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doi:10.1182/blood-2002-07-2104
Prepublished online December 27, 2002;
Francesco Dazzi
Mauro Krampera, Sarah Glennie, Julian Dyson, Diane Scott, Ruthline Laylor, Elizabeth Simpson and
memory antigen-specific T cells to their cognate peptide
Bone marrow mesenchymal stem cells inhibit the response of naive and
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20036.
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BONE MARROW MESENCHYMAL STEM CELLS INHIBIT THE RESPONSE
OF NAÏVE AND MEMORY ANTIGEN-SPECIFIC T CELLS TO THEIR
COGNATE PEPTIDE.
Running title: T cell inhibition by mesenchymal stem cells
Mauro Krampera§, Sarah Glennie§, Julian Dyson*, Diane Scott*, Ruthline Laylor,
Elizabeth Simpson*, and Francesco Dazzi
§MK and SG equally contributed to this work
Department of Immunology and *Transplantation Biology, Imperial College Faculty of
Medicine, Hammersmith Hospital, London, UK
MK current address is:
Dipartimento di Medicina Clinica e Sperimentale, Cattedra di Ematologia, Policlinico
G.B. Rossi, P.le L.A. Scuro 10, 37134 VERONA, Italy
This work has been funded by the Leukaemia Research Fund.
MK was recipient of an AIRC-FIRC Fellowship (“L Fontana e M Lionello”)
Word counts: Text 5138
Abstract 246
Scientific heading: Transplantation
Address for correspondence:
Francesco Dazzi
Department of Immunology
Imperial College Faculty of Medicine
Hammersmith Hospital
Du Cane Road
London W12 0NN
Tel +44 208 383 2134
FAX +44 208 742 9335
Email: f.dazzi@ic.ac.uk
Copyright (c) 2002 American Society of Hematology
Blood First Edition Paper, prepublished online December 27, 2002; DOI 10.1182/blood-2002-07-2104
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Abstract
Mesenchymal stem cells (MSC) have been recently shown to inhibit T-cell proliferation
to polyclonal stimuli. We characterized the effect of MSC of bone marrow origin on the
T-cell response of naïve and memory T cells to their cognate antigenic epitopes. The
immune response to murine male transplantation antigens, HY, was selected because the
peptide identity and MHC restriction of the immunodominant epitopes are known.
C57BL/6 female mice immunized with male cells were the source of memory T cells,
whereas C6 mice transgenic for HY-specific T-cell receptor provided naïve T cells.
Responder cells were stimulated in vitro with male spleen cells or HY peptides in the
presence or absence of MSC. MSC inhibited HY-specific naïve and memory T cells in a
dose-dependent fashion and affected cell proliferation, cytotoxicity, and the number of
IFN producing HY peptide-specific T cells. However, MSC inhibitory effect did not
selectively targeted antigen-reactive T cells. When MSC were added to the T-cell cultures
in a Transwell system or MSC were replaced by MSC culture supernatant, the inhibitory
activity was abrogated. T-cell reactivity was also restored if MSC were removed from the
cultures. The expression of MHC molecules, the presence in culture of antigen presenting
cells (APC) or of CD4+/CD25+ regulatory T cells were not required for MSC to inhibit.
We conclude that MSC inhibit naïve and memory T cell responses to their cognate
antigens. Overall our data suggest that MSC physically hinder T cells from the contact
with APC in a non-cognate fashion.
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Introduction
Bone marrow (BM) stroma contains multipotential non-hematopoietic progenitor cells
capable of differentiating into various tissues of mesenchymal origin. First identified for
their ability to differentiate into bone and adipocytes1, further studies have demonstrated
that mesenchymal stem cells (MSC) can also differentiate, under appropriate in vitro
conditions, to form chondrocytes, tenocytes, skeletal myocytes, neurons, and cells of
visceral mesoderm2-5. MSC have been isolated in different species and are present at a
low frequency (1/105) in adult BM but they appear to constitute approximately one-third
of the initial adherent bone marrow-derived stromal colonies in vitro. They are
characterized by the absence of hematopoietic markers (CD45-/CD34-/CD14-) and the
expression of a specific pattern of adhesion molecules (CD106+/CD54+/SH2+/SH3+).
Through these molecules, MSC interact with hematopoietic stem cells and influence their
homing and differentiation. Evidence for a definite role of BM stroma in T-cell
development comes from the observation that the BM microenvironment provides
appropriate support for T cells to develop in the absence of the thymus6 and that the
majority of T cells adhering to BM stroma display an immature phenotype7. Furthermore,
after bone marrow transplantation, BM stromal cells appear to migrate to the thymus
where they participate in the positive selection of thymocytes8,9. In addition to providing
critical cell-cell contact, MSC produce growth factors for hematopoiesis and may attract
infused hematopoietic stem cells to the marrow by inducing homing receptors. In a
NOD/SCID system Peled et al. showed that stromal derived factor 1 (SDF-1) and its
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receptor CXCR4 enhance CD34+ cell migration and engraftment in marrow10. Therefore
the BM microenvironment greatly influences the developmental potential of
hematopoietic stem cells. These characteristics have generated substantial interest in the
transplantation setting where MSC may be a promising cell type for tissue engineering,
cellular therapy, and somatic gene therapy applications. Studies already conducted in
both humans and animal models have demonstrated that MSC are capable of long-term
engraftment and in vivo differentiation11,12.
Great potential for MSC would be derived from the observation that they can exert an
immunoregulatory activity. Their effects, especially on mature T lymphocytes, have not
yet been defined. It has recently been reported that MSC can inhibit T-cell proliferation
induced in a mixed lymphocyte reaction or by non-specific mitogens13. Of particular
interest is the observation that in vivo administration of MSC in baboons significantly
prolongs the survival of MHC-mismatched skin grafts14. Since these properties might
open attractive possibilities in the field of hematopoietic as well as solid organ
transplantation, better characterization of MSC immunoregulatory activity and the
elucidation of its mechanism are crucial. In view of the lack of MHC class I and II on
MSC, it is difficult to ascribe specific T-cell receptor/MHC/peptide interactions to their
mechanism of immunoregulation. However, MSC might selectively inhibit T cells which
have encountered antigen, sparing those which have not been activated by T-cell receptor
engagement. The susceptibility of naïve and memory T cells to immunoregulatory
stimuli15 could have profound implications when considering potential clinical
applications of MSC. We addressed these questions utilizing an animal model in which
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the effect of MSC of bone marrow origin on the immune responses to various peptide
epitopes of the transplantation antigen HY was evaluated. In this system the peptide
identity and the MHC restriction of the immunodominant epitopes are known16,17. We
found that inhibition requires the presence of MSC in culture and MSC-T cell contact.
Both naïve and memory cells are subjected to MSC mediated suppression but MSC do
not appear to selectively target antigen-reactive T cells. MSC inhibitory effect does not
require the presence of antigen-presenting-cells and is not mediated through
CD4+/CD25+ regulatory T cells. The implications of these results are discussed.
MATERIALS AND METHODS
Mice
C57BL/6 (H2b), BALB.B (H2b), and CBA (H2k) mice were purchased form Olac Harlen
(Bicester, UK). CBA/Ca, C57BL/6-Thy1.1, and C3H.SW (H2b) mice were bred by CBS
at the Hammersmith Hospital. Mice were used between age 7 and 10 weeks of age.
C6 TCRhigh mice (C6) are transgenic for the V8 and V11 T-cell receptor chains from
the HY-specific CD8+ T cell clone, C6, specific for the HY antigen-derived peptide
TENSGKDI presented by H2Kk (HY Kk)17,18. The strain is on a CBA/Ca background.
Spleen cells from these mice were used as a source of HY-specific naïve T cells. HY-
specific memory T cells were obtained by immunizing C57BL/6 female mice by
intraperitoneal (ip) injection of 5x106 syngeneic male splenocytes.
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Generation of MSC
MSC were generated from BALB.B (H2b), C3H.SW (H2b), or CBA (H2k) mice. BM
cells, collected by flushing femurs and tibias with medium, were cultivated in 6-well
plates or 25 cm2 flasks (Costar, Cambridge, MA) at a concentration of 106/ml nucleated
cells in Dulbecco's modified Eagle medium (DMEM), with high glucose concentration,
GLUTAMAX ITM (GibcoBRL, UK), 10% heat-inactivated adult bovine serum (Labtech
International, Sussex, UK), 100 U/ml penicillin and 100 µg/ml streptomycin
(GibcoBRL). No cytokines were added at any stage. Cultures were incubated at 37oC in a
5% CO2 atmosphere. After 72 hours, non-adherent cells were removed. When 70-80%
confluent, adherent cells were trypsinized (0.05% trypsin at 37o C for 5 min), harvested,
and expanded in larger flasks. A homogenous cell population was obtained after 3 to 5
weeks of culture. Before further expansion and experimental use, MSC were tested for
their ability to differentiate into adipocytes and osteoblasts19. Adipocytic differentiation
was induced by 10-8 M dexamethasone, whereas 10-8 M dexamethasone, 50 g/mL
ascorbic acid and 10 mM -glycerophosphate were used for osteoblastic differentiation.
Oil-red-O and von Kossa dyes were employed to identify adipocytes and osteoblasts,
respectively. More than 90% of the cells differentiated depending on the time left in
culture with the differentiating agent. The identity of MSC was also confirmed by
immunophenotypic criteria based on the expression of CD106, and the absence of
hematopoietic (with anti-CD45, -CD14, -CD11c antibodies) or endothelial cell (with anti-
CD31 antibodies) markers. All antibodies were purchased from Pharmingen, Oxford,
UK. MSC were maintained in culture for no more than 15 in vitro passages. The
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proportion of CD45+ cell in the MSC preparations used in the various experiments never
exceeded 3% CD45+ cells.
In selected experiments the 3T3-F442A mesenchymal stem cell line (from a H2d mouse),
a kind gift of Dr. H Green (Harvard Medical School, Boston, MA), was used. The cells
were cultivated in DMEM containing 10% heat-inactivated adult bovine serum
(GibcoBRL) according to the conditions originally described 20.
T-cell cultures
All cultures were carried out in RPMI 1640 supplemented with 5x10-5M 2ME, 10% FCS,
GLUTAMAX ITM (GibcoBRL, Life Technologies Ltd, UK), 100 U/ml penicillin and 100
g/ml streptomycin. In some experiments an anti-TGF-1-2-3 monoclonal blocking
antibody (Genzyme, Cambridge MA) was added to the cultures at a concentration of 1
and 10 µg/ml21.
Depletion of CD25+ cells
Depletion of CD25+ cells was performed using MiniMacs system (Miltenyi Biotec,
Camberley, UK). Biotinylated anti-CD25 antibodies (7D4 clone, BD PharMingen,
Germany) were added to spleen cell suspensions at a concentration of 1µl/30x106
cells/ml and incubated at 4O C for 20 min. After extensive washing with PBS, cells were
incubated with Mini-MACS Streptavidin MicroBeads (Mylteni Biotech) for 15 min and
subject to passage through selection columns in a magnetic field. FACS analysis of the
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eluted fraction (CD25-) stained with a PE labeled anti-CD25 (PC61 clone) showed
CD25+ cells to be less than 1%.
Antigenic peptides
Stimulator cells were pulsed with 1000 ng/ml of Kk-restricted (TENSGKDI) HY
peptide17 (encoded by the Smcy gene) or with the Db-restricted Smcy (KCSRNRQYL)22,
the Uty (WMHHNMDLI)23 encoded, and the Ab-restricted Dby encoded
(NAGFNSNRANSSRSS)24 HY peptides at 37oC for 2 hours. Unpulsed irradiated CBA or
C57BL/6 female splenocytes were used as negative controls.
T-cell proliferation assay
T-cell proliferation assays were performed in round-bottom 96-well plates (Costar,
Cambridge, MA) in a total volume of 0.2 ml RPMI 1640. A total of 0.5 µCi of [3H]-
thymidine (ICN, Costa Mesa, CA) was added into each well 2 (naïve transgenic T cells)
or 3 (memory T cells) days later as required, and cells were harvested onto glass fiber
filters using an LKB 96 well-harvester (Wallac Oy, Turku, Finland) after an additional 24
hours. [3H] thymidine uptake was measured on an LKB Betaplate counter (Wallac Oy).
The results are expressed as mean cpm for triplicate cultures (standard errors were
routinely <10%).
CD3/CD28 stimulation
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CD28/CD3-coated Dynabeads (Dynal, Bromborough, U.K.) were prepared by
conjugating 5µg of CD28 and CD3 antibodies (both from Pharmingen) to 107 beads,
according to the manufacturer’s instructions. For proliferation, 1-5x105 responder T cells
were incubated with 1-5x104 CD3/CD28 coated Dynabeads/well. The cells were
cultivated in a total volume of 200µl in flat-bottomed 96 well plates and assessed for
proliferation 3 days later.
T-cell cytotoxicity
Effector cells were incubated in 96-well plates with 51Cr-labelled peptide-pulsed RMA-S
target cells at E:T ratios of 50:1, 17:1, 5:1 and 1.5:1. After 4 h, 100 µl of supernatant was
collected and 51Cr release was measured using a gamma counter. Results were calculated
from a 12 point regression curve resolved at an E:T ratio of 10:1. The percentage of lysis
was calculated from the formula 100 x (E - M)/(T - M), where E is the experimental
release, M is the spontaneous release in the presence of medium alone, and T is the
maximum release in the presence of 5% Triton X-100.
Flow cytometric analysis
In addition to the monoclonal antibodies listed in a previous paragraph, MSC were also
stained with CD80 (B7-1), CD86 (B7-2), class I (H2) and class II (H2A) major
histocompatibility complex (MHC) molecules, all purchased from Pharmingen. For
immunophenotype analysis, MSC were detached using trypsin/EDTA, washed and
resuspended at 106/ml. One hundred microlitres of cell suspension were incubated at
+4oC for 15 min with mouse unconjugated immunoglobulins (Sigma Immunochemicals,
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Poole, UK), followed by incubation with the specific antibody at +4oC for 30 minutes.
Cells were washed with PBS containing 1% bovine serum albumin and 0.1% sodium
azide (PBSAS). At least 10,000 events were analyzed by flow cytometry (FACScan,
Becton Dickinson) using Cell Quest software.
Antigen specific T-cell responses were measured by enumerating IFN producing T cells
in response to HY peptides. Irradiated syngeneic splenocytes, pulsed with 10 nM of HY
peptides at 37oC for 2 hours, and then washed, were added to responder T cells at a 1:1
ratio. Prior to peptide pulsing, stimulator cells were T-cell-depleted with anti-mouse pan-
T Dynabeads (Dynal, Bromborough, U.K.). Non peptide-pulsed, irradiated syngeneic
splenocytes were used as the negative control. During the last 4 hours of stimulation,
Brefeldin A (Sigma) was added at a concentration of 10 µg/ml to block cytokine
secretion. At the end of the cultures, the cells were stained with anti-CD4 and -CD8
(Pharmingen). Subsequently, the cells were washed with PBS and fixed/permeabilized
with a commercial solution (Permeabilizing solution, Becton Dickinson) for 10 minutes.
After further washing, the cells were stained with anti-IFN antibody (Pharmingen) for
30 minutes at R/T. At least 100,000 events in the lymphocyte gate were analyzed at
FACScan.
CD8+ T cells expressing H2b restricted HY-specific T-cell receptors were analyzed by
using soluble/MHC peptide tetramers. Tetramers were produced by refolding H2-Db
heavy chain with 2microglobulin and HY peptides WMHHNMDLI (encoded by the Uty
gene) or KCSRNRQYL (encoded by the Smcy gene)22,23, as previously described16.
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Tetramers were conjugated with PE-labeled ExtrAvidin-R-PE complex (Sigma). Before
staining, cells were depleted of the B-cell fraction by negative selection with anti-mouse
pan-B Dynabeads (Dynal).
Transwell cultures
Splenocytes (15x106/ml) from HY-immunized C57BL/6 female mice were cultivated
with irradiated syngeneic male splenocytes (15x106/ml) in the lower chamber of a 24 mm
diameter Transwell® plate with a 0.3 µm pore size membrane (Costar, Cambridge, MA).
Autologous or allogeneic MSC (2x105) were seeded onto the transwell membrane of the
inner chamber 1-2 hours before the beginning of the culture. Control cultures did not
contain MSC or MSC were added directly to the secondary MLR. After 7 days, viable
cells were evaluated for antigen-specific intracellular IFN and frequency of peptide:
MHC tetramer+/CD8+ T cells.
In selected experiments, MSC were replaced with supernatant from MSC cell cultures. A
day before being split, MSC culture supernatant was harvested, centrifuged, and filtered
through a 0.2µm Millipore filter.
RESULTS
MSC fail to stimulate T cells in secondary H2-mismatched MLR
Initial experiments showed MSC to be poor stimulators of secondary mixed leukocyte
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reactions (MLR). Their ability to function as antigen presenting cells (APC) was tested
by using MSC as stimulators of secondary allogeneic mixed leukocyte reaction (MLR).
Spleen cells (2x105/well) from CBA (H-2k) mice were used as responder cells in primary
cultures stimulated with irradiated BALB.B spleen cells (5x105/well). Cells harvested at
day 7 from these primary cultures were then seeded at 2x105/well and re-stimulated with
graded numbers (102, 103, 104 per well) of irradiated (60cGy) MSC of BALB.B origin.
BALB.B splenocytes (2x105/well) were used as stimulators in the control cultures. The
limit of the maximum number of MSC (104/well) was chosen because at this
concentration MSC occupy on a 96-well plate the approximately same surface as 2x105
splenocytes, the number producing the best stimulation of secondary MLR (not shown).
No proliferation was detected at any of the MSC doses employed (Figure 1). No
difference was seen whether the MSC were irradiated or not. This lack of stimulator cell
activity was consistent with their phenotype: MSC expressed neither MHC class I nor
class II molecules. The analysis for co-stimulatory molecules showed that they did not
express CD86, although they were positive for CD80 (Figure 2). To assess whether the
lack of MHC class I expression was the major factor for the inability of MSC to present
alloantigens, MSC were pre-treated with IFN prior to their use as stimulators of
secondary MLR. Despite the induction of MHC class I expression (Figure 2) no
proliferation was observed (Figure 1). MHC class II was not expressed following IFN
pretreatment
MSC inhibit naïve and memory antigen-specific T cells
Since, despite the expression of MHC class I and some co-stimulatory molecules, MSC
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failed to induce T-cell stimulation, we tested whether this could be attributed to an
inhibitory effect on T-cell activation. The effect of addition of MSC to cultures of both
naïve and memory HY-specific T cells in the presence of their cognate peptides was
tested. Splenocytes from C6 mice transgenic for a H2-Kk-restricted HY-specific T-cell
receptor were used as a source of naïve T cells and cultivated in the presence of irradiated
syngeneic (CBA, H2k) spleen cells pulsed with the cognate Kk restricted HY peptide (HY
KkSmcy) as APC. Graded doses of MSC were added as third party cells for the duration
of the in vitro stimulation. After 24 hours CD8+ cells were analyzed for IFN by
intracellular staining (Figure 3B). Parallel cultures were assessed for T-cell proliferation
after 48 hours (Figure 3A). Both HY peptide specific IFN production and T cell
proliferation were inhibited. Inhibition was dependent on the number of MSC in culture.
The proliferative activity was significantly inhibited at a MSC/responder (M/R) ratio as
low as 1:104. At the highest ratio (1:101), T-cell proliferation and the percentage of IFN
producing CD8+ T cells were reduced by 85% and 53%, respectively.
Splenocytes from wild type female C57BL/6 (H2b) mice immunized with syngeneic male
spleen cells were used as a source of HY specific memory T cells. They were
restimulated in vitro with syngeneic male spleen cells. Graded doses of MSC were added
as a third party to the MLR. After 7 days, the cells were harvested and restimulated with
H2b-restricted HY peptide- (HY DbSmcy, HY DbUty, HY AbDby) pulsed female spleen
cells and assessed for T-cell proliferation (Figure 4A), number of HY peptide specific
IFN-positive (Figure 4B) and HY DbSmcy and HY DbUty tetramer-positive CD8+ T
cells (Figure 4C). To measure HY-specific cytotoxic activity, cells were harvested after
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5 days and tested against HY peptide (DbSmcy and DbUty) pulsed RMA-S cell line in a
51Cr-release assay (Figure 4D). Proliferation, number of IFN producing and tetramer-
positive CD8+ cells, and the cytotoxic activity of these HY specific memory T cells were
inhibited by MSC in a dose dependent fashion. It is interesting to note that the effector
functions (IFN- production and cytotoxicity) of memory T cells appeared to be more
susceptible to inhibition than antigen-specific T-cell expansion (proliferation and
expansion of tetramer-positive T cells). Furthermore, the percentage of inhibition
observed on IFN production is greater than that observed on cytotoxic activity, thus
suggesting that MSC could selectively interfere with certain pathways of T-cell
activation/function.
MSC do not specifically target antigen-reactive T cells
Although MSC can inhibit antigen-specific immune responses, it is unlikely that this
results from the recognition of the cognate peptide presented by MSC because they lack
expression of MHC molecules. Nevertheless, MSC might specifically interact with and
inhibit only the T cells which become activated after encountering antigen. To address
this question we utilized as responder cells a population containing different proportions
of HY-specific T cells but we kept the number of MSC fixed. Memory spleen cells from
HY-immune female C57BL/6 mice were stimulated in vitro with male cells. After 7 days,
the cultures, which contained an average of 50% of DbUty tetramer-positive CD8+ T
cells (Figure 5A) were used for restimulation either directly or diluted 1:2 or 1:10 with
splenocytes from naïve female mice to reduce the concentration of HY-specific T cells to
25 or 5%, respectively. Naïve and memory cells were from mice polymorphic for the
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Thy1 allele (Thy1.1+ and Thy1.2+, respectively) in order to be able to confine the analysis
to the memory T-cell population. Responder cells were restimulated with H2b-restricted
HY peptide- (HY DbSmcy, HY DbUty, HY AbDby) pulsed female spleen cells and
assessed for number of HY peptide specific IFN-positive Thy1.2+ cells generated in the
presence or absence of a fixed number of MSC. In this system the ratio between MSC
and T cells remained the same but the ratio between MSC and HY-specific T cells varied.
The inhibitory effect of MSC on HY-specific memory T cells after in vitro expansion is
significant but not complete. By reducing the ratio between antigen-specific and non-
antigen-specific T cells of up to 10 fold, we would expect a higher inhibition if MSC
exclusively targeted antigen-specific T cells. However, no difference in the magnitude of
inhibition was observed at different ratios between MSC and HY-specific T cells (Figure
5B).
MSC inhibitory effect is not MHC-dependent
MSC do not constitutively express MHC molecules, but they can do so after IFN
treatment. In the cultures in which MSC were added to the MLR as third party, we
observed that after 7 days they expressed MHC class I molecules, probably as a result of
IFN production in the culture (data not shown). Although the presence of MHC class I
on MSC does not appear to induce the proliferation of allogeneic T cells in MHC
mismatched cultures (Figure 1), we tested whether MHC class I molecule expression
was required for the inhibitory effects on naïve and memory HY specific T cells to be
exerted. For these experiments, we used the 3T3-F442A MSC line of BALB/c origin,
which does not express MHC molecules even after IFN treatment (data not shown). No
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difference in the inhibitory activity was detected in comparison with H2b matched
BALB.B MSC in terms of proliferation and IFN production by naive (Figure 6A) or
memory (Figure 6B) responder cells. These findings were consistent with the
observation that C6 splenocytes (H2k) can be inhibited by H2b MSC (Figure 3).
MSC inhibitory effect is transient
The fact that MHC expression on MSC does not influence the MSC mediated inhibition
suggests that it also does not require the T cells to recognize antigen on MSC. Moreover,
our data shows that MSC do not exclusively target antigen-reactive T cells. It remained to
be determined whether the MSC induced persistent non-responsiveness or whether their
inhibitory effect was transient. To address this question, splenocytes from C6 mice were
stimulated with the Kk-restricted HY peptide in the presence or absence of MSC as a
third party. Twenty-four hours later, non-adherent cells were harvested and depleted of
CD106+ cells to exclude the interference of any residual MSC. The CD106-/non-adherent
C6 cells were re-stimulated with the HY peptide without MSC. IFN producing CD8+ T
cells were enumerated 24 hours later. The results are shown in Figure 7. Although the
presence of MSC in the first 24hr culture inhibited the first antigenic stimulation, when
MSC were removed the response to the HY peptide was restored. These findings
demonstrate that MSC inhibit T cells only when they are present in culture, but the effect
is reversed when MSC are removed.
MSC inhibitory effect requires cell contact
The MSC inhibitory effect could be mediated by soluble factors or could require cell
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contact to be exerted. The fact that the inhibitory activity was dose-dependent and
transient (ie entirely dependent on the presence of MSC) was in support of the latter
hypothesis. MSC culture supernatant was added (1:1 dilution) to the cultures in which
memory HY-specific T cells were stimulated with syngeneic male spleen cells. After 7
days the numbers of HY DbSmcy tetramer-positive CD8+ T cells were evaluated. In
parallel, cells were stimulated with HY peptide pulsed APC for 6 hrs to assess the
numbers of IFN+/CD8+ T cells. The results are reported in Figure 8. No difference in
the number of tetramer positive T cells was detected (Figure 8A) and the number of
IFN+/CD8+ T cells was not reduced in the presence of MSC supernatant (Figure 8B).
The necessity of cell contact for the inhibitory effect was confirmed by experiments in a
transwell system. MSC were added to the inner chamber and the MLR was cultivated
separately in the lower chamber. When the MSC were not in contact with the MLR, the
inhibition of antigen-specific T cells was much reduced (Figure 8A and 8B). Consistent
with a need for cell-cell contact and the absence of an effect of soluble factors, we did not
find any IL-10 in the MSC culture supernatant as evaluated by ELISA; further, addition
of anti-TGF blocking antibody had no influence on inhibitory activity even when used
at concentrations 10 fold higher (10µg/ml) than those described by others13 (data not
shown).
MSC inhibitory effect is neither dependent on antigen-presenting cells nor on
CD4+/CD25+ regulatory T cells
MSC inhibitory effect could be exerted directly on effector T cells or mediated via
different splenocyte types present in culture. To address this question, we asked whether
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antigen presenting cells (APC) were required for the inhibitory effect to be exerted. We
stimulated T cell proliferation with CD3/CD28 coated beads. This type of stimulation
circumvents the need for APC, as antibodies interact directly with T cells to activate
them. When MSC were added to the cultures they potently inhibited spleen cell
proliferation in response to the antibody coated beads (Figure 9A). The results were
confirmed using a HY DbUty-specific CD8+ T-cell clone (CTL-10)25 as responder cells
(Figure 9B)
Alternatively, MSC might exert their inhibitory effect via regulatory T cells. Much
attention has recently been paid to a distinct CD4+ T cell subset expressing the CD25
molecule which appears to actively suppress T cell activation26. In order to ascertain
whether CD4+/CD25+ regulatory T cells are required for the MSC mediated inhibition,
spleen cells from C6 mice were depleted of CD25+ cells and stimulated with H2k-
restricted HY peptide- (HY KkSmcy) pulsed female spleen cells in the presence or
absence of MSC and 24 hours later assessed for the number of HY peptide specific IFN-
positive CD8+ cells. CD25-depleted and CD25-replete C6 T cells were equally inhibited
by MSC (50% vs 44% = not statistically significant) (Fig 10).
Discussion
Transplantation has been one of the major advances in medicine during the last few
decades. BM transplantation in particular, can cure a variety of malignancies by
exploiting the graft-versus-tumor effect exerted by the lymphocytes contained in the
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donor BM preparation. Major problems remain with the lack of suitable BM donors. The
option of increasing donor-recipient histoincompatibility is associated with a high risk of
both graft rejection and/or graft-versus-host disease, a situation in which more powerful
and selective strategies to diminish immune responses following transplantation would be
desirable. There has recently been enormous interest in the stem cells contained in the
BM since it appears that they can differentiate into lineages other than hematopoietic 27-
32. BM also contains rare stem cells which differentiate into mesenchymal lineage cells
which not only have multipotential differentiation ability33 but they also appear to
modulate immune responses in vitro. This property could have a substantial impact in
transplantation since infusion of MSC in conjunction with the donor organ or bone
marrow might favor engraftment. Administration of MSC has been reported to prolong
donor skin graft survival in non-human primates14. The ability of MSC to suppress
immune responses could also be harnessed to reduce GvHD, the idea underlying a
multicentric clinical trial in which patients with advanced hematologic malignancies
receive donor MSC at the time of haemopoietic stem cell infusion as a prophylaxis for
GvHD. So far, the reported overall incidence of acute and chronic GvHD appears to be
significantly lower in the group infused with MSC as compared to the controls (p=0.002
and p=0.02, respectively)34. However, the characteristics and the mechanisms of this
inhibition are entirely unknown.
Here we show that BM derived MSC have a profound inhibitory effect on activation of T
cells by their cognate peptides in vitro. This inhibition, which affects both naïve and
memory T cells, is manifest in antigen-specific proliferation, IFN production and
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cytotoxic activity. Physiologic inhibition of T cells is considered to occur by deletion,
anergy, or suppression35. Whereas in the first case T cells die, in the other situations T
cells remain alive but are unable to respond to antigen. Here we show that the inhibitory
effect exerted by MSC on naïve T cells is already detectable at 24 hours but it is relieved
once MSC are removed from the culture, so they are not deleted (Fig 7). However, in the
cultures in which MSC are incubated with memory T cells for 7 days which are then
harvested and re-stimulated with antigen, T cells remain unable to respond (Fig 4). In the
case of naïve antigen reactive T cells, which recover responsiveness when separated after
24 hours with MSC, it seems unlikely that their non-responsiveness in the continued
presence of MSC is due to anergy, since 24 hours is a reasonable time-frame for anergy
induction36. The alternative hypotheses are that MSC induce T-cell suppression either
directly by soluble factors or indirectly by preventing T-cell activation. The requirement
of cell contact for MSC to display their inhibition suggests they may operate by
preventing T cell activation. The absence of IL-10 in MSC culture supernatants and the
inability of anti-TGF blocking antibodies to inhibit the MSC effect (data not shown) are
in accord with such a hypothesis. It is not yet clear whether naïve are more difficult to
anergize than memory T cells15. Although the inhibition of IFN production seems more
pronounced on memory T cells than in naïve T cells, the precursor frequency of
responder T cells differ in the two populations. At the time of culture with MSC the
numbers of antigen-specific T cells were higher in the naïve transgenic splenocytes than
in the memory HY primed splenocytes. The proportion of HY-specific T cells in spleens
of immunized female mice is 10%16 compared with greater than 90% in naïve C6
transgenic mice18.
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A crucial question is whether the inhibitory effect depends on the presence of cognate
antigen. The evidence that MSC do not require MHC molecules to inhibit T-cell
responses (Fig 6) strongly argues against a cognate mechanism but does not rule out the
possibility that MSC inhibit specifically the cells which are activated by interaction with
antigen. However, it is the ratio between MSC and total T cells rather than with antigen-
specific T cells which influences the inhibition (Fig 5). These results favor the notion that
MSC do not specifically target T cells which encounter antigen. Since neither APC (Fig
9) nor CD4+/CD25+ regulatory T cells (Fig 10) are required for the inhibition to occur,
overall our data suggest that MSC physically hinder T cells from the contact with APC in
a non-cognate fashion.
Two papers have recently reported that MSC inhibit T-cell proliferation induced by non-
specific mitogens or in polyclonal, polyepitope MLR13,14. Ours is the first demonstration
of the inhibitory effect of MSC on T-cell response to cognate peptide. It is noteworthy
that our findings differ from those reported by Di Nicola et al13 since in their work the
effect of MSC appears to be mediated by soluble factors, and in particular by TGF-
and/or HGF, and does not require cell contact. Although this discrepancy might merely
be explained by the fact that experiments were carried out in different species, our MSC
have been generated in a different way. It is possible that BM stromal cells are different
from MSC in this respect. These differences may also explain the much higher inhibitory
efficiency of our MSC: an effect is clearly visible when the ratio between MSC and
responder T cells is as low as 1:103 as compared to ratio of 1:1 required to see an effect
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with stromal cells of Di Nicola et al. However, it is possible that soluble factors may have
a small role also in our system (Fig 8).
Regardless of the mechanisms underlying the MSC inhibitory effect, the physiological
role of this is unclear. It is well known that mesenchymal elements play a major role in T-
cell ontogeny in the thymus. Since we observed that cell contact is required, MSC might
provide a niche in the bone marrow in which lymphocytes are suppressed or deleted.
However, the effect of MSC may not be specific to the immune system but have a more
general “inhibitory” activity on protein synthesis and/or the cell cycle in a variety of cell
types.
Our findings clearly have possible therapeutic implications. Because of their
differentiation ability and susceptibility to stable gene transduction, MSC represent an
attractive target to develop gene therapy. The evidence that MSC do not present
alloantigen and that they do not require MHC expression to exert their inhibitory effect,
suggests that they can be derived from a donor irrespective of their MHC haplotype and
prepared as an “off-the-shelf” reagent for any patient. Here we show that MSC have a
profound inhibitory effect on T-cell responses to transplantation antigens and may thus be
used to control host-versus-graft (HvG) and GvHD, especially in those situations in
which bone marrow transplantation is performed following reduced intensity
conditioning. More importantly, we have demonstrated that not only naïve but also
antigen-experienced (memory) T cells can be inhibited. The current ongoing clinical
trials involve the infusion of MSC at the time of bone marrow transplant34. When this
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procedure is used for patients with leukemia, it is possible that the inhibitory effect may
also jeopardize the graft-versus-leukemia effect which is crucial for a successful
outcome. This potential complication needs to be considered against the probability that,
according to our data, MSC have the potential ability to inhibit even ongoing GvHD.
References
1. Fridenstein A. Stromal bone marrow cells and the hematopoietic
microenvironment. Arkh Patol. 1982;44:3-11
2. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult
human mesenchymal stem cells. Science. 1999;284:143-147
3. Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow
mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve. 1995;18:1417-1426
4. Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone
marrow stromal cells differentiate into neurons. J Neurosci Res. 2000;61:364-370
5. Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM. Purification
and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood.
2001;98:2615-2625
6. Dejbakhsh-Jones S, Jerabek L, Weissman IL, Strober S. Extrathymic
maturation of alpha beta T cells from hemopoietic stem cells. J Immunol. 1995;155:3338-
3344
7. Barda-Saad M, Rozenszajn LA, Globerson A, Zhang AS, Zipori D. Selective
adhesion of immature thymocytes to bone marrow stromal cells: relevance to T cell
lymphopoiesis. Exp Hematol. 1996;24:386-391
For personal use only. by guest on June 12, 2013. bloodjournal.hematologylibrary.orgFrom
8. Li Y, Hisha H, Inaba M, et al. Evidence for migration of donor bone marrow
stromal cells into recipient thymus after bone marrow transplantation plus bone grafts: A
role of stromal cells in positive selection. Exp Hematol. 2000;28:950-960
9. Suniara RK, Jenkinson EJ, Owen JJ. An essential role for thymic mesenchyme
in early T cell development. J Exp Med. 2000;191:1051-1056
10. Peled A, Petit I, Kollet O, et al. Dependence of human stem cell engraftment
and repopulation of NOD/SCID mice on CXCR4. Science. 1999;283:845-848
11. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-
derived mesenchymal cells engraft and stimulate growth in children with osteogenesis
imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci U S A.
2002;99:8932-8937
12. Pereira RF, Halford KW, O'Hara MD, et al. Cultured adherent cells from
marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated
mice. Proc Natl Acad Sci U S A. 1995;92:4857-4861
13. Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal
cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic
stimuli. Blood. 2002;99:3838-3843
14. Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells
suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp
Hematol. 2002;30:42-48
15. Marelli-Berg FM, Weetman A, Frasca L, et al. Antigen presentation by
epithelial cells induces anergic immunoregulatory CD45RO+ T cells and deletion of
CD45RA+ T cells. J Immunol. 1997;159:5853-5861
16. Millrain M, Chandler P, Dazzi F, Scott D, Simpson E, Dyson PJ.
Examination of HY response: T cell expansion, immunodominance, and cross-priming
revealed by HY tetramer analysis. J Immunol. 2001;167:3756-3764
17. Scott DM, Ehrmann IE, Ellis PS, et al. Identification of a mouse male-
specific transplantation antigen, H-Y. Nature. 1995;376:695-698
For personal use only. by guest on June 12, 2013. bloodjournal.hematologylibrary.orgFrom
18. Chai JG, Vendetti S, Bartok I, et al. Critical role of costimulation in the
activation of naive antigen-specific TCR transgenic CD8+ T cells in vitro. J Immunol.
1999;163:1298-1305
19. Kitano Y, Radu A, Shaaban A, Flake AW. Selection, enrichment, and culture
expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling
adherent bone marrow cells. Exp Hematol. 2000;28:1460-1469
20. Morikawa M, Nixon T, Green H. Growth hormone and the adipose
conversion of 3T3 cells. Cell. 1982;29:783-789
21. Veiby OP, Jacobsen FW, Cui L, Lyman SD, Jacobsen SE. The flt3 ligand
promotes the survival of primitive hemopoietic progenitor cells with myeloid as well as B
lymphoid potential. Suppression of apoptosis and counteraction by TNF-alpha and TGF-
beta. J Immunol. 1996;157:2953-2960
22. Markiewicz MA, Girao C, Opferman JT, et al. Long-term T cell memory
requires the surface expression of self-peptide/major histocompatibility complex
molecules. Proc Natl Acad Sci U S A. 1998;95:3065-3070
23. Greenfield A, Scott D, Pennisi D, et al. An H-YDb epitope is encoded by a
novel mouse Y chromosome gene. Nat Genet. 1996;14:474-478
24. Scott D, Addey C, Ellis P, et al. Dendritic cells permit identification of genes
encoding MHC class II-restricted epitopes of transplantation antigens. Immunity.
2000;12:711-720
25. King TR, Christianson GJ, Mitchell MJ, et al. Deletion mapping by
immunoselection against the H-Y histocompatibility antigen further resolves the Sxra
region of the mouse Y chromosome and reveals complexity of the Hya locus. Genomics.
1994;24:159-168
26. Shevach EM. CD4+ CD25+ suppressor T cells: more questions than answers.
Nat Rev Immunol. 2002;2:389-400
27. Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by
bone marrow-derived myogenic progenitors. Science. 1998;279:1528-1530
For personal use only. by guest on June 12, 2013. bloodjournal.hematologylibrary.orgFrom
28. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted
myocardium. Nature. 2001;410:701-705
29. Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential
source of hepatic oval cells. Science. 1999;284:1168-1170
30. Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells
from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest.
2002;109:1291-1302
31. Brazelton TR, Rossi FM, Keshet GI, Blau HM. From marrow to brain:
expression of neuronal phenotypes in adult mice. Science. 2000;290:1775-1779
32. Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. Turning blood
into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science.
2000;290:1779-1782
33. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal
stem cells derived from adult marrow. Nature. 2002;418:41-49
34. Frassoni F LM, Bacigalupo A, Gluckman E, Rocha V, Bruno B, Lazarus H,
Devine S, Holland K, McCarthy P, Curtin P, Maziarz R, Shpall E, Mosely AM.
Expanded mesenchymal stem cells (MSC), co-infused with HLA identical hemopoietic
stem cell transplants, reduce acute and chronic graft versus host disease: a matched pair
analysis. Bone Marrow Transplant. 2002;29:75
35. Van Parijs L, Abbas AK. Homeostasis and self-tolerance in the immune
system: turning lymphocytes off. Science. 1998;280:243-248
36. Jenkins MK, Schwartz RH. Antigen presentation by chemically modified
splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J Exp
Med. 1987;165:302-319
For personal use only. by guest on June 12, 2013. bloodjournal.hematologylibrary.orgFrom
Legends to Figures
Figure 1. MSC do not stimulate allogeneic T cells.
The ability of MSC to function as antigen presenting cells was tested by using MSC as
stimulators of secondary allogeneic MLR. Spleen cells from CBA (H2k) mice, stimulated
in primary cultures with BALB.B (H2b) spleen cells (2x105/well), were re-stimulated
with (A): BALB.B or (B): autologous CBA splenocytes as stimulators in the control
cultures and with (C): graded numbers (102, 103, 104 per well) of irradiated (60cGy)
(black bars) or non irradiated (white bars) MSC of BALB.B origin. (D): In a separate
experiment BALB.B MSC were pre-treated with IFN (100U/ml for 48 hrs) before being
used in the cultures. (E): Proliferation of MSC alone not irradiated. Cultures were
performed in triplicate and the results reported are the average of 3 experiments of
identical design. The bars show the standard deviation.
*: statistically significant (at least p<0.01) vs control cultures (stimulation with syngeneic
spleen cells) in all experiments
Figure 2. Immunophenotype of BM MSC
MSC were analysed for the expression of CD106, CD80, and MHC class I molecules in
standard conditions (A) or after 48h-culture with 100 U/ml IFN(B).
Figure 3. MSC inhibit the response of naïve antigen-specific T cells in a dose-
dependent fashion.
Splenocytes from C6 mice transgenic for a Kk-restricted HY-specific T-cell receptor were
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stimulated with irradiated syngeneic (CBA, H-2k) spleen cells pulsed with the cognate
HY KkSmcy (TENSGKDI) p eptide. Graded doses of MSC were added as third party cells
for the duration of the in vitro stimulation. After 24 hours CD8+ cells were analyzed for
IFN by intracellular staining (B). T-cell proliferation was assessed after 48 hours (A).
Results are shown for cells cultured in the presence (solid lines) or absence (dotted lines)
of the HY peptide. The results reported are the average of 3 experiments of identical
design. T-cell proliferation cultures were performed in triplicate and results averaged.
The bars show the standard deviation.
MSC/R: MSC to responder T cells ratio.
*: statistically significant (at least p<0.01) vs control cultures without MSC in all
experiments
Figure 4. MSC inhibit the response of antigen-specific memory T cells in a dose-
dependent fashion.
Splenocytes from normal, non transgenic female C57BL/6 (H-2b) mice immunized with
syngeneic male spleen cells were restimulated in vitro with syngeneic male spleen cells.
Graded doses of MSC were added as a third party to the MLR. After 7 days, the cells
were harvested and restimulated with female spleen cells pulsed with the HY peptides
HY DbSmcy (KCSRNRQYL), HY DbUty (WMHHNMDLI), and HY AbDby
(NAGFNSNRANSSRSS) and then assessed for T-cell proliferation (A), numbers of HY-
specific IFN+ cells (B) and HY DbSmcy and HY DbUty tetramer-positive CD8+ T cells
(C). HY-specific cytotoxic activity was tested against HY peptide (HY DbSmcy and HY
DbUty) pulsed RMA-S cell line in a 51Cr-release assay (D). The results refer to the
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cultures in the presence (solid lines) or absence (dotted lines) of the HY peptide. The
results reported are the average of 5 experiments of identical design. T-cell proliferation
(2 experiments) were performed in triplicate and results averaged. The bars show the
standard deviation.
MSC/R: MSC to responder T cells ratio.
*: statistically significant (at least p<0.01) vs control cultures without MSC in all
experiments
Figure 5. MSC do not specifically target antigen-reactive T cells
Spleen cells from normal Thy1.2 C57BL/6 HY-immune female mice were stimulated
with syngeneic male cells. After 7 days the proportion of HY-TCR+ T cells were 50%
(A). These responder cells were used as such or mixed with splenocytes from Thy1.1
C57BL/6 naïve female mice at a 1:2 or 1:10 ratio. 5x106 cells of the different mixtures
were stimulated with equal number of HY peptide (DbSmcy, DbUty, AbDby) pulsed
syngeneic female cells in the presence or absence of a fixed number (105) of MSC cells.
After 24 hours the Thy1.2+ cell population was assessed for numbers of peptide-specific
IFN+ cells. The figure (B) reports the percentages of inhibition exerted by MSC on the
different mixtures of responder cells.
Figure 6. MSC inhibitory effect is MHC-independent.
Spleen cells from C6 Tg (naïve) (A) or normal C57BL/6 HY-immune (memory) (B) mice
were stimulated with HY KkSmcy peptide or syngeneic male cells respectively, as
described previously, in the presence or absence of the 3T3-F442A MSC line which does
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not express MHC class I molecules even after IFN treatment. T-cell proliferation (cpm)
and percentage of IFN producing CD8+ T cells were evaluated. The results refer to the
cultures in the presence (white bars) or absence (black bars) of HY antigen. The results
reported are the average of 3 experiments of identical design. T-cell proliferation cultures
were performed in triplicate and results averaged. The bars show the standard deviation.
*: statistically significant (at least p<0.01) vs control cultures without MSC in all
experiments.
Figure 7. The MSC inhibitory effect is transient
(A): Spleen cells from C6 Tg (naïve) mice were stimulated for 24 hrs with HY KkSmcy
(TENSGKDI) peptide-pulsed APC (black bar). Cells were then harvested, washed and
restimulated with the same peptide for a further 24 hours (white bar). (B): The same
responder cells (C6 Tg) were stimulated for 24 hrs with HY KkSmcy peptide-pulsed APC
in the presence of MSC (MSC/R ratio = 1:10) (black bar). Cells were then harvested,
depleted of MSC by CD106 antibodies and Dynabeads before restimulation with the
same peptide (white bar). The Y-axis reports the percentage of IFN producing CD8+ T
cells. The results reported are the average of 3 experiments of identical design. The bars
show the standard deviation.
*: Statistically significant (at least p<0.01) vs. control cultures containing MSC (A, black
bar) in all experiments. The comparison between the restimulation of C6 cells with HY
KkSmcy peptide (white bars) previously stimulated with the same peptide in the presence
(B) or absence (A) of MSC was not statistically significant. The bars show the standard
deviation.
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Figure 8. MSC inhibitory effect requires cell contact
HY-specific memory T cells from immunized B6 female were stimulated in vitro with
syngeneic male spleen cells (B6) in the presence of MSC (B6+MSC) or supernatant from
MSC cultures (B6+MSC-SN). In parallel, responder T cells were also stimulated with
syngeneic male spleen cells in the presence of MSC in a Transwell® system (B6+MSC-
T/W). The percentage of HYDbSmcy and HYDbUty tetramer-positive CD8+ T cells (A)
and/or HY-specific IFN-positive CD8+ T cells (B) are shown for each culture. The
results reported are the average of 4 identical experiments. The bars show the standard
deviation.
*: Statistically significant (at least p<0.01) vs. control cultures (B6)
Figure 9. MSC inhibitory effect does not require antigen presenting cells
Proliferative response of B6 splenocytes (A) or HY-specific T-cell clone CTL-1025 (B) to
CD3/CD28-coated beads in the presence or absence of MSC. One representative
experiment is shown of three performed. The background counts for unstimulated spleen
and T-cell clone was less than 1% and less than 5%, respectively. The scale of the y-axis
on the left and on the right is different.
*: statistically significant (at least p<0.01) vs control cultures without MSC in all
experiments.
Figure 10. MSC inhibitory effect does not require CD4+/CD25+ regulatory T cells
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5x106 CD25-depleted or CD25-replete C6 splenocytes were stimulated in vitro with HY
KkSmcy peptide pulsed female cells in the presence or absence of MSC (105). After 24
hours HY-specific IFN-positive CD8+ T cells were enumerated. MSC inhibition on
CD25-negative responders was 50% as compared to 44% in the control CD25-replete
cultures (not statistically significant). The bars show the standard deviation.
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