Activated T cells modulate immunosuppression by embryonic-and bone marrow-derived mesenchymal stromal cells through a feedback mechanism

Cytotherapy, 2012; 14: 274–284

Activated T cells modulate immunosuppression by embryonic- and bone marrow-derived mesenchymal stromal cells through a feedback mechanism WENYU LIN1,2, STEVE K. W. OH1, ANDRE B. H. CHOO1,3 & ANDREW J. T. GEORGE2 Institute, Agency for Science, Technology and Research (A∗STAR), Singapore, of Immunobiology, Department of Medicine, Imperial College London, London, United Kingdom, and 3Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore 1Bioprocessing Technology

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2Section

Abstract Background aims. Human embryonic stem cell (hESC)-derived mesenchymal stromal cells (MSC) (hESC-MSC) are an alternative source of MSC to bone marrow (BM)-derived MSC (BM-MSC), which are being investigated in clinical trials for their immunomodulatory potential. hESC-MSC have the advantage of being consistent because each batch can be generated from hESC under defined conditions. In contrast, BM-MSC have a limited proliferative capacity. Methods. The ability to suppress the proliferation of anti-CD3/CD28-stimulated CD4 ⫹ T cells by hESC-MSC was compared with adult BM-MSC and neonatal foreskin fibroblast (Fb). Results. hESC-MSC suppress the proliferation of CD4 ⫹ T cells in both contact and transwell systems, although inhibition is less in the transwell system. hESC-MSC are approximately 2-fold less potent (67 cells/100 T cells) than BM-MSC and Fb (37 and 34 cells/100 T cells, respectively) at suppressing T-cell proliferation by 50% in a transwell [inhibitory concentration(IC)50]. The anti-proliferative effect is not contact-dependent but requires the presence of factors such as interferon (IFN)-γ produced by activated T cells. IFN-γ induces the expression of indoleamine-2,3-dioxygenase (IDO) in hESC-MSC, BM-MSC and Fb, contributing to their immunosuppressive property. Conclusions. The feedback loop between MSC or Fb and activated T cells may limit the immunosuppressive effects of MSC and Fb to sites containing ongoing immunologic or inflammatory responses where activated T cells induce the up-regulation of IDO and immunomodulatory properties of MSC and Fb. These data demonstrate that hESC-MSC may be evaluated further as an allogeneic cell source for therapeutic applications requiring immunosuppression. Key Words: embryonic stem cells, fibroblasts, immunosuppression, indoleamine-2,3-dioxygenase, interferon, mesenchymal stromal cells, T cells

Introduction Mesenchymal stromal cells (MSC) or MSC-like cells have been generated from human embryonic stem cells (hESC) by retroviral transduction (1), coculture with murine stromal cells (2), spontaneous differentiation (3,4) and addition of growth factors (5). These hESC-derived MSC (hESC-MSC) are an alternative source of MSC that can be derived consistently from hESC (5). The hESC-MSC are more resistant to senescence and can be propagated for at least 80 population doublings (5), compared with 30–40 for bone marrow (BM)-derived MSC (BMMSC) (6,7). The hESC-MSC also have a higher proliferative rate, with a doubling time of 1–5 days

(1,3,5) compared with 1.3 days at primary culture to 8–15 days at the third passage for BM-MSC (6). The use of hESC-MSC obviates the need for invasive procedures to isolate MSC from sites such as BM and synovial fluid. Besides being used in cell replacement therapy because of their multipotency, MSC possess immunomodulatory properties. BM-MSC are being developed for the treatment of steroid refractory graft-versus-host disease, Crohn’s disease, multiple sclerosis and osteoarthritis (8,9). The effects of BM-MSC on T cells have been studied extensively, although BM-MSC also interact with other immune cells such as dendritic cells (10) and B cells (11).

Author contributions: WL, conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; SKWO, ABHC and AJTG, conception and design, final approval of manuscript. Correspondence: Wenyu Lin, Stem Cell Group, Bioprocessing Technology Institute, 20 Biopolis Way, 06–01, Centros, Singapore 138668. E-mail: wenyu. [email protected]. Professor Andrew J. T. George, Section of Immunobiology, Department of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK. E-mail: [email protected]. (Received 23 March 2011; accepted 5 May 2011) ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2011.635853

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Activated T cells modulate immunosuppression by hESC-MSC and BM-MSC BM-MSC suppress T-cell responses in vitro in mixed lymphocyte reactions (MLR) (12–15) and following mitogen activation (16). BM-MSC modulate the response via both soluble factors and cell–cell contact-mediated mechanisms (10,17–21). Interferon (IFN)-γ is a key molecule in the suppressive activity of BM-MSC (22). Neutralizing anti-IFN-γ receptor antibody inhibits the suppressive activity of BM-MSC on CD4 ⫹ and CD8 ⫹ T cells and natural killer (NK) cells (17). IFN-γ induces the tryptophancatabolizing enzyme indoleamine-2,3-dioxygenase (IDO) in BM-MSC, contributing to the suppressive effect on T cells (15,18). IDO operates both by the depletion of essential tryptophan (23,24) and the production of toxic kynurenines (25,26). Only limited studies have described the immunologic profile of hESC-MSC, and the precise mechanisms by which they suppress T-cell responses are not known. hESC-MSC express HLA class I, but not HLA class II, molecules (4,27) and are negative for co-stimulatory molecules CD40 (4), CD80 (4,27) and CD86 (27). hESC-MSC suppress T-cell proliferation in MLR and following stimulation with anti-CD3/CD28 beads, with the suppressive effect being seen in a transwell system (4,27). In this study, we compared hESC-MSC with BMMSC and neonatal foreskin fibroblasts (Fb), which have also been shown to be immunomodulatory (28,29). We show that hESC-MSC, similar to BM-MSC and Fb, suppress T-cell proliferation without direct cell–cell contact. This is partly because of the induction of IDO by IFN-γ released by activated T cells. Our findings suggest that hESC-MSC may serve as an alternative source of MSC for immunosuppressive therapy.

Methods Cell culture Passage 5–8 BM-MSC (PT-2501; Lonza, Walkersville, MD, USA) from four donors were cultured in MSC growth medium (MSCGM; MSC basal medium with MSC growth supplement, glutamine, penicillin and streptomycin; Lonza), as described by the manufacturer. hESC-MSC (a gift of S. K. Lim, Institute of Medical Biology, A*STAR, Singapore) were derived from the hESC cell line HuES9 (human embryonic stem HUES Facility, Cambridge, MA, USA), as described previously (5). Passage 15–22 (after derivation from hESC) hESC-MSC were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM glutamine, 1% non-essential amino acids (NEAA) and 10% fetal bovine serum (FBS) (Thermo Scientific-Hyclone, Cramlington, UK). Passage 9–22 human foreskin Fb (BJ-10; ATCC, Manassas, VA, USA) were

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grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin. Frozen peripheral blood mononuclear cells (PBMC) were purchased from Allcells (Emeryville, CA, USA). All isolatedT-cell cultures were maintained in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin. All media and supplements were from Invitrogen-Gibco (CA, USA) unless stated otherwise. Flow cytometry MSC and Fb, 1 ⫻ 105, were stained with mouse antihuman CD29, CD34, CD44, CD45, CD73, CD90 and CD105 (Lab Vision Corporation, CA, USA) or with appropriate isotype controls (Biolegend, San Diego, CA, USA) for 30 min at 4°C. All the antibodies were purchased from BD Biosciences (CA, USA) unless stated otherwise. The cells were resuspended in 100 μL 1:500 rabbit anti-mouse–fluorescein isothiocyanate (FITC) antibody (Dako, Glostrup, Denmark) at 4°C for 15 min. Cells were resuspended in 1.25 μg/mL propidium iodide in 1% bovine serum albumin (BSA)/phosphate-buffered saline (PBS) and analyzed using a FACSCalibur (BD Biosciences). Osteogenic, adipogenic and chondrogenic differentiation The differentiation protocols were adapted from Liu et al. (30). Cells were plated in a 24-well plate and grown until confluent. Osteogenic differentiation medium contained 100 nM dexamethasone, 10 mM β-glycerophosphate and 50 μM ascorbate-2phosphate. Adipogenic differentiation medium contained 0.5 mM isobutyl methylxanthine, 1 μM dexamethasone, 10 μM insulin and 200 μM indomethacin. Fresh media were replenished every 3 days for 2–4 weeks. For chondrogenic differentiation, 2 ⫻ 105 cells were pipetted into a 15-mL polypropylene tube and centrifuged at 200 g for 5 min. The pellet was cultured at 37°C with 5% CO2 in 500 μL chondrogenic medium that contained 10 ng/mL transforming growth factor (TGF)-β3 (R&D Systems, Minneapolis, MN, USA), 10 μM dexamethasone, 50 μM ascorbate2-phosphate, 4 mM proline, 1 mM minimum essential medium (MEM) sodium pyruvate and 1% insulin– transferrin–selenous acid (ITS) ⫹ Premix (BD Biosciences). The medium was replaced every 3–4 days for 28 days. All supplements were from Sigma-Aldrich (MO, USA) unless stated otherwise. All cultures were rinsed with PBS and fixed in 10% formaldehyde (Sigma-Aldrich). Oil red O, alizarin red and alcian blue stains were used to identify adipocytes, osteoblasts and chondrocytes, respectively.

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Mitomycin C-treatment of MSC and Fb MSC and Fb were mitomycin C-treated (10 μg/mL) for 2.5 h and then cryopreserved in 90% FBS and 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich).

The percentage of cohort divided is the percentage of cohort cells found in division number 1 onwards. It gives an indication of the percentage of cells that have divided: ∞

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Purification of

CD4 ⫹

T cells

Frozen PBMC were thawed and resuspended in 500 μL RPMI-1640; 30 μL CD14 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were added and incubated for 30 min at 4°C on a rotator. The cells were washed, resuspended in 1 mL PBS, and passed through a magnetic column. The CD14– fraction was resuspended in 1 μg/mL anti-CD8 (BD Biosciences) antibody and 0.5 μg/mL anti-CD14, anti-CD19, anti-CD33, anti-CD56 (Millipore-Chemicon International, MA, USA) and anti-CD16 antibodies (BD Biosciences) in RPMI-1640, and left on a rotator for 30 min at 4°C. Following another round of negative selection using biomag goat anti-mouse IgG beads (Qiagen, Duesseldorf, Germany), the remaining cells (80–95% CD4 ⫹ CD8–) were used in T-cell proliferation assays. Carboxyfluorescein diacetate succinimidyl ester labeling CD4 ⫹ T cells were resuspended at 1 ⫻ 106 cells/mL in warm PBS. Carboxyfluorescein diacetate succinimidyl ester (CFSE) (CellTrace CFSE cell proliferation kit; Invitrogen) was added at a final concentration of 0.5 μM. Cells were incubated at 37°C for 5 min, followed by the addition of ice-cold culture medium. Cells were washed once with RPMI-1640 and twice more with PBS. CFSE analysis Cell proliferation was analyzed using FlowJo software (TreeStar, CA, USA). Cells that were originally stained with CFSE at the start of the experiment were called the cohort cells (supplementary Figure 1). Cells that had divided i times were in division number i. After i divisions, each cohort cell had given rise to 2i daughter cells. If there was no cell death, the number of cohort cells that would have generated the daughter cell population in division number i could be determined by: No. of cohort cells in division i ⫽

N (i ) ) 2i ⫽1 % cohort divided ⫽ i∞ ⫻100% N (i ) ( i ) ∑ 2 i⫽0

∑(

N(i) 2i

where N(i) is the number of events in division i. The percentage of cells divided is the percentage of cells found in division number 1 onwards (supplementary Figure 1).

The proliferation index is the average number of cell divisions undergone by the cohort cells that have divided (it ignores cells in division number 0). ∞

N (i ) ) 2i i⫽1 Proliferation index ⫽ ∞ N (i ) ( i ) ∑ 2 i⫽1

∑ (i ⫻

The CFSE indices (percentage of cells divided, percentage of cohort divided and proliferation index) were calculated and normalized to the values of the positive control, which was with stimulated CD4 ⫹ T cells alone. Normalized CFSE index ⫽ CFSE index in the presence of MSC / Fb ⫻100% CFSE index with stimulated T cells alone

Contact experiments Mitomycin C-treated hESC-MSC, BM-MSC and Fb were seeded at 0.05, 0.5 and 5 ⫻ 104/well in a 96-well clear flat-bottomed microtiter plate (Corning, NY, USA) and allowed to attach overnight before the addition of CFSE-labeled CD4 ⫹ T cells (5 ⫻ 104/ well) with or without 0.2 μL/well anti-CD3/CD28coated beads (Invitrogen-Dynal Biotech). The plates were incubated at 37°C for up to 5 days. Transwell experiments Mitomycin C-treated hESC-MSC, BM-MSC and Fb, 2.8 ⫻ 105, were added to the bottom chamber of a 24-well plate transwell (Corning); 2.8 ⫻ 105 CFSElabeled CD4 ⫹ T cells were added to the top chamber (0.4 μM pore-size) the next day in the presence or absence of 1.12 μL anti-CD3/CD28-coated beads. After 5 days, CFSE-labeled cells were analyzed by flow cytometry. Preparation of supernatants Supernatants were collected from cultures of hESCMSC, BM-MSC, Fb and anti-CD3/CD28-stimulated

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A

CD34

No. of cells

CD44

CD45

CD29

96.20

97.33

CD73

CD90

96.78

CD105

MSC markers hESC-MSC

B

BM-MSC

Fb

C Osteogenic

Adipogenic

% positive

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98.41

97.84

1.45

0.83

Chondrogenic MSC markers

Figure 1. Phenotype and differentiation potential of hESC-MSC, BM-MSC and Fb. (A) The surface antigen expression of CD34, CD45, CD29, CD44, CD73, CD90 and CD105 on hESC-MSC was determined by flow cytometry. The profile shown is representative of five independent experiments. (B) Comparative surface antigen expression by flow cytometry. The mean ⫾ SEM of four independent experiments is shown. (C) The trilineage differentiation potential was assessed by culturing the cells for at least 2 weeks in osteogenic, adipogenic and chondrogenic medium and then staining the cells with alizarin red, Oil red O and alcian blue, respectively. Scale bars ⫽ 100 μm.

CD4 ⫹ T cells only and transwell cultures of MSC or Fb with stimulated CD4 ⫹ T cells. Mitomycin-C treated-MSC and –Fb, 2.8 ⫻ 105, were plated and cultured in the presence or absence of an equal number of stimulated CD4 ⫹ T cells in 700 μL RPMI-1640 for 5 days and the supernatants filtered before use. Secondary T-cell proliferation assay One-hundred and fifty microliters of supernatant were added to 50 μL 5 ⫻ 104 CFSE-labeled CD4 ⫹ T cells suspended in fresh RPMI-1640. CD4 ⫹ T cells were stimulated with 0.2 μL anti-CD3/CD28-coated beads for 5 days. Metabolite analysis The collected supernatants were analyzed electrochemically for glucose, glutamine, lactate and ammonia using BioProfile 100 Plus (Nova Biomedical Corporation, MA, USA).

Western blotting Protein lysates harvested after 5 days of incubation in the presence or absence of neutralizing antibody against IFN-γ (BD Biosciences) were separated by gel electrophoresis and electroblotted onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA, USA). The membranes were probed with mouse anti-human IDO antibody, 1:500 dilution (Millipore, billerica, MA, USA), or rabbit anti-human β-actin antibody, 1:2000 dilution (Abcam, Cambridge, UK). Secondary antibody labeling was performed using goat anti-mouse horseradish peroxidase (HRP) or goat anti-rabbit HRP (Dako) diluted 1:10 000. Chemiluminescent detection was done using Immobilon Western chemiluminescent HRP substrate (Millipore) or Amersham ECL plus Western blotting detection reagents (GE Healthcare, Uppsala, Sweden) and Lumi-Film chemiluminescent detection film (Roche, Mannheim, Germany) to visualize proteins. β-actin was used as the loading control.

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Detection of IDO activity IDO activity was determined by measuring the concentration of kynurenines in the supernatants using a colorimetric assay as described previously (31). Statistical analysis Results are expressed as mean ⫾ SEM. The ANOVA test and the unpaired t-test were used to test the probability of significant differences.

Characterization of hESC-MSC, BM-MSC and Fb hESC-MSC were negative for CD34 and CD45 but positive for CD29, CD44, CD73, CD90 and CD105 (Figure 1A). The expression of these surface markers on hESC-MSC was comparable to BM-MSC and Fb (Figure 1B). The cells were evaluated for their ability to differentiate into osteoblasts, adipocytes and chondrocytes (Figure 1C). Only BM-MSC could be induced to differentiate into all three lineages. hESC-MSC

A

hESC-MSC inhibit the proliferation of CD4 ⫹ T cells in a dose-dependent manner Proliferation of CFSE-labeled CD4 ⫹ T cells stimulated by anti-CD3/CD28-coated beads in the presence of increasing numbers of hESC-MSC, BMMSC and Fb was assessed after 5 days. The degree of suppression of CD4 ⫹ T-cell proliferation was greatest at an MSC/Fb:T-cell ratio of 1:1 (Figure 2A–C). The CFSE indices calculated showed that hESCMSC, BM-MSC and Fb could prevent both the overall population of T cells (Figure 2A), and the cohort T cells (Figure 2B) that were initially stained with CFSE, from dividing. In addition, hESC-MSC, BM-MSC and Fb slowed down the division rate of dividing cells (Figure 2C). However, BM-MSC and Fb were more suppressive than hESC-MSC. Similar results were obtained with another hESC-MSC line

C

B

D

Control (CD4+ T cells without MSC/Fb)

Day 3 Day 4 Day 5

hESC-MSC

BM-MSC

Fb

% of maximum

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Results

could only differentiate into adipocytes, albeit less efficiently than BM-MSC. Fb, as expected, failed to differentiate into any of the lineages. Therefore the hESC-MSC were MSC-like because they exhibit limited differentiation capability.

CFSE intensity

Figure 2. hESC-MSC, BM-MSC and Fb inhibit T-cell proliferation in a contact system. Normalized CFSE indices. (A) Percentage of cells divided. (B) Percentage of cohort divided. (C) Proliferation index of CFSE-labeled T cells in the presence or absence of different numbers of MSC or Fb, as determined on day 5 by flow cytometry. The CFSE indices were normalized to the CFSE indices of anti-CD3/ CD28-stimulated CD4 ⫹ T cells in the absence of MSC or Fb. The mean ⫾ SEM of at least four independent experiments is shown. ∗P ⬍ 0.05, ∗∗P ⬍ 0.01, ∗∗∗P ⬍ 0.001 compared with control CD4 ⫹ T cells without MSC or Fb. (D) Kinetics of T-cell proliferation in the presence or absence of an equal number of MSC or Fb, as determined by flow cytometry on days 3, 4 and 5. Proliferation profiles are representative of four independent experiments.

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Figure 3. hESC-MSC, BM-MSC and Fb inhibit T-cell proliferation without cell–cell contact in a transwell system. Normalized CFSE indices. (A) Percentage of cells divided. (B) Percentage of cohort divided. (C) Proliferation index of CFSE-labeled T cells in the presence or absence of an equal number of MSC or Fb separated by a 0.4-μm semi-permeable membrane, as determined on day 5 by flow cytometry. Anti-CD3/CD28-stimulated T cells were incubated in the top chamber while MSC or Fb were seeded in the bottom one. The CFSE indices were normalized to the CFSE indices of anti-CD3/CD28-stimulated CD4 ⫹ T cells in the absence of MSC or Fb. The mean ⫾ SEM of at least eight experiments is shown. ∗∗∗P ⬍ 0.001 compared with CD4 ⫹ T cells without MSC or Fb.

(data not shown).The effect of both MSC types and Fb on the proliferation of CD4 ⫹ T cells was also evaluated at different time-points (days 3, 4 and 5) to determine the kinetics of T-cell suppression. The extent of inhibition by both MSC types and Fb was most evident on day 5 (Figure 2D). Cell–cell contact is not important in mediating the suppressive effect T-cell proliferation in the top chamber of a transwell was inhibited by all the MSC and Fb in the bottom chamber (P ⬍ 0.001) (Figure 3). These results indicated that hESC-MSC, similar to MSC and Fb, mediated their suppressive effect via soluble factors. hESC-MSC are less potent than BM-MSC and Fb at suppressing T-cell proliferation To quantify accurately the potency of T-cell suppression among the various cells, the inhibitory

concentration (IC50, the number of adherent MSC or Fb required per 100 CD4 ⫹ T cells for the suppression of T-cell proliferation by 50%) was determined. A plot of the proliferation index (normalized to CD4 ⫹ T cells alone) against the number of adherent MSC or Fb was generated, in order to determine the number of cells required to suppress the proliferation of 2.8 ⫻ 105 T cells by 50% (supplementary Figure 2). In the transwell, the IC50 of hESC-MSC, BM-MSC and Fb was 66.6 ⫾ 2.7, 37.3 ⫾ 8.8 and 33.9 ⫾ 11.6 cells, respectively (Figure 4A). hESC-MSC were approximately 2-fold less potent than BM-MSC and Fb at suppressing T-cell proliferation. The IC50 in transwells was greater than that in a contact setting (P ⬍ 0.05 for hESC-MSC) (Figure 4A). More MSC and Fb was required to produce the same degree of suppression in transwells than in the contact setting. Close proximity of MSC or Fb with T cells may favor local suppression by paracrine factors. Alternatively, cell–cell contact may enhance the suppressive effect of both MSC types and Fb.

Figure 4. hESC-MSC are less potent than BM-MSC and Fb. (A) The IC50 (the number of adherent MSC or Fb required to inhibit the proliferation of T cells by 50%) was determined in both the contact and transwell systems. The mean ⫾ SEM of at least three independent experiments is shown. ∗∗P ⬍ 0.01 when the IC50 in the contact system was compared with the IC50 in the transwell. (B) Supernatants collected from transwells were more suppressive. hESC-MSC, BM-MSC and Fb were incubated for 5 days in the absence or presence of an equal number of anti-CD3/CD28-stimulated T cells in a transwell. Supernatants were also collected from wells of stimulated T cells alone and RPMI medium without any cells. The supernatants were added at 0.75 ⫻ total volume to a secondary T-cell culture. The proliferation index was normalized to that of anti-CD3/CD28-stimulated CD4 ⫹ T cells in the absence of MSC or Fb in the secondary culture. The mean ⫾ SEM of four independent experiments is shown.

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Presence of activated T cells induces the suppressive effect of MSC and Fb

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To determine whether soluble factors produced by the cells were responsible for the suppressive effect, supernatants were obtained from cultures of hESCMSC, BM-MSC and Fb alone or transwell cultures with activated CD4 ⫹ T cells. The supernatants from cultures of both MSC types and Fb alone had no

significant effect on T-cell proliferation (Figure 4B). However, supernatants in which MSC or Fb were incubated with activated T cells inhibited T-cell proliferation (Figure 4B). Therefore, the presence of activated T cells was required for the suppressive effect of hESC-MSC, BM-MSC and Fb. To determine whether the inhibition of T-cell proliferation was the result of the depletion of nutrients or the accumulation of toxic metabolites, we

Figure 5. Role of IDO in the suppressive effect of hESC-MSC, BM-MSC and Fb. (A) Western blots showing the expression of IDO by MSC (HM, hESC-MSC and BM, BM-MSC) and Fb in the presence of stimulated CD4 ⫹ T cells. MSC or Fb lysates were collected from cultures of MSC or Fb alone (alone) and transwells (transwell) after 5 days. T-cell lysates were collected from transwells in the absence (T cells-T) or presence of MSC or Fb (T cells- HM, BM, Fb). Results are representative of three experiments. (B) Increased concentrations of tryptophan metabolites, kynurenines, in transwells when activated T cells were cultured with MSC or Fb for 5 days. The mean ⫾ SEM of triplicates of three independent experiments is shown. ∗∗P ⬍ 0.01, ∗∗∗P ⬍ 0.001 when concentrations of kynurenines were compared among the supernatants collected from transwells or cultures of MSC, Fb or T cells only. (C) The addition of 1-MT, a competitive inhibitor of IDO, reversed the suppressive effect of MSC or Fb on T-cell proliferation in the transwell system. The mean ⫾ SEM of three independent experiments is shown. ∗P ⬍ 0.05, ∗∗P ⬍ 0.01, ∗∗∗P ⬍ 0.001 when compared with the normalized proliferation index of stimulated T cells in the presence of the respective MSC or Fb with vehicle added.

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determined the concentrations of glutamine, glucose, ammonium and lactate concentrations electrochemically. The concentrations of these molecules were similar in all the supernatants (data not shown).

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MSC and Fb express functional IDO IDO has been shown to contribute to the immunosuppressive property of BM-MSC (18,28). We therefore determined whether IDO is expressed by hESC-MSC. None of the cells showed basal expression of IDO, as detected by Western blotting (Figure 5A, alone). However, in the presence of stimulated T cells, both MSC types and Fb expressed IDO (Figure 5A, transwell). In contrast, stimulated T cells did not express IDO in the presence or absence of MSC or Fb (Figure 5A, T cells). This showed that IDO expression by both MSC types and Fb was induced by factors produced by stimulated CD4 ⫹ T cells. IDO activity was assessed by measuring the concentration of kynurenines in the supernatants. Consistent with the results obtained by Western blotting, IDO activity was not seen in cultures of any of the cells alone, and was only up-regulated in the transwells when MSC or Fb were cultured with T cells (Figure 5B). Therefore the IDO induced in both MSC types and Fb by factors released by T cells was functionally active. To investigate whether IDO is important in the suppressive effect of MSC and Fb, a competitive inhibitor of IDO, 1-methyltryptophan (1-MT), was added. 1-MT reversed the suppression of T-cell proliferation by hESC-MSC, BM-MSC and Fb (Figure 5C), indicating that IDO was responsible in part for the suppressive effect of both MSC types and Fb. IDO expression and the suppressive effect of MSC and Fb is dependent on IFN-γ IFN-γ produced by T cells can induce the expression of IDO. Blocking antibody to IFN-γ reduced the suppressive effect of hESC-MSC, BM-MSC and Fb (Figure 6A), and also inhibited the expression of IDO in transwell cultures (Figure 6B). This was associated with a reduced level of kynurenines in the transwell supernatants (Figure 6C). Thus activated T cells induced IFN-γ-dependent IDO expression in MSC and Fb, contributing to the suppressive effect of hESC-MSC, BM-MSC and Fb. Discussion The immunomodulatory properties of BM-MSC make them attractive candidates for cell therapy

Figure 6. IFN-γ induces the suppressive effect of hESC-MSC, BM-MSC and Fb in a transwell system. (A) Abrogation of MSC or Fb suppression of T-cell proliferation with blocking antibody to IFN-γ. The mean ⫾ SEM of three independent experiments is shown. ∗∗∗P ⬍ 0.001 when the normalized proliferation index was compared with the corresponding isotype control. (B) Western blots showing the down-regulation of IDO expression by MSC and Fb in the presence of anti-IFN-γ antibody. MSC or Fb lysates were collected from transwells with activated T cells in the presence of isotype or anti-IFN- γ antibody. The results from hESC-MSC are shown and are representative of three sets of experiments. (C) Negligible amounts of tryptophan metabolites, kynurenines, in transwells with the addition of anti-IFN-γ antibody. The mean ⫾ SEM of triplicates of three independent experiments is shown.

requiring immunosuppression. We have investigated whether hESC-MSC can be an alternative to BMMSC for immunosuppression. hESC-MSC expressed similar surface antigens and were able to differentiate into adipocytes. However, they failed to differentiate into osteoblasts and chondrocytes. The hESC-MSC used in the current study has been shown previously to undergo trilineage differentiation, albeit less

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efficiently into osteoblasts (5). On the other hand, hESC-MSC derived by Trivedi & Hematti (4) could differentiate into all three lineages. According to the International Society for Cell Therapy, MSC should be plastic adherent, express specific cell-surface markers and differentiate into adipocytes, osteoblasts and chondrocytes (32). Therefore, the hESC-MSC used in the current study are MSC-like because they exhibit limited differentiation capability. The differences in hESC-MSC differentiation efficiency suggest that the derivation protocols may affect their multipotency, and further studies could determine whether the method of derivation affects the immunomodulatory functions of hESC-MSC derived from the same parental hESC. Although the hESC-MSC exhibited limited differentiation capability, they could still suppress the proliferation of anti-CD3/CD28-stimulated CD4 ⫹ T cells. Previous studies have shown that this immunosuppressive property is independent of their differentiation ability and shared by cells of stromal origin (28,29). As reported previously (29,33), Fb, which did not differentiate into any of the three lineages, could also inhibit the proliferation of T cells in this study. Transwell experiments suggested that hESCMSC could mediate their suppressive effects without cell–cell contact, and that soluble mediators were involved. These results corroborate previous studies where hESC-MSC has been reported to suppress T-cell proliferation in a transwell system (4,27). Likewise, BM-MSC and Fb have been reported to suppress T-cell proliferation in a transwell system (4,17,27,28). The IC50 showed that twice the number of hESCMSC compared with BM-MSC were required to achieve the same degree of suppression. While this

might appear to limit their clinical utility, the ability to expand them readily in vitro may compensate for this (5). The inhibition by supernatants collected from transwell cultures of hESC-MSC, BM-MSC or Fb with activated T cells was less than that produced when MSC or Fb were present continuously with T cells. Soluble factors may require continuous production and replenishment, or they could have been diluted by the addition of 0.25 ⫻ normal medium, or they might operate best locally (as might be expected for tryptophan degradation by IDO) (24,34). Factors produced by activated T cells potentiate the immunosuppressive effect of MSC and Fb. Various groups have demonstrated the involvement of IDO in the suppressive activity of BM-MSC and Fb (15,17,20,28). This is the first time that hESC-MSC have been shown to mediate the immunosuppressive effect via IDO. In a similar manner to BM-MSC (15) and Fb (28), IDO was not expressed by hESC-MSC basally but could be up-regulated upon incubation with activated T cells. Previous studies have shown that expression of IDO in BM-MSC is dependent on IFN-γ (15) and that the addition of anti-IFN-γ antibodies reverses the inhibitive effect of BM-MSC and Fb (17,28). The role of IFN-γ produced by activated T cells in mediating the suppressive effect of hESC-MSC is supported by the observation that neutralizing antibody to IFN-γ partially abrogated the suppressive effect on T-cell proliferation and prevented induction of IDO. In conclusion, our data indicate that hESC-MSC can suppress anti-CD3/CD28-stimulated CD4 ⫹ T-cell proliferation and that they use similar mechanisms to BM-MSC and Fb. IFN-γ plays a critical role in inducing immunosuppression in both MSC

Figure 7. A model showing the possible feedback loop between MSC or Fb and T cells. Activated T cells induce the suppressive effect of MSC or Fb through factors, for example IFN-γ, released upon activation. The suppressive effect can be because of the up-regulation of factors like IDO in MSC and Fb or the release of suppressive factors by MSC and Fb. Subsequently, the proliferation of T cells is inhibited and restored to the normal state. The suppressive effect of MSC or Fb then ceases.

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Activated T cells modulate immunosuppression by hESC-MSC and BM-MSC types and Fb. IFN-γ released by activated T cells up-regulates the expression of IDO by MSC and Fb, leading to the inhibition of T-cell proliferation. We speculate that this feedback loop (Figure 7) may maintain a balance between inflammation and immunomodulation when MSC and Fb are administered for immunosuppressive therapy. The immunomodulatory property of MSC and Fb may be induced in a paracrine manner upon encountering activated T cells at the intended site of action. Once the inflammatory response has been attenuated, the immunosuppressive effect of MSC and Fb will be decreased in the absence of continued activation of T cells. These findings support further evaluation of hESC-MSC for their therapeutic potential.

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Acknowledgments We would like to acknowledge Dr Jerry Chan for critically reviewing the manuscript and Dr Sai Kiang Lim for providing hESC-MSC for this study. This work was supported by the Agency for Science, Technology and Research (A∗STAR). Disclosure of interest: The authors indicate no potential conflicts of interest.

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