highest levels of osteopontin were found in allogeneic stem cell transplant recipients at the
time of transplantation. The levels were high even compared to myeloma patients who had received
both induction therapy and stem cell mobilization, and they were not significantly changed by stem
cell transplantation. This observation indicates that high osteopontin concentrations is one of the
characteristics of the pro-inflammatory state induced by conditioning therapy and underlying disease
in allogeneic stem cell transplant recipients. This pro-inflammatory cytokine balance is considered as
an important basis for development of GVHD [45], and osteopontin blockade is shown to reduce CD8+
T-cell mediated GVHD in mice [46]. Our findings suggest greater importance of the osteopontin
level in the patient compared to the donor and stem cell graft. The osteopontin levels during
conditioning therapy and allogeneic stem cell transplantation in humans and the possible importance
for development of GVHD should be studied in further detail in order to evaluate osteopontin as a
possible therapeutic target in graft versus host disease.
Previous studies have demonstrated that G-CSF has immunomodulatory effects and can suppress
T lymphocytes [62].Such effects are probably important in allotransplant recipients receiving peripheral
blood stem cell grafts because the frequency of GVHD is similar for bone marrow and mobilized
peripheral blood stem cell grafts even though a higher frequency would be expected for the blood
grafts due to their larger number of T cells in these grafts [62]. The molecular mechanisms behind
this are not known, but our present study suggests that effects of osteopontin on immunocompetent
cells may be a part of the G-CSF-induced immunomodulation in healthy stem cell donors. A better
understanding of the mechanisms behind the G-CSF associated immunomodulation will be important
for the future development of therapeutic strategies to target graft T cells and thereby reduce the risk
of severe GVHD without reducing the graft versus leukemia reactivity.
4. Materials and Methods
4.1. Stem Cell Donors and Allotransplant Recipients
All studies were conducted in accordance with the Declaration of Helsinki and approved by the
local ethics committee (REK III No. 126.01, Regional Committee for Medical and Health Research
Ethics of Western Norway: 2008/1580, 2011/996, 2011/1237, 2011/1241, and 2013/634) and donors
and patients were included after signing a written informed consent. The present studies included
(i) 22 consecutive healthy human leukocyte antigen matched (HLA-matched), related, allogeneic
Int. J. Mol. Sci. 2016, 17, 1158
11 of 17
stem cell donors; (ii) 15 consecutive autologous stem cell donors, all patients with newly-diagnosed
symptomatic multiple myeloma; (iii) 16 allogeneic stem cell transplant recipients; and (iv) 15 healthy
platelet donors (Table 2). The allogeneic stem cell donors did not differ from myeloma patients
and healthy platelet donors with regard to age, gender distribution, or initial peripheral blood
leukocyte count.
4.2. Stem Cell Mobilization in Healthy Donors and Myeloma Patients
The matched related donors received stem cell mobilizing with human non-glycosylated G-CSF
10 µg/kg per day for four days before stem cell harvesting. Initial induction therapy for the myeloma
patients was two cycles of either intravenous cyclophosphamide 1 g/m2 on day 1 at four weeks
intervals (14 patients) or bortezomib 1.3 mg/m2 on days 1, 4, 8, and 11 at a three-week interval
(one patient); both regimens were combined with dexamethasone 40 mg orally on days 1–4 and 9–12.
All myeloma patients either responded to the treatment or had stable disease, and stem cells were,
thereafter, mobilized with intravenous cyclophosphamide 2 g/m2 followed by G-CSF 5 µg/kg/day.
Peripheral blood leukocyte counts were significantly higher in healthy stem cell donors compared
to myeloma patients immediately before stem cell harvesting (p < 0.001, Table 2), but the peripheral
blood concentration of CD34+ cells did not differ significantly between groups.
4.3. Apheresis Procedures
Stem cell quantification was started on day 4 or 5 of G-CSF stimulation for stem cell donors
and myeloma patients, respectively. For the myeloma patients this corresponded to day 10 after the
start of cyclophosphamide. Stem cell harvest was performed when the stem cell count exceeded
15–20 ˆ 103 /mL. Large-volume leukapheresis with four times processing of the total blood volume
on a Cobe Spectra cell separator, version 7 (Cobe Laboratories, Gloucester, UK) was used for
nine of the healthy stem cell donors and all the myeloma patients; the other 13 healthy stem cell
donors were harvested with a Spectra Optia cell separator, version 9 (Terumo BCT Inc., Lakewood,
CO, USA). The automated mononuclear cells (MNC) procedure was used in accordance with the
instructions from the manufacturer. The yield of CD34+ cells per kg bodyweight obtained by
apheresis and the white blood cell count in the apheresis product did not differ significantly between
groups. Finally, single-donor platelet concentrates from unstimulated healthy volunteer donors were
prepared with a Fenwal Amicus cell separator (Baxter Healthcare Corp., Deerfield, IL, USA) and
leukocyte-reduction provided by elutriation. The platelets were suspended in 37% plasma and 63%
platelet additive solution (T-sol, Baxter Healthcare Corp.) as described in detail previously [63,64].
4.4. Allogeneic Stem Cell Transplantation
Eleven of the 16 allotransplant recipients were diagnosed with acute myeloid leukemia (AML),
three with acute B cell lymphoblastic leukemia (B-ALL), one with myelofibrosis and one with
myelodysplastic syndrome (MDS). All leukemia patients were in complete hematological remission
at the time of transplantation. The patients received (i) myeloablative conditioning with intravenous
busulfan plus cyclophosphamide and mesna (14 patients); or (ii) reduced intensity conditioning
with intravenous fludarabine plus busulfan (two patients). All patients were transplanted with
G-CSF mobilized peripheral blood stem cell grafts derived from HLA-matched family donors and
received graft versus host disease (GVHD) prophylaxis with cyclosporine A, plus methotrexate.
Neutrophil reconstitution was defined as neutrophil counts exceeding 0.2/0.5 ˆ 109 /L for at least three
consecutive days, and platelet reconstitution as at least three consecutive days with stable platelet
counts exceeding 20/50 ˆ 109 /L.
Int. J. Mol. Sci. 2016, 17, 1158
12 of 17
4.5. Preparation of Plasma and Peripheral Blood Mononuclear Cells (PBMC)
4.5.1. Blood Sampling
Venous blood samples from the allogeneic stem cell donors were collected (A) prior to G-CSF
stimulation at the time of the pre-transplant evaluation (median 20.5 days before apheresis). For the
three study groups undergoing apheresis, blood samples were also drawn (B) in the morning
immediately before apheresis, (C) immediately after apheresis, and (D) approximately 24 h after
start of apheresis. All venous blood samples from allotransplant recipients were collected between
07:00 and 09:00. Samples for plasma preparation were collected into Vacuette 9NC tubes and samples
for cell preparation into acid-citrate-dextrose solution A (ACD-A) tubes with sodium citrate and
acid-citrate-dextrose solution A as anticoagulants (Greiner Bio-One GmbH, Kremsmünster, Austria).
Samples from stem cell allo- and autografts and platelet concentrates were transferred to plastic tubes
without additives.
4.5.2. Preparation of Plasma Samples
The blood samples were centrifuged at 2000ˆ g (myeloma patients and platelet donors) or
1310ˆ g (allotransplant recipients) for ten minutes at room temperature within 30 min of sampling.
The supernatants were immediately transferred to plastic tubes, frozen, and stored at ´70 ˝ C
until analyzed.
4.5.3. Preparation of PBMC Samples
After isolation by density gradient separation (Lymphoprep, AXIS-SHIELD PoC AS, Oslo,
Norway; specific density: 1.077 g/mL), PBMC were dissolved in RPMI 1640 medium supplemented
with 2 mmol/L L-glutamine, penicillin 100 IE/mL, streptomycin 0.1 mg/mL (Sigma-Aldrich, St. Louis,
MO, USA), and 20% fetal bovine serum (FBS, Biowest, Nuaillé, France). 10% dimethyl sulfoxide
(DMSO, Sigma-Aldrich, St. Louis, MO, USA) was used as cryoprotectant, and the vials were stored in
liquid nitrogen at ´150 ˝ C after gradual cooling to ´80 ˝ C in Mr. Frosty Freezing Container (Thermo
Fisher Scientific, Waltham, MA, USA).
4.6. Analysis of Plasma Osteopontin and G-CSF Concentrations
Plasma osteopontin levels were determined by enzyme-linked immuno-sorbent assays (ELISA)
(Quantikine ELISA Human Osteopontin (OPN) Immunoassay from R&D Systems, Minneapolis,
MN, USA). Plasma G-CSF concentrations were determined by Luminex analyses (R&D Systems,
Minneapolis, MN, USA). All samples were analyzed in duplicates, strictly according to the
manufacturer’s instructions.
4.7. Flow Cytometry Analyses
PBMC were thawed in a 37 ˝ C water bath, dissolved in supplemented RPMI 1640 medium, and
incubated for one hour (37 ˝ C, a humidified atmosphere of 5% CO2 ) before incubation with near-IR
fluorescent reactive dye (LIVE/DEAD Fixable Dead Cell Stain Kits, Molecular Probes, Eugene, OR,
USA) for 30 min to determine cell viability. After washing in phosphate-buffered saline (PBS) with
1% bovine serum albumin fraction V (BSA, Roche Diagnostics GmbH, Mannheim, Germany) the cells
were incubated for 20 min with the following mouse anti-human monoclonal antibodies: CD3-PE-Cy7
(SK7), CD4-PerCP-Cy5.5 (RPA-T4), CD8-V500 (RPA-T8), CD19-PerCP-Cy5.5 (SJ25C1), CD45-RA-V450
(HI100), and CD24-PE-Cy7 (ML5) (all from Becton Dickinson Biosciences-BD Pharmingen, San Diego,
CA, USA), rat CD44-Ax 488 (IM7) and mouse CD49b-FITC (P1E6-C5) (both from BioLegend, San Diego,
CA, USA), mouse CD38-PB (HIT2; EXBIO, Prague, Czech Republic) and goat LAG-3-PE (FAB2319P;
R&D Systems, Minneapolis, MN, USA). Eight-color flow cytometry analysis was performed using a
FACS Canto II flow cytometer (Becton Dickinson Biosciences-Immunocytometry Systems; San Jose,
Int. J. Mol. Sci. 2016, 17, 1158
13 of 17
CA, USA). Acquisition of 30,000 CD3+ T cells or 10,000 CD19+ B cells per sample was endeavored,
and cytometer performance was monitored daily with Cytometer Setup and Tracking Beads (Becton
Dickinson Biosciences-BD Pharmingen, San Diego, CA, USA). The data were analyzed with FlowJo
software version X (FlowJo LLC, Ashland, OR, USA).
4.8. Analysis of T-Cell Proliferation by 3 H-Thymidine Incorporation
PBMC were cultured in 96-well microtiter plates (5 ˆ 104 cells per well, 190 µL medium per
well), the culture medium being X-vivo10® with 100 µg/mL gentamycin (BioWhittaker, Walkersville,
MA, USA). The T cells were activated by anti-CD3 (clone CLB-T3/4.E, 1XE, PeliCluster, Sanquin,
Amsterdam, The Netherlands; final concentration 316 ng/mL) and anti-CD28 (clone: CLB-CD28/1,
15E8 PeliCluster; final concentration 842 ng/mL). The corresponding control antibodies were
purchased from R&D Systems (Abingdon, UK). The medium was supplemented with recombinant
human osteopontin 50 ng/mL (R&D Systems, Minneapolis, MN, USA) and eventually recombinant
human G-CSF 10 pg/mL (PeproTech EC Ltd., Rocky Hill, NJ, USA). After three days of culture
3 H-thymidine (280 kBq per well added in 20 µL of saline; TRA 310, Amersham International,
Amersham, UK) was added and cultures harvested 18 h later. The median count per minute (cpm) of
nuclear radioactivity for triplicate cultures was used for all calculations.
4.9. Statistical Analyses
The statistical analyses were performed by the standard computer software package IBM SPSS
Statistics 22 (IBM Corporate, Armonk, NY, USA). The Wilcoxon’s test for paired samples was applied
for analyses of paired observations, and the independent samples Mann-Whitney U test for comparison
of groups. The covariance between different continuous variables was studied with simple linear
regression analyses with one way analysis of variance (ANOVA).
Acknowledgments: The study received financial support from the Norwegian Cancer Society and Helse-Vest.
We thank the staff at Section for Cell Therapy, Department of Immunology and Transfusion Medicine, Haukeland
University Hospital for help with sample collection and preparation. The technical assistance of Kristin Paulsen
and Karen Marie Hagen is gratefully acknowledged.
Author Contributions: Øystein Bruserud, Guro Kristin Melve and Elisabeth Ersvaer conceived and designed the
experiments; Guro Kristin Melve and Elisabeth Ersvaer performed the experiments; Guro Kristin Melve analyzed
the data; Øystein Bruserud, Tor Hervig, Çiğdem Akalın Akkök, Einar K. Kristoffersen and Guro Kristin Melve
contributed reagents/materials/analysis tools; Guro Kristin Melve, Øystein Bruserud, Çiğdem Akalın Akkök,
Aymen Bushra Ahmed, Elisabeth Ersvaer, Einar K. Kristoffersen and Tor Hervig wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
Abbreviations
G-CSF
CD
HLA
GVHD
OPN
HSC
HSCT
MFI
PBMC
LAG-3
Tr1 cells
3H
MNC
Granulocyte colony-stimulating factor
Cluster of differentiation
Human leukocyte antigen
Graft versus host disease
Osteopontin
Hematopoietic stem cell
Hematopoietic stem cell transplantation
Mean fluorescence intensity
Peripheral blood mononuclear cells
Lymphocyte activation gene 3
T regulatory type 1 cells
Tritiated hydrogen
Mononuclear cells
Int. J. Mol. Sci. 2016, 17, 1158
AML
B-ALL
MDS
ACD-A
FBS
DMSO
Near-IR
PBS
BSA
14 of 17
Acute myeloid leukemia
B cell lymphoblastic leukemia
Myelodysplastic syndrome
Acid-citrate-dextrose solution A
Fetal Bovine Serum
Dimethyl sulfoxide
Near-infrared
Phosphate-buffered saline
Bovine Serum Albumin
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