Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive T cells
2005; Elsevier BV; Volume: 33; Issue: 8 Linguagem: Inglês
10.1016/j.exphem.2005.05.002
ISSN1873-2399
AutoresMargaret E. Groh, Basabi Maitra, Emese Szekely, Omer N. Koç,
Tópico(s)Cancer Cells and Metastasis
ResumoHuman bone marrow-derived mesenchymal cells (MSCs) are precursors of nonhematopoietic mesenchymal cells of the bone marrow microenvironment.MSCs were shown to inhibit alloreactive T lymphocytes, but the mechanism and mediators of this effect are not fully understood. Here we describe a novel interaction between blood monocytes and bone marrow-derived, culture-expanded MSCs, which results in inhibition of T-lymphocyte activation. We found that CD14+ monocytes from blood activate MSCs to secrete inhibitory molecules that lead to inhibition of alloreactive T cells. This cellular communication is not contact-dependent, but rather is mediated by soluble factors that include interleukin (IL)-1β. MSC-mediated inhibition of alloreactive T lymphocytes is associated with downregulation of activation markers CD25, CD38, and CD69 detected both in CD4+ and CD8+ T lymphocytes. The cytokines secreted by MSCs that mediate T-cell inhibition include transforming growth factor-β1, but not IL-10. The interaction between blood monocytes and the MSCs represents a unique immune regulatory paradigm that can potentially be exploited in clinic. Human bone marrow-derived mesenchymal cells (MSCs) are precursors of nonhematopoietic mesenchymal cells of the bone marrow microenvironment. MSCs were shown to inhibit alloreactive T lymphocytes, but the mechanism and mediators of this effect are not fully understood. Here we describe a novel interaction between blood monocytes and bone marrow-derived, culture-expanded MSCs, which results in inhibition of T-lymphocyte activation. We found that CD14+ monocytes from blood activate MSCs to secrete inhibitory molecules that lead to inhibition of alloreactive T cells. This cellular communication is not contact-dependent, but rather is mediated by soluble factors that include interleukin (IL)-1β. MSC-mediated inhibition of alloreactive T lymphocytes is associated with downregulation of activation markers CD25, CD38, and CD69 detected both in CD4+ and CD8+ T lymphocytes. The cytokines secreted by MSCs that mediate T-cell inhibition include transforming growth factor-β1, but not IL-10. The interaction between blood monocytes and the MSCs represents a unique immune regulatory paradigm that can potentially be exploited in clinic. Human mesenchymal stem cells (MSC) are derived from bone marrow and are a uniform population of highly proliferative cells with the ability to differentiate along osteogenic, chondrogenic, and adipogenic lineages [1Pittenger M. Mackay A. Beck S. et al.Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17560) Google Scholar]. MSCs and their progeny support hematopoietic cells in the bone marrow by providing scaffolding and soluble and membrane-bound survival and growth signals. An emerging body of data indicates that MSCs inhibit T-lymphocyte activation and proliferation induced by mitogens, recall antigens, and alloantigens [2Di Nicola M. Carlo-Stella C. Magni M. et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002; 99: 3838-3843Crossref PubMed Scopus (2588) Google Scholar, 3Tse W.T. Pendleton J.D. Beyer W.M. Egalka M.C. Guinan E.C. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.Transplantation. 2003; 75: 389-397Crossref PubMed Scopus (1263) Google Scholar, 4Krampera M. Glennie S. Dyson J. et al.Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide.Blood. 2003; 101 (Epub 2002 Dec 27): 3722-3729Crossref PubMed Scopus (1359) Google Scholar, 5Le Blanc K. Tammik C. Rosendahl K. Zetterberg E. Ringden O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells.Exp Hematol. 2003; 31: 890-896Abstract Full Text Full Text PDF PubMed Scopus (1344) Google Scholar, 6Maitra B. Szekely E. Gjini K. et al.Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation.Bone Marrow Transplant. 2004; 33: 597-604Crossref PubMed Scopus (413) Google Scholar, 7Aggarwal S. Pittenger M. Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood. 2005; 105 (Epub 2004 Oct 19): 1815-1822Crossref PubMed Scopus (3523) Google Scholar, 8Beyth S. Borovsky Z. Mevorach D. et al.Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T cell unresponsiveness.Blood. 2005; 105 (Epub 2004 Oct 28): 2214-2219Crossref PubMed Scopus (860) Google Scholar] in vitro. To date, the mechanism of MSC-mediated inhibition of T-lymphocyte activation has not been fully understood and is likely to be complex and involve multiple pathways. Most groups reported that this effect is mediated by soluble factors, yet others claim that cell-to-cell contact is necessary. Secretion of transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), or prostaglandin E2 by MSCs, and MSC-mediated enzymatic depletion of tryptophan are proposed mechanisms of T-lymphocyte suppression [2Di Nicola M. Carlo-Stella C. Magni M. et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002; 99: 3838-3843Crossref PubMed Scopus (2588) Google Scholar, 7Aggarwal S. Pittenger M. Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood. 2005; 105 (Epub 2004 Oct 19): 1815-1822Crossref PubMed Scopus (3523) Google Scholar, 9Meisel R. Zibert A. Laryea M. Gobel U. Daubener W. Dilloo D. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation.Blood. 2004; 103 (Epub 2004 Mar 4): 4619-4621Crossref PubMed Scopus (1350) Google Scholar]. More recently MSCs were found to have multiple effects on both T cells and antigen-presenting cells (APCs) [7Aggarwal S. Pittenger M. Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood. 2005; 105 (Epub 2004 Oct 19): 1815-1822Crossref PubMed Scopus (3523) Google Scholar, 8Beyth S. Borovsky Z. Mevorach D. et al.Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T cell unresponsiveness.Blood. 2005; 105 (Epub 2004 Oct 28): 2214-2219Crossref PubMed Scopus (860) Google Scholar]. Human MSCs were shown to decrease tumor necrosis factor secretion from mature type 1 dendritic cells, increase interleukin (IL)-10 secretion from mature type 2 dendritic cells, decrease interferon secretion of TH1 cells and increase secretion of IL-4 from TH2 cells. In addition, it was found that MSCs increased the proportion of CD4+CD25+ regulatory cells in a mixed lymphocyte reaction (MLR), although the significance of this proportional increase was not clear. MSCs were also suggested to block APC maturation and activation necessary for allogeneic T-cell activation and proliferation. These data indicate a complex interplay between MSCs, T cells, APCs, and inflammatory cells. Here we report that MSCs inhibit activation of alloreactive lymphocytes by soluble factors found in culture-conditioned medium. Importantly, we found that MSCs must be activated by blood CD14+ monocytes to become immunosuppressive. This reaction between MSCs and CD14+ cells appears to be mediated by IL-1β. Activated MSCs secrete TGF-β1, which is partially responsible for inhibition of T lymphocytes. This interaction between MSCs and monocytes is novel and represents a potential target for immune regulation that can be exploited clinically [10Koç O.N. Lazarus H.M. Mesenchymal stem cells: heading into the clinic.Bone Marrow Transplant. 2001; 27: 235-239Crossref PubMed Scopus (198) Google Scholar, 11Le Blanc K. Rasmusson I. Sundberg B. et al.Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.Lancet. 2004; 363: 1439-1441Abstract Full Text Full Text PDF PubMed Scopus (2225) Google Scholar]. Human MSCs were obtained as described previously [6Maitra B. Szekely E. Gjini K. et al.Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation.Bone Marrow Transplant. 2004; 33: 597-604Crossref PubMed Scopus (413) Google Scholar] from fresh bone marrow aspirates and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing low glucose (Invitrogen, Carlsbad, CA, USA) with 1% antibiotic/antimycotic and 10% fetal bovine serum from defined lots screened for maximum growth and multipotentiality (Invitrogen). Bone marrow was aspirated (10–30 mL) under local anesthesia from healthy volunteer donors after a consenting process approved by the Institutional Review Board of the University Hospitals of Cleveland. Blood mononuclear cells (MNCs) were prepared from normal volunteer blood by density centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden). Cell counts and viability were measured using dye exclusion analysis under bright-field microscopy. Subpopulations of MNCs (CD14+, CD8+, CD19+) were isolated from peripheral blood using positive selection with monoclonal antibodies conjugated to magnetic microbeads (Miltenyi Biotec, Inc., Auburn, CA, USA). Cells were stained with the following conjugated monoclonal antibodies and corresponding isotype controls: phycoerythrin- conjugated mouse anti-human CD3 and CD38, fluorescein isothiocyanate-conjugated mouse anti-human CD3, CD 25, and CD8, APC-conjugated mouse anti-human CD45, Peridium Chlorophyll Protein (PCP)-conjugated mouse anti-human CD69 and CD4 (BD Biosciences Pharmingen, San Diego, CA, USA). Cells were analyzed on a Beckman Coulter XL-MCL flow cytometer (Miami, FL, USA). EliSpot analysis was performed as described previously [6Maitra B. Szekely E. Gjini K. et al.Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation.Bone Marrow Transplant. 2004; 33: 597-604Crossref PubMed Scopus (413) Google Scholar]. Briefly EliSpot 96-well plates (Polyfiltronics, Rockland MA, USA) were coated with capture antibody for human interferon-γ (IFN-γ) (2G1; Endogen, Woburn, MA, USA). A two-way mixed lymphocyte reaction was initiated using peripheral blood MNCs from two unrelated adult donors (150,000 cells each) in a total of 200 μL medium. Control wells contained each donor's cells with and without phytohemagglutinin (PHA) 10 μg/mL final concentration (Sigma, St. Louis, MO, USA). Test wells contained conditioned medium as described in figure legends for each experiment. In general, a minimum of triplicate wells for each condition was analyzed. All experimental conditions were repeated at least twice and standard deviations were determined for all replicates. After 24 hours, plates were washed and incubated with biotinylated detection antibody (B133.5; Endogen, 4 mg/mL) overnight at 4°C. Streptavidin-Horse Radish Peroxidase (HPR) (Dako, Carpenteria, CA, USA) was then added for 2 hours at room temperature. IFN-γ spots were visualized after incubation with 3-amino-9-ethylcarbazole (Pierce, Rockford, IL, USA). Spots in each well were quantified using computer-assisted EliSpot image analyzer (Immunospot Cellular Technology, Cleveland, OH, USA). Cell-free supernatants of cells cultured under various conditions were analyzed for cytokine content using the Quantikine ELISA Kit for TGF-β1, IL-10, HGF, and Activin A (R&D Systems, Inc., Minneapolis, MN, USA). Cell-free supernatants were analyzed in triplicate according to manufacturer's instructions. Confluent monolayers of MSCs were cultured in the presence of blood MNCs (approximately 5 × 105 to 1 × 106 cells per cm2) separated by trans-well chambers (Corning, Acton, MA, USA) and incubated for 24 hours. The cell-free supernatant was harvested and analyzed for its ability to inhibit alloreactive lymphocytes in the IFN-γ EliSpot assay. Recombinant human cytokines IL-1β, IL-2, IL-3, IL-10, and granulocyte colony-stimulate factor (G-CSF) were purchased from R&D Systems, Inc. Granulocyte macrophage colony-stimulating factor (GM-CSF) was purchased from Immunex (Seattle, WA, USA). Anti-TGF-β1,2,3 (clone 1D11), anti-TGF-β1 (clone 9016), recombinant anti-human IL-1 receptor (AF269) and anti-human IL-10 receptor (clone 37607) antibodies were purchased from R&D Systems, Inc. Anti-TGF-β, anti-IL-1 receptor, and anti-IL-10 receptor antibodies were selected for their ability to neutralize human bioactivity of its target cytokine and were used at concentrations twice the documented 50% neutralizing dose (ND50). Results are expressed as mean ± standard deviation or standard error of the mean. Differences between experimental conditions were analyzed by t-test (paired when possible). A p value of < 0.05 was considered statistically significant. Human MSCs inhibit activation of human lymphocytes stimulated by allogeneic blood mononuclear cells (MNCs) and PHA. We observed robust upregulation of T-lymphocyte activation antigens CD25, CD38, and CD69 after PHA stimulation. This upregulation was significantly inhibited at 24 hours both in CD3+CD4+ and CD3+CD8+ lymphocytes when PHA stimulation was conducted in the presence of an adherent layer of MSCs obtained from an unrelated donor (Fig. 1). Although there was a variation in percent expression of activation markers, downregulation was consistently observed for all antigens in both CD4+ and CD8+ lymphocytes subsets. We also measured activation and IFN-γ secretion of human blood lymphocytes by allogeneic human blood MNCs (MLR) using EliSpot assay and found consistent inhibition of this reaction by addition of MSCs derived from donors not related to either MLR donors (third party). We evaluated the variability of this inhibition using MSCs prepared from 11 different donors and 11 independent MLRs (Fig. 2). A large variation was seen in the number of IFN-γ spots formed per 300,000 blood MNCs tested in each experiment (183 ± 140 spots, range 67–480, Fig. 2) due to the expected variation in the numbers of alloreactive cells present in each specimen. Addition of MSCs resulted in a consistent inhibition of the MLR in every experiment. Despite a large variation in the number of spots observed in each MLR, the percent inhibition by MSCs was consistent between experiments (71% ± 14%, range 48–92%). A direct relationship was seen between the number of alloreactive lymphocytes at baseline and the percent inhibition by MSCs (Fig. 2, inset). This observation suggests that the degree of inhibition was dependent on the ratio of alloreactive lymphocytes present and the number of MSCs. In these experiments, the number of MSCs was kept constant (150,000) for each two-way MLR (300,000 total MNCs) that had variable numbers of alloreactive T-cells.Figure 2MSCs inhibit alloreactive T-cell activation. (A) Allogeneic T-cell activation was measured by interferon-γ (IFNg) EliSpots generated in 11 independent experiments without and with addition of 11 different third party MSCs. Insert: Relationship between the number of baseline EliSpots (alloreactive T cells) to percent inhibition mediated by a fixed dose of MSCs. All experimental conditions were examined in triplicate wells. Percent inhibition was obtained by direct comparison to corresponding control MLR. In all cases, the inhibition was statistically significant, p < 0.0001.View Large Image Figure ViewerDownload (PPT) Cell-free supernatant obtained from near confluent layers of human MSCs (conditioned culture medium) had no inhibitory activity on allogeneic MLRs, and had stimulatory activity in some experiments. However, the MLR was significantly inhibited by addition of cell-free CCM obtained from near confluent layers of human MSCs mixed with human blood MNCs (Fig. 3). This inhibitory effect was observed consistently when MSCs were preactivated with blood MNCs obtained either from the same donor as the MLR or unrelated third-party donor. We found that the MSC and blood MNC CCM became inhibitory within 12 hours and reached maximal potency by 24 hours (data not shown). Furthermore, this activation step was not contact-dependent because conditioned medium from MSCs cocultured directly with third-party MNCs had comparable inhibition to conditioned medium harvested from the cultures separated by a transwell system (Fig. 3). To examine further the activation process of MSCs by blood MNCs, cell-free supernatants were sequentially transferred from individual cultures of MSCs and blood MNCs as shown in Figure 4 insert. Culture medium was incubated for 24 hours in each condition and the final supernatant was tested on an allogeneic MLR EliSpot-IFN-γ assay. Inhibitory activity was observed when the medium was conditioned first by the blood MNCs and then the MSCs, but not with the reverse sequence (Fig. 4) confirming a necessary activation step of MSCs by soluble factors generated by blood MNCs.Figure 4MSCs-mediated immunosuppression requires activation of MSCs by soluble factors secreted by blood mononuclear cells (MNCs). The effect of serially transferred cell-free culture conditioned medium on MLR EliSpot-interferon-γ (IFN-γ) formation was measured. Conditioned medium was harvested after sequential exposure to (24 hours each) MSCs and blood MNC or in the reverse order (insert). The final cell-free supernatant in each case was examined for percent inhibition of MLR EliSpot-IFN-γ and compared to readout from the direct addition of MSCs as shown. All conditions described were examined in triplicate within each experiment and data pooled from three separate experiments. ∗p < 0.0001 vs control MLR. IFNg = interferon-γ.View Large Image Figure ViewerDownload (PPT) Next, we determined the subpopulation of cells in blood responsible for MSC activation. MSCs were cocultured with enriched populations of CD8+, CD14+, and CD19+ cells separately for 24 hours. Cell-free supernatants from these cultures were tested for their ability to inhibit alloreactivity using EliSpot-IFN-γ assay. Only the supernatant from MSC and CD14+ cell co-culture inhibited the T-cell activation (Fig. 5). Furthermore, when the CD14-negative fraction of the blood cells was analyzed in the same fashion, no inhibitory effect was observed. Human MSCs express receptors for IL-1β and their stimulation with this cytokine causes significant changes in cytokine levels in their medium. We postulated that CD14+ monocytes may be activating MSCs by their ability to secrete IL-1β. IL-1β was added to MSC cultures for 24 hours and cell-free supernatant was analyzed for its ability to inhibit T-cell activation in allogeneic MLR EliSpot-IFN-γ assay (Fig. 6). Cell-free supernatant obtained from IL-1β–activated MSCs had inhibitory effect on alloreactivity, whereas GM-CSF–, G-CSF–, and IL-3–activated MSC supernatant did not have such activity. IL-1β added directly to MLR had no effect (data not shown). Activation of MSCs by blood MNCs and CD14+ cells was blocked in some experiments (2 of 4) in the presence of IL-1 receptor antibody. We detected immunosuppressive cytokines, such as TGF-β, HGF, and Activin A in MSC-conditioned medium. There was either no change (HGF and Activin A) or only an additive increase (TGF-β) in the concentration of these cytokines when MSCs were cocultured with blood MNCs (data not shown). In particular, TGF-β concentrations were in the range of 0.3 to 0.6 ng/mL, even in MSC and blood MNC cocultures and TGF-β was mostly in protein-bound form, thought to be biologically inactive. These concentrations of TGF-β are at the low end of the concentrations we found to be inhibitory in allogeneic MLRs (Fig. 7A). In order to determine further if these low concentrations of TGF-β1 had a role in MSC-mediated inhibition of alloreactivity, we used neutralizing antibodies to TGF-β (at ND50 concentrations). The first antibody used neutralized all isoforms of TGF-β (β1, β2, and β3, clone 1D11) and the second was specific to the β1 isoform only (clone 9016.1). We found a significant, but incomplete, reversal of the MSC-mediated inhibition of alloreactivity using both antibodies, confirming the role of TGF-β1 in this process (Fig. 7B). These same antibodies added directly to MLR gave EliSpot readout comparable to that of control wells (data not shown). We did not detect human IL-10 in culture supernatants of MSCs but there was a significant induction of IL-10 in cocultures of MSCs and blood MNCs compared to MNCs alone (Fig. 8A). However, addition of IL-10 receptor antibodies did not reverse the inhibition of MLR mediated either by direct addition of MSCs or by addition of activated MSC culture conditioned medium (Fig. 8B). Here we describe a novel interaction between blood monocytes and bone marrow-derived, culture-expanded MSCs. The CD14+ monocytes activate MSCs to secrete inhibitory molecules that lead to inhibition of alloreactive T cells (Fig. 9). This cellular communication is not contact-dependent, but rather is mediated by soluble factors that include IL-1β. Although MSCs were recently shown to influence the pattern of cytokine secretion from APCs and T cells [7Aggarwal S. Pittenger M. Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood. 2005; 105 (Epub 2004 Oct 19): 1815-1822Crossref PubMed Scopus (3523) Google Scholar], our results are the first to indicate a critical activating role for APCs in MSC-mediated T-cell inhibition. This interaction represents a potential target for investigation and manipulation of auto- and alloimmune disorders. It is tempting to postulate that MSCs may play an important immunoregulatory role in the bone marrow microenvironment to create an immune-privileged site in which primitive stem cells and APCs coexist. A recent observation in patients with aplastic anemia indicate diminished immunosuppressive potential of MSCs obtained from these patients compared to normal donors, implicating MSCs and their immunoregulatory function in the pathogenesis of this autoimmune disorder [12Bacigalupo A. Valle M. Podesta M. et al.The suppressive effect of bone marrow mesenchymal stem cells on T cell activation is deficient in patients with severe aplastic anemia.Blood. 2004; 104: 147aGoogle Scholar]. We have investigated the range and consistency of MSC-mediated T-cell inhibition by testing MSCs from 11 different healthy volunteers on 11 independent sets of alloreactive T lymphocytes from unrelated healthy volunteers. As expected, we observed a relatively wide range of alloreactive T lymphocytes in a group of donors as measured by IFN-γ readout on EliSpot assay. However, the addition of unrelated human MSCs (third party) to the assay resulted in a consistent inhibition of each reaction. These data represent the largest human cohort analysis of MSC-mediated T-cell inhibition reported thus far. It is possible that this interaction between MSCs and T cells may be altered in various disease states and there may be polymorphisms in the general population that affect the degree of this interaction. Population-based studies on MSC function may potentially uncover links to various pathologic processes and disease risk. A number of laboratories have reported on the inhibitory effects of MSCs on T-lymphocyte activation and proliferation [2Di Nicola M. Carlo-Stella C. Magni M. et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002; 99: 3838-3843Crossref PubMed Scopus (2588) Google Scholar, 3Tse W.T. Pendleton J.D. Beyer W.M. Egalka M.C. Guinan E.C. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.Transplantation. 2003; 75: 389-397Crossref PubMed Scopus (1263) Google Scholar, 4Krampera M. Glennie S. Dyson J. et al.Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide.Blood. 2003; 101 (Epub 2002 Dec 27): 3722-3729Crossref PubMed Scopus (1359) Google Scholar, 5Le Blanc K. Tammik C. Rosendahl K. Zetterberg E. Ringden O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells.Exp Hematol. 2003; 31: 890-896Abstract Full Text Full Text PDF PubMed Scopus (1344) Google Scholar, 6Maitra B. Szekely E. Gjini K. et al.Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation.Bone Marrow Transplant. 2004; 33: 597-604Crossref PubMed Scopus (413) Google Scholar, 13Djouad F. Plence P. Bony C. et al.Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals.Blood. 2003; 102 (Epub 2003 Jul 24): 3837-3844Crossref PubMed Scopus (996) Google Scholar]. We have studied the effects of MSCs on activation and IFN-γ secretion using EliSpot readout and found comparable suppressive activity. We have previously measured the effect of MSCs on MLR using 3H-incorporation readout in a cell proliferation assay with identical results. We have chosen to use the EliSpot assay because of its convenience and reproducibility and presented all data using EliSpot readout for consistency and comparison between experiments. MSC-mediated inhibition of alloreactive T lymphocytes was also associated with the downregulation of activation markers CD25, CD38, and CD69 both in CD4+ and CD8+ T-lymphocytes consistent with recent observations by others [14Le Blanc K. Rasmusson I. Gotherstrom C. et al.Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes.Scand J Immunol. 2004; 60: 307-315Crossref PubMed Scopus (295) Google Scholar]. Most investigators reported that T-cell inhibition by human MSCs is mediated by soluble factors. However, Krampera et al. [4Krampera M. Glennie S. Dyson J. et al.Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide.Blood. 2003; 101 (Epub 2002 Dec 27): 3722-3729Crossref PubMed Scopus (1359) Google Scholar] observed that mouse MSC-mediated inhibition of MLR is contact-dependent and suggested that MSCs physically hinder T lymphocytes from interacting with APCs. These differences may be species-specific or due to differences in experimental methods. Our results clearly show that T-cell inhibitory effects of MSCs can be transferred by cell-free conditioned culture medium after activation of MSCs. Conflicting observations exist regarding soluble factors involved in MSC-mediated T-cell inhibition. Di Nicola et al. [2Di Nicola M. Carlo-Stella C. Magni M. et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002; 99: 3838-3843Crossref PubMed Scopus (2588) Google Scholar] suggested that HGF and TGF-β1 are important, while Aggarwal et al. [7Aggarwal S. Pittenger M. Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood. 2005; 105 (Epub 2004 Oct 19): 1815-1822Crossref PubMed Scopus (3523) Google Scholar] showed that MSC-derived prostaglandin E2 is a mediator, and Meisel et al. [9Meisel R. Zibert A. Laryea M. Gobel U. Daubener W. Dilloo D. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation.Blood. 2004; 103 (Epub 2004 Mar 4): 4619-4621Crossref PubMed Scopus (1350) Google Scholar] proposed enzymatic depletion of tryptophan as a mechanism of T-lymphocyte suppression. Tse et al. could not show a role for IL-10, TGFβ, or prostaglandin E2 in this process [3Tse W.T. Pendleton J.D. Beyer W.M. Egalka M.C. Guinan E.C. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.Transplantation. 2003; 75: 389-397Crossref PubMed Scopus (1263) Google Scholar]. These results should be reevaluated in the context of our findings, which indicate that presence of monocytes or other APCs in the experimental conditions can greatly influence the results. It is likely that when in contact with T cells and APCs, MSCs secrete an array of factors that influences T-cell responses either directly or indirectly through APCs. Our data show that TGF-β1 plays an important role in T-cell inhibition, but likely not the sole mediator of this effect. Limited in vivo data exist to describe the in vivo activity of MSCs on T cells. Prolonged skin allograft survival was observed in nonhuman primates when MSCs were infused [15Bartholomew A. Sturgeon C. Siatskas M. et al.Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.Exp Hematol. 2002; 30: 42-48Abstract Full Text Full Text PDF PubMed Scopus (1930) Google Scholar]. In a remarkable case report, haploidentical human MSCs greatly attenuated severe graft-vs-host disease after allogeneic stem cell transplantation [11Le Blanc K. Rasmusson I. Sundberg B. et al.Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.Lancet. 2004; 363: 1439-1441Abstract Full Text Full Text PDF PubMed Scopus (2225) Google Scholar]. This immunosuppressive effect appears to be nonspecific and potentially detrimental to tumor immunity [13Djouad F. Plence P. Bony C. et al.Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals.Blood. 2003; 102 (Epub 2003 Jul 24): 3837-3844Crossref PubMed Scopus (996) Google Scholar]. The immunosuppressive properties of MSCs in vitro and in vivo afford potential therapeutic avenues. Intravenous infusion of autologous and allogeneic MSCs have been demonstrated to be both feasible and safe [16Koç O.N. Gerson S.L. Cooper B.W. et al.Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy.J Clin Oncol. 2000; 18: 307-316PubMed Google Scholar, 17Koç O.N. Day J. Nieder M. Gerson S.L. Lazarus H.M. Krivit W. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH).Bone Marrow Transplant. 2002; 30: 215-222Crossref PubMed Scopus (573) Google Scholar] and preclinical and clinical testing of MSC therapy in immune disorders is warranted. We conclude that MSCs require activation by CD14+ blood monocytes to exert their T-cell inhibitory effects. The activation signal includes IL-1β and leads to secretion of T-cell inhibitory molecules by MSCs that include TGF-β1 but not IL-10. The complete mechanism of MSC-mediated T-cell suppression likely employs multiple, perhaps over- lapping pathways. Elucidation of these events will further our understanding of the activity of human MSCs and their potential therapeutic role. This research was supported by National Institutes of Health, R01HL62360 (PI: ON Koç) and the Case Comprehensive Cancer Center of Case Western Reserve University and the University Hospitals of Cleveland (P30 CA43703), Flow Cytometry and the Hematopoietic Stem Cell Core Facilities, and partially supported by the Center for Stem Cell and Regenerative Medicine with a grant from Ohio Biomedical Research and Technology Trust, Ohio Department of Development.
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