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The immunosuppressive effects of human bone marrow-derived mesenchymal stem cells target T cell proliferation but not its effector function
Cellular Immunology 251 (2008) 131–136
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Cellular Immunology j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y c i m m
The immunosuppressive effects of human bone marrow-derived mesenchymal stem cells target T cell proliferation but not its effector function Rajesh Ramasamy a,b,*, Chih Kong Tong a, Heng Fong Seow a, Sharmili Vidyadaran a, Francesco Dazzi b a b
Immunology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Stem Cell Biology Section, Kennedy Institute of Rheumatology and Division of Investigative Sciences, Imperial College of London, UK
a r t i c l e
i n f o
Article history: Received 1 February 2008 Accepted 16 April 2008 Available online 27 May 2008 Keywords: Mesenchymal stem cells T cells Immunosuppression
a b s t r a c t Mesenchymal stem cells (MSC) are non-haematopoietic stem cells that are capable of differentiating into tissues of mesodermal origin. MSC play an important role in supporting the development of fetal and adult haematopoiesis. More recently, MSC have also been found to exhibit inhibitory effect on T cell responses. However, there is little information on the mechanism of this immunosuppression and our study addresses this issue by targeting T cell functions at various level of immune responses. We have generated MSC from human adult bone marrow (BM) and investigated their immunoregulatory func tion at different phases of T cell responses. MSC showed the ability to inhibit mitogen (CD3/CD28 micro beads)-activated T cell proliferation in a dose-dependent manner. In order to evaluate the specificity of this immunosuppression, the proliferation of CD4+ and CD8+ cells were measured. MSC equally inhibit CD4+ and CD8+ subpopulations of T cells in response to PHA stimulation. However, the antiproliferative effect of MSC is not due to the inhibition of T cell activation. The expression of early activation markers of T cells, namely CD25 and CD69 were not significantly altered by MSC at 24, 48 and 72 h. Furthermore, the immunosuppressive effect of MSC mainly targets T cell proliferation rather than their effector function since cytotoxicity of T cells is not affected. This work demonstrates that the immunosuppressive effect of MSC is exclusively a consequence of an anti-proliferative activity, which targets T cells of different sub populations. For this reason, they have the potential to be exploited in the control of unwanted immune responses such as graft versus host disease (GVHD) and autoimmunity. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Mesenchymal stem cells (MSC) constitute a rare non-haema topoietic population in the adult bone marrow (BM) which can be defined according to its ability to self-renew and differentiate into tissues of mesodermal origin (osteocytes, adipocytes, chondro cytes) [1,2]. They are progenitors of bone marrow stroma and thus play a crucial role in supporting haematopoiesis [3,4] by provid ing haematopoietic progenitors the necessary cytokines and cellmediated signals to self-renew and/or differentiate [5]. There is abundant evidence that not only does the mesenchymal-derived microenvironment affect the differentiation of haematopoietic pro genitors, but also function of mature cells such as lymphocytes. The idea of investigating the effect of MSC on T cell responses comes from the notion that MSC contribute to thymic epithelial cells which are essential in T cell positive selection [6,7]. In fact, a preferential migration of infused donor MSC into recipient thymus after bone marrow transplantation (BMT) has also been observed * Corresponding author. Address: Immunology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Fax: +603 8941 3802. E-mail address: r.ra[email protected] (R. Ramasamy). 0008-8749/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2008.04.009
[7]. Initial studies addressing the immunological properties of MSC showed that they not only fail to stimulate allogeneic T cells but they also exhibit an active immunosuppressive effect. MSC have been tested for their immunosuppressive activity on T cell responses triggered by alloantigen [8–10], peptide antigens [11], mitogens [8,12] and CD3/CD28 antibody [11,12]. The data showed that MSC suppress T cell responses to polyclonal stimuli, poly-epi tope mixed lymphocyte reactions, and their cognate peptide in a dose-dependent fashion. The inhibition does not appear to be antigen-specific [13] and targets both primary and secondary T cell responses [11], but may still exert some selectivity because it appears to discriminate between cellular responses to alloanti gens and recall antigens [10]. The lack of antigen specificity is also supported by the evidence that T cell suppression is not cognate dependent because it can be observed using class I-negative MSC [11] and can be exerted by MSC of different MHC origin from the target T cells [14]. Although these results indicate that MSC affect proliferation of T cells, their effect on T cell activation remains to be elucidated. Some studies have shown that human MSC prevent the expression of CD25, CD38 and CD69 on PHA stimulated CD4+ T cells [15,16]. Others have observed that MSC actually induce a slight increase in
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CD25+ T cells [17]. Murine data suggest that MSC have little or no effect on the expression of activation marker on T cells [11]. Since these are early markers of T cell activation, their reduction at day 3 of co-cultivation with MSC could be the consequence of reduced T cell proliferation. One of the major effector mechanisms of the immune system is cytotoxic T cells (CTL). CD8+ CTL recognize target cells by inter acting with the peptide presented in association with MHC class I molecules. Following the interaction, CTL release perforins and kill the target cell. Human MSC have been shown to inhibit CTL activity when added at the beginning of cultures. However, reduced CTL effector function may be attributable to the diminished prolifera tion of CTL following cultivation with MSC [18,19]. In line with this, murine studies have observed that MSC reduce the number of anti gen-specific IFN-c+/CD8+ cells [20]. T cell responses consist of sequenced phases commencing from cellular activation, proliferation and effector functions. Cellular proliferation is being a vital phase whereby activated T cells are amplified into sufficient copies of antigen-specific T cells in order to perform effector function as cytotoxic cells. Taken together the importance of above phases in the T cell life span, this study spe cifically investigates the effect of MSC on various steps in T cell responses. 2. Materials and methods 2.1. Generation of human mesenchymal stem cell (MSC) 10–20 ml bone marrow samples were obtained from harvests of normal bone marrow donors, ranging in age from 20 to 50 years. All samples were obtained with written, informed consent in accordance with the Hammersmith Hospital and Queen Char lotte’s Hospital Ethical Committee requirements. Mononuclear cells from BM aspirates were isolated using density Ficoll-Paque gradient separation (Amersham-Phamarcia, Piscataway, NJ, USA) and seeded at 5–15 £ 106 cells/25 cm2 in MSC complete medium. The cells were incubated at 37 °C in a humidified 5% CO2 atmo sphere and allowed to adhere for 72 h and non-adherent cells were then removed. The medium was changed twice a week. When the cells were 80–90% confluent, adherent cells were tryp sinized (0.05% trypsin, Gibco BRL) at 37 °C for 5 min and replated in 25 or 75 cm2 flasks (BD Falcon, MA, USA). After passage 3, a morphologically homogenous population of adherent cells was obtained. During the expansion, the medium was changed every 4–5 days. The cells were analyzed for cell surface markers before use in various experiments. 2.2. T cells Buffy coats from healthy donors for T cell experiments were pur chased from the National Blood Service (NBS, Colindale Edgware London). The buffy coats were diluted with RPMI and layered on Ficoll-Paque for density gradient separation. The mononuclear cells thus obtained were cryopreserved in freezing medium (10% DMSO, and 90% heat-inactivated FBS) and thawed for each experi ments. PBMC were cultured in complete T cell medium which con sist of RPMI 1640 (Gibco-BRL,) supplemented with 10% human AB serum (Sigma–Aldrich) and 1% penicillin/streptomycin/amphoteri cin (Gibco-BRL). 2.3. Polyclonal stimulation of PBMC In mitogen-induced proliferation assays, responder PBMC (0.5 £ 105 cells) were cultured with either phytohemagglutinin (PHA) at 5–10 lg/ml (Sigma–Aldrich, USA), and 0.2 ll/well antiCD3/28 microbeads (Dynal, Wirral, UK). The proliferation assays
were pulsed at day 3 for 18 h with 0.037 MBq/well 3H TdR and har vested on day 4. 2.4. Antigen-specific T cell expansion PBMC from buffy coats were cultivated with cytomegalovirus (CMV, NLVPMVATV), or Epstein–Barr virus (EBV, RAKFKQLL) antigenic peptides (Proimmune, Oxford, UK). Buffy coats were selected based on the presence of HLA-B8 EBV or HLA-A2 CMVspecific CD8-positive T cells identified by the positive staining with PE-conjugated EBV HLA-B8 or CMV HLA-A2 tetramers (Pro immune, Oxford, UK). Peptide-driven T cell expansion was carried out in the presence of 10 lg/ml MHC class I viral peptide (Proim mune), 50 U/ml interleukin 2 (IL-2) (Roche), 50 U/ml Interleukin 7 (IL-7) (Roche, Pitsacaway, NJ) in T cell media in 24 or 6 well plates for 7 days. 2.5. Intracellular cytokine detection and tetramer staining After 7 days culture in which T cells were stimulated with the viral peptide, expanded T cells were harvested, washed with RPMI, and 1.0 £ 106 of the T cells were restimulated for 6 h with previ ously irradiated and peptide pulsed autologous PBMC or T2 cells at 1:2 ratio. The T2 cell line is HLA-A2 positive and defective in intracellular peptide loading of class I molecules that makes the cells present extracellular peptide coupled with HLA-A2 MHC I molecules. Non-peptide pulsed and irradiated autologous PBMC or T2 cells were used as negative controls. Brefeldin A (BFA; Sigma– Aldrich) was added at 10 lg/ml in the last 4 h of culture of a 6 h pep tide restimulation to block the Golgi apparatus to inhibit cytokine secretion. Restimulated T cells were washed with PBS and labelled with APC-conjugated anti-CD8 monoclonal antibody for 15 min at 4 °C and then washed with PBS. Then the cells were permeabilized with 500 ll of 1£ Permeabilising solution (BD, Pharmingen) for 10 min at room temperature. After the permeabilization, the cells were washed with cytokine buffer (0.5 nM Sodium Azide and 1 mg/ ml bovine serum albumin (Sigma–Aldrich) in PBS and blocked by technical grade anti-mouse IgG antibody for 15 min in room tem perature. After the wash, the cells were incubated with FITC-conju gated anti-interferon-c antibody or with FITC-conjugated isotype antibody as a control. 2.6. 3H-Thymidine assay Cell proliferation was measured by tritiated thymidine (3H-TdR) (Amersham, Buckingham, UK) incorporation, which reflects the percentage of cells in S phase of the cell cycle. Cultures in 96-well plates were pulsed with 3H-TdR (0.037 MBq/well [0.5 lCi/well] dur ing the final 18 h of incubation. At the end of incubation the cells were harvested onto glass fibre filter mats (Perkin-Elmer) using a 96-well plate automated cell harvester (Skatron, Lier, Norway), scintillation fluid was added and thymidine incorporation was measured by liquid scintillation spectroscopy on a beta counter (Wallac). The results were expressed in counts per minute (cpm) or percentage of control proliferation. 2.7. CFSE assay In certain experiments, cell proliferation was also measured by the cell membrane-bound dye CFSE (Sigma). Division of CFSE labelled cells will reflected by a reduction in CFSE intensity. Cells were washed in PBS prior to staining and 5 £ 106 cells were incu bated with 1.7 lM of CFSE in 10 ml PBS at room temperature for 8 min. Then cells were washed once with FCS followed by second washing with complete medium. Cells were analysed by flow cytometry analysis.
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3. Results 3.1. MSC inhibit polyclonally stimulated PBMC The effect of MSC on T cell proliferation was evaluated by add ing MSC as third party to PBMC in the presence of various stim uli. T cell proliferation induced by anti-CD3/CD28-stimulation was measured by 3H-thymidine uptake after a 3-day culture. MSC inhibited anti-CD3/CD28 stimulated PBMC proliferation in a dosedependent fashion (Fig. 1). The same results were obtained when MSC were added to PHA or allogenic (mixed lymphocytes reaction, MLR) stimulated PBMC [data not shown]. 3.2. MSC inhibit both CD4- and CD8-positive T cells To investigate whether the inhibitory effect of MSC was con fined to a specific T cell subpopulation, CFSE-labelled PBMC were cultured with PHA in the presence or absence of MSC and the proliferation of CD4-positive and CD8-positive cells assessed at different time points. Using this method system the number of T cell division can be correlated to the reduction in CFSE intensity. T cell proliferation could be detected after 48 h incubation. The MSC induced inhibition of cell proliferation was greatest at 96 h. MSC equally inhibited proliferation of both CD4-positive and CD8positive T cells (87% vs. 90%, respectively) (Fig. 2). 3.3. MSC inhibit antigen-specific T cell responses In order to determine the effect of MSC on antigen-specific T cell proliferation and function, MSC were added at ratio of 1:10 of PBMC from HLA-A2+ and CMV seropositive individuals. PBMC were stimulated with HLA-A2 restricted pp65 CMV SEQUENCE peptide. Antigen-specific T cell expansion was measured by enumerating CMV HLA-A2-pp65 tetramer positive T cells. IFN-c+ T cells were enumerated upon further restimulation with CMV peptide. HLAA2 pp65 tetramer+/CD8+ and pp65-specific/IFN-c+ T cell expansion was inhibited by MSC (Fig. 3). T cell proliferation and the percent age of IFN-c producing T cells were reduced by 95% in comparison with culture without MSC.
Fig. 2. Both CD4- and CD8-positive T cells are inhibited by MSC. Two million PBMC were co-cultured with (red histogram) or without (purple histogram) MSC at 1:10 of MSC:T cell ratio for 24, 48, 72 and 96 h and stimulated with PHA. At the end of the incubation period, the cells were collected and stained with CD4 (A) and CD8 (B) antibodies analysed by a flow cytometer. Non-stimulated PBMC with and without MSC were used as negative controls in this assay. This figure represents CD4 and CD8 cell proliferation on 96 h. Unstimulated PBMC cocultured with MSC did not pro liferate. Data show a representative result from three experiments.
this early effect on T cells, CD3-/CD28-stimulated PBMC were cul tured with or without MSC at a 1:10 ratio in 24-well plates. The expression of CD25 and CD69 was assessed by flow cytometry at 24, 48 and 72 h. The percentages of CD25- and CD69-positive cells were increased in stimulated T cells regardless of the presence or absence of MSC at 24 and 48 h (Fig. 4). Thus, the inhibitory mecha nism of MSC does not affect the activation status of T cells. 3.5. MSC do not affect cytotoxic effector function of T cells
3.4. MSC do not prevent the priming of T cell activation Antigen or mitogen stimulation of T cells rapidly induces the upregulation of activation markers CD25, CD69 and protein synthe sis. To test whether the immunosuppressive effect of MSC prevents
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Our data have shown that MSC profoundly inhibit T cell pro liferation and the number of IFN-c antigen-specific T cells upon cognate antigen re-challenge. However the dramatic reduction in IFN-c+/CD8+ cells may be attributable to the failure of T cell expansion. To overcome this question, we tested the effect of MSC in inhibiting the killing ability of cytotoxic CD8+ T cells. WT126 specific T cells were expanded in the presence of WT126 peptide for 7 days. For cytotoxic assay, T2 cells pulsed with WT126 pep tide and loaded with radioactive chromium were used as target cells. WT126-specific T cells (effector cells) and WT126 pulsed T2 cells (target cells) were mixed at different ratios. The result shows that the cytotoxicity of WT126-specific T cells to lyse target cells, WT126 pulsed T2 cells was unaffected in the present of MSC at dif ferent ratios (Fig. 5). 4. Discussion
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Much evidence have indicated that MSC exert an immunosup pressive effect on T cell activity [8,11]. The key component of the immune system is T cells and it plays a major role in adaptive immunity. T cell responses consist of a sequence of cellular acti vation, proliferation and effector function. The initial work by Di Nicola explored the ability of MSC to inhibit mitogen stimulated T cell proliferation [8]. This study has led to further research and the immunosuppressive effect of MSC were tested using various
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Fig. 3. MSC inhibit the expansion and IFN-c production of antigen-specific CD8+/CMV HLA-A2-pp65+ T cells. Two million PBMC positive for the CMV HLA-A2-pp65 were cul tured with MSC at 1:10 ratio for 7 days. Flowcytometry density plots show CMV HLA-A2-pp65 tetramer staining and IFN-c secretion when rechallenged with peptide pulsed T2 cells in the presence (B) and absence (A) of MSC. This is a representive result from three different experiments.
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Fig. 4. MSC do not prevent T cell priming upon stimulation. Two million PBMC were cultured with MSC at a 1:10 ratio and with anti-CD3/28 microbeads as stimulators at different time points. At the end of incubation, CD4+ (A) and CD8+ (B) T cells were harvested and labelled for CD25 and CD69 antibodies and assessed by flowcytometry. The results show mean of two independent experiments.
modes of T cell stimulation, including allogenic or antigen-specific stimulation [14,20]. However, most studies were limited at examin ing immunosuppressive effects of MSC at one or two phases of a T cell responses. Various phases of T cell responses such as activa tion, proliferation and effector function require different cellular
and molecular mechanisms. Therefore it is important to dissect the effect of MSC at these phases. We found that MSC were unable to stimulate resting allogeneic T cells proliferation and IFN-c produc tion despite normal expression of MHC class I (data not shown). This deficit may be related to the fact that MSC do not express co-
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Effector:Target ratios Fig. 5. MSC did not affect effector function of T cells. WT126-specific cells were expanded for 6 days in the presence of WT-126 peptide and feeder cell layer. At day 7, CTL were retrieved and incubated with Cr-56-labelled WT126-pulsed T2 cells in the presence and absence of MSC for 6 h incubation. The effect of MSC on effector function as measured by their ability to lyse target cells was measured by a-reader and expressed in percentage.
stimulatory molecules (CD80 and CD86) [data not shown], which are crucial for providing the necessary second signal for T cell activation. However, neither IFN-c pre-treatment [21] nor retro viral-mediated MSC transduction with co-stimulatory molecules confers the ability to induce a T cell proliferative response upon MSC [22,23]. When MSC were added to T cells stimulated by mitogen, T cell proliferation was reduced in a dose-dependent manner. The inhibi tory effects of MSC do not target any specific T cell subpopulations since both CD4+ and CD8+ positive T cells were equally affected. Furthermore the inhibitory effect of MSC also can be extended to a more defined antigen-specific T cell population. The expansion of antigen-specific T cells and the ability to secrete IFN-c against cog nate antigen re-challenge were dramatically reduced when MSC were cocultured with CMV serotype positive T cells and relevant antigen peptides. These proliferation assays confirm that MSC not only inhibit antigen-specific T cells but also other cells within the T cell subpopulations. Therefore, MSC exert a non-specific immuno suppressive activity on T cells. In line with this, others have shown that MSC inhibit both naïve and memory T cells responses in a mouse model [11]. The effect of the inhibition was further charac terised by dissecting T cell activation and effector function. As a consequence of TCR engagement, T cells are activated and express CD25 which is the a chain of IL-2 receptor and CD69. CD25 expres sion is required for autocrine IL-2-mediated clonal expansion while CD69 expression enhances the activation and serves as a co-stimulatory molecule [24]. We observed that MSC do not signif icantly alter the expression of these early activation markers at 24 and 48 h. While increased CD25 expression could be derived from increasing regulatory T cells however, the simultaneous increase in CD69 co-expression argues against this hypothesis. A small decline in the percentage of CD25+ cells was noticed at 72 h and this might be the consequence of an anti-proliferative effect induced by MSC, hence the reduced number of activated T cells. Although MSC exerted a profound anti proliferative effect on T cells, their effector function was not prevented. The ability of WT-126 specific CTL to lyse WT-126 peptide pulsed T2 cells was unaffected in the presence of MSC. These data are consistent with the findings of Rasmusson et al. who showed that lympholysis of target PBMC was not affected when MSC were added at effector phase or at the third day of 6-day MLR [18]. In their experiments when MSC were cultured at the beginning of the MLR they inhib ited CTL cytotoxicity. However, the inhibition of cytotoxicity could be explained by the fact that MSC might have prevented CTL gen
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eration/expansion during the culture. In accordance with this, we have observed that the number of IFN-c+ T cells at the 7th day of antigen-specific stimulation was dramatically reduced as a result of the prevented expansion (Fig. 3). Our data strongly support the fact that the inhibitory effect of MSC on T cell responses is confined to cellular proliferation rather then its effector function which does not require cell proliferation. However, it is still possible that the incubation time of CTL with MSC was too short for MSC to exert an inhibitory effect. The immunosuppressive activity of MSC may be exploited for therapeutic purposes. Since MSC can produce an anti-proliferative effect on T cells, they promise to be an attractive therapy for graft versus host disease (GVHD). Pathophysiology of GVHD is initiated by host-specific donor T cells clonal proliferation whereby the early inhibition host-specific T cell expansion would be ideal for GVHD amelioration. Although this concept is still in a premature stage, a few phase I and II clinical trials have shown that infusion of MSC appears to suppress GVHD without inducing any toxicity [25,26]. A potential disadvantage of MSC as treatment for GVHD is that they may also suppress graft versus leukaemia (GVL) leading to an increased risk of leukaemia relapse. However, clinical trials have shown that infusion of MSC appears to suppress GVHD with out interfering with the GVL effect [27,28]. If this were true, the pro tective effect of MSC on GVHD might be ascribable to their tissue repair activity. In fact, the T cells responsible for GVL and GVHD are virtually the same and require a major expansion. In this perspec tive, one could expand in vitro mature leukaemia-reactive or virusspecific T cells and administer them to patients without the risk of MSC interfering with their effector function [29,30]. Acknowledgment This project was funded by Science Fund (Project No: 02-01-04SF0028) Ministry of Science, Technology and Innovation (MOSTI), Malaysia. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellimm.2008.04.009. References [1] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [2] E.E. Horwitz, Clarification of the nomenclature for MSC: the international society for cellular therapy position statement, Cytotherapy 7 (2005) 393– 395. [3] L.M. Calvi, G.B. Adams, K.W. Weibrecht, J.M. Weber, D.P. Olson, M.C. Knight, R.P. Martin, E. Schipani, P. Divieti, F.R. Bringhurst, et al., Osteoblastic cells regu late the haematopoietic stem cell niche, Nature 425 (2003) 841–846. [4] J. Zhang, C. Niu, L. Ye, H. Huang, X. He, W.G. Tong, J. Ross, J. Haug, T. Johnson, J.Q. Feng, et al., Identification of the haematopoietic stem cell niche and con trol of the niche size, Nature 425 (2003) 836–841. [5] F. Dazzi, R. Ramasamy, S. Glennie, S. Jones, I. Roberts, The role of mesenchymal stem cells in haemopoiesis, Blood Rev. 20 (2005) 161–171. [6] A.A. Rzhaninova, S.N. Gornostaeva, D.V. Goldshtein, Isolation and phenotyp ical characterization of mesenchymal stem cells from human fetal thymus, Bull. Exp. Biol. Med. 139 (2005) 134–140. [7] R.K. Suniara, E.J. Jenkinson, J.J. Owen, An essential role for thymic mesenchyme in early T cell development, J. Exp. Med. 191 (2000) 1051–1056. [8] M. Di Nicola, C. Carlo-Stella, M. Magni, M. Milanesi, P.D. Longoni, P. Matteucci, S. Grisanti, A.M. Gianni, Human bone marrow stromal cells suppress T-lympho cyte proliferation induced by cellular or nonspecific mitogenic stimuli, Blood 99 (2000) 3838–3843. [9] W.T. Tse, J.D. Pendleton, W.M. Beyer, M.C. Egalka, E.C. Guinan, Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation, Transplantation 75 (2003) 389–397. [10] J.A. Potian, H. Aviv, N.M. Ponzio, J.S. Harrison, P. Rameshwar, Veto-like activ ity of mesenchymal stem cells: functional discrimination between cellular responses to alloantigens and recall antigens, J. Immunol. 171 (2003) 3426– 3434.
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nomodulatory activity of human bone marrow mesenchymal stem cells, Stem Cells (2005) 2005–2008. [22] N.J. Zvaifler, L. Marinova-Mutafchieva, G. Adams, C.J. Edwards, J. Moss, J.A. Bur ger, R.N. Maini, Mesenchymal precursor cells in the blood of normal individu als, Arthritis Res. 2 (2002) 477–488. [23] M.K. Majumdar, M. Keane-Moore, D. Buyaner, W.B. Hardy, M.A. Moorman, K.R. McIntosh, J.D. Mosca, Characterization and functionality of cell surface molecules on human mesenchymal stem cells, J. Biomed. Sci. 10 (2003) 228– 241. [24] D. Sancho, M. Gomez, F. Sanchez-Madrid, CD69 is an immunoregulatory molecule induced following activation, Trends Immunol. 26 (2005) 136– 140. [25] H.M. Lazarus, O.N. Koc, S.M. Devine, P. Curtin, R.T. Maziarz, H.K. Holland, E.J. Shpall, P. McCarthy, K. Atkinson, B.W. Cooper, et al., Cotransplantation of HLAidentical sibling culture-expanded mesenchymal stem cells and hematopoi etic stem cells in hematologic malignancy patients, Biol. Blood Marrow Trans plant. 11 (2005) 389–398. [26] E.M. Horwitz, D.J. Prockop, L.A. Fitzpatrick, W.W. Koo, P.L. Gordon, M. Neel, M. Sussman, P. Orchard, J.C. Marx, R.E. Pyeritz, et al., Transplantability and ther apeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta, Nat. Med. 5 (1999) 309–313. [27] L. Seung Tae, J. Joon Ho, C. June-Won, K. Jin Seok, M. Ho-Young, H. Jee Sook, K. Yun Woong, S. You, Treatment of hight risk acute myelogenous leukaemia by myeloblative chemotherapy followed by co-infusion of T cell-depleted hae matopoietic stem cells and culture-expanded marrow mesenchymal stem cell from a related donor with one fully mismatched human leucocytes antigen haplotype, Br. J. Haematol. (2002) 1128–1131. [28] K. Le Blanc, I. Rasmusson, B. Sundberg, C. Gotherstrom, M. Hassan, M. Uzunel, O. Ringden, Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells, Lancet 363 (2004) 1439–1441. [29] S. Gottschalk, H.E. Heslop, C.M. Rooney, Adoptive immunotherapy for EBVassociated malignancies, Leuk. Lymphoma 46 (2005) 1–10. [30] J.J. Molldrem, P.P. Lee, C. Wang, K. Felio, H.M. Kantarjian, R.E. Champlin, M.M. Davis, Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia, Nat. Med. 6 (2000) 1018–1023.
Contents lists available at ScienceDirect
Cellular Immunology j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y c i m m
The immunosuppressive effects of human bone marrow-derived mesenchymal stem cells target T cell proliferation but not its effector function Rajesh Ramasamy a,b,*, Chih Kong Tong a, Heng Fong Seow a, Sharmili Vidyadaran a, Francesco Dazzi b a b
Immunology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Stem Cell Biology Section, Kennedy Institute of Rheumatology and Division of Investigative Sciences, Imperial College of London, UK
a r t i c l e
i n f o
Article history: Received 1 February 2008 Accepted 16 April 2008 Available online 27 May 2008 Keywords: Mesenchymal stem cells T cells Immunosuppression
a b s t r a c t Mesenchymal stem cells (MSC) are non-haematopoietic stem cells that are capable of differentiating into tissues of mesodermal origin. MSC play an important role in supporting the development of fetal and adult haematopoiesis. More recently, MSC have also been found to exhibit inhibitory effect on T cell responses. However, there is little information on the mechanism of this immunosuppression and our study addresses this issue by targeting T cell functions at various level of immune responses. We have generated MSC from human adult bone marrow (BM) and investigated their immunoregulatory func tion at different phases of T cell responses. MSC showed the ability to inhibit mitogen (CD3/CD28 micro beads)-activated T cell proliferation in a dose-dependent manner. In order to evaluate the specificity of this immunosuppression, the proliferation of CD4+ and CD8+ cells were measured. MSC equally inhibit CD4+ and CD8+ subpopulations of T cells in response to PHA stimulation. However, the antiproliferative effect of MSC is not due to the inhibition of T cell activation. The expression of early activation markers of T cells, namely CD25 and CD69 were not significantly altered by MSC at 24, 48 and 72 h. Furthermore, the immunosuppressive effect of MSC mainly targets T cell proliferation rather than their effector function since cytotoxicity of T cells is not affected. This work demonstrates that the immunosuppressive effect of MSC is exclusively a consequence of an anti-proliferative activity, which targets T cells of different sub populations. For this reason, they have the potential to be exploited in the control of unwanted immune responses such as graft versus host disease (GVHD) and autoimmunity. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Mesenchymal stem cells (MSC) constitute a rare non-haema topoietic population in the adult bone marrow (BM) which can be defined according to its ability to self-renew and differentiate into tissues of mesodermal origin (osteocytes, adipocytes, chondro cytes) [1,2]. They are progenitors of bone marrow stroma and thus play a crucial role in supporting haematopoiesis [3,4] by provid ing haematopoietic progenitors the necessary cytokines and cellmediated signals to self-renew and/or differentiate [5]. There is abundant evidence that not only does the mesenchymal-derived microenvironment affect the differentiation of haematopoietic pro genitors, but also function of mature cells such as lymphocytes. The idea of investigating the effect of MSC on T cell responses comes from the notion that MSC contribute to thymic epithelial cells which are essential in T cell positive selection [6,7]. In fact, a preferential migration of infused donor MSC into recipient thymus after bone marrow transplantation (BMT) has also been observed * Corresponding author. Address: Immunology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Fax: +603 8941 3802. E-mail address: r.ra[email protected] (R. Ramasamy). 0008-8749/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2008.04.009
[7]. Initial studies addressing the immunological properties of MSC showed that they not only fail to stimulate allogeneic T cells but they also exhibit an active immunosuppressive effect. MSC have been tested for their immunosuppressive activity on T cell responses triggered by alloantigen [8–10], peptide antigens [11], mitogens [8,12] and CD3/CD28 antibody [11,12]. The data showed that MSC suppress T cell responses to polyclonal stimuli, poly-epi tope mixed lymphocyte reactions, and their cognate peptide in a dose-dependent fashion. The inhibition does not appear to be antigen-specific [13] and targets both primary and secondary T cell responses [11], but may still exert some selectivity because it appears to discriminate between cellular responses to alloanti gens and recall antigens [10]. The lack of antigen specificity is also supported by the evidence that T cell suppression is not cognate dependent because it can be observed using class I-negative MSC [11] and can be exerted by MSC of different MHC origin from the target T cells [14]. Although these results indicate that MSC affect proliferation of T cells, their effect on T cell activation remains to be elucidated. Some studies have shown that human MSC prevent the expression of CD25, CD38 and CD69 on PHA stimulated CD4+ T cells [15,16]. Others have observed that MSC actually induce a slight increase in
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CD25+ T cells [17]. Murine data suggest that MSC have little or no effect on the expression of activation marker on T cells [11]. Since these are early markers of T cell activation, their reduction at day 3 of co-cultivation with MSC could be the consequence of reduced T cell proliferation. One of the major effector mechanisms of the immune system is cytotoxic T cells (CTL). CD8+ CTL recognize target cells by inter acting with the peptide presented in association with MHC class I molecules. Following the interaction, CTL release perforins and kill the target cell. Human MSC have been shown to inhibit CTL activity when added at the beginning of cultures. However, reduced CTL effector function may be attributable to the diminished prolifera tion of CTL following cultivation with MSC [18,19]. In line with this, murine studies have observed that MSC reduce the number of anti gen-specific IFN-c+/CD8+ cells [20]. T cell responses consist of sequenced phases commencing from cellular activation, proliferation and effector functions. Cellular proliferation is being a vital phase whereby activated T cells are amplified into sufficient copies of antigen-specific T cells in order to perform effector function as cytotoxic cells. Taken together the importance of above phases in the T cell life span, this study spe cifically investigates the effect of MSC on various steps in T cell responses. 2. Materials and methods 2.1. Generation of human mesenchymal stem cell (MSC) 10–20 ml bone marrow samples were obtained from harvests of normal bone marrow donors, ranging in age from 20 to 50 years. All samples were obtained with written, informed consent in accordance with the Hammersmith Hospital and Queen Char lotte’s Hospital Ethical Committee requirements. Mononuclear cells from BM aspirates were isolated using density Ficoll-Paque gradient separation (Amersham-Phamarcia, Piscataway, NJ, USA) and seeded at 5–15 £ 106 cells/25 cm2 in MSC complete medium. The cells were incubated at 37 °C in a humidified 5% CO2 atmo sphere and allowed to adhere for 72 h and non-adherent cells were then removed. The medium was changed twice a week. When the cells were 80–90% confluent, adherent cells were tryp sinized (0.05% trypsin, Gibco BRL) at 37 °C for 5 min and replated in 25 or 75 cm2 flasks (BD Falcon, MA, USA). After passage 3, a morphologically homogenous population of adherent cells was obtained. During the expansion, the medium was changed every 4–5 days. The cells were analyzed for cell surface markers before use in various experiments. 2.2. T cells Buffy coats from healthy donors for T cell experiments were pur chased from the National Blood Service (NBS, Colindale Edgware London). The buffy coats were diluted with RPMI and layered on Ficoll-Paque for density gradient separation. The mononuclear cells thus obtained were cryopreserved in freezing medium (10% DMSO, and 90% heat-inactivated FBS) and thawed for each experi ments. PBMC were cultured in complete T cell medium which con sist of RPMI 1640 (Gibco-BRL,) supplemented with 10% human AB serum (Sigma–Aldrich) and 1% penicillin/streptomycin/amphoteri cin (Gibco-BRL). 2.3. Polyclonal stimulation of PBMC In mitogen-induced proliferation assays, responder PBMC (0.5 £ 105 cells) were cultured with either phytohemagglutinin (PHA) at 5–10 lg/ml (Sigma–Aldrich, USA), and 0.2 ll/well antiCD3/28 microbeads (Dynal, Wirral, UK). The proliferation assays
were pulsed at day 3 for 18 h with 0.037 MBq/well 3H TdR and har vested on day 4. 2.4. Antigen-specific T cell expansion PBMC from buffy coats were cultivated with cytomegalovirus (CMV, NLVPMVATV), or Epstein–Barr virus (EBV, RAKFKQLL) antigenic peptides (Proimmune, Oxford, UK). Buffy coats were selected based on the presence of HLA-B8 EBV or HLA-A2 CMVspecific CD8-positive T cells identified by the positive staining with PE-conjugated EBV HLA-B8 or CMV HLA-A2 tetramers (Pro immune, Oxford, UK). Peptide-driven T cell expansion was carried out in the presence of 10 lg/ml MHC class I viral peptide (Proim mune), 50 U/ml interleukin 2 (IL-2) (Roche), 50 U/ml Interleukin 7 (IL-7) (Roche, Pitsacaway, NJ) in T cell media in 24 or 6 well plates for 7 days. 2.5. Intracellular cytokine detection and tetramer staining After 7 days culture in which T cells were stimulated with the viral peptide, expanded T cells were harvested, washed with RPMI, and 1.0 £ 106 of the T cells were restimulated for 6 h with previ ously irradiated and peptide pulsed autologous PBMC or T2 cells at 1:2 ratio. The T2 cell line is HLA-A2 positive and defective in intracellular peptide loading of class I molecules that makes the cells present extracellular peptide coupled with HLA-A2 MHC I molecules. Non-peptide pulsed and irradiated autologous PBMC or T2 cells were used as negative controls. Brefeldin A (BFA; Sigma– Aldrich) was added at 10 lg/ml in the last 4 h of culture of a 6 h pep tide restimulation to block the Golgi apparatus to inhibit cytokine secretion. Restimulated T cells were washed with PBS and labelled with APC-conjugated anti-CD8 monoclonal antibody for 15 min at 4 °C and then washed with PBS. Then the cells were permeabilized with 500 ll of 1£ Permeabilising solution (BD, Pharmingen) for 10 min at room temperature. After the permeabilization, the cells were washed with cytokine buffer (0.5 nM Sodium Azide and 1 mg/ ml bovine serum albumin (Sigma–Aldrich) in PBS and blocked by technical grade anti-mouse IgG antibody for 15 min in room tem perature. After the wash, the cells were incubated with FITC-conju gated anti-interferon-c antibody or with FITC-conjugated isotype antibody as a control. 2.6. 3H-Thymidine assay Cell proliferation was measured by tritiated thymidine (3H-TdR) (Amersham, Buckingham, UK) incorporation, which reflects the percentage of cells in S phase of the cell cycle. Cultures in 96-well plates were pulsed with 3H-TdR (0.037 MBq/well [0.5 lCi/well] dur ing the final 18 h of incubation. At the end of incubation the cells were harvested onto glass fibre filter mats (Perkin-Elmer) using a 96-well plate automated cell harvester (Skatron, Lier, Norway), scintillation fluid was added and thymidine incorporation was measured by liquid scintillation spectroscopy on a beta counter (Wallac). The results were expressed in counts per minute (cpm) or percentage of control proliferation. 2.7. CFSE assay In certain experiments, cell proliferation was also measured by the cell membrane-bound dye CFSE (Sigma). Division of CFSE labelled cells will reflected by a reduction in CFSE intensity. Cells were washed in PBS prior to staining and 5 £ 106 cells were incu bated with 1.7 lM of CFSE in 10 ml PBS at room temperature for 8 min. Then cells were washed once with FCS followed by second washing with complete medium. Cells were analysed by flow cytometry analysis.
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3. Results 3.1. MSC inhibit polyclonally stimulated PBMC The effect of MSC on T cell proliferation was evaluated by add ing MSC as third party to PBMC in the presence of various stim uli. T cell proliferation induced by anti-CD3/CD28-stimulation was measured by 3H-thymidine uptake after a 3-day culture. MSC inhibited anti-CD3/CD28 stimulated PBMC proliferation in a dosedependent fashion (Fig. 1). The same results were obtained when MSC were added to PHA or allogenic (mixed lymphocytes reaction, MLR) stimulated PBMC [data not shown]. 3.2. MSC inhibit both CD4- and CD8-positive T cells To investigate whether the inhibitory effect of MSC was con fined to a specific T cell subpopulation, CFSE-labelled PBMC were cultured with PHA in the presence or absence of MSC and the proliferation of CD4-positive and CD8-positive cells assessed at different time points. Using this method system the number of T cell division can be correlated to the reduction in CFSE intensity. T cell proliferation could be detected after 48 h incubation. The MSC induced inhibition of cell proliferation was greatest at 96 h. MSC equally inhibited proliferation of both CD4-positive and CD8positive T cells (87% vs. 90%, respectively) (Fig. 2). 3.3. MSC inhibit antigen-specific T cell responses In order to determine the effect of MSC on antigen-specific T cell proliferation and function, MSC were added at ratio of 1:10 of PBMC from HLA-A2+ and CMV seropositive individuals. PBMC were stimulated with HLA-A2 restricted pp65 CMV SEQUENCE peptide. Antigen-specific T cell expansion was measured by enumerating CMV HLA-A2-pp65 tetramer positive T cells. IFN-c+ T cells were enumerated upon further restimulation with CMV peptide. HLAA2 pp65 tetramer+/CD8+ and pp65-specific/IFN-c+ T cell expansion was inhibited by MSC (Fig. 3). T cell proliferation and the percent age of IFN-c producing T cells were reduced by 95% in comparison with culture without MSC.
Fig. 2. Both CD4- and CD8-positive T cells are inhibited by MSC. Two million PBMC were co-cultured with (red histogram) or without (purple histogram) MSC at 1:10 of MSC:T cell ratio for 24, 48, 72 and 96 h and stimulated with PHA. At the end of the incubation period, the cells were collected and stained with CD4 (A) and CD8 (B) antibodies analysed by a flow cytometer. Non-stimulated PBMC with and without MSC were used as negative controls in this assay. This figure represents CD4 and CD8 cell proliferation on 96 h. Unstimulated PBMC cocultured with MSC did not pro liferate. Data show a representative result from three experiments.
this early effect on T cells, CD3-/CD28-stimulated PBMC were cul tured with or without MSC at a 1:10 ratio in 24-well plates. The expression of CD25 and CD69 was assessed by flow cytometry at 24, 48 and 72 h. The percentages of CD25- and CD69-positive cells were increased in stimulated T cells regardless of the presence or absence of MSC at 24 and 48 h (Fig. 4). Thus, the inhibitory mecha nism of MSC does not affect the activation status of T cells. 3.5. MSC do not affect cytotoxic effector function of T cells
3.4. MSC do not prevent the priming of T cell activation Antigen or mitogen stimulation of T cells rapidly induces the upregulation of activation markers CD25, CD69 and protein synthe sis. To test whether the immunosuppressive effect of MSC prevents
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Our data have shown that MSC profoundly inhibit T cell pro liferation and the number of IFN-c antigen-specific T cells upon cognate antigen re-challenge. However the dramatic reduction in IFN-c+/CD8+ cells may be attributable to the failure of T cell expansion. To overcome this question, we tested the effect of MSC in inhibiting the killing ability of cytotoxic CD8+ T cells. WT126 specific T cells were expanded in the presence of WT126 peptide for 7 days. For cytotoxic assay, T2 cells pulsed with WT126 pep tide and loaded with radioactive chromium were used as target cells. WT126-specific T cells (effector cells) and WT126 pulsed T2 cells (target cells) were mixed at different ratios. The result shows that the cytotoxicity of WT126-specific T cells to lyse target cells, WT126 pulsed T2 cells was unaffected in the present of MSC at dif ferent ratios (Fig. 5). 4. Discussion
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Much evidence have indicated that MSC exert an immunosup pressive effect on T cell activity [8,11]. The key component of the immune system is T cells and it plays a major role in adaptive immunity. T cell responses consist of a sequence of cellular acti vation, proliferation and effector function. The initial work by Di Nicola explored the ability of MSC to inhibit mitogen stimulated T cell proliferation [8]. This study has led to further research and the immunosuppressive effect of MSC were tested using various
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Fig. 3. MSC inhibit the expansion and IFN-c production of antigen-specific CD8+/CMV HLA-A2-pp65+ T cells. Two million PBMC positive for the CMV HLA-A2-pp65 were cul tured with MSC at 1:10 ratio for 7 days. Flowcytometry density plots show CMV HLA-A2-pp65 tetramer staining and IFN-c secretion when rechallenged with peptide pulsed T2 cells in the presence (B) and absence (A) of MSC. This is a representive result from three different experiments.
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Fig. 4. MSC do not prevent T cell priming upon stimulation. Two million PBMC were cultured with MSC at a 1:10 ratio and with anti-CD3/28 microbeads as stimulators at different time points. At the end of incubation, CD4+ (A) and CD8+ (B) T cells were harvested and labelled for CD25 and CD69 antibodies and assessed by flowcytometry. The results show mean of two independent experiments.
modes of T cell stimulation, including allogenic or antigen-specific stimulation [14,20]. However, most studies were limited at examin ing immunosuppressive effects of MSC at one or two phases of a T cell responses. Various phases of T cell responses such as activa tion, proliferation and effector function require different cellular
and molecular mechanisms. Therefore it is important to dissect the effect of MSC at these phases. We found that MSC were unable to stimulate resting allogeneic T cells proliferation and IFN-c produc tion despite normal expression of MHC class I (data not shown). This deficit may be related to the fact that MSC do not express co-
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Effector:Target ratios Fig. 5. MSC did not affect effector function of T cells. WT126-specific cells were expanded for 6 days in the presence of WT-126 peptide and feeder cell layer. At day 7, CTL were retrieved and incubated with Cr-56-labelled WT126-pulsed T2 cells in the presence and absence of MSC for 6 h incubation. The effect of MSC on effector function as measured by their ability to lyse target cells was measured by a-reader and expressed in percentage.
stimulatory molecules (CD80 and CD86) [data not shown], which are crucial for providing the necessary second signal for T cell activation. However, neither IFN-c pre-treatment [21] nor retro viral-mediated MSC transduction with co-stimulatory molecules confers the ability to induce a T cell proliferative response upon MSC [22,23]. When MSC were added to T cells stimulated by mitogen, T cell proliferation was reduced in a dose-dependent manner. The inhibi tory effects of MSC do not target any specific T cell subpopulations since both CD4+ and CD8+ positive T cells were equally affected. Furthermore the inhibitory effect of MSC also can be extended to a more defined antigen-specific T cell population. The expansion of antigen-specific T cells and the ability to secrete IFN-c against cog nate antigen re-challenge were dramatically reduced when MSC were cocultured with CMV serotype positive T cells and relevant antigen peptides. These proliferation assays confirm that MSC not only inhibit antigen-specific T cells but also other cells within the T cell subpopulations. Therefore, MSC exert a non-specific immuno suppressive activity on T cells. In line with this, others have shown that MSC inhibit both naïve and memory T cells responses in a mouse model [11]. The effect of the inhibition was further charac terised by dissecting T cell activation and effector function. As a consequence of TCR engagement, T cells are activated and express CD25 which is the a chain of IL-2 receptor and CD69. CD25 expres sion is required for autocrine IL-2-mediated clonal expansion while CD69 expression enhances the activation and serves as a co-stimulatory molecule [24]. We observed that MSC do not signif icantly alter the expression of these early activation markers at 24 and 48 h. While increased CD25 expression could be derived from increasing regulatory T cells however, the simultaneous increase in CD69 co-expression argues against this hypothesis. A small decline in the percentage of CD25+ cells was noticed at 72 h and this might be the consequence of an anti-proliferative effect induced by MSC, hence the reduced number of activated T cells. Although MSC exerted a profound anti proliferative effect on T cells, their effector function was not prevented. The ability of WT-126 specific CTL to lyse WT-126 peptide pulsed T2 cells was unaffected in the presence of MSC. These data are consistent with the findings of Rasmusson et al. who showed that lympholysis of target PBMC was not affected when MSC were added at effector phase or at the third day of 6-day MLR [18]. In their experiments when MSC were cultured at the beginning of the MLR they inhib ited CTL cytotoxicity. However, the inhibition of cytotoxicity could be explained by the fact that MSC might have prevented CTL gen
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eration/expansion during the culture. In accordance with this, we have observed that the number of IFN-c+ T cells at the 7th day of antigen-specific stimulation was dramatically reduced as a result of the prevented expansion (Fig. 3). Our data strongly support the fact that the inhibitory effect of MSC on T cell responses is confined to cellular proliferation rather then its effector function which does not require cell proliferation. However, it is still possible that the incubation time of CTL with MSC was too short for MSC to exert an inhibitory effect. The immunosuppressive activity of MSC may be exploited for therapeutic purposes. Since MSC can produce an anti-proliferative effect on T cells, they promise to be an attractive therapy for graft versus host disease (GVHD). Pathophysiology of GVHD is initiated by host-specific donor T cells clonal proliferation whereby the early inhibition host-specific T cell expansion would be ideal for GVHD amelioration. Although this concept is still in a premature stage, a few phase I and II clinical trials have shown that infusion of MSC appears to suppress GVHD without inducing any toxicity [25,26]. A potential disadvantage of MSC as treatment for GVHD is that they may also suppress graft versus leukaemia (GVL) leading to an increased risk of leukaemia relapse. However, clinical trials have shown that infusion of MSC appears to suppress GVHD with out interfering with the GVL effect [27,28]. If this were true, the pro tective effect of MSC on GVHD might be ascribable to their tissue repair activity. In fact, the T cells responsible for GVL and GVHD are virtually the same and require a major expansion. In this perspec tive, one could expand in vitro mature leukaemia-reactive or virusspecific T cells and administer them to patients without the risk of MSC interfering with their effector function [29,30]. Acknowledgment This project was funded by Science Fund (Project No: 02-01-04SF0028) Ministry of Science, Technology and Innovation (MOSTI), Malaysia. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellimm.2008.04.009. References [1] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [2] E.E. Horwitz, Clarification of the nomenclature for MSC: the international society for cellular therapy position statement, Cytotherapy 7 (2005) 393– 395. [3] L.M. Calvi, G.B. Adams, K.W. Weibrecht, J.M. Weber, D.P. Olson, M.C. Knight, R.P. Martin, E. Schipani, P. Divieti, F.R. Bringhurst, et al., Osteoblastic cells regu late the haematopoietic stem cell niche, Nature 425 (2003) 841–846. [4] J. Zhang, C. Niu, L. Ye, H. Huang, X. He, W.G. Tong, J. Ross, J. Haug, T. Johnson, J.Q. Feng, et al., Identification of the haematopoietic stem cell niche and con trol of the niche size, Nature 425 (2003) 836–841. [5] F. Dazzi, R. Ramasamy, S. Glennie, S. Jones, I. Roberts, The role of mesenchymal stem cells in haemopoiesis, Blood Rev. 20 (2005) 161–171. [6] A.A. Rzhaninova, S.N. Gornostaeva, D.V. Goldshtein, Isolation and phenotyp ical characterization of mesenchymal stem cells from human fetal thymus, Bull. Exp. Biol. Med. 139 (2005) 134–140. [7] R.K. Suniara, E.J. Jenkinson, J.J. Owen, An essential role for thymic mesenchyme in early T cell development, J. Exp. Med. 191 (2000) 1051–1056. [8] M. Di Nicola, C. Carlo-Stella, M. Magni, M. Milanesi, P.D. Longoni, P. Matteucci, S. Grisanti, A.M. Gianni, Human bone marrow stromal cells suppress T-lympho cyte proliferation induced by cellular or nonspecific mitogenic stimuli, Blood 99 (2000) 3838–3843. [9] W.T. Tse, J.D. Pendleton, W.M. Beyer, M.C. Egalka, E.C. Guinan, Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation, Transplantation 75 (2003) 389–397. [10] J.A. Potian, H. Aviv, N.M. Ponzio, J.S. Harrison, P. Rameshwar, Veto-like activ ity of mesenchymal stem cells: functional discrimination between cellular responses to alloantigens and recall antigens, J. Immunol. 171 (2003) 3426– 3434.
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