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Extended report
Human bone marrow mesenchymal stem cells and chondrocytes promote and/or suppress the in vitro proliferation of lymphocytes stimulated by interleukins 2, 7 and 15 C Bocelli-Tyndall,1,2 L Bracci,1,3 S Schaeren,1 C Feder-Mengus,1 A Barbero,1 A Tyndall,2 G C Spagnoli1 c Additional data (Supplementary file 1) are published online only at http:// ard.bmj.com/content/vol68/ issue8 1
Institute of Surgical Research and Hospital Management and Department of Biomedicine, University Hospital Basel, Basel, Switzerland; 2 Department of Rheumatology, University of Basel, Basel, Switzerland; 3 Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita`, Rome, Italy Correspondence to: Dr C Bocelli-Tyndall, University Dept of Rheumatology, Felix Platter Spital, Burgfelderstrasse 101, 4012 Basel, Switzerland;
[email protected] Accepted 10 July 2008 Published Online First 22 July 2008
ABSTRACT Objectives: To investigate whether human bone marrowderived mesenchymal stem cells (BM-MSCs) and articular chondrocytes (ACs) affect the in vitro proliferation of T lymphocytes and peripheral blood mononuclear cells (PBMCs) driven by the homeostatic interleukin (IL)2, IL7 and IL15 cytokines binding to the common cytokine receptor c-chain (cc) in the absence of T cell receptor (TCR) triggering. Methods: PBMCs, total T cells and T cell subsets (CD4+ and CD8+) were stimulated with IL2, IL7 or IL15 and exposed to cultured BM-MSCs and ACs at varying cell:cell ratio either in contact or in transwell conditions. Lymphocyte proliferation was measured by 3H-thymidine uptake or by flow cytometry of carboxyfluorescein succinimidyl ester (CFSE)-labelled lymphocytes. Results: MSCs and ACs enhanced and inhibited lymphocyte proliferation depending on the extent of lymphocyte baseline proliferation and on the MSC/AC to lymphocyte ratio. Enhancement was significant on poorly proliferating lymphocytes and mostly at lower MSC/AC to lymphocyte ratio. Suppression occurred only on actively proliferating lymphocytes and at high MSC/AC to lymphocyte ratio. Neither enhancement nor inhibition required cell–cell contact. Conclusions: There is a dichotomous effect of MSCs/ ACs on lymphocytes proliferating in response to the homeostatic IL2, IL7 and IL15 cytokines likely to be encountered in homeostatic and autoimmune inflammatory conditions. The effect is determined by baseline lymphocyte proliferation, cell:cell ratio and is dependent on soluble factor(s). This should be taken into account when planning cellular therapy for autoimmune disease (AD) using stromal-derived cells such as MSCs.
In vitro expanded mesenchymal stem cells isolated from bone marrow (BM-MSCs) or other sources (eg, fat) are being investigated for their antiproliferative properties affecting haematopoietic and tissue somatic cells. These properties have been demonstrated in vitro,1 in vivo in animal models of autoimmune disease,2–4 acute inflammation5 and renal ischaemia6 and in humans suffering from acute graft versus host disease.7 MSCs are also being considered for the treatment of autoimmune disease (AD) in humans.8 Primary cultures of chondrocytes and skin fibroblasts share the antiproliferative properties of BM-MSCs.9–11 1352
Multiple factors appear to be involved in the antiproliferative response of the MSCs: transforming growth factor (TGF)b1,12 indoleamine 2,3 dioxygenase (IDO) enzyme,13 soluble histocompatibility antigen class I G (HLA-G),14 nitric oxide15 and Toll-like receptor (TLR) ligands.16 17 However, BM-MSCs may also stimulate the proliferation of somatic cells such as haematopoietic stem cells,18 tumour cells19 and, sporadically, healthy lymphocytes20–22 in mixed lymphocyte reactions (MLR) or following anti-CD3e mAb stimulation. The modulatory effects of the MSCs on lymphocyte proliferation have been so far tested on mitogen-stimulated or antigen-stimulated lymphocytes (ie, via T cell receptor triggering). In vivo, however, activation of a range of T lymphocytes much wider than that responding to a specific antigen and responsible for the maintenance and survival of T lymphocyte subpopulations, occurs in the homeostatic process. Several homeostatic cytokines including interleukin (IL)2, IL7 and IL15, signal T lymphocyte proliferation and survival by binding to the common cytokine receptor cchain (cc, CD132), activating well known signal transduction mitogenic pathways.23–25 BM stromal cells participate in the BM and the thymus niche in the differentiation of haematopoietic precursors into the myeloid and lymphoid lineages.18 In vitro, expanded MSCs function as stromal support in the differentiation of haematopoietic stem cells in long-term culture-initiating cell (LTC-IC) assays.26 In vivo, BM stromal cells participate in peripheral homeostatic niches by controlling the population size of T lymphocytes.27 28 Interestingly, the same cytokines are also present in the inflammatory milieu of AD,29–31 presently the target of MSC therapy. We investigated whether BM-MSCs affect the proliferation of lymphocytes stimulated in vitro by IL2, IL7 and IL15 cytokines in the absence of T cell receptor (TCR) triggering. We show here for the first time that in vitro expanded human MSCs are able to inhibit and to enhance very effectively the proliferation of peripheral blood mononuclear cells (PBMCs) and purified T lymphocytes primed by these cytokines. The type of response induced by the MSCs depends principally on the baseline proliferation of the lymphocytes and on the MSC to lymphocyte ratio. Suppression and stimulation of proliferation are mediated by soluble factor(s) as Ann Rheum Dis 2009;68:1352–1359. doi:10.1136/ard.2008.094003
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Extended report shown by transwell experiments. These features are shared by cultured articular chondrocytes (ACs) previously shown to suppress the activity on proliferating lymphocytes in vitro.9–11 These in vitro results describe a new and significant stimulatory property of MSCs in the modulation of the lymphocyte proliferative function in response to homeostatic cytokines, which should be taken into account in clinical applications.
MATERIALS AND METHODS Cell cultures BM-MSC and AC primary cultures were established as previously described.9 21
PBMC and T lymphocyte isolation PBMCs were purified as previously described.21 Total T lymphocytes, CD4+ and CD8+ T lymphocytes were isolated by immunoselection with specific magnetic beads.
Cytokine (IL2, IL7 and IL15)-induced proliferation of PBMC and T lymphocytes with or without MSCs or ACs Lymphocyte proliferation induced by cytokines (10 ng/ml) was measured at day 7 by either [3H]thymidine uptake or flow cytometry of 1–2 mM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained lymphocytes as previously described.21
Transwell experiments CFSE-labelled total T lymphocytes or PBMCs were added to either the bottom or top well of 24-well or 12-well transwell plates (0.4 mm pore size). MSCs or ACs were added to the corresponding complementary well. See also online Supplementary file 1.
RESULTS BM-MSCs suppress and enhance the proliferation of lymphocytes induced with IL2, IL7 or IL15 cytokines PBMCs, total T cells and purified CD4+ and CD8+ lymphocytes were stimulated with IL2, IL7 or IL15 at 10 ng/ml concentration in the presence or absence of BM-MSCs and the proliferation induced by each cytokine was measured by [3H]thymidine uptake. In most experiments MSCs were irradiated but identical results were also obtained when they were not treated (data not shown). As controls, MSCs and lymphocytes were incubated together at the same cell ratios, ranging between 1:2 and 1:50, without cytokines. Furthermore, non-irradiated BM-MSCs alone were also incubated with each cytokine to measure their proliferative response. Neither of these controls showed evidence of significant proliferation. Results from multiple experiments performed by using PBMCs, total T lymphocytes, CD8+ and CD4+ T cells from at least four donors per cell population, expressed as 3H-counts/ min (cpm) absolute values (standard error of the mean (SEM)) are reported in fig 1. The proliferation activities of the lymphocyte populations induced by IL2, IL7 or IL15 were significantly different. Despite donor dependent variations, clear patterns of responsiveness emerged. IL15 induced the highest proliferation in all lymphocyte populations (fig 1). IL2 induced a proliferation higher than IL7 in PBMCs and CD8+T cells, whereas IL7 was more effective than IL2 on total T cells and CD4+T cells (fig 1). The addition of MSCs generated two distinct patterns of modulation of lymphocyte proliferation. A significant decrease of proliferation was induced at high MSC to lymphocyte ratio (1:2). The highest inhibition was observed with IL7; all
Figure 1 Proliferation of peripheral blood mononuclear cells (PBMCs), total T lymphocytes, CD4+ and CD8+ T cells measured by 3H-thymidine uptake, in the presence of interleukin (IL)2, IL7 and IL15 cytokines (10 ng/ml) and bone marrow-derived mesenchymal stem cells (BM-MSCs) at second to third expansion passage. Measurements were performed at day 7 of MSC–lymphocyte coculture. Each bar represent the proliferation of 100 000 responder cells in the presence of IL2, IL7 or IL15 and increasing numbers of BM-MSCs. Data are reported as counts/min (cpm) (standard error (SE)) (y axis). MSC to lymphocyte ratios are reported on the x axis (0 = no MSC). Asterisks above each line joining two experimental groups indicate a p(0.05. In each panel, n represents the number of experiments performed. Ann Rheum Dis 2009;68:1352–1359. doi:10.1136/ard.2008.094003
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Extended report lymphocyte populations were also affected at a 1:10 ratio with residual proliferations ranging between 26% and 43% of baseline proliferation value. With IL15 this range was between 38% and 50% and with IL2 between 45% and 70%. Proliferation induced by these cytokines was inhibited only at a 1:2 MSC:responder cell ratio. The reduction was significant (p,0.05) for PBMCs, total T, CD4+ and CD8+ cells with IL7 and IL15, for PBMCs, total T and CD8+ but not CD4+ with IL2 (p.0.05). In contrast, a significant (p,0.05) enhancement of proliferation above baseline values was induced in all lymphocyte populations stimulated with IL2 (p = 0.05 for CD4+, p = 0.045 for CD8+ and p = 0.05 for PBMCs and total T) and IL15 (p = 0.014 for CD4+, p = 0.016 for CD8+, p = 0.004 for PBMCs and p = 0.033 for total T) at a low MSC to lymphocyte ratio (1:50). The average increase above baseline proliferation was in the range of 26% to 100% with IL2 and 12% to 50% with IL15. Overall, no significant stimulation (p.0.05) occurred with IL7. Importantly, proliferation of PBMCs and T lymphocytes from two donors was also enhanced by autologous MSCs, suggesting that the effects of MSCs are not related to alloantigenic stimulation (data shown in fig 1).
Proliferation of CFSE-labelled lymphocytes in the presence of MSCs in cell–cell contact and transwell conditions To assess whether the enhancing or inhibitory effects of MSCs on cytokine-driven lymphocyte proliferation were mediated by soluble factors or required cell–cell contact, responder cells were labelled with CFSE and their proliferation, expressed as percentage of dividing cells in the presence or absence of unlabelled MSCs, in contact or in transwell conditions, was
measured by flow cytometry. Based on the results shown in fig 1, PBMCs and total T cells, were considered as representative of all lymphocyte populations investigated previously. Figures 2–5 illustrate the response variation observed in cell– cell contact and transwell conditions. In fig 2, IL15-driven proliferation of total T lymphocytes in contact with the MSCs, is enhanced at MSC to lymphocyte ratios of 1:50 and 1:10, but it is inhibited at the 1:2 ratio with residual proliferation accounting for 18% of baseline value. However, upon IL2 stimulation, the same lymphocytes showed very low baseline proliferation, which was enhanced by the MSCs at all ratios. With regard to IL7, on average MSCs exerted only inhibitory effects on cytokine-driven lymphoproliferation (figs 1 and 3A). However, when individual donor lymphocytes, responded poorly to cytokine stimulation, MSCs also enhanced IL7-driven proliferation (fig 3B). Figure 3 represents the two patterns of responsiveness of T cells from two different donors to IL7 and MSCs in cell–cell contact conditions. In the experiment reported in panel A, the lymphocytes are moderately proliferating and no significant enhancement but rather a significant inhibition of proliferation to almost baseline values at the two highest MSC to lymphocyte ratios can be observed. In contrast, in the experiment depicted in panel B, lymphocyte baseline proliferation is minimal: the addition of the MSCs induces a significant lymphoproliferation at all ratios but the highest. Next, we utilised a transwell system in which the MSCs were placed in the lower chamber and PBMCs in the top chamber at a 1:50 MSC to PBMC ratio. These experiments clearly indicated that the effects of MSCs on cytokine-driven proliferation of PBMCs are cell–cell contact independent (fig 4).
Figure 2 Proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labelled total T lymphocytes in the presence of interleukin (IL)2 or IL15 and bone marrow-derived mesenchymal stem cells (BM-MSCs) measured by flow cytometry. Measurements were performed at day 7 of MSC–lymphocyte coculture. The MSC to lymphocyte ratios are indicated above the panels. T cell proliferation data obtained in the presence of specific cytokines but in the absence of BM-MSCs are reported in left panels. The bars indicate the gate in which the proliferating cells are counted. The number above represents the percentage of dividing cells estimated by using FlowJo software (http://www.flowjo.com/). 1354
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Extended report
Figure 3 Proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labelled total T lymphocytes in the presence of interleukin (IL)7 and bone marrow-derived mesenchymal stem cells (BM-MSCs) measured by flow cytometry. Lymphocytes in panels (A) and (B) are from different donors. Measurements were performed at day 7 of MSC–lymphocyte coculture. The MSC to lymphocyte ratios are indicated above the panels. In each panel left histograms refer to lymphoproliferation in the presence of IL7 but in the absence of BM-MSC. The bars indicate the gate in which the proliferating cells are counted. The numbers above represent percentage of dividing cells estimated by using FlowJo software (http://www.flowjo.com/). In transwell plates, the floor of the top chamber has a smaller area (approximately 1/10 in a 24 well plate) than that of the lower, resulting in a different density of the adhering MSCs when plated in each of the wells, though leaving the MSC to lymphocyte ratio unchanged. We investigated whether changing the location of the MSCs would alter their effect on the proliferating lymphocytes. The results are shown in fig 5. Active proliferation of lymphocytes, especially following IL15 stimulation, was not significantly enhanced by cell–cell contact or, in transwell conditions, the MSCs being either in the top or in the bottom well at the MSC to PBMC ratio of 1:50. Inhibition was observed in all conditions at the highest (1:5) MSC to PBMC ratio. A similar pattern was also observed with IL2-stimulated PBMCs (data not shown).
significant enhancement of lymphocyte proliferation was observed with IL2 at a 1:50 AC to lymphocyte ratio (fig 6A). In agreement with these data, inhibition of cytokine-driven lymphoproliferation by ACs was also observed by using CFSElabelled lymphocytes when these proliferated actively (data not shown). However, as seen with the MSCs, when individual donor lymphocytes showed a low response to cytokine stimulation, ACs were able to enhance as well as inhibit the proliferation of CFSE-labelled lymphocytes, as exemplified in fig 6B by IL15-driven lymphocytes. Similar results were also obtained in parallel experiments with IL2-driven and IL7-driven total T and PBMCs (data not shown).
DISCUSSION ACs also suppress and stimulate lymphocyte proliferation induced by IL2, IL7 and IL15 Primary AC cultures from three different donors were also tested to assess their capacity to enhance or inhibit the proliferation of PBMCs or total T cells induced by IL2, IL7 or IL15. AC to lymphocyte ratios were the same as used with BMMSCs and proliferation was measured by 3H-thymidine uptake. Significant inhibition of the proliferation of total T lymphocytes (fig 6A) or PBMCs from the same donors (data not shown) was observed with all cytokines at the AC to lymphocyte 1:2 ratio. The same was also observed at the 1:10 ratio with IL7 and IL15. Average residual lymphoproliferation was 31% with IL2, 28% with IL7 and 17% with IL15. In these experiments a Ann Rheum Dis 2009;68:1352–1359. doi:10.1136/ard.2008.094003
MSCs are currently being tested in clinical trials in graft versus host disease (GVHD), in several ADs including Crohn disease and multiple sclerosis, and are being considered for inflammatory rheumatic AD. The rationale for infusing large numbers of ex vivo expanded MSCs in acute inflammatory clinical settings such as GVHD is based largely on their in vitro antiproliferative properties on mitogen-stimulated and antigen-stimulated lymphocytes, on animal models and on anecdotal case reports. In vivo, MSC-derived stromal cells participate in haematopoiesis in the BM18 32 and in the regulation of the population size of T lymphocytes. In these settings, the lymphocytes proliferate through the mediation of ‘‘homeostatic’’ cytokines, including IL2, IL7 and IL1533 in response to different stimuli (eg, 1355
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Extended report
Figure 4 Enhancement of proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labelled peripheral blood mononuclear cells (PBMCs) in the presence of interleukin (IL)2, IL7 and IL15 and bone marrow-derived mesenchymal stem cells (BM-MSCs) in either cell–cell contact or transwell (tw) conditions. Measurements were performed at day 7 of MSC and lymphocyte coculture. In these experiments MSCs were cultured in the bottom wells. MSC to PBMC ratios are indicated in brackets. The bars indicate the gate in which the proliferating cells are counted. The number above represents the percentage of dividing cells estimated by using FlowJo software (http://www.flowjo.com/). lymphocyte depletion during aplasia following haematopoietic stem cell transplantation).34 35 These cytokines function as growth and survival factors for the lymphocytes.33 36 Furthermore, IL2 and IL15 provide signals for survival or death to T cells depending on cytokine concentrations.37 By contrast, the same cytokines in an AD setting may enhance autoaggressive reactions, and high levels of IL7 and IL15 have been detected in inflammation sites.29–31 BM-MSCs do not produce nor have receptors for IL2. In contrast, they can produce IL15 and IL7, for which they also have receptors.38 39 We have detected the expression of the IL7 receptor a chain (CD127) on cultured MSCs only after exposure to the cytokine. Notably, no differential proliferation of MSCs cultured in the 1356
presence or absence of any of the above cytokines was observed (data not shown). The current studies did not determine whether in our experimental conditions MSCs secrete IL7 and IL15. This would nevertheless account for negligible amounts of cytokines as compared to those added exogenously (10 ng/ml). We also investigated whether BM-MSCs modulate the proliferation of lymphocytes induced with IL2, IL7 and IL15 as already described with anti-CD3e mAb-induced proliferation.21 The lymphocyte proliferative response to all cytokines was variable and donor dependent, but not correlated with health state or age. This high variability was also observed using lower (5–1.25 ng/ml) IL2, IL7 and IL15 cytokine concentrations on Ann Rheum Dis 2009;68:1352–1359. doi:10.1136/ard.2008.094003
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Extended report
Figure 5 Inhibition of proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labelled peripheral blood mononuclear cells (PBMCs) in the presence of interleukin (IL)7 and IL15 and bone marrow-derived mesenchymal stem cells (BM-MSCs) in either cell–cell contact or transwell (tw) conditions. MSCs are either in the bottom well, or alternatively in the top well as reported in the Results section. MSC to PBMC ratios are indicated in the panels. Measurements were performed at day 7 of MSC–lymphocyte coculture. The bars indicate the gate in which the proliferating cells are counted. Numbers above bars represents the estimated percentage of dividing cells.
CD8+ and CD4+ T cells from healthy donors (CFM, unpublished results). In our experiments the MSCs alone did not induce proliferation of either allogeneic or autologous lymphocytes (data included but not shown). Ann Rheum Dis 2009;68:1352–1359. doi:10.1136/ard.2008.094003
When the lymphocytes were induced to proliferate by any of the three tested cytokines, the addition of the MSCs resulted either in enhancement or/and in suppression of lymphocyte proliferation. Suppression (with 50% to 10% residual proliferation) 1357
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Extended report
Figure 6 Enhancement and inhibition of proliferation of total T lymphocytes and peripheral blood mononuclear cells (PBMCs) in the presence of interleukin (IL)2, IL7 and IL15 cytokines and increasing number of articular chondrocytes (ACs) at second or third expansion passage. Measurements were performed at day 7 of AC and lymphocyte coculture. A. Proliferation measured by 3H-thymidine uptake of total T lymphocytes in the presence of IL2, IL7 and IL15 cytokines (10 ng/ml) and ACs at third expansion passage. Each bar represent the proliferation of 100 000 responder cells in the presence of IL2, IL7 or IL15 and increasing numbers of ACs. Data are expressed as counts/min (cpm) (standard error (SE) (y axis). The AC to lymphocyte ratio is reported on the x axis (0 = no AC). In each panel n represents the number of individual experiments performed. B. Proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labelled total T cells in the presence of IL15 and increasing numbers of ACs. AC to lymphocyte ratios are indicated on top of the panels. preferentially occurred on actively proliferating lymphocytes with any cytokine and only at high MSCs to lymphocyte ratio (1:2) or (1:5) (fig 5) as also observed with anti-CD3e mAb-stimulated lymphocytes.21 Notably, the MSCs also consistently suppressed IL7-induced lymphoproliferation, irrespective of the lymphocyte population tested, at a 1:10 MSC to lymphocyte ratio. However, a lower baseline lymphoproliferation can be enhanced by MSCs at a 1:50 ratio but suppressed at a 1:2 ratio. In the extreme case of very poor baseline lymphocyte proliferation, the addition of MSCs induces a proliferative ‘‘rescue’’ of the apparently resting lymphocytes, which may occur at most (fig 3B) or all (fig 2, IL2) MSC to lymphocyte ratios under investigation. Our transwell results show that inhibition and enhancement of lymphocyte proliferation can occur without cell–cell contact (figs 4 and 5). In addition, the swapping of compartments, which have very different adhesion surface areas, between the MSCs and the lymphocytes resulting in cell density (but not cell:cell ratio) changes does not cause significant differences in either enhancement or suppression, suggesting that cell density plays no major role. These results show that at least some soluble factors, not yet identified, are involved in suppression and stimulation of the lymphocytes by the MSCs. Ongoing studies will address the nature of the soluble factors involved in the observed effects and of the role of specific T cell subsets. By analogy with experiments performed previously with antiCD3e mAb-stimulated lymphocytes and ACs, we tested whether cultured ACs had the same properties as the BMMSCs on lymphocytes induced to proliferate with Il 2, IL7 and IL15. Our results show that they are very effective inhibitors of 1358
the proliferation of cytokine-activated lymphocytes as efficiently as upon anti-CD3e mAb activation.21 However, they can also induce a very robust enhancement of proliferation in cytokine-activated but poorly proliferating lymphocytes (fig 6B), as observed with BMMSCs. Cultured ACs lose the differentiated phenotype and dedifferentiate.40 41 Replenishing ‘‘stem’’ cells are present in every tissue as a form of cellular homeostasis, and it is possible that the properties that we see exerted in vitro on lymphocytes also extend to other proliferating cells in tissues in vivo. This experimental lymphocyte proliferation system emphasises the ability of the BM-MSCs and ACs to enhance as well as to inhibit in a homeostatic fashion cytokine-induced T lymphocyte proliferation. The response is the result of crosstalk between cell populations, largely mediated in our experiments by soluble factors. The concept of a balance of opposing effects22 and that inhibition by the MSCs occurs in response to the signalling of proliferating lymphocytes11 is supported by our data. Enhancement of lymphoproliferation induced by the MSCs in vitro occurs mostly at low MSC to lymphocyte ratios. This has been suggested to also occur with TCR-stimulated lymphocytes.20–22 A low ratio of MSC to target cell is probably closer to an in vivo situation than the very high MSC to lymphocyte ratio required to suppress proliferating cells in vitro. It is not known how many of the in vivo infused cells reach their destination, preferentially inflammation sites in animal models.42 Patients suffering from some autoimmune inflammatory rheumatic diseases have localised high levels of IL7 and IL15 in inflammation sites.29 30 If these sites become the destination of in vivo infused MSCs, it would be difficult to predict their effects, whether suppressive or stimulatory on T cell proliferation. Ann Rheum Dis 2009;68:1352–1359. doi:10.1136/ard.2008.094003
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Extended report Acknowledgements: We would like to acknowledge Professor Francesco Dazzi for critical reading of the manuscript.
19.
Funding: This study was partially funded by a grant of the Swiss National Science Foundation to GCS.
20.
Competing interests: None. 21.
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Human bone marrow mesenchymal stem cells and chondrocytes promote and/or suppress the in vitro proliferation of lymphocytes stimulated by interleukins 2, 7 and 15 C Bocelli-Tyndall, L Bracci, S Schaeren, et al. Ann Rheum Dis 2009 68: 1352-1359 originally published online July 22, 2008
doi: 10.1136/ard.2008.094003
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