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The Journal of Immunology, 2007, 179, 1549 -1558
Copyright © 2007 by The American Association of Immunologists, Inc.
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Regulation of MHC Class II Expression and Antigen Processing in Murine and Human Mesenchymal Stromal Cells by IFN-{gamma}, TGF-beta, and Cell Density1

Raphaëlle Romieu-Mourez2, Moïra François2, Marie-Noëlle Boivin, John Stagg and Jacques Galipeau3

Department of Medicine and Oncology, Sir Mortimer B. Davis Jewish General Hospital and Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mesenchymal stromal cells (MSC) possess immunosuppressive properties, yet when treated with IFN-{gamma} they acquire APC functions. To gain insight into MSC immune plasticity, we explored signaling pathways induced by IFN-{gamma} required for MHC class II (MHC II)-dependent Ag presentation. IFN-{gamma}-induced MHC II expression in mouse MSC was enhanced by high cell density or serum deprivation and suppressed by TGF-beta. This process was regulated by the activity of the type IV CIITA promoter independently of STAT1 activation and the induction of the IFN regulatory factor 1-dependent B7H1/PD-L1 encoding gene. The absence of direct correlation with the cell cycle suggested that cellular connectivity modulates IFN-{gamma} responsiveness for MHC II expression in mouse MSC. TGF-beta signaling in mouse MSC involved ALK5 and ALK1 TGF-betaRI, leading to the phosphorylation of Smad2/Smad3 and Smad1/Smad5/Smad8. An opposite effect was observed in human MSC where IFN-{gamma}-induced MHC II expression occurred at the highest levels in low-density cultures; however, TGF-beta reduced IFN-{gamma}-induced MHC II expression and its signaling was similar as in mouse MSC. This suggests that the IFN-{gamma}-induced APC features of MSC can be modulated by TGF-beta, serum factors, and cell density in vitro, although not in the same way in mouse and human MSC, via their convergent effects on CIITA expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mesenchymal stromal cells (MSC)4 are bone marrow-derived mesenchymal progenitors thought to give rise to cells that constitute the hemopoietic microenvironment. MSC are classically expanded in vitro from the plastic-adherent cell population from bone marrow aspirates. MSC can serve as precursors for the generation of a variety of nonhemopoietic tissues, including bone, cartilage, muscle, and neural elements. As a result, they have been extensively studied as potential tools in regenerative medicine (1, 2). Also of great interest are the immunosuppressive properties of MSC, especially in the repression of allogeneic immune responses (3, 4, 5, 6, 7) and of autoimmune diseases (8). Soluble factors produced by MSC, either constitutively or in response to a contact-dependent activation by lymphoid cells, have been suggested to play a role in immunosuppression (9). In contrast, MSC up-regulate the expression of MHC class I (MHC I) and class II (MHC II) molecules in response to proinflammatory stimulus such as IFN-{gamma}. We have shown that MSC activated by IFN-{gamma} acquire MHC II-mediated Ag presentation (10), and this observation has been confirmed independently (11). We also observed that MHC-mismatched MSC implanted in normal mice lead to an alloimmune response and to their rejection (12), and this was observed as well in nonmyeloablative allogeneic bone marrow transplantation in mice (13). In addition, MSC fail to prevent acute graft-versus-host disease (GVHD) in a mouse model of allogeneic transplantation (14), an observation in stark contrast to the promising data obtained in early-phase human clinical trials (3, 4, 5). How can we conciliate the apparent paradox of MSC behaving as immune suppressor cells as well as conditional APC? In an attempt to decipher this enigma, we investigated the molecular mechanisms underlying IFN-{gamma}-induced MHC II-mediated APC functions in MSC. Our observations made in mouse and human MSC suggest that cell culture parameters, such as cell density, serum factors, and TGF-beta, can markedly modulate priming of MSC responsiveness to IFN-{gamma} for the up-regulation of MHC II expression and Ag processing, providing insights on how the immune plasticity of MSC can be readily manipulated. The implications could be of relevance for the use of MSC as a cellular pharmaceutical in modulation of immune response in health and disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
MSC culture and treatment

Mouse primary MSC were obtained from bone aspirates of 4- to 6-wk-old BALB/c or C57BL/6 mice like previously shown (12). Both femurs and tibias from each leg were isolated and cleaned from any remaining flesh. Bones were then flushed with complete medium (high glucose DMEM, 10% FBS, and 100 U/ml penicillin and streptomycin (Pen/Strep; Wisent Technologies) to extract the bone marrow, which was plated for 5 days. Fresh complete medium was added every 3–4 days until the culture reached 80% confluence. In routine maintenance culture, mouse MSC were seeded at 500 cells/cm2 every 4 days since they reached confluence after 7 days. Human primary MSC were provided to us by D. J. Prockop (Tulane University, New Orleans, LA) and maintained in {alpha}-MEM, 2 mM L-glutamine (Wisent Technologies), 16.5% FBS (Atlanta Biologicals), and 100 U/ml Pen/Strep. Donors 240L, 5066R and 5068L were a 24 year-old male, 22 year-old female, and, 24 year-old male, respectively. Karyotypes of MSC from donor 240L has been established by D. J. Prockop and colleagues. For this task, cells were cultured and harvested using standard cytogenetic procedures. Twenty metaphases were examined and normal karyotypes were observed, in the absence of consistent numerical or structural chromosome anomalies (D. J. Prockop, personal communication). Human MSC were expanded for one to six passages by plating cells every 7 days at either 100 or 2000 cells/cm2, as indicated. Both human and mouse MSC populations were tested for the absence of CD31+ or CD45+ cells, expression of CD34, CD44, CD73, CD90, and CD105, and ability to differentiate into adipocytes and osteocytes as described elsewhere (12). Recombinant human (rh) and mouse (rm) IFN-{gamma} were purchased from BioSource International and rhTGF-beta1 was obtained from R&D Systems.

Cell lines

C57BL/6 mouse DC2.4 dendritic cells (DC) and chicken egg white albumin (OVA)-specific I-Ab-restricted MF2.2D9 CD4+ T hybridoma cell line were provided by K. L. Rock (University of Massachusetts, Worcester, MA) (15) and cultured in RPMI 1640, 10% FBS, and 100 U/ml Pen/Step and 50 µM 2-ME. Mouse BLK CL.4 (ATCC TIB-81) fibroblastic cells and bEnd.3 (ATCC CRL-2299) endothelial cells were purchased from American Type Culture Collection. Primary HUVEC were obtained from Cambrex.

Flow cytometry analysis

The following Abs were used for flow cytometry analysis: PE-conjugated anti-mouse CD45 (clone 30-F11), CD105 (clone MJ7/18; eBioscience), CD80 (clone 16-10A1), Db or Kb (MHC I, clones KH95 or AF6-88.5), I-Ab (MHC II, clone AF6-120.1), or B7-H1 (clone MIH5; eBioscience); FITC-conjugated anti-human CD105 (clone 8E11; Chemicon International); biotin-coupled anti-human CD90 (clone 5E10) or CD45 (clone HI30); PE-conjugated anti-human CD31 (clone WM-59) or CD73 (clone AD2); allophycocyanin-conjugated anti-human CD34 (clone 581) or CD44 (clone G44-26); PerCP-conjugated anti-human HLA-DR Ab (MHC II, clone L243), and isotypic controls. Except where indicated, Abs were purchased from BD Biosciences. For cell cycle analysis of mouse MSC, surface staining of I-Ab was first performed and cells were fixed with 1% paraformaldehyde (PFA), washed with PBS and with the BD Biosciences Perm/Wash solution, and stained overnight with 7-aminoactinomycin D (BD Biosciences) in the presence of 50 µg/ml DNase-free RNase (Roche). Flow cytometry analysis was performed on 20,000 events using a FACSCalibur cytometer and data were analyzed using CellQuest software (BD Biosciences).

MHC II-restricted Ag-processing assay

Cell cultures were incubated with 2 mg/ml OVA (Sigma-Aldrich) and 10 U/ml (3 ng/ml) rmIFN-{gamma} for 24 h. Afterward, cells were lifted by incubation with trypsin, washed and fixed with 1% PFA, and seeded in 96-well plates at 105 or 3 x 104 cells/well. I-Ab-restricted OVA-specific MF2.2D9 T hybridoma cells (105) were added in a total volume of 200 µl. After 24 h, the levels of IL-2 in the supernatant were quantified by ELISA (eBioscience). In other assays, nonfixed cells were coincubated with MF2.2D9 cells (105) in 96-well plates in the presence of 2.5-0.625 mg/ml OVA and 10 U/ml rmIFN-{gamma} for 24 h, and supernatants were processed as described above.

Growth response to TGF-beta

MSC were seeded in 96-well plates and treated or not with 4–40 pM (0.1–1 ng/ml) rhTGF-beta1 for 3 days before performing a MTT assay using the Cell Titer96 reagent (Promega) to measure cell viability. Conversion of tetrazolium compound to a 490-nm absorbing formazan product was measured on an ELX800 microplate reader (Bio-Tek Instruments).

Immunoblot analysis

Cell pellets were resuspended in Cell Lysis Buffer (Sigma-Aldrich) complemented with protease inhibitors (Roche). Protein concentration of whole cell extracts (WCE) was determined using a Bradford protein assay (Bio-Rad). Equal amounts of WCE (20–40 µg) were run on a 4–20% Tris-glycine gel (Invitrogen Life Technologies) and transferred onto a 0.45-µm polyvinylidene fluoride membrane (Millipore). Primary Abs were specific for: phospho-Stat1 (Tyr401; Cell Signaling), phospho-Smad2/Smad3 (Ser465/467; Cell Signaling), phospho-Smad1/Smad5/Smad8 (Ser463/465; Cell Signaling), HLA-DR (BRA22; Santa Cruz Biotechnology) or {alpha}-Tubulin (Tu-02; Santa Cruz Biotechnology). Secondary Abs were either HRP-conjugated sheep anti-rabbit IgG or rabbit anti-mouse IgG (GE Healthcare) and were revealed using the ECL Advance solution (GE Healthcare). Immunoblots were exposed to films (Kodak BioMax MR films) for 5 s to 10 min. Figures show scans of films with exposures below saturating levels of the chemoluminescent signal.

RT-PCR

DNA-free total RNA was prepared using the RNeasy kit with DNase digestion (Qiagen). RNA (2 µg) was reverse transcribed with the avian myeloblastosis virus reverse transcriptase (Roche Applied Science) using the random primer p(dN)6. An aliquot of one-fiftieth of the resulting cDNA was used for PCR amplification in a 20-µl reaction volume containing the Thermus aquaticus Taq polymerase (Upstate Biotechnology). Type-specific primers for mouse C2ta type I, type III, and type IV were designed by Pai et al. (16). GAPDH primers were as described previously (17). Other mouse primers were: Acvrl1 (ALK1, NM_009612.1) forward 1613-ACA CCCACCATCCCTAACC-1631 and reverse 1667-ACCAGCACTCTCT CATCATCTG-1688 (amplicon 76 bp); Tgfbr1 (ALK5, NM_009370.2) forward 1236-AAATTGCTCGACGCTGTTCT-1255 and reverse 1280-GGTACAAGATCATAATAAGGCAACTG-1305 (amplicon 70 bp); and ActB (beta-actin, NM_007393.1) forward 1010-TGACAGGATGCAGAAGGAGA-1029 and reverse 1067-CGCTCAGGAGGAGCAATG-1084 (amplicon 75 bp). These primers were designed using the Roche Applied Science Universal Probe Library web site and had an annealing temperature of 58°C.

Real-time PCR

Quantitative PCR assays for CIITA and beta-actin mRNA were performed in duplicate using the Universal Probe Library system (Roche Applied Science). Primers used for determination of total mouse CIITA mRNA levels (AF100709.1 in the region shared by all isoforms) were: forward 2939-GATGTGGAAGACCTGGATCG-2958 and reverse 2982-TGCATCTTCTGAGGGGTTTC-3001 and were used with the Universal Probe Library 110. Primers specific for mouse ActB (beta-actin) were as described above and used with Universal Probe Library 106. Quantitative PCR assays specific for mouse Gapdh were performed using the SYBR Green I reagent (Roche Applied Science) as previously detailed (17). PCR efficiency corrections were determined for all primers by establishing standard PCR curves performed on a mixture of cDNA pooled from IFN-{gamma}-activated macrophages, splenocytes, and MSC. Data were collected using the LightCycler 2.0 Real-Time PCR System (Roche Applied Science) and analyzed using the relative quantification (based on the relative expression of target genes vs beta-actin or GAPDH as reference genes) with a normalized calibrator and with an efficiency correction method on the LightCycler analysis software. The absence of genomic DNA contamination was demonstrated routinely by analysis of PCR performed with total RNA and with each of the primer sets.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of cell density on IFN-{gamma}-induced MHC II expression and Ag processing in murine MSC

Primary mouse MSC were isolated and characterized as previously described (12). Only MSC populations negative for CD31+ or CD45+ cells, expressing CD34, CD44, CD73, CD90, and CD105, and displaying osteogenic and adipogenic differentiation ability were selected for further characterizations (data not shown). Because it has been reported that cell density markedly influences the potential mesenchymal plasticity of MSC (18, 19), we tested whether it would affect immune plasticity, as manifested by MHC II expression, as well. Mouse C57BL/6 MSC were plated at high (12,000–16,000 cells/cm2) or low (550 cells/cm2) density for 2–3 days and then activated with IFN-{gamma} for 24 h. At the end of this period, MSC cultures reached 100% and 10–20% confluence, respectively (Fig. 1A, bottom panels). Proliferation rates of MSC were unaffected by the addition of IFN-{gamma} and showed an ~15- and 4-fold increase in cell number after 4 days in cultures initially plated at 550 and 12,000 cells/cm2, respectively (data not shown). Flow cytometry analysis demonstrated that the up-regulation of MHC II molecules by IFN-{gamma} at 100 U/ml (Fig. 1A) or 10 U/ml (data not shown) occurred mostly in high cell density MSC cultures. By contrast, there was no difference in MHC I or B7-H1/PD-L1 expression levels up-regulated by IFN-{gamma} in low- and high-cell density cultures; in addition, expression of endoglin (CD105) was unchanged by IFN-{gamma} or cell density. Two other C57BL/6 as well as one BALB/c MSC preparations were tested for IFN-{gamma}-induced MHC II and B7-H1 expression in cells plated at high or low density, and expression patterns were similar to those described above (data not shown).


Figure 1
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  		FIGURE 1. Expression of MHC II and MHC II-mediated Ag processing in high- or low-density mouse MSC cultures. A, Flow cytometry analyses of MSC cultured at different cell densities. C57BL/6 MSC were plated at 12,000 or 550 cells/cm2 for 3 days and activated or not with 100 U/ml rmIFN-{gamma} for 24 h. Cells were analyzed by flow cytometry for expression of CD45, CD105, Db, I-Ab, CD80, and B7-H1. Plots show isotype control IgG-staining profiles for IFN-{gamma}-stimulated cells (dotted line) vs specific Ab-staining profiles (thin line for nonstimulated cells and thick line for IFN-{gamma}-stimulated cells). Isotype control-staining profiles for nonstimulated cells were similar to the ones of IFN-{gamma}-stimulated cells (data not shown). Bottom panel, Phase-contrast MSC cultures at harvesting (original magnification, x100). B, APC assay. C57BL/6 MSC, BLK CL.4 cells, or DC2.4 (APC) were plated at 12,000 or 550 cells/cm2 for 3 days and exposed or not to 100 U/ml rmIFN-{gamma} and 2 mg/ml OVA for 24 h, harvested by trypsinization, and fixed with PFA. APC (105 or 3 x 104 cells) were then cocultured in 96-well plates with OVA-specific I-Ab-restricted CD4+ T hybridoma cells (MF2.2D9; 105 cells). After 20 h, supernatants were collected and tested for IL-2 release by CD4+ T hybridoma cells using ELISA. Means of triplicates ± SDs of one of three representative experiments are shown.

 
Next, MHC II-mediated Ag processing was tested. C57BL/6 mouse MSC cultures at different cell densities were incubated for 24 h with IFN-{gamma} and OVA as a source of soluble exogenous Ag. Positive controls included the C57BL/6 DC2.4 immortalized DC line, which displays constitutive APC functions. APC were fixed with PFA and exposed to the CD4+ MF2.2D9 T hybridoma cells that are specific for an OVA C-terminal epitope (OVA323–339, ISQAVHAAHAEINAGR) presented in the context of I-Ab molecules. T hybridoma cells are not dependent on costimulation signals for activation and respond to the presence of specific Ag-MHC complexes on APC by the production of IL-2. Levels of IL-2 were measured supernatants from APC and T hybridoma cells cocultures by ELISA. It revealed that high-density MSC cultures were more easily primed for MHC II-mediated processing of OVA when activated with IFN-{gamma} compared with low-density cultures (Fig. 1B). As expected, low- and high- density cultures of IFN-{gamma}-activated BLK CL.4 fibroblasts did not process OVA, although they expressed MHC II (data not shown). Thus, MSC from high-density cell cultures display increased responsiveness to IFN-{gamma} for MHC II expression and Ag processing.

IFN-{gamma}-induced MHC II expression in mouse MSC is not correlated with the cell cycle and is increased by serum starvation or cell connectivity

To test whether MHC II up-regulation by IFN-{gamma} was dependent on the cell cycle status of MSC, IFN-{gamma}-activated MSC from low- and high-cell density cultures were subjected to analysis of MHC II expression and DNA content levels by flow cytometry. High-density cultures accumulated cells mostly in the G0-G1 and G2-M phases (Fig. 2A, left panel). Low-density MSC cultures contained asynchronous cells and the higher proportion of cells in the S phase, which is most likely linked to optimal cell culture conditions since cells in the S phase were less numerous after serum deprivation in low-density MSC cultures. As described above, low-density MSC cultures have a blunted IFN-{gamma}-induced MHC II expression response compared with high-density cell cultures (Fig. 2A, right panel). In both, however, the proportion of cells in the G0-G1, S, or G2-M phases expressing MHC II was similar. Surprisingly, IFN-{gamma}-induced MHC II expression was strongly up-regulated by the removal of serum factors, and this effect occurred regardless of the cell cycle status. Accordingly, serum-starved low-density MSC cultures exhibited increased ability to process soluble OVA and present I-Ab-restricted OVA epitopes to CD4+ T hybridomas (Fig. 2B). Thus, the ability of mouse MSC to express MHC II in response to IFN-{gamma} is not directly related to the cell cycle but can be up-regulated by high cell density, possibly through increased cell connectivity, or the removal of serum factors.


Figure 2
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  		FIGURE 2. Absence of correlation between cell cycle and IFN-{gamma}-induced MHC II expression in high- or low-density mouse MSC cultures. A, Flow cytometry analyses of high- vs low-cell density or serum-starved mouse MSC cultures. C57BL/6 MSC were plated at 12,000 or 550 cells/cm2 in complete medium. Where indicated, medium was replaced with serum-free medium 2 days later. Twenty-four hours later, cells were left untreated or treated with 30 U/ml rmIFN-{gamma} for an additional day. Cells were harvested by trypsinization and incubated with PE-conjugated anti-I-Ab Ab or PE-conjugated mouse IgG2a isotype (negative) control Ab. Cells were then fixed, permeabilized, counterstained with 7-aminoactinomycin D (7AAD) for DNA content, and analyzed by flow cytometry. Cell cycle phase is denoted as G0-G1, S, and G2-M. B, APC assay on serum-starved mouse MSC cultures. C57BL/6 MSC or BLK CL.4 cells were plated at 550 cells/cm2. Where indicated, medium was replaced with serum-free medium 2 days later. Twenty-four hours later, all cell cultures were treated with 30 U/ml rmIFN-{gamma} in the presence or absence of 2 mg/ml OVA for an additional day and processed as described in Fig. 1B.

 
Signaling pathways involved in IFN-{gamma}-induced MHC II expression in murine MSC

To study the regulation of the IFN-{gamma} response, a major downstream event to the activation of the IFN-{gamma} receptor, namely, STAT1 activation, was assessed. The phosphorylation of STAT1 was detected as early as 10 min after IFN-{gamma} stimulation in high- and low-confluent MSC cultures, as seen in immunoblots on WCE using a phospho-specific Ab (Fig. 3A). STAT1 phosphorylation was monitored in cells exposed to decreasing concentrations of IFN-{gamma} and an identical response was observed in high- and low-confluent MSC cultures (Fig. 3B). Consistent with this finding, the IFN regulatory factor 1 (IRF-1)-dependent B7-H1 encoding gene was also unaffected by cell density (Fig. 1A), and these results suggest that IFN-{gamma}R-mediated activation of STAT1 and IRF-1 are not impaired in low-density mouse MSC cultures.


Figure 3
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  		FIGURE 3. IFN-{gamma}-induced STAT1 phosphorylation and expression of CIITA in high- or low-density mouse MSC cultures. A, STAT1 phosphorylation. C57BL/6 MSC were plated at 12,000 or 550 cells/cm2 in complete medium for 3 days. Cells were left untreated or treated with 30 U/ml rmIFN-{gamma} for 10 or 20 min. Samples of WCE were run on a SDS-PAGE gel and subsequently subjected to immunoblot analysis of STAT1 phosphorylation at residue Tyr701 or {alpha}-tubulin expression, as a control. B, Same as A, except that cells were left untreated or treated with different doses of rmIFN-{gamma} (0.15–10 U/ml) for 20 min. C, CIITA expression. C57BL/6 MSC or BLK CL.4 cells were plated at 12,000 or 550 cells/cm2 in complete medium for 3 days. Splenocytes and peritoneal macrophages were harvested from C57BL/6 mice. Cells were left untreated or treated with 30 U/ml rmIFN-{gamma} for 18 h. Total DNase-treated RNA was prepared and processed for quantitative RT-PCR analysis of CIITA mRNA levels with primers and probe common for all isoforms. Levels of CIITA were obtained by normalizing to GAPDH levels. Similar results were obtained when using beta-actin as a reference gene for normalization (data not shown). D, Same as C, except that RNA were processed for RT-PCR analysis of mRNA expression of CIITA forms I, III, and IV and beta-actin as a control. Twenty-five, 30, 35, and 40 cycles were conducted for each amplification and PCR products were analyzed by electrophoresis on 3% agarose gels. Photographs show ethidium bromide fluorescence of PCR products before reaching saturation levels for CIITA form IV and beta-actin and at 40 cycles for CIITA forms I and III. Negative controls to test genomic DNA contamination included PCR performed with RNA samples not subjected (–) to reverse transcription (RT).

 
The regulation of MHC II expression and Ag processing occurs predominantly at the transcriptional level and the CIITA protein is a potent activator for MHC II gene transcription (20). CIITA mRNA expression was assessed in MSC by quantitative RT-PCR using primers and a probe common for all forms. With a difference to primary splenocytes or peritoneal macrophages, MSC did not express constitutively CIITA (Fig. 3C). Total CIITA mRNA expression was induced by IFN-{gamma}; however, levels were reduced by ~4-fold in low-density compared with high-density culture cells (Fig. 3C). Of note, a reduction of CIITA mRNA levels was also observed when BLK CL.4 fibroblasts were plated at a low-compared with a high-cell density. This suggests that the observed regulation of MHC II expression by cell density in mouse MSC may occur in other cell types. To define promoter elements responsible for IFN-{gamma} induction of the CIITA gene, we used the type-specific primers for CIITA types I, III, and IV designed by Pai et al. (16). Type I and type III CIITA mRNA were detected, as expected, in ex vivo mouse splenocytes. They also expressed type IV CIITA mRNA independently of activation with rmIFN-{gamma} (Fig. 3D), which may reflect a basal production of IFN-{gamma}. By contrast, none of the CIITA isoforms was expressed in unstimulated MSC and only the type IV was present in IFN-{gamma}-activated cells (Fig. 3D). Therefore, CIITA up-regulation occurs mostly via the activation of promoter IV in MSC stimulated with IFN-{gamma}. In addition, CIITA expression is regulated by cell density and is likely a key regulator of the regulation of MHC II expression in MSC.

TGF-beta signaling pathways and effects on IFN-{gamma} induction of MHC II expression in murine MSC

Various factors such as IL-1beta, IL-4, TGF-beta, and IL-10 have been shown to block the up-regulation of MHC II expression induced by IFN-{gamma} in various cell types, e.g., astroglioma cells or DC (21, 22, 23, 24, 25). We examined the response of MSC to TGF-beta with respect to cell viability, signaling, and regulation of MHC II expression. MSC were moderately sensitive to either growth increase or inhibition by TGF-beta at high- or low-cell density, respectively (Fig. 4A). Mammals express one TGF-beta type II receptor (TGF-betaRII) and two TGF-betaRI, i.e., ALK5 and ALK1/TSR-I. Although ALK5 is ubiquitously expressed, ALK1 was observed predominantly present in blood vessels, mesenchyme of the lung, submucosal layer of the stomach and intestines, and at sites of epithelial and mesenchymal interactions during mouse embryo development (26) and in vitro in human endothelial cell lines (27). In this study, expression of ALK5 and ALK1 mRNA was assessed in primary MSC, splenocytes, and peritoneal macrophages as well as in the bEnd.3 endothelial and BLK CL.4 fibroblast cell lines by semiquantitative RT-PCR assays. ALK5 mRNA was expressed in all tested cell types (Fig. 4B). ALK1 mRNA levels appeared, as expected, increased in bEnd.3 cells compared to other cell types. In addition, observed results suggested that levels of ALK1 transcripts were increased by low- or high-density culture conditions in MSC or BLK CL.4 cells, respectively. These results prompted us to probe downstream events to TGF-betaR binding. ALK5 signals through receptor-regulated Smad2/Smad3 (Smad2/3) while ALK1 activates receptor-regulated Smad1/Smad5/Smad8 (Smad1/5/8) (28). WCE from TGF-beta1-activated MSC, BLK CL.4, and bEnd.3 cells were subjected to immunoblot analysis of receptor-regulated Smad activation using phospho-specific Abs. Low-density MSC cultures, compared with high-density cultures, demonstrated increased sensitivity to TGF-beta pertaining to the phosphorylation of both Smad2/3 and Smad1/5/8 (Fig. 4C). A different response was detected in BLK CL.4 cells, in which only high-cell density cultures displayed a TGF-beta-induced, dose-dependent Smad1/5/8 phosphorylation, while low-cell density cultures displayed basal phosphorylation levels (Fig. 4D). Lastly, we tested whether TGF-beta regulates MHC II expression and Ag processing in mouse MSC. Flow cytometry data showed that IFN-{gamma}-induced expression of surface MHC II was inhibited by pretreatment with TGF-beta in MSC, while levels of MHC I, B7-H1, and CD80 were unchanged (Fig. 5A). Accordingly, MHC II-restricted processing of soluble OVA and presentation to T hybridoma cells was reduced by TGF-beta (Fig. 5B). Quantitative RT-PCR assays demonstrated that TGF-beta pretreatment resulted in a ~3-fold down-regulation of IFN-{gamma}-induced CIITA mRNA levels (Fig. 5C). TGF-beta-mediated inhibition of CIITA and MHC II induction by IFN-{gamma} was independent of STAT1 because its phosphorylation was not inhibited by TGF-beta (Fig. 5D). Thus, TGF-beta signaling, which involves ALK5, ALK1, Smad2/3, and Smad1/5/8, does not affect IFN-{gamma}-R activity and STAT1 phosphorylation. Nonetheless, it inhibits IFN-{gamma}-induced MHC II-restricted Ag presentation in MSC and this effect correlates with the decrease in CIITA and MHC II levels.


Figure 4
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  		FIGURE 4. TGF-beta signaling in mouse MSC. A, TGF-beta growth effect. C57BL/6 MSC were plated in 96-well plates at the indicated densities in complete medium for 2 days. Cells were left untreated or treated with 40 pM rhTGF-beta1 for 3 days and subjected to a MTT cell viability assay measured by tetrazolium reduction to a 490-nm absorbing formazan compound. Shown are the means of triplicates ± SDs of one of three representative experiments. A two-way ANOVA with a Bonferroni post test were used to compare replicate means (*, p < 0.05; **, p < 0.01; ***, p < 0.001). B, ALK1 and ALK5 expression. C57BL/6 MSC or BLK CL.4 cells were plated at 12,000 or 550 cells/cm2 in complete medium for 3 days. Splenocytes and peritoneal macrophages were harvested from C57BL/6 mice. bEnd.3 endothelial cells were harvested at 80% confluence. Total RNA was prepared from adherent live cells and processed for RT-PCR analysis of mRNA expression of ALK5, ALK1, and gapdh as a control. Twenty-five, 30, 35, and 40 cycles were conducted for each amplification and PCR products were analyzed by electrophoresis on 3% agarose gels. Photographs show ethidium bromide fluorescence of PCR products obtained before reaching saturation levels. Amplicon sizes of ALK5, ALK1, and gapdh were 70, 76, and 550 bp, respectively. Primers specific for ALK5 formed dimers (indicated by *) that migrated on a 3% agarose gel slightly below specific PCR product and that were visible in most samples, including in PCR performed in the absence of cDNA (ddH2O lane, top panel). C, TGF-beta signaling. C57BL/6 MSC were plated at 12,000 or 550 cells/cm2 in complete medium for 3 days. bEnd.3 cells were grown to 80% confluence. Cells were serum-starved for 2 h and left untreated or treated with rhTGF-beta1 (0.3–30 pM) for 45 min. Samples of WCE were run on a SDS-PAGE gel and subsequently subjected to immunoblot analysis of Smad2 dually phosphorylated at residues Ser465 and Ser467 (top panel), Smad1 dually phosphorylated at residues Ser463 and Ser465, as well as Smad5 and Smad8 phosphorylated at equivalent sites (middle panel) or {alpha}-tubulin expression as a control (bottom panel). D, Same as C, except that BLK CL.4 cells were analyzed.

 

Figure 5
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  		FIGURE 5. TGF-beta-mediated modulation of IFN-{gamma}-induced signaling and MHC II processing in mouse MSC. A, Flow cytometry analyses. C57BL/6 MSC were plated at 12,000 cells/cm2 for 2 days and pretreated or untreated with 40 pM rhTGF-beta1 for 24 h before 30 U/ml rmIFN-{gamma} was added, where indicated, for another 24 h. Cells were then analyzed by flow cytometry for Kb, I-Ab, B7-H1, and CD80 expression. B, APC assay. C57BL/6 MSC were plated at the indicated densities for 2 days and treated or untreated with 40 pM rhTGF-beta1. The next day, cells were harvested by trypsinization and cocultured in 96-well plates with OVA-specific I-Ab-restricted CD4+ T hybridoma cells (MF2.2D9; 105 cells) in the presence of 30 U/ml rmIFN-{gamma} and OVA (0–2.5 mg/ml). After 20 h, supernatants were collected and tested for IL-2 release by CD4+ T hybridoma cells using ELISA. Means of triplicates ± SDs of one two representative experiments are shown. C, CIITA expression. C57BL/6 MSC were plated at 12,000 or 550 cells/cm2 for 2 days and treated or untreated with 40 pM rhTGF-beta1. The next day, cells were left untreated or treated with 30 U/ml rmIFN-{gamma} for 16 h and processed for determination of CIITA mRNA levels as described in Fig. 3C. D, STAT1 phosphorylation. C57BL/6 MSC were plated at 12,000 cells/cm2 for 2 days, pretreated or untreated with 40 pM rhTGF-beta1 for 24 h, and stimulated or not with rmIFN-{gamma} (0.15–10 U/ml) for 20 min. Samples of WCE were analyzed for STAT1 phosphorylation as described in Fig. 3A.

 
Regulation of growth, differentiation and IFN-{gamma}-induced MHC II expression in human MSC

Human MSC were established from three normal donors. Flow cytometry analyses showed that cells were negative for CD31, CD45, and CD34 and expressed CD44, CD73, CD90, and CD105 (Fig. 6A). In addition, when exposed to specific differentiation-inducing agents, human MSC acquired an adipogenic (data not shown) or osteogenic phenotype (see Fig. 6B). Mouse MSC can grow well in culture for extended periods, get confluent, and still behave like stem/progenitor cells. In contrast, human MSC become senescent after several in vitro passages. At early passages, single-derived human MSC clones become heterogeneous as they expand. They contain small, round, or spindle-shaped (fibroblast like) rapidly self-renewing MSC (RS-MSC) and larger, slowly renewing MSC (SR-MSC) (18). RS-MSC have the greatest potential for multilineage differentiation (18) and in vivo engraftment (19). In agreement with these reports, we observed that early passage human MSC plated at high-cell density (2000 cells/cm2) did not reach confluence after a 7-day culture period but lost RS-MSC while SR-MSC became large flat cells and acquired a mature phenotype refractory to osteogenic differentiation compared with cells plated at 100 cells/cm2 (Fig. 6B and data not shown). As we described above in mouse MSC, TGF-beta induced a partial growth inhibition in human MSC unless cells were overconfluent (cell density ≥20,000 cells/cm2, Fig. 6C) and its signaling involved Smad1/5/8 and Smad2/3 (Fig. 6D). We next investigated the effects of TGF-beta and cell density on MHC II levels in human MSC. First, we tested whether IFN-{gamma} up-regulates the total expression of MHC II or the trafficking to the cell surface of a preexisting pool of MHC II molecules, as previously suggested (29). MSC from two donors were exposed to IFN-{gamma} for 24 h and WCE were subjected to immunoblot analysis of total MHC II levels. Results showed that MHC II expression was not constitutive and depended on activating stimulus, e.g., IFN-{gamma} (Fig. 7A). In addition, TGF-beta inhibited the up-regulation of IFN-{gamma}-induced MHC II (Fig. 7B). However, with difference to mouse MSC, human MSC cultures plated at ≥2000 cells/cm2, which contained less FSClowSSClow small cells (R2 population, Fig. 7C), displayed decreased levels of IFN-{gamma}-induced MHC II expression compared with cells plated at 100–200 cells/cm2, as observed by flow cytometry (Fig. 7C) and immunoblot (Fig. 7D) analyses. Thus, cell proliferation, differentiation, and IFN-{gamma}-induced MHC II levels are regulated by TGF-beta and cell density in human MSC, although not in the same way as in mouse MSC.


Figure 6
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  		FIGURE 6. Proliferation, differentiation, and TGF-beta signaling pathways in human MSC (donor 240L). A, Expression of MSC markers. Cells (passage 1) were cultured at 100 cells/cm2 for 7 days and analyzed for the expression of CD31, CD45, CD34, CD44, CD73, CD90, and CD105 by flow cytometry. Plots show isotype control IgG-staining profiles (dotted line) vs specific Ab-staining profiles (thick line). B, Initial cell density affects MSC differentiation. Cells were cultured for two passages at 100 or 2000 cells/cm2 and equal numbers of cells (105) were seeded in 6-well plates and subjected to a bone differentiation assay that was revealed by Alizarin Red staining. C, Growth response to TGF-beta. Passage two MSC (plated in routine at 100 cells/cm2) were seeded at the indicated cell density in 96-well plates for 3 days. Cells were then left untreated or treated with rhTGF-beta1 (4 or 40 pM) for 3 days and subjected to a MTT cell viability assay as described in Fig. 4A. D, TGF-beta signaling. MSC cultured from passages one to five at either 100 or 2000 cells/cm2, as indicated, or HUVEC were serum-starved for 2 h and left untreated or treated with rhTGF-beta1 (1–30 pM) for 45 min. Samples of WCE were run on a SDS-PAGE gel and subsequently subjected to immunoblot analysis of Smad2 dually phosphorylated at residues Ser465 and Ser467 (top panel), Smad1 dually phosphorylated at residues Ser463 and Ser465, as well as Smad5 and Smad8 phosphorylated at equivalent sites (middle panel) or {alpha}-tubulin expression as a control (bottom panel).

 

Figure 7
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  		FIGURE 7. Effect of TGF-beta and cell density on IFN-{gamma}-induced MHC II expression in human MSC. A, Expression of HLA-DR in unstimulated and IFN-{gamma}-stimulated cells. Passage two MSC from donors 5066R and 5068L (plated at passage one at 2000 cells/cm2) were plated at 2000 cells/cm2 for 2 days and stimulated or not with rhIFN-{gamma} (10–200 U/ml). Samples of WCE were then analyzed by immunoblot for the expression of HLA-DR (MHC II). B, Effect of TGF-beta on IFN-{gamma}-induced MHC II expression. Passage five MSC (donor 240L), routinely cultured at 100 cells/cm2, were plated at 500 cells/cm2 for 3 days. rhTGF-beta1 (10 pM) was then added to cultures for 24 h before the addition of 30 U/ml rhIFN-{gamma} for another 24 h. HLA-DR expression was analyzed as in A. C, Effect of cell density on HLA-DR expression. Passage five MSC (donor 240L) plated at 100 (left panel) or 2000 cells/cm2 (right panel) from passages one to four (p1-> p4) were seeded at 4000 and 200 cells/cm2, respectively, for 4 days before being activated or not with 30 U/ml rhIFN-{gamma} for 24 h. Cell morphology and surface HLA-DR expression were then assessed by flow cytometry. The R1, R2, and R3 gates represent the entire population, small cells (FSClowSClow, RS-MSC-enriched population) and large granular cells (SR-MS-enriched population), respectively. D, Cells were treated as in C, except that HLA-DR expression was determined by immunoblot on WCE samples.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Divergent observations have been reported on the use of MSC as immunomodulatory cells. Suppression of in vitro and in vivo immune reactions by MSC has been substantially documented, as detailed in a recent review (9). Nonetheless, we observed that MSC are able to induce in vivo allogeneic immune reactions (12) or to acquire MHC II-mediated Ag presentation when activated with IFN-{gamma} (10). Nauta et al. (13) reported that allogeneic mouse bone marrow implantation was favored by coinjection of host MSC but inhibited by donor MSC (13). In line with these divergent observations, expression of MHC II expression on human or mouse MSC was alternatively observed to be constitutive (11, 30), up-regulated (10, 12, 29, 31) or not (32) by IFN-{gamma} or inhibited by IFN-{gamma} (11). Some of these discrepancies regarding the immune plasticity of MSC may be related to underrecognized variable: cell culture conditions, including the cell density and resultant connectivity of MSC. The behavior of human MSC, and to a much lesser extent of rodent MSC, seems sharply dependent on cell density. Indeed, MSC late passages or cultures grown for extended times at high density lose RS-MSC, and SR-MSC become large flat cells as they acquire a mature phenotype unfavorable to multilineage differentiation, replication, and in vivo engraftment (18, 19, 33). We therefore studied the effect of MSC density on MHC II expression and Ag processing. We observed that MHC II expression and mediated Ag processing induced by IFN-{gamma} were down-regulated by low-cell density in mouse C57BL/6 (Fig. 1) or BALB/c (data not shown) MSC. This plasticity appears rapidly regulated because differences were observed after plating cells at either low or high density for only 4 days. Surprisingly, an opposite effect was observed in human MSC where IFN-{gamma}-induced MHC II expression occurred at the highest levels in low-density cultures (Fig. 7, C and D). In both cases, however, this suggests that MHC II-related immunostimulatory functions in MSC are critically regulated by culture conditions such as cell density.

Using mouse MSC, we investigated possible mechanisms involved in the cell density-dependent regulation of MHC II expression. C57BL/6 MSC cultures plated at 550 cells/cm2 were in log phase of growth at the time of harvesting 4 days later, whereas cultures plated at 12,000 cells/cm2 reached the stationary phase and confluence. Despite these differences, IFN-{gamma}-induced MHC II expression occurred regardless of the cell cycle in mouse MSC (Fig. 2A). Transfer of conditioned medium from confluent cells to dividing cells or vice versa did not change the regulation of MHC II expression, suggesting that secreted factors do not play a role in this response (data not shown). This raises the possibility that contact-dependent receptors modulate APC functions in mouse MSC. Candidate receptors known to be expressed by MSC and deemed to play a role in Ag presentation include: CD44, a glycosaminoglycan present in extracellular matrixes and in association with cell surfaces that acts as a receptor for hyaluronate (34); cadherins mediating Ca2+-dependent cell-cell adhesion, or connexins in intercellular gap junctions. MSC have been shown to express several connexins, including connexin 43, and form functional gap junction intercellular communications when maintained at high confluence (35, 36), a feature shared by professional APC and proposed to be involved in immune activation (37, 38).

MHC II Ag presentation pathways are specific to the so-called professional APC of hematological origin, i.e., DC, macrophages, and B lymphocytes. We know very little about mechanisms involved in the turnover at the cell surface of MHC II molecules. Immature DC display fully efficient MHC II Ag-presenting machinery; however, the recycling of surface MHC II molecules is significantly faster compared with mature DC (39). Le Blanc et al. (29) observed that nonactivated MSC express cytoplasmic MHC II molecules and that IFN-{gamma} triggers their transport to the cell surface. In our hands, intracellular MHC II expression by resting human MSC was absent. We observed that MHC II expression is inducible and depends on activating stimulus, e.g., IFN-{gamma}. Nevertheless, our previous flow cytometry analysis of MHC II expression on MSC clonal populations demonstrated that a small percentage displayed a constitutive surface expression of MHC II (10), suggesting that MSC preparations may vary in the proportion of such cells. The majority of cell types activated with IFN-{gamma} up-regulate MHC II via the activation of CIITA promoter IV (40). Yet, very few of them are capable of MHC II-mediated Ag presentation. Some intestinal, skin, and thymic cortical epithelial cells can mediate MHC II Ag presentation to immunoregulatory gut T cells. The human T82 intestinal epithelial cell line acquires MHC II Ag processing after IFN-{gamma} exposure. In these cells, the transcription of genes encoding MHC II as well as the MHC II chaperones HLA-DM and invariant chain relies on STAT1-mediated IFN-{gamma} transduction and induction of CIITA (41). In this study, we observed that IFN-{gamma}-induced MHC II expression and Ag processing was switched on by CIITA expression subsequently to CIITA promoter IV activation in MSC.

CIITA promoter IV is regulated by STAT1, IRF-1, and upstream transcription factor 1 transcription factors (20). IL-1beta and TGF-beta were found to down-regulate its activity, yet they do not interfere with STAT1 and/or IRF-1 activation (21, 22, 24, 25). Similarly, we observed that cell density and TGF-beta signaling critically regulated CIITA expression and MHC II Ag processing in MSC, whereas they had no effect on IFN-{gamma}-induced STAT1 phosphorylation nor on the expression of B7-H1. B7-H1 expression was described dependent on the STAT1-induced IRF-1 transcription factor (42). No clear model has been suggested to account for the antagonistic regulation of transcriptional responses induced by IFN-{gamma} and TGF-beta. We observed that TGF-beta signaling in human and mouse MSC may involve ALK5 and Smad2/3 as well as ALK1 and Smad1/5/8. A study suggested that the activation of ALK1 up-regulates the expression of STAT1 and thus interferes with IFN-{gamma} signaling in HUVEC (43). Other authors attributed the inhibition of CIITA promoter IV by TGF-beta to the activation of ALK5 and Smad3 transcription factor; however, no putative Smad3 binding site has been found (24). One hypothesis is that STAT1 and Smad3 compete for interaction with limiting amounts of cotranscriptional factors such as p300/CBP (44). Further characterizations will be necessary to elucidate molecular factors involved in the cross-talk between IFN-{gamma} and TGF-beta signaling in mouse and human MSC.

In summary, the molecular mechanisms governing the immune plasticity of MSC described in this study could be of relevance for their use in clinical settings and may help explain the disparity between outcome in murine models of disease and what has been observed in vitro and in vivo in humans. In the only mouse study so far describing the effect of MSC on GVHD arising from allogeneic bone marrow transplantation (alloBMT), donor MSC were cultured at high-cell density (6000 cells/cm2) (14). Based on our observations, these cells would be intrinsically primed to be responsive to inflammatory stimulus and adopt MHC II-mediated APC-like features. Indeed, it is well described that conditioning regimens and alloBMT increase plasma levels of inflammatory cytokines, such as IFN-{gamma}, IL-1, and TNF-{alpha} (45). Therefore, i.v. infusion of MSC primed for APC response in an inflammatory allogeneic environment may not display suppressive features, such as attenuating GVHD and may indeed elicit a MHC II-dependent immune response. In contrast, some research groups that have dealt with human MSC in Phase II clinical trials to treat or prevent GVHD used cells that were cultured at high densities (4000–6000 cells/cm2) (4, 5). Because we show that human MSC do not appear to be primed for a MHC II-mediated APC phenotype by high-cell culture densities, a distinguishing feature relative to mouse MSC, it is conceivable that human MSC may indeed retain a suppressive phenotype in the setting of alloBMT. Our investigations also lead us to speculate that tight control of in vitro culture conditions or TGF-beta pretreatment may skew the MSC immune phenotype even more so toward a gain of immunosuppression and thus influence the outcome of their infusion in vivo. This would improve their use as an anti-inflammatory and suppressive regulatory cell in the setting of life-threatening GVHD and morbid autoimmune ailments as well.


   Acknowledgments
 
We thank Anna Derjuga (McGill University) for assistance in the setting up of real-time PCR assays, Drs. Margaret Wolfe and Darwin J. Prockop (Tulane University) for the distribution of human MSC and useful information on their phenotype, and Dr. Nicoletta Eliopoulos (McGill University) for helpful comments on this manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by grants from Canadian Institutes of Health Research (MOP-15017), the Fond de Recherche en Santé du Québec, and the Canadian Stem Cell Network. Back

2 R.R.-M. and M.F. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Jacques Galipeau, Department of Medicine and Oncology, Sir Mortimer B. Davis Jewish General Hospital and Lady Davis Institute for Medical Research, McGill University, 3755 Cote Sainte-Catherine Road, Montreal, Quebec, Canada. E-mail address: jacques.galipeau{at}mcgill.ca Back

4 Abbreviations used in this paper: MSC, mesenchymal stem cell (mesenchymal stromal cell); MHC I, MHC class I; MHC II, MHC class II; GVHD, graft-versus-host disease; Pen/Strep, penicillin/streptomycin; PFA, paraformaldehyde; recombinant human; rm, recombinant mouse; DC, dendritic cell; WCE, whole cell extract; RS-MSC, rapidly self-renewing MSC; SR-MSC, slowly renewing MSC; alloBMT, allogeneic bone marrow transplantation. Back

Received for publication February 22, 2007. Accepted for publication May 22, 2007.


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