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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-07-2104.
TRANSPLANTATION
From the Department of Immunology and Transplantation
Biology, Imperial College Faculty of Medicine, Hammersmith Hospital,
London, United Kingdom.
Mesenchymal stem cells (MSCs) have been recently shown to inhibit
T-cell proliferation to polyclonal stimuli. We characterized the effect
of MSCs of bone marrow origin on the T-cell response of naive 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 major histocompatibility complex (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 naive T cells.
Responder cells were stimulated in vitro with male spleen cells or HY
peptides in the presence or absence of MSCs. MSCs inhibited HY-specific
naive and memory T cells in a dose-dependent fashion and affected cell
proliferation, cytotoxicity, and the number of interferon Bone marrow (BM) stroma contains multipotential
nonhematopoietic progenitor cells capable of differentiating into
various tissues of mesenchymal origin. First identified for their
ability to differentiate into bone and adipocytes,1
further studies have demonstrated that mesenchymal stem cells (MSCs)
can also differentiate, under appropriate in vitro conditions, to form chondrocytes, tenocytes, skeletal myocytes, neurons, and cells of
visceral mesoderm.2-5 MSCs 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 BM-derived stromal colonies in vitro. They are
characterized by the absence of hematopoietic markers (CD45 Great potential for MSCs 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 MSCs can inhibit T-cell proliferation induced in a
mixed lymphocyte reaction (MLR) or by nonspecific mitogens.13 Of particular interest is the observation that
in vivo administration of MSCs in baboons significantly prolongs the
survival of major histocompatibility complex (MHC)-mismatched skin
grafts.14 Because 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 molecules on MSCs, it is difficult to ascribe specific T-cell receptor (TCR)/MHC/peptide interactions to their mechanism of immunoregulation. However, MSCs
might selectively inhibit T cells that have encountered antigen, sparing those that have not been activated by TCR engagement. The
susceptibility of naive and memory T cells to immunoregulatory stimuli15 could have profound implications when
considering potential clinical applications of MSCs. We addressed these
questions using an animal model in which the effect of MSCs of BM
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
known.16,17 We found that inhibition requires the presence
of MSCs in culture and MSC-T-cell contact. Both naive and memory cells
are subjected to MSC-mediated suppression but MSCs do not appear to
selectively target antigen-reactive T cells. The MSC inhibitory effect
does not require the presence of antigen-presenting cells (APCs) and is
not mediated through CD4+/CD25+ regulatory T
cells. The implications of these results are discussed.
Mice
C6 TCRhigh mice (C6) are transgenic for the V Generation of MSCs
In selected experiments the 3T3-F442A MSC 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 (Gibco BRL) according to the conditions originally described.20 T-cell cultures All cultures were carried out in RPMI 1640 supplemented with 5 × 10 5 M 2-mercaptoethanol (2-ME), 10% fetal calf
serum (FCS), GLUTAMAX I (Gibco BRL, Life Technologies, United
Kingdom), 100 U/mL penicillin, and 100 µg/mL streptomycin. In
some experiments an anti-transforming growth factor  (TGF-1-2-3) monoclonal blocking antibody (Genzyme, Cambridge, MA) was added to the cultures at a concentration of 1 and 10 µg/mL.21
Depletion of CD25+ cells Depletion of CD25+ cells was performed using MiniMacs system (Miltenyi Biotec, Camberley, United Kingdom). Biotinylated anti-CD25 antibodies (7D4 clone; BD Pharmingen, Germany) were added to spleen cell suspensions at a concentration of 1 µl/30 × 106 cells/mL and incubated at 4°C for 20 minutes. After extensive washing with phosphate-buffered saline (PBS), cells were incubated with Mini-MACS streptavidin MicroBeads (Miltenyi Biotech) for 15 minutes and subjected to passage through selection columns in a magnetic field. Fluorescence-activated cell sorting (FACS) analysis of the eluted fraction (CD25) stained with a phycoerythrin
(PE)-labeled anti-CD25 (PC61 clone) showed CD25+ cells to
be less than 1%.
Antigenic peptides Stimulator cells were pulsed with 1000 ng/mL 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 37°C 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) in a total volume of 0.2 mL RPMI 1640. A total of 0.5 µCi (0.0185 MBq) [3H]-thymidine (ICN, Costa Mesa, CA) was added into each well 2 (naive 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. Uptake of [3H]-thymidine was measured on an LKB Betaplate counter (Wallac Oy). The results are expressed as mean counts per minute (cpm) for triplicate cultures (SEs were routinely < 10%).CD3/CD28 stimulation CD28/CD3-coated Dynabeads (Dynal, Bromborough, United Kingdom) were prepared by conjugating 5 µg CD28 and CD3 antibodies (both from Pharmingen) to 107 beads, according to the manufacturer's instructions. For proliferation, 1 to 5 × 105 responder T cells were incubated with 1 to 5 × 104 CD3/CD28-coated Dynabeads/well. The cells were cultivated in a total volume of 200 µL 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-labeled peptide-pulsed RMA-S target cells at effect-to-target (E/T) ratios of 50:1, 17:1, 5:1, and 1.5:1. After 4 hours, 100 µL supernatant was collected and 51Cr release was measured using a 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 × (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 "Generation of MSCs," MSCs were also stained with CD80 (B7-1), CD86 (B7-2), class I (H2), and class II (H2A) MHC molecules, all purchased from Pharmingen. For immunophenotype analysis, MSCs were detached using trypsin/EDTA (ethylenediaminetetraacetic acid), washed and resuspended at 106/mL. The 100 µL cell suspension was incubated at 4°C for 15 minutes with mouse unconjugated immunoglobulins (Sigma Immunochemicals, Poole, United Kingdom), followed by incubation with the specific antibody at 4°C 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, Heidelberg, Germany) using Cell Quest software.Antigen-specific T-cell responses were measured by enumerating
interferon 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 Transwell cultures Splenocytes (15 × 106/mL) from HY-immunized C57BL/6 female mice were cultivated with irradiated syngeneic male splenocytes (15 × 106/mL) in the lower chamber of a 24-mm diameter Transwell plate with a 0.3-µm pore size membrane (Costar). Autologous or allogeneic MSCs (2 × 105) were seeded onto the Transwell membrane of the inner chamber 1 to 2 hours before the beginning of the culture. Control cultures did not contain MSCs or MSCs 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-positive/CD8+ T cells.
In selected experiments, MSCs were replaced with supernatant from MSC cell cultures. A day before being split, MSC culture supernatant was harvested, centrifuged, and filtered through a 0.2-µm Millipore filter.
MSCs fail to stimulate T cells in secondary H2-mismatched MLR Initial experiments showed MSCs to be poor stimulators of secondary MLRs. Their ability to function as APCs was tested by using MSCs as stimulators of secondary allogeneic MLR. Spleen cells (2 × 105/well) from CBA (H-2k) mice were used as responder cells in primary cultures stimulated with irradiated BALB.B spleen cells (5 × 105/well). Cells harvested at day 7 from these primary cultures were then seeded at 2 × 105/well and restimulated with graded numbers (102, 103, 104/well) of irradiated (60 cGy) MSCs of BALB.B origin. BALB.B splenocytes (2 × 105/well) were used as stimulators in the control cultures. The limit of the maximum number of MSCs (104/well) was chosen because at this concentration MSCs occupy on a 96-well plate the approximately same surface as 2 × 105 splenocytes, the number producing the best stimulation of secondary MLR (not shown). No proliferation was detected at any of the MSCs doses used (Figure 1). No difference was seen whether the MSCs were irradiated or not. This lack of stimulator cell activity was consistent with their phenotype: MSCs expressed neither MHC class I nor class II molecules. The analysis for costimulatory 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 MSCs to present alloantigens, MSCs were pretreated with IFN- prior to their
use as stimulators of secondary MLRs. 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 naive and memory antigen-specific T cells Because, despite the expression of MHC class I and some costimulatory molecules, MSCs 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 MSCs to cultures of both naive 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 naive 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 APCs. Graded doses of MSCs 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 MSCs in culture.
The proliferative activity was significantly inhibited at an
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 MSCs were added as a third party to the MLR. After 7 days, the cells were harvested and
restimulated with H2b-restricted HY peptide-pulsed (HY
DbSmcy, HY DbUty, HY
AbDby) female spleen cells and assessed for
T-cell proliferation (Figure 4A), number
of HY peptide-specific IFN-
MSCs do not specifically target antigen-reactive T cells Although MSCs can inhibit antigen-specific immune responses, it is unlikely that this results from the recognition of the cognate peptide presented by MSCs because they lack expression of MHC molecules. Nevertheless, MSCs might specifically interact with and inhibit only the T cells that become activated after encountering antigen. To address this question we used as responder cells a population containing different proportions of HY-specific T cells but we kept the number of MSCs 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 naive female mice to reduce the concentration of HY-specific T cells to 25% or 5%, respectively. Naive and memory cells were from mice polymorphic for the Thy1 allele (Thy1.1+ and Thy1.2+, respectively) to be able to confine the analysis to the memory T-cell population. Responder cells were restimulated with H2b-restricted HY peptide-pulsed (HY DbSmcy, HY DbUty, HY AbDby) female spleen cells and assessed for number of HY peptide-specific IFN-+ Thy1.2+
cells generated in the presence or absence of a fixed number of MSCs.
In this system the ratio between MSCs and T cells remained the same,
but the ratio between MSCs and HY-specific T cells varied. The
inhibitory effect of MSCs 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 up to
10-fold, we would expect a higher inhibition if MSCs exclusively
targeted antigen-specific T cells. However, no difference in the
magnitude of inhibition was observed at different ratios between MSCs
and HY-specific T cells (Figure 5B).
MSC inhibitory effect is not MHC-dependent MSCs do not constitutively express MHC molecules, but they can do so after treatment with IFN-. In the cultures in which MSCs 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 molecules on MSCs 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 naive 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 difference in the inhibitory activity
was detected in comparison with H2b-matched BALB.B MSCs 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 MSCs
(Figure 3).
MSC inhibitory effect is transient The fact that MHC expression on MSCs does not influence the MSC-mediated inhibition suggests that it also does not require the T cells to recognize antigen on MSCs. Moreover, our data show that MSCs do not exclusively target antigen-reactive T cells. It remained to be determined whether the MSCs induced persistent nonresponsiveness or whether the 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 MSCs as a third party. Twenty-four hours later, nonadherent cells were harvested and depleted of CD106+ cells to exclude the interference of any residual MSCs. The CD106/nonadherent
C6 cells were restimulated with the HY peptide without MSCs.
IFN--producing CD8+ T cells were enumerated 24 hours
later. The results are shown in Figure 7.
Although the presence of MSCs in the first 24-hour culture inhibited
the first antigenic stimulation, when MSCs were removed the response to
the HY peptide was restored. These findings demonstrate that MSCs
inhibit T cells only when they are present in culture, but the effect
is reversed when MSCs are removed.
MSC inhibitory effect requires cell contact The MSC inhibitory effect could be mediated by soluble factors or could require cell contact to be exerted. The fact that the inhibitory activity was dose-dependent and transient (ie, entirely dependent on the presence of MSCs) 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 APCs for 6 hours 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. MSCs were added to the inner
chamber and the MLR was cultivated separately in the lower chamber.
When the MSCs were not in contact with the MLR, the inhibition of
antigen-specific T cells was much reduced (Figure 8A-B). Consistent
with a need for cell-cell contact and the absence of an effect of
soluble factors, we did not find any interleukin 10 (IL-10) in the MSC
culture supernatant as evaluated by enzyme-linked immunosorbent assay
(ELISA); further, addition of anti-TGF-blocking antibody had no
influence on inhibitory activity even when used at concentrations
10-fold higher (10 µg/mL) than those described by
others13 (data not shown).
MSC inhibitory effect is neither dependent on APCs nor on CD4+/CD25+ regulatory T cells The 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 APCs 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 APCs because antibodies interact directly with T cells to activate them. When MSCs 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 an HY DbUty-specific CD8+ T-cell clone (CTL-10)25 as responder cells (Figure 9B).
Alternatively, MSCs 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 activation.26 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-pulsed (HY
KkSmcy) female spleen cells in the presence or
absence of MSCs and 24 hours later assessed for the number of HY
peptide-specific IFN-
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 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 or graft-versus-host disease (GVHD), 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 because it appears that they can differentiate into lineages other than hematopoietic.27-32 BM also contains rare stem cells that differentiate into mesenchymal lineage cells that not only have multipotential differentiation ability33 but also appear to modulate immune responses in vitro. This property could have a substantial impact in transplantation because infusion of MSCs in conjunction with the donor organ or BM might favor engraftment. Administration of MSCs has been reported to prolong donor skin graft survival in nonhuman primates.14 The ability of MSCs 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 MSCs at the time of hemopoietic 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 MSCs as compared to the controls (P = .002 and P = .02, respectively).34 However, the characteristics and the mechanisms of this inhibition are entirely unknown. Here we show that BM-derived MSCs have a profound inhibitory effect on
activation of T cells by their cognate peptides in vitro. This
inhibition, which affects both naive and memory T cells, is manifest in
antigen-specific proliferation, IFN- A crucial question is whether the inhibitory effect depends on the presence of cognate antigen. The evidence that MSCs do not require MHC molecules to inhibit T-cell responses (Figure 6) strongly argues against a cognate mechanism but does not rule out the possibility that MSCs inhibit specifically the cells that are activated by interaction with antigen. However, it is the ratio between MSCs and total T cells rather than with antigen-specific T cells that influences the inhibition (Figure 5). These results favor the notion that MSCs do not specifically target T cells that encounter antigen. Because neither APCs (Figure 9) nor CD4+/CD25+ regulatory T cells (Figure 10) are required for the inhibition to occur, overall our data suggest that MSCs physically hinder T cells from the contact with APCs in a noncognate fashion. Two papers have recently reported that MSCs inhibit T-cell
proliferation induced by nonspecific mitogens or in polyclonal, polyepitope MLRs.13,14 Ours is the first demonstration of
the inhibitory effect of MSCs on T-cell response to cognate peptide. It
is noteworthy that our findings differ from those reported by Di Nicola
et al13 because in their work the effect of MSCs appears
to be mediated by soluble factors, and in particular by TGF- Regardless of the mechanisms underlying the MSC inhibitory effect, the physiologic role of this is unclear. It is well known that mesenchymal elements play a major role in T-cell ontogeny in the thymus. Because we observed that cell contact is required, MSCs might provide a niche in the BM in which lymphocytes are suppressed or deleted. However, the effect of MSCs may not be specific to the immune system but have a more general "inhibitory" activity on protein synthesis 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, MSCs represent an attractive target to develop gene therapy. The evidence that MSCs 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 MSCs 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 BM transplantation is performed following reduced intensity conditioning. More importantly, we have demonstrated that not only naive but also antigen-experienced (memory) T cells can be inhibited. The current ongoing clinical trials involve the infusion of MSCs at the time of BM transplantation.34 When this 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, MSCs have the potential ability to inhibit even ongoing GVHD.
Submitted July 22, 2002; accepted December 16, 2002.
Prepublished online as Blood First Edition Paper, December 27, 2002; DOI 10.1182/blood-2002-07-2104.
Supported by the Leukaemia Research Fund. M.K. was recipient of an AIRC-FIRC (Associazione Italiana Ricerca Cancro-Federazione Italiana Ricerca Cancro) Fellowship ("L Fontana e M Lionello").
M.K. and S.G. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Francesco Dazzi, Department of Immunology, Imperial College Faculty of Medicine, Hammersmith Hospital, Du Cane Rd, London W12 0NN, United Kingdom; e-mail: f.dazzi{at}ic.ac.uk.
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Top
Abstract
Introduction
8 and
V
) or absence
(
)
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 SD.
MSC/R indicates MSC-to-responder T cells ratio. Asterisk signifies
statistically significant (at least P < .01) versus
control cultures without MSCs in all experiments.
) or absence (
