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First published online October 25, 2007
Stem Cells Vol. 26 No. 1 January 2008, pp. 89 -98
doi:10.1634/stemcells.2007-0151; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS

Immunosuppression by Embryonic Stem Cells

Cody A. Kocha,b, Pedro Geraldesa,b, Jeffrey L. Platta,b,c,d

aTransplantation Biology Program and
Departments of bImmunology,
cSurgery, and
dPediatrics, Mayo Clinic College of Medicine, Rochester, Minnesota, USA

Key Words. Stem cells • T cells • Dendritic cells

Correspondence: Correspondence: Jeffrey L. Platt, M.D., Transplantation Biology, 2-66 Medical Sciences Building, Mayo Clinic, 200 1st Street SW, Rochester, Minnesota 55905, USA. Telephone: 507-538-0313; Fax: 507-284-4957; e-mail: platt.jeffrey{at}mayo.edu

Received on February 28, 2007; accepted for publication on October 19, 2007.

First published online in STEM CELLS EXPRESS  October 25, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem cells or their progeny inevitably differ genetically from those who might receive the cells as transplants. We tested the barriers to engraftment of embryonic stem cells and the mechanisms that determine those barriers. Using formation of teratomas as a measure of engraftment, we found that semiallogeneic and fully allogeneic embryonic stem cells engraft successfully in mice, provided a sufficient number of cells are delivered. Successfully engrafted cells did not generate immunological memory; unsuccessfully engrafted cells did. Embryonic stem cells reversibly, and in a dose-dependent manner, inhibited T-cell proliferation to various stimuli and the maturation of antigen-presenting cells induced by lipopolysaccharide. Inhibition of both was owed at least in part to production of transforming growth factor-β by the embryonic stem cells. Thus, murine embryonic stem cells exert "immunosuppression" locally, enabling engraftment across allogeneic barriers.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Originating from the inner cell mass of the embryo, embryonic stem cells represent the primordial stem cells from which all tissues, including germ cells, derive [1–4]. Embryonic stem cells have two generally recognized properties: (a) they can proliferate indefinitely in culture, and (b) they can differentiate into any type of cell in the body, including germ cells [2, 4–9]. The latter two characteristics have generated widespread interest in the potential use of the cells in tissue engineering and repair, and recent studies support their use [10–13]. Embryonic stem cells have been proposed for use in repairing damaged myocardium [14, 15]. Behfar et al. [16, 17] and Hodgson et al. [18] found that murine embryonic stem cells injected into infracted rat myocardium differentiate into cardiomyocytes and improve contractile performance of the infracted heart; however, teratomas formed in some treated mice. Kehat et al. [19] developed a culture system in which human embryonic stem cells could be driven to differentiate into cardiomyocytes ex vivo. Limiting this approach is the possibility that embryonic stem cells might generate teratomas [20], although teratoma formation might be controlled by manipulation of the complement system [21].

One other important limitation to use of embryonic stem cells is that these cells might and their progeny certainly would express major and/or minor transplantation antigens incompatible with the host (in the case of nuclear cloning, these antigens are encoded by mitochondrial DNA). If embryonic stem cells or cells derived from them were to provoke intense immune responses, as mature allogeneic cells do, the immune responses might destroy or impair the function of the transplanted cells. Consistent with this concern, Kofidis et al. [22] detected an alloimmune response in mice in which labeled embryonic stem cells were implanted in ischemic myocardium. Swijnenburg et al. [23] found that murine embryonic stem cells transplanted into injured myocardium provoke infiltration of T cells, B cells, and macrophages, and the transplanted cells and their progeny disappear over a period of weeks, presumably because of this response.

On the other hand, instead of immunity, embryonic stem cells might induce a state of tolerance. Fandrich et al. [24] found that allogeneic rat embryonic stem cell-like cells injected into the portal vein induce a state of tolerance that allows enduring survival of cardiac allografts of the same major histocompatibility complex (MHC) types as the embryonic stem cells, but not third-party cardiac allografts.

Although some would advocate therapeutic use of differentiated embryonic stem cells rather than undifferentiated cells because of potential tumor formation, some applications, particularly regeneration and immune modulation, might require undifferentiated cells. Accordingly, we investigated whether and how embryonic stem cells would interact with the allogeneic and semiallogeneic immune system. Given the difficulty of studying the immune response to allogeneic or semiallogeneic transplants of human embryonic stem cells, we asked whether embryonic stem cells incite or modify cell-mediated immune responses and by what mechanism(s).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Derivation and Culture of Embryonic Stem Cells
The C57BL/6, BALB/c x 129 (Open Biosystems, Huntsville, AL, http://www.openbiosystems.com), R1 (generously provided by A. Nagy, Mount Sinai Hospital, Toronto, ON, Canada) [25], and 129SvJ (generously provided by R. Bram, Mayo Clinic, Rochester, MN) embryonic stem cell lines were cultured as previously described [16]. Embryonic stem cells were considered undifferentiated on the basis of the expression of Oct-4, measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), and SSEA-1, measured by flow cytometry. Embryonic stem cell lines were periodically tested for their ability to form teratomas in immunodeficient RAG2–/–/{gamma}c–/– mice (Taconic Farms, Germantown, NY, http://www.taconic.com).

Transplantation of Embryonic Stem Cells, B16 Melanoma Cells, and Skin Grafts
Embryonic stem cells were harvested using 0.05% trypsin/0.53 mM EDTA (100x; Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) in phosphate-buffered saline (PBS). Feeder cells were depleted by adherence to untreated tissue culture plates for 45 minutes in embryonic stem cell medium. B16 melanoma cells (generously provided by R. Vile, Mayo Clinic, Rochester, MN) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and passaged when 80% confluent. The ability of B16 melanoma cells to form tumors was tested periodically in immunodeficient RAG2–/–/{gamma}c–/– mice. Embryonic stem cells and B16 melanoma cells were suspended in PBS in the desired numbers and injected subcutaneously between the scapulae or intravenously in the external jugular vein using a 25-gauge needle. Skin transplants were performed as previously described [26]. Bandages were removed after 5 days, and skin grafts were evaluated daily thereafter for rejection. A skin graft was considered rejected when 100% of the graft was necrotic.

Histologic Analysis of Teratomas
Tissue samples were snap-frozen, sectioned, and fixed as previously described [27]. Tissue sections were stained with biotin-conjugated mouse anti-H-2Kb, phycoerythrin-conjugated mouse anti-H-2Kd, hamster anti-mouse CD3{varepsilon} (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml), rat anti-mouse Foxp3 (eBioscience Inc., San Diego, http://www.ebioscience.com), or mouse anti-I-Ab (American Type Culture Collection, Manassas, VA, http://www.atcc.org) diluted in PBS containing 5% bovine serum albumin. Bound antibodies were detected using fluorescein-conjugated anti-biotin antibodies (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), rabbit polyclonal antibodies to phycoerythrin (Biogenesis, Kingston, NH, http://www.biogenesis.co.uk/index), fluorescein-conjugated goat anti-hamster IgG, or fluorescein-conjugated rabbit anti-mouse IgG2a (MP Biomedicals, Irvine CA, http://www.mpbio.com), and slides were visualized as described previously [28].

Analysis of Apoptosis and the Expression of Costimulatory Molecules by Flow Cytometry
Apoptosis of T cells in mixed leukocyte cultures was measured using an annexin V-phycoerythrin apoptosis detection kit (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) according to the manufacturer's instructions. Binding of antibodies and annexin V and exclusion of 7-aminoactinomycin D (7-AAD) were measured on a FACScan flow cytometer and analyzed using CellQuest software (BD Biosciences). The expression of MHC class II and costimulatory molecules on antigen-presenting cells was studied as previously described [29].

Mixed Leukocyte Cultures
Mixed leukocyte cultures were performed as previously described [29, 30], with irradiated (2,000 rads) embryonic stem cells added in various numbers. Results for mixed leukocyte cultures supplemented with leukemia inhibitory factor were similar to cultures containing leukemia inhibitory factor. In some experiments, human transforming growth factor (TGF)-β1, TGF-β sRII/Fc, or neutralizing antibodies were added to cell cultures (R&D Systems Inc., Minneapolis, http://www.rndsystems.com). In other experiments, T cells were stimulated with hamster anti-mouse CD3{varepsilon} antibodies (5 µg/ml) bound to the plates and soluble hamster anti-mouse CD28 antibodies (5 µg/ml) (eBioscience) or concanavalin A (0.5 µg/ml).

Measurement of Natural Killer Cell-Mediated Cytotoxicity
Natural killer (NK) cells from female C3H/HeJ mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) were activated in vivo by the injection of 100 µg of poly(I:C) as previously described [31]. Activated murine NK cells were isolated using the NK Cell Isolation Kit (Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com) according to the manufacturer's instructions. Isolated NK cells were used in a 4-hour 51Cr-release assay as previously described [32], with mouse C57BL/6 embryonic stem cells or YAC-1 cells (generously provided by Paul Leibson, Mayo Clinic, Rochester, MN) as targets.

Expression and Secretion of TGF-β
Total RNA from embryonic stem cells was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA, http://www1.qiagen.com) according to the manufacturer's instructions. Two nanograms of total RNA was used to synthesize cDNA using the Thermoscript RT-PCR system (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's instructions. The following primers were used in PCRs: β-actin, AGC CAT GTA CGT AGC CAT CC and TTT GAT GTC ACG CAC GAT TT; TGF-β1, GCC CTG GAC ACC AAC TAT TGC T and AGG CTC CAA ATG TAG GGG CAG G. The products were fractionated on 1% agarose-Tris borate-EDTA gels, stained with ethidium bromide, and quantitated using the Gel Doc 2000 and Quantity One software (Bio-Rad, Hercules, CA, http://www.bio-rad.com).

The bioactivity of TGF-β in conditioned culture medium was determined on the basis of the proliferative response of mink lung epithelial cell line Mv1Lu (American Type Culture Collection) as previously described [33]. Mv1Lu cells (2 x 104) were plated in 96-well flat-bottomed tissue culture plates in Dulbecco's modified Eagle's medium supplemented with 5% FCS and allowed to adhere. After the cells were attached, the culture medium was exchanged for conditioned medium, and the cells were incubated for 20 hours. Latent TGF-β was activated by acidification with 1 M HCl for 5 minutes followed by normalization of pH to 7.4 with 1 M NaOH. Mv1Lu cells were then pulsed with 1 µCi of 3H-thymidine for 4 hours and harvested with 0.05% trypsin/0.53 mM EDTA for 5 minutes at 37°C. The cells were collected on filtermats with a 96-well cell harvester (Tomtec, Hamden, CT, http://www.tomtec.com), and the radioactivity was measured using a Wallac scintillation counter (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com). The amount of TGF-β secreted into the medium was measured using a Quantikine murine TGF-β1 sandwich enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) according to the manufacturer's instructions.

Statistical Analysis
Results are expressed as mean ± SEM. An unpaired two-tailed Student's t test was used to compare means, with a p value <0.05 considered significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Engraftment of Embryonic Stem Cells Across Semiallogeneic and Fully Allogeneic Histocompatibility Barriers
We first asked whether murine embryonic stem cells spontaneously engraft across semiallogeneic or fully allogeneic MHC barriers. To test engraftment, we inspected grafted mice for teratomas, as undifferentiated embryonic stem cells form teratomas in immunodeficient mice over a period of a few weeks [1, 3]. After teratomas were first observed (~2 weeks), the tumors were monitored until they reached a size of 300 mm2 (the maximum size compatible with humane care) (~3–4 weeks after tumors were first noted), to exclude the possibility that an immune response might eventually control tumor growth.

To test engraftment of semiallogeneic embryonic stem cells, 5 million BALB/c x 129 embryonic stem cells were injected subcutaneously in the interscapular region of BALB/c mice, and the formation of teratomas was monitored. Each of 10 semiallogeneic grafts formed a teratoma, indicating that engraftment had occurred (Fig. 1A). To test engraftment of fully allogeneic embryonic stem cells, 5 million C57BL/6 embryonic stem cells were injected into BALB/c mice. Three of 10 fully allogeneic grafts formed teratomas within 21 days. The fraction of embryonic stem cell grafts accepted across fully allogeneic barriers increased when larger numbers cells were transplanted. For example, 90% of grafts consisting of 20 million embryonic stem cells from C57BL/6 mice generated teratomas in BALB/c mice within 10 days (Fig. 1A). On the other hand, 1 million embryonic stem cells from C57BL/6 mice never formed teratomas in BALB/c mice. These results show that allogeneic embryonic stem cells can engraft if a sufficient number of cells are provided.


Figure 1
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  		Figure 1. Engraftment of ES cells transplanted across semiallogeneic and fully allogeneic major histocompatibility (MHC) barriers and skin graft survival after transplantation of ES cells. (A): Survival of ES cell grafts. Various numbers of C57BL/6 (allogeneic) or BALB/c x 129 (semiallogeneic) ES cells were injected subcutaneously into BALB/c mice (n = 10 each), and the formation of teratomas was monitored as a marker of engraftment. As controls, 10 C57BL/6 mice were injected with C57BL/6 (syngeneic) ES cells, and 5 BALB/c mice were grafted with C57BL/6 (allogeneic) skin. These results show that ES cells can engraft across semiallogeneic barriers and even fully allogeneic MHC barriers, but the latter depends on the number of cells transferred. (B): Survival of skin grafts in mice "sensitized" with ES cells. ES cells from C57BL/6 mice (20 x 106 cells) were injected subcutaneously into the interscapular region of BALB/c mice (n = 5). Four weeks later, skin grafts from C57BL/6 mice were placed on those mice in which teratomas had formed, and the fate of the skin grafts was monitored. Teratomas were surgically removed at the time of skin grafting to prevent further tumor growth. Control mice (n = 5) were injected with PBS in lieu of ES cells. The results show that subcutaneous injection of ES cells induces neither tolerance, evidenced by prolonged survival of skin grafts, nor a second-set response, evidenced by hastened rejection of skin grafts; rather, the rate of rejection was comparable to that observed in naïve (control) mice. (C): Survival of skin grafts performed in conjunction with i.v. injection of ES cells. To confirm that after migrating through the blood, ES cells do not induce tolerance, ES cells from C57BL/6 mice (1 x 106 cells) were injected into the external jugular vein of BALB/c mice (n = 5), and the mice were grafted immediately with C57BL/6 skin. Control mice (n = 5) were injected intravenously with PBS in lieu of ES cells at the time of skin grafting. These results show that i.v. injection of ES cells does not significantly modify the outcome of skin allografts. (D): Role of regulatory T cells in the survival of allogeneic teratomas. Murine spleens (positive control) or teratomas formed after injection of 20 x 106 C57BL/6 ES cells into BALB/c mice were removed after 5 weeks and stained with anti-Foxp3 antibodies. Photomicrographs show that fully allogeneic teratomas do not express Foxp3. Magnification, x200. Abbreviations: ES, embryonic stem; PBS, phosphate-buffered saline.

 
Potential Evasion or Exhaustion of Immune Responses
We questioned whether successful engraftment of embryonic stem cells in an allogeneic host might reflect evasion or exhaustion of potential responder cells by rapidly proliferating allogeneic cells. To test this possibility, we injected 5 million or 20 million rapidly proliferating allogeneic B16 melanoma cells subcutaneously into BALB/c mice and monitored tumor growth, reasoning that if acceptance of the graft was attributable to overwhelming of the immune response by proliferating allogeneic cells, then B16 melanoma cells (H-2Kb), which proliferate more rapidly than embryonic stem cells, would also engraft. However, although grafts of 5 million or 20 million B16 melanoma cells initially formed small tumors, these tumors were rejected at ~15 days (data not shown). These results suggest that embryonic stem cells do not exhaust the immune response, as some tumors do, but possess other properties that allow the cells to evade immunity.

Potential of Embryonic Stem Cells to Induce Tolerance
We next asked whether successful engraftment of embryonic stem cells across semiallogeneic or fully allogeneic barriers reflects a state of allogeneic tolerance. To address that question, we injected 20 million C57BL/6 embryonic stem cells subcutaneously into BALB/c mice, waited 4 weeks, and transplanted C57BL/6 skin onto the flanks of animals that had developed teratomas, reasoning that if tolerance had been induced, the skin grafts would be accepted (or survival would be prolonged). To prevent further tumor growth that might compromise the well-being of the recipients, the teratomas were surgically removed at the time of skin grafting. As Figure 1B shows, mice in which teratomas formed after the injection of embryonic stem cells rejected skin grafts at the same time as mice that had not received embryonic stem cells. Hence, the engraftment of allogeneic embryonic stem cells did not induce a robust state of allogeneic tolerance.

To further exclude the possibility that removing the teratomas had abrogated tolerance, we injected 20 million C57BL/6 embryonic stem cells into BALB/c mice and grafted C57BL/6 skin at the first evidence of teratoma formation without removing the teratomas. Mice with teratomas and thus with accepted embryonic stem cell grafts rejected skin grafts at a median of 13 days (range, 12–15 days), a time similar to that of mice that had not received embryonic stem cell grafts (median, 14 days; range, 13–15 days; p > .05).

To exclude the possibility that failure of embryonic stem cells to induce tolerance might reflect failure of the cells to enter blood vessels in sufficient numbers, we administered embryonic stem cells directly into blood and then monitored for immunological tolerance. BALB/c mice given 1 million C57BL/6 embryonic stem cells into the external jugular vein rejected skin grafts from C57BL/6 mice at a median of 14 days, whereas control mice injected with PBS at the time of skin grafting rejected grafts at a median of 12 days (Fig. 1C). To address the possibility that injection of embryonic stem cells at the time of skin grafting did not allow time for the establishment of chimerism and tolerance, 1 million C57BL/6 embryonic stem cells were injected in the external jugular vein at 14 or 30 days prior to skin grafting. Injection of C57BL/6 embryonic stem cells at 14 or 30 days prior to skin grafting did not prolong skin graft survival (data not shown).

To explore the possibility that embryonic stem cells induce tolerance by recruitment and activation of regulatory T cells, we tested teratomas that formed after the transplantation of either 20 million C57BL/6 or 20 million BALB/c x 129 embryonic stem cells into BALB/c mice for the expression of Foxp3, a transcription factor expressed by regulatory T cells. C57BL/6 (Fig. 1D) and BALB/c x 129 (not shown) teratomas contained only scattered cells and sometimes no cells expressing Foxp3, whereas normal mouse spleen, serving as a positive control, contained numerous Foxp3-positive cells.

Potential of Immunologic Ignorance
Formation of teratomas from allogeneic embryonic stem cells could reflect failure of the recipient to recognize the foreign cells. Murine embryonic stem cells do not express MHC class I or MHC class II antigens [34], which we verified by flow cytometry at multiple different passage numbers (data not shown). However, teratomas derived from C57BL/6 embryonic stem cells expressed H-2Kb, and teratomas derived from BALB/c x 129 embryonic stem cells expressed both H-2Kb and H-2Kd (Fig. 2A). Teratomas derived from either C57BL/6 or BALB/c x 129 embryonic stem cells did not express appreciable levels of MHC class II molecules (data not shown). Thus, engraftment of embryonic stem cells and formation of teratomas cannot be ascribed to lack of expression of MHC class I antigens, nor does the action of NK cells on embryonic stem cells expressing MHC in a decreased or an aberrant way explain control of teratoma formation. Allogeneic embryonic stem cells formed tumors in allogeneic recombinase-deficient mice (not shown), and as Figure 2D shows, embryonic stem cells resisted lysis by sygeneic and allogeneic NK cells.


Figure 2
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  		Figure 2. The role of natural killer cells and immune ignorance in the survival of ES cell grafts. Whether natural killer cells or immune ignorance might account for the engraftment of ES cells across semiallogeneic and fully allogeneic major histocompatibility (MHC) differences was explored. (A): Expression of MHC class I on semiallogeneic and fully allogeneic teratomas. Teratomas formed after injection of 5 x 106 BALB/c x 129 ES cells or 20 x 106 C57BL/6 ES cells were removed after 5 weeks and stained with anti-H-2Kb, anti-H-2Kd antibodies or secondary (2°) antibodies alone. Photomicrographs show that semiallogeneic and fully allogeneic teratomas express MHC class I antigen. Magnification, x100. (B): Rejection of skin grafts by mice in which teratomas did not form after injection of ES cells. BALB/c mice were injected subcutaneously with 5 x 106 C57BL/6 ES cells (n = 5), 1 x 106 C57BL/6 splenocytes (n = 5), or PBS (n = 5). Four weeks later, mice in which teratomas had not formed were grafted with C57BL/6 skin, and the survival of the skin grafts was monitored. Mice previously injected with allogeneic ES cells and in which teratomas did not form rejected allogeneic skin grafts with second-set kinetics, indicating that these mice had been sensitized by the ES cells. (C): Infiltration of T cells in teratomas formed from syngeneic, semiallogeneic, and fully allogeneic ES cells. Teratomas formed after injection of 5 x 106 C57BL/6 ES cells in C57BL/6 mice (syngeneic), 5 x 106 BALB/c x 129 ES cells (semiallogeneic), or 20 x 106 C57BL/6 ES cells (allogeneic) in BALB/c mice were removed after 5 weeks and stained with anti-CD3{varepsilon} antibodies. Photomicrographs show that T cells of the recipient infiltrated semiallogeneic and fully allogeneic teratomas compared with the syngeneic control. Magnification, x100. (D): Susceptibility of ES cells to lysis by natural killer cells. C57B46 ES cells and YAC cells were combined with natural killer cells from C3H/HeJ mice in various ratios. The target cells were prelabeled with 51Cr, and percentage of specific lysis was determined after 4 hours. Abbreviations: ES, embryonic stem; PBS, phosphate-buffered saline.

 
To exclude the possibility that immune ignorance allows formation of teratomas from allogeneic embryonic stem cells, we asked whether mice engrafted with allogeneic embryonic stem cells mount a second-set response when challenged with another graft (allogeneic skin) and whether T cells invade allogeneic teratomas. Five million C57BL/6 embryonic stem cells were injected subcutaneously into BALB/c mice, and 4 weeks later, C57BL/6 skin was grafted onto those mice in which teratomas had not formed. Mice in which teratomas had not formed rejected skin grafts at a median of 7 days (the same time as mice injected with 1 million C57BL/6 splenocytes), whereas mice injected with PBS instead of embryonic stem cells rejected skin at a median of 14 days (Fig. 2B). Teratomas derived from both C57BL/6 and BALB/c x 129 embryonic stem cells growing in BALB/c mice contained infiltrates of T cells, as identified by staining for CD3{varepsilon} (Fig. 2C). Thus, the immune system is not ignorant of embryonic stem cell grafts.

Embryonic Stem Cells Inhibit T-Cell Proliferative Responses
Since smaller numbers of embryonic stem cells elicited alloimmunity and larger numbers did not, we questioned whether embryonic stem cells might act as an immunosuppressive agent. To address that question, we asked whether embryonic stem cells modify the proliferation of T cells to various stimuli. As shown in Figure 3A, R1 embryonic stem cells inhibited proliferation of BALB/c CD4+ T cells in a dose-dependent fashion. Similar results were obtained using 129SvJ embryonic stem cells. The inhibition of T-cell proliferation did not depend on the sharing of an MHC between the embryonic stem cells and either the responding T cells or the dendritic cells (data not shown), nor did this result likely reflect cell crowding, because similar results were observed across a broad range of numbers of stimulator and responder cells (not shown), nor was suppression of proliferation restricted to alloimmune responses, as R1 embryonic stem cells suppressed proliferation of CD4+ BALB/c T cells stimulated with plate-bound anti-CD3{varepsilon} antibodies and soluble anti-CD28 antibodies (Fig. 3B). Similar results were obtained with 129SvJ and C57BL/6 embryonic stem cells and when T cells were stimulated with soluble Concanavalin A (data not shown).


Figure 3
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  		Figure 3. Impact of ES cells on the proliferative responses of T cells. Whether ES cells suppress the proliferative responses of T cells to stimulation with allogeneic cells or with anti-CD3 and anti-CD28 was tested. The results were determined as percentage inhibition compared with cultures from which ES cells were omitted. (A): The impact of ES cells on the proliferative T-cell response to allogeneic dendritic cells. BALB/c CD4+ T cells (50,000 cells) were incubated with C57BL/6 dendritic cells (5,000 cells) in the presence of various numbers of R1 ES cells for 3 days, and proliferation was measured on the basis of incorporation of tritiated thymidine as described in Materials and Methods. The results, expressed as the mean ± SEM of five separate experiments, show that proliferation of T cells is suppressed in the presence of ES cells. (B): The impact of ES cells on the proliferation of T cells stimulated with anti-CD3{varepsilon} and anti-CD28 antibodies. BALB/c CD4+ T cells (50,000 cells) and various numbers of R1 ES cells were added to 96-well plates coated with anti-CD3{varepsilon} antibodies (5 µg/ml) and incubated in medium containing soluble anti-CD28 antibodies (5 µg/ml). After 3 days, the proliferation of the T cells was measured on the basis of incorporation of tritiated thymidine as described in Materials and Methods. The results, expressed as the mean ± SEM of three separate experiments, show that ES cells suppress T-cell proliferation stimulated by anti-CD3{varepsilon} and anti-CD28 antibodies. Abbreviation: ES, embryonic stem.

 
Embryonic Stem Cells Do Not Induce Apoptosis or Anergy in T Cells
We next asked whether embryonic stem cells evade rejection by triggering anergy or apoptosis of T cells. As Figure 4A shows, BALB/c CD4+ T cells cultured with embryonic stem cells proliferated normally when restimulated, indicating that they were not anergic and that the suppression of proliferation caused by embryonic stem cells was reversible. Furthermore, as Figure 4B shows, embryonic stem cells fully inhibited proliferation of CD4+ T cells in the presence of IL-2, indicating that suppression was not caused by anergy. Furthermore, T cells exposed to R1 embryonic stem cells had the same level of apoptosis (defined as annexin V+/7-AAD–) as T cells cultured in the absence of embryonic stem cells (Fig. 4C).


Figure 4
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  		Figure 4. Mechanism(s) of suppression of T-cell responses by ES cells. The question of whether ES cells reversibly suppress the proliferation of T cells or whether they induce anergy and apoptosis was tested. (A): Reversibility of suppression T-cell proliferation following culture with ES cells. BALB/c CD4+ T cells (50,000 cells) were incubated with allogeneic C57BL/6 dendritic cells (5,000 cells) in the presence of various numbers of R1 ES cells or without ES cells (control). After 3 days, CD4+ T cells were reisolated from the cultures using anti-mouse CD4 microbeads and a magnetic cell sorting separator. The reisolated CD4+ T cells were restimulated with fresh allogeneic C57BL/6 dendritic cells (in the absence of ES cells) for 3 days, and T-cell proliferative responses were measured on the basis of incorporation of tritiated thymidine as described in Materials and Methods. The results, representative of three separate experiments, show that suppression of T-cell proliferation by ES cells is reversible. (B): Impact of IL-2, as an index of anergy, on the T-cell responses in the presence of ES cells. Whether IL-2 would overcome suppression was used as a test to exclude anergy as a mechanism by which ES cells suppress T-cell responses. BALB/c CD4+ T cells (50,000 cells) were cultured with allogeneic C57BL/6 dendritic cells (5,000 cells) in the presence of various numbers of R1 ES cells in the presence (experimental) or absence (control) of exogenous IL-2 for 3 days, and T-cell proliferative responses were measured as described in Materials and Methods. The results, expressed as the mean ± SEM of three separate experiments, show that exogenous IL-2 does not reverse the suppression of T-cell proliferation by ES cells, indicating that suppression is not owed to anergy. (C): Apoptosis of T cells incubated with ES cells. BALB/c CD4+ T cells (50,000 cells) were incubated with C57BL/6 dendritic cells (5,000 cells) in the presence of various numbers of R1 ES cells for 3 days. Control cultures did not contain ES cells. Cells were stained with fluorescein isothiocyanate-conjugated anti-CD4, phycoerythrin-conjugated annexin V, and 7-aminoactinomycin D (7-AAD). T cells undergoing apoptosis were identified as CD4+/annexin V+/7-AAD–. The results, expressed as the mean ± SEM of three separate experiments, show that ES cells do not induce apoptosis of T cells. Abbreviations: ES, embryonic stem; IL, interleukin.

 
Embryonic Stem Cells Suppress T-Cell Proliferation in a Contact-Independent Manner
Embryonic stem cells might suppress T-cell responses locally or regionally [35, 36]. Regional suppression might depend on cell contact or on the establishment of a concentration gradient of a suppressive substance. To test whether embryonic stem cells must contact the T cells they suppress, medium conditioned for 48 hours by various combinations of embryonic stem cells, dendritic cells, T cells, or fibroblasts was added (1:1 with fresh medium) in cultures containing BALB/c CD4+ T cells and irradiated C57BL/6 dendritic cells, and proliferation was measured. As Figure 5A shows, medium conditioned by embryonic stem cells, but not medium conditioned by dendritic cells, T cells, or fibroblasts, inhibited T-cell proliferation in a dose-dependent manner. As Figure 5B shows, embryonic stem cells also inhibited proliferation of BALB/c CD4+ T cells in a dose-dependent manner even when separated from responder and stimulator cells by a semipermeable membrane.


Figure 5
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  		Figure 5. The nature of cell-cell interactions through which ES cells suppress T-cell proliferative responses and the contribution of TGF-β. Whether ES cells suppress T-cell proliferative responses by a contact-dependent or contact-independent mechanism was tested by determining whether medium conditioned by ES cells or ES cells separated from T cells by a porous membrane suppress T-cell proliferation. (A): Impact of medium conditioned by ES cells on proliferation of T cells in mixed leukocyte cultures. Medium conditioned by various numbers of R1 ES cells, by C57BL/6 murine embryonic fibroblasts (25,000 cells), by BALB/c CD4+ T cells (50,000 cells), or by C57BL/6 dendritic cells (50,000 cells) for 48 hours was added to mixed leukocyte cultures (1:1 with fresh medium) containing BALB/c CD4+ T cells (50,000 cells) and C57BL/6 dendritic cells (5,000 cells). T-cell proliferative responses were measured by incorporation of tritiated thymidine after 3 days as described in Materials and Methods. The results, expressed as the mean ± SEM of five separate experiments, show that medium conditioned by ES cells suppresses T-cell proliferative responses. (B): T-cell proliferative responses in transwell mixed leukocyte cultures in the presence of ES cells. BALB/c CD4+ T cells (250,000 cells) and allogeneic C57BL/6 dendritic cells (25,000) were cultured in the top chamber of 24-well plates separated by a membrane (0.4-µm pores) from various numbers of R1 ES cells in the bottom chamber. After 3 days, proliferation was measured on the basis of incorporation of tritiated thymidine as described in Materials and Methods. The results show that ES cells suppress T-cell proliferative responses even when separated by a membrane from the mixed cultures. (C): Expression and secretion of TGF-β by ES cells. The expression of TGF-β was verified by RT-polymerase chain reaction (inset). TGF-β in medium conditioned by ES cells was measured by a bioassay. Medium conditioned by R1 ES cells or BALB/c CD4+ T cells and C57BL/6 dendritic cells for 48 hours was added to cultures of mink lung epithelial cells (Mv1Lu cell line; 2 x 104 cells) grown in 96-well plates with (total) or without (active) acid activation of latent TGF-β. After 20 hours, proliferation of the epithelial cells was measured on the basis of incorporation of tritiated thymidine as described in Materials and Methods. The results, expressed as the mean ± SEM of three separate experiments, show that ES cells secrete biologically active TGF-β. (D): Relative impact of TGF-β secreted by ES cells on T-cell proliferative responses in mixed leukocyte cultures. CD4+ T cells (50,000 cells) from BALB/c mice were cultured with allogeneic C57BL/6 dendritic cells (5,000 cells) in the presence of various numbers of R1 ES cells with or without TGF-β sRII/Fc fusion protein (5 µg/ml), which blocks the interaction of TGF-β with TGF-β receptors. T-cell proliferative responses were measured after 3 days on the basis of incorporation of tritiated thymidine as described in Materials and Methods. The results, expressed as the mean ± SEM of three separate experiments, show that TGF-β sRII/Fc fusion protein partly reverses suppression of T-cell proliferative responses caused by ES cells, indicating that TGF-β contributes to that suppression. Abbreviations: ES, embryonic stem; RT, reverse transcription; TGF-B, transforming growth factor-β.

 
Contribution of TGF-β to Inhibition of T-Cell Proliferative Responses by Embryonic Stem Cells
Among the soluble substances that might reversibly suppress T-cell responses are nitric oxide [37], catabolites of tryptophan [38–40], prostaglandins [41], and TGF-β. Inhibitors of nitric oxide synthase, tryptophan metabolism, and prostaglandin synthesis failed to prevent or even decrease the suppression of T-cell proliferative responses by embryonic stem cells (data not shown). However, embryonic stem cells have been shown to produce TGF-β [42]. We verified by RT-PCR that embryonic stem cells express mRNA for TGF-β (Fig. 5C, inset). As Figure 5C shows, embryonic stem cells not only expressed TGF-β mRNA but also secreted TGF-β into culture medium, as measured by bioassay. The amount of TGF-β secreted into the medium by irradiated C57BL/6 embryonic stem cells over a period of 72 hours was measured by ELISA. As Table 1 shows, C57BL/6 embryonic stem cells secreted TGF-β into the culture medium in direct relation to the number of cells in the cultures. TGF-β sRII/Fc fusion protein blocked R1 embryonic stem cell inhibition of BALB/c CD4+ T-cell proliferation by 58.0% ± 5.2%. Inhibition was reversed to a similar extent when a TGF-β neutralizing antibody was added to the cultures (data not shown). Thus, the secretion of TGF-β by embryonic stem cells has significant consequences for cell-mediated immune responses and is at least partially responsible for the inhibition of T-cell proliferation.


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  		Table 1. TGF-β1 secreted by embryonic stem cells

 
Impact of Embryonic Stem Cells on the Maturation of Antigen-Presenting Cells
We next questioned whether the suppression of T-cell proliferation by products of embryonic stem cells might reflect an effect of the embryonic stem cells on antigen-presenting cells. To address this question, we treated immature bone marrow-derived antigen-presenting cells with medium conditioned by R1 embryonic stem cells for 3 days and measured the expression of MHC class II (I-Ab), CD40, CD80, and CD86 by flow cytometry after stimulation with lipopolysaccharide (LPS) for 24 hours. As Figure 6A shows, medium conditioned by embryonic stem cells prevented induction of MHC class II, CD40, CD80, and CD86 expression by LPS. Addition of TGF-β to the medium inhibited maturation of dendritic cells to a similar extent. Consistent with the importance of TGF-β, TGF-β sRII/Fc partly blocked inhibition of the maturation of antigen-presenting cells by medium conditioned by embryonic stem cells (Fig. 6A).


Figure 6
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  		Figure 6. Impact of embryonic stem cells on maturation of antigen-presenting cells and activation of T cells. Whether embryonic stem cells modify the maturation of dendritic cells and/or directly suppress T-cell responses was tested. (A): Impact of medium conditioned by embryonic stem cells on expression of major histocompatibility (MHC) class II and costimulatory molecules by antigen-presenting cells. Dendritic cells were derived from bone marrow cells by culturing bone marrow for 3 days in medium supplemented with granulocyte macrophage-colony-stimulating factor (10 ng/ml). Immature dendritic cells so obtained were incubated in medium conditioned for 48 hours by R1 embryonic stem cells with or without TGF-β sRII/Fc fusion protein (5 µg/ml) or medium supplemented with TGF-β1 (20 ng/ml) with or without TGF-β sRII/Fc fusion protein for an additional 3 days. After these treatments, the dendritic cells were stimulated with LPS (10 ng/ml) for 24 hours, washed, and stained with fluorescein isothiocyanate-anti-mouse MHC class II (I-Ab), -CD40, -CD80, and -CD86, and the binding of the antibodies was measured by fluorescence-activated cell sorting. The results show that medium conditioned by embryonic stem cells suppresses the expression of MHC class II and costimulatory molecules on antigen-presenting cells and that this impact is inhibited by TGF-β sRII/Fc fusion protein and hence mediated at least in part by TGF-β. (B): Direct suppression of T-cell proliferative responses by medium conditioned by embryonic stem cells. CD4+ T cells (50,000 cells) from BALB/c mice were cultured with antigen-presenting cells (5,000 cells) treated as listed in the figure with various combinations of CM, TGF-β, and/or TGF-β sRII/Fc (as described above). Proliferation of the CD4+ T cells was measured after 3 days on the basis of incorporation of tritiated thymidine as described in Materials and Methods. The results of two separate experiments, one of which is illustrated, show that medium conditioned by embryonic stem cells inhibits the ability of antigen-presenting cells to stimulate T-cell proliferation, and this inhibition is reversed by TGF-β sRII/Fc fusion protein, indicating that it is mediated, at least in part by TGF-β. Abbreviations: CM, conditioned medium; LPS, lipopolysaccharide; TGF, transforming growth factor.

 
We next asked whether treating antigen-presenting cells with medium conditioned by embryonic stem cells inhibits the ability of the antigen-presenting cells to stimulate T-cell proliferation. To address this question, antigen-presenting cells treated with medium conditioned by R1 embryonic stem cells were stimulated for 24 hours with LPS. After stimulation with LPS, the cells were washed and added to cultures of BALB/c CD4+ T cells, and proliferative responses were measured. As Figure 6B shows, stimulation of T-cell proliferation by antigen-presenting cells was inhibited by treatment with medium conditioned by embryonic stem cells compared with antigen-presenting cells not treated with conditioned medium. The ability of antigen-presenting cells to stimulate proliferation of T cells was partially, but not completely, restored by the addition of TGF-β sRII/Fc to the conditioned medium.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem cells or cells differentiated from them have been proposed to repair damaged myocardium, and their efficacy has been demonstrated in animal models [14, 15, 17]. However, embryonic stem cells might be allogeneic with respect to those who would be treated. Whether embryonic stem cells would provoke an alloimmune response and survive in the face of such a response is not well-characterized. Here we report that embryonic stem cells differ from mature cells and tissues in their ability to stimulate and/or be targeted by alloimmunity—and indeed, they engraft in semiallogeneic and fully allogeneic hosts under conditions in which other cellular grafts are rejected. Reversible immunosuppression by allogeneic murine embryonic stem cells may explain how the cells can form tumors, as previously observed for syngeneic embryonic stem cell grafts [43], and why the risk of tumor formation is a direct function of the number of cells engrafted.

The formation of tumors by larger numbers of embryonic stem cells might occur if grafted cells expand in a way that precludes control by the alloimmune response. Jones et al. [44] transferred various numbers of H-2Kb-specific CD8+ T cells followed by H-2Kb cardiac, skin, or islet grafts into athymic T-cell-depleted CBA mice (H-2k) and found that larger numbers of T cells had to be transferred to reject cardiac allografts than skin or islet allografts that were of smaller mass. He et al. [45] placed one or two male C57BL/6 skin grafts onto female C57BL/6 mice and found that the addition of a second skin graft significantly prolonged survival of the two grafts and that female C57BL/6 mice did not reject large male C57BL/6 cardiac grafts but did reject small cardiac grafts. Thus, the successful engraftment of larger numbers of allogeneic embryonic stem cells might reflect the failure of the recipients to generate sufficient numbers of alloreactive T cells to reject the grafts. However, for several reasons, we think this mechanism does not explain the acceptance of larger numbers of allogeneic embryonic stem cells. First, transplants consisting of large numbers of allogeneic B16 melanoma cells in BALB/c mice were rejected in all cases, albeit after a brief period of tumor growth. Second, recipients of small embryonic stem cell grafts were able to mount accelerated alloimmune responses, whereas recipients of larger numbers of cells were not. We have found that formation of teratomas from smaller numbers of embryonic stem cells in syngeneic systems is controlled by the complement system [21]; this evasion might allow alloimmunity to ensue if the stem cells are semiallogeneic or fully allogeneic. It is also possible that natural killer cells may protect against tumor formation by small numbers of embryonic stem cells. Stern et al. [46] reported that murine natural killer cells lysed embryonal cell lines. However, we found that natural killer cells do not lyse syngeneic or allogeneic murine embryonic stem cells in in vitro. Consistent with this observation, Drukker et al. [47] found that human embryonic stem cells were only minimally lysed by natural killer cells; thus, NK cells probably cannot prevent the formation of tumors. On the basis of these findings and work presented here, we would contend that larger numbers of allogeneic embryonic stem cells evade early control by complement and may engraft because of dose-dependent local immunosuppression delivered by the embryonic stem cells.

Some have suggested that embryonic stem cells might survive in allogeneic hosts by inducing tolerance and, furthermore, that embryonic stem cells might be administered for that purpose [24]. However, murine embryonic stem cells administered in subcutaneous or i.v. sites did not induce tolerance in the systems we used. In light of our results, we think it possible that the state of tolerance observed by Fandrich et al. [24] after injection of embryonic stem cells into the portal circulation could reflect "portal tolerance" [48], rather than the type of cell administered. Although skin allografts might be less amenable to tolerance (e.g., more immunogenic), embryonic stem cells have failed to induce tolerance to other types of grafts. Magliocca et al. [49] were unable to induce tolerance to cardiac allografts by injecting murine embryonic stem cells MHC-matched for the cardiac allograft via the portal vein.

We found that formation of teratomas by allogeneic and semiallogeneic embryonic stem cells depends on the number of cells transplanted. Smaller numbers of embryonic stem cells sensitized recipients, leading to memory immune responses and rapid rejection of skin grafts MHC-matched to the embryonic stem cells. Consistent with our observations, Swijnenburg et al. [23] reported that 1 x 106 allogeneic murine embryonic stem cells injected into infracted mouse hearts initially form teratomas but then appear to recruit an inflammatory cell infiltrate, leading to rejection weeks later. Similarly, Nussbaum et al. [20] reported that 5 x 105 allogeneic murine embryonic stem cells transplanted into infracted hearts initially form teratomas but then recruit an inflammatory cell infiltrate and undergo rejection. However, we observed that as the number of embryonic stem cells exceeded the numbers used by Swijnenburg et al. [23] and Nussbaum et al. [20], neither sensitization nor rejection occurred.

Rather, our results suggest that allogeneic and semiallogeneic murine embryonic stem cells in sufficient numbers suppress immune responses locally, at least in part by production of TGF-β. Although murine embryonic stem cells are clearly immunosuppressive, the ability of human embryonic stem cells to engraft across allogeneic and semiallogeneic MHC barriers remains unknown.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This work was supported by NIH Grant HL52297.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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