Costimulation Blockade Induces Tolerance to HESC Transplantedto the Testis and Induces Regulatory T-Cells to HESC Transplantedinto the Heart

KARL-HENRIK GRINNEMO,a RAMI GENEAD,b MAKIKO KUMAGAI-BRAESCH,c AGNETA ANDERSSON,b

CHRISTIAN DANIELSSON,b AGNETA MÅNSSON-BROBERG,b GORAN DELLGREN,a ANNE-MARIE STROMBERG,d

HENRIK EKBERG,e OUTI HOVATTA,d CHRISTER SYLVEN,b MATTHIAS CORBASCIOf

aDepartment of Molecular Medicine and Surgery, Division of Cardiothoracic Surgery and Anesthesiology,bDepartment of Medicine, Division of Cardiology, cDepartment of Clinical Immunology, and dDepartment ofGynecology, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden; eDepartment of Nephrologyand Transplantation, Lund University, Lund, Sweden; fDepartment of Heart Diseases, Cardiothoracic Surgery,Haukelands University Hospital, Bergen, Norway

Key Words. Human embryonic stem cells • Foxp3� T-cells • Tolerance • CTLA4Ig • anti-CD40L • anti-LFA-1

ABSTRACT

In order to study the ability of costimulation blockade toinduce tolerance to human embryonic stem cells (HESC),severe combined immunodeficient (SCID), and immuno-competent C57BL/6 mice treated with costimulation block-ade received intratesticular and intramyocardial HESCtransplants. All SCID mice with intratesticular HESC trans-plants developed teratoma. When SCID mice were trans-planted intramyocardially, only two of five mice developedteratoma-like tumors. C57BL/6 mice transplanted intrates-ticularly and treated with costimulation blockade all devel-oped teratoma and were surrounded by CD4�CD25�Foxp3�

T-cells, while isotype control treated recipients rejected theirgrafts. Most C57BL/6 mice transplanted intramyocardially

and treated with costimulation blockade demonstrated lym-phocytic infiltrates 1 month after transplantation, whereasone maintained its graft. Isolation of regulatory T-cells fromintramyocardial transplanted recipients treated with co-stimulation blockade demonstrated specificity toward undif-ferentiated HESC and down-regulated naive T-cell activa-tion toward HESC. These results demonstrate thatcostimulation blockade is sufficiently robust to induce toler-ance to HESC in the immune-privileged environment of thetestis. HESC specific regulatory T-cells developed to HESCtransplanted to the heart and the success of transplantationwas similar to that seen in SCID mice. STEM CELLS 2008;26:1850–1857

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

INTRODUCTION

Human embryonic stem cells (HESC) are pluripotent stem cellswith an inborn capacity for self-renewal and differentiation [1,2]. These characteristics make HESC an attractive source ofcells for cardiac repair, but obstacles still remain to be overcomebefore these cells can be used for cardiomyoplasty in a clinicalsetting. The conversion of undifferentiated HESC into cardio-myocytes is an inefficient process, where 15–25% of HESCdifferentiate into cardiomyocytes in vitro, depending on whichprotocol is used [3, 4]. There is a severe risk of teratoma

development as demonstrated by studies conducted with mouseESC injected into syngeneic mice [5]. Mouse and human ESCshare several genomic and proteonomic features [6]. However ifthe results described by Kolossov and co-workers working withmouse ESC can be extrapolated to HESC is still unresolved.

Apart from these obstacles, another major challenge will beto overcome immune rejection of the implanted cells. This canbe achieved by manipulating both the HESC and the recipient.Originally immuno-surgery was used in the derivation process,fetal calf serum (FCS) was a constituent of the culture mediumand HESC were cultured on mouse fibroblast feeder cells [1, 2].HESC were exposed to animal constituents in every step, which

Author contributions: K.-H.G.: conception and design, administrative support, provision of study material, collection and assembly of data,data analysis and interpretation, manuscript writing and final approval of manuscript; R.G.: conception and design, administrative support,collection and assembly of data and manuscript writing; M.K.-B. and C.D.: conception and design, collection and assembly of data, dataanalysis and interpretation, and manuscript writing; A.A. and A.M.-B.: collection and assembly of data, data analysis and interpretation; G.D.and C.S.: conception and design, administrative support, manuscript writing, final approval of manuscript and financial support; A.-M.S.:provision of study material; H.E.: financial support and final approval of manuscript; O.H.: administrative support, provision of studymaterial, manuscript writing and final approval of manuscript; M.C.: conception and design, data analysis and interpretation, financialsupport, manuscript writing and final approval of manuscript.

Correspondence: Karl-Henrik Grinnemo, M.D., Ph.D., Department of Molecular Medicine and Surgery, Division of Cardiothoracic Surgeryand Anaesthesiology, Karolinska University Hospital, S-171 76, Stockholm, Sweden. Telephone: �46-8-51770000; Fax: �46-8-51773437;e-mail: karl-enrik.grinnemo@karolinska.se Received February 4, 2008; accepted for publication April 15, 2008; first published online inSTEM CELLS EXPRESS May 8, 2008; available online without subscription through the open access option. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2008-0111

EMBRYONIC STEM CELLS

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constitutes both a risk of transmitting infections and the acqui-sition of xenogeneic antigens which might augment immunerejection [7]. To minimize the involvement of animal productsin the derivation and culture process, multiple steps have beenimplemented. Mechanical isolation of the inner cell mass [8],culturing on post-natal human skin fibroblast feeder cells [9],and utilizing a culturing medium with serum replacement in-stead of FCS [10, 11] will reduce the exposure of HESC toanimal products.

The immunogenicity of HESC is relatively uncharted terri-tory. In recent articles published by Li et al. [12] and Drukker etal. [13], in vitro and in vivo data was presented demonstratingthat HESC possess immune-privileged properties and thus canbe transplanted over the xenogeneic [12] and allogeneic barriers[13]. Their conclusions contrast to our findings when workingwith a similar cell line where HESC induce a robust immuneresponse in both allogeneic and xenogeneic settings [14]. Ac-cording to our data, the use of HESC-derived cells for cardio-myoplasty would require chronic immunosuppression, whichsubstantially increases the likelihood of developing opportunis-tic infections [15], cancer [16], and morbidity secondary tochronic drug toxicity.

Two possible approaches to overcome the immunologicalbarriers without using immunosuppression are to employ eithersomatic cell nuclear transfer (SCNT) or induced human pluri-potent stem cells. In the process of SCNT, the nucleus of theoocyte is removed and replaced by the genetic material from asomatic cell of the graft recipient. The oocyte cytoplasm enablesthe donor genome from a somatic cell to support embryonicdevelopment by a process called reprogramming [17]. The em-bryo is cultured to the blastocyst stage from which the embry-onic stem cell lines are derived. The HESC lines producedexpress the recipients transplantation antigens, except for theantigens derived from the mitochondria of the embryo donor.Successful production of embryonic stem cell lines throughSCNT from both mice [18, 19] and cattle [20] have beenreported. In humans, early blastocysts have been producedthrough SCNT [21], but the embryo failed to establish an HESCline. The alternative strategy is to transfect the human somaticcell with up to four different transcription factors and therebyreprogramming the somatic cells into induced pluripotent stemcells (iPS) [22–25]. Although human iPS cells are similar toHESC they are not identical. There is a difference in the geneexpression between iPS cells and HESC [24]. Furthermore, it isstill too early to speculate whether iPS cells have the samepotential as HESC to generate the different cell types in thehuman organism. All of the groups that created iPS used viralvectors to deliver the transcription factors into the human so-matic cells. Analysis of the reprogrammed iPS cells showed thatthese cells have on average 20 or more retroviral integrationsites [24, 25]. This is not acceptable from a clinical point ofview because of the high-risk of retroviral insertional mutagen-esis. It is, therefore, too early to say if iPS or SCNT can replacethe allogeneic HESC and their progeny in regenerative medi-cine.

Another attractive option to circumvent immune rejectionand chronic immunosuppression is to induce immunologicaltolerance toward the transplanted HESC by using costimulationblockade.

CTLA4Ig is a fusion protein consisting of the extra cellulardomain of CTLA4 and a human IgG1. CTLA4Ig binds to the B7family of costimulatory molecules expressed on dendritic cellsduring activation. By binding these molecules, reacting T-cellsdo not receive the necessary secondary signals to initiate T-cellactivation, which leads to anergy and apoptosis of reactingT-cells [26–28]. Anti-CD40L binds to CD40L, a moleculeexpressed on T-cells which is a central regulatory signal in the

immune system when initiating an immune response. Inhibitionof this signal leads to prevention of T-cell memory developmentand effector cell activation, B-cell differentiation and isotypeswitching, as well as macrophage activation [29]. Anti-LFA-1blocks the adhesion molecule LFA-1, which is ubiquitouslyexpressed on nearly every cell in the immune system. It has apivotal role in leukocyte function and the formation of theimmunological synapses between T-cells and dendritic cells andT-cells and their targets [30]. By administering these threesubstances at the time of transplantation, tolerance can be in-duced to allogeneic and xenogeneic tissues and organs [31].This state of tolerance seems to be mediated partially by regu-latory T-cells that inhibit naive T-cells directed toward thegrafted cells.

In an attempt to further clarify these issues, the present studywas designed to test the tumorigenicity and immunogenicity ofHESC transplanted into both the myocardium and testis ofimmunocompetent and immunodeficient mice. Furthermore, wehypothesized that the successful strategy to induce tolerancetoward grafts transplanted over the xenogeneic barrier usingcostimulation blockade might be applicable to HESC. Thereforewe tested whether or not a short course treatment with anti-CD40L/anti-LFA-1 and CTLA4Ig could induce long-term ac-ceptance of HESC implanted into mice and whether or not therewas a difference in engraftment between testis and the myocar-dium.

MATERIALS AND METHODS

Derivation and Culture of Human ESCHESC line HS401 was derived and cultured at the Fertility Unit,Department of Gynecology, Karolinska University Hospital [9, 11].The HESC line was derived from supernumerary blastocysts do-nated by couples undergoing fertility treatment. The HESC werecultured on post-natal human skin fibroblast feeder cells in serumreplacement medium [9]. The colonies were split mechanicallywithout the use of enzymes and replated onto new human fibroblastfeeder cells every 5–7 days. The HS401 expressed markers typicalof undifferentiated HESC, namely alkaline phosphatase, stage-spe-cific embryonic antigen (SSEA-4), tumor-related antigen (TRA1–60, TRA 1–81), and octamer-binding transcription factor-4(OCT-4), and formed teratoma when injected into severe combinedimmunodeficient (SCID) mice. For the implantation experiments,the HS401 colonies were divided manually with metal needles on astereo-microscope (Nikon �1500 [Nikon, Inc., Melville, NY, http://www.nikonusa.com]) and loaded into syringes (200.000 HESC in15 �l culture medium).

Animals and ReagentsEight-week-old male C57BL/6 mice (B and K Universal AB, Sol-lentuna, Sweden, http://www.bku.com) along with eight week oldmale SCID/Beige (C.B.-17/GbmsTac-SCID-bgDFN7; M and B,Taconic, Denmark, http://www.taconic.com) remains were used.The Animal Care Committee of Karolinska University Hospitalapproved all procedures.

The active costimulation blocking reagents used were anti-LFA-1 (clone M17/5.2), anti-CD40L (clone MR1), CTLA4 Ig,andtheir respective isotype control antibodies: rat IgG2b (clone 9A2),hamster IgG1 and human IgG1, (Bioexpress, West Lebanon, NH,http://www.bioexpress.com). The C57BL/6 mice treated with activecostimulation blockade (costimulation blockade treated group), re-ceived 0.5 mg anti-CD40L, 0.2 mg anti-LFA-1, and 0.5 mgCTLA4-Ig given intraperitoneally (ip) every other day for 8 days.The control mice received corresponding amount of the isotypecontrol reagents (isotype control treated group).

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Anesthesia and Post-Operative CareThe mice were anesthetized with a mixture of Midazolam (5 mg/kg;F. Hoffmann-La Roche Ltd., Basel, Switzerland, http://www.roche.com), Medetomidine (0.1 mg/kg; Orion Corporation, Espoo, Fin-land, http://www.orion.fi), Fentanyl (0.3 mg/kg; B. Braun MedicalAG, Seesatz, Switzerland, http://www.bbraun.ch) and given ip. Inthe group where HESC were injected into the myocardium, the micewere tracheotomized, and the ventilation was maintained using aZoovent ventilator, model CWC600AP, (B and K Universal). Theventilation was not supported when the cells were injected into thetestis of the mice. The anesthesia was reversed by an ip injection ofFlumazenil (0.1 mg/kg; F. Hoffmann-La Roche Ltd) and Atipam-ezol hydrochloride (5 mg/kg; Orion Corp). For post-operative an-algesia, Buprenorphin hydrochloride (Schering-Plough Corp., Ken-ilworth, U.K., http://www.sgp.com) was given ip used.

Cell Transplantation and Induction ofImmunological ToleranceThrough an incision in the distal part of the abdominal wall, throughthe linea alba, the right testis was exposed and 200.000 HESC wereinjected under the testis capsule of SCID mice (n � 6) and C57BL/6mice, which were randomized to receive either costimulation block-ade (n � 6) or isotype control reagents (n � 6). The mice wereeuthanized after 8 weeks, and the testis was harvested and analyzedhistochemically.

The testis is described to be an immune-privileged organ [32].Although the HESC survive in a testis from an immunocompetentmouse treated with costimulation blockade, the result might bedifferent if the HESC are transplanted to the heart. To further studythis hypothesis, we injected another set of mice with the sameamount of HESC through a left thoracotomy [14], into the myocar-dium of SCID mice (n � 5) and C57BL/6 mice, which were againrandomized to costimulation blockade (n � 8) or isotype controltreatment (n � 8). Two mice from both the costimulation blockadetreated and the isotype control treated groups were euthanized after4 weeks, whereas the remaining mice were euthanized after 8 weeks(i.e., SCID n � 5, C57BL/6 costimulation blockade treated n � 6;C57BL/6 isotype control treated n � 6).

Because of failure to induce teratoma and the appearance ofcellular infiltrates at 1 month, a second set of transplants wasperformed. Costimulation blockade or their isotype controls, both atthe time of transplantation and 3 weeks later (n � 5 in each group),were injected. We named these groups costimulation blockade x2and isotype control x2, respectively. The mice were euthanized after8 weeks.

Detection of Transplanted Cells and HistologicalEvaluation of the Immune ResponseThe hearts and testes from the euthanized mice were freeze-sec-tioned into 5 �m thick sections, and the site of injection wasidentified by hematoxylin and eosin staining. In case of teratomaformation, these sections were further evaluated searching for struc-tures representing the three germ layers. The HESC and theirdifferentiated progeny were traced using fluorescence in situ hy-bridization technique (FISH), which has previously been described[33]. In brief, fluorescence inmarked DNA-probes hybridize to totalhuman DNA of viable cells, labeling the whole nucleus red. Thesections with surviving HESC derived cells in the heart were furtherstained for the cardiomyocyte markers �-actinin (clone EA-53;Sigma-Aldrich Corp, St. Louis, http://www.sigmaaldrich.com) andDesmin (clone DE-R-11; Dako Cytomation, Glostrup, Denmark,http://www.dakocytomation.com), together with TRA 1–60 (cloneTRA-1–60, Chemicon International Inc., Temecula, CA, http://www.chemicon.com) which is a marker for undifferentiated HESC.After fixation with 4% formaldehyde and blocking with 5% rabbitserum (X0902; Dako Cytomation) for 30 minutes, the slides wereincubated with the respective primary antibody over night in ahumidified chamber. The sections were then incubated with fluo-rescence-labeled rabbit anti-mouse immunoglobulins/ FITC (DakoCytomation) and visualized in the fluorescence microscope (Olym-pus BX60; Olympus Optical Ltd, Tokyo, http://www.olympus-global.com). As positive controls we used human heart (Desmin and

�-actinin) and HESC in the myocardium of SCID/beige mice,which were euthanized and freeze-sectioned directly after injection(TRA 1–60).

The inflammatory response was evaluated by hematoxylin andeosin staining together with immunohistochemical analysis. Afterfixation with 4% formaldehyde (Histolab Products Ltd, Gothen-burg, Sweden, http://www.histolab.se) and blocking with 5% rabbitserum (code No. X0902; Dako Cytomation) and 5% mouse serum(code no. X0910; Dako Cytomation) in TBS (stock solution 10xconcentration: 87.66 g NaCl, 60.55 g Tris diluted to 1,000 ml indistilled water, pH: 7.4). The sections were subsequently incubatedover night in a humidified chamber with the primary antibodies.These included rat anti-mouse CD3 (clone KT3), rat anti-mouseCD4 (clone YTS191.1), rat anti-mouse CD8 (clone KT15), ratanti-mouse CD11b (clone 5C6) (Serotec, Oslo, Norway, http://www.ab-direct.com) and rat anti-mouse Foxp3 (clone FJK-16s;eBioscience, San Diego, http://www.ebioscience.com). The slideswere then incubated with the secondary antibodies rabbit anti-rat-IgG (FITC code No. F0234; Dako Cytomation) for 2 hours in ahumidified chamber and mounted with an anti-fading reagent con-taining 4,6-diamidino-2-phenylindole (DAPI) before visualizationin the fluorescence microscope (Olympus BX60). Spleens fromC57BL/6 were used as positive controls, whereas hearts from SCID/beige mice served as negative controls for the primary antibodies.

Mixed Leukocyte ReactionSplenocytes from naive C57BL/6 mice, together with C57BL/6mice from the costimulation blockade treated and the isotype con-trol groups, were used for this experiment. The spleens were ho-mogenized and the CD4� T-cells were separated using a CD4�

T-cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany,http://www.miltenyibiotec.com) as previously described [14]. Thepurity of the CD4� T-cells was more than 95%, as estimated byfluorescence-activated cell sorter analysis (FACS). CD4�CD25�

T-cells from C57BL/6 mice of the costimulation blockade andisotype control groups were separated according to the manufactur-er’s protocol using a CD25 MicroBead Kit (Miltenyi Biotec) andthe purity was estimated to 70% by FACS analysis.

The dendritic cells (DC) were prepared from the bone marrowof the femurs of syngenic C57BL/6 mice according to the protocolpreviously described [14].

The DC (1 � 106) from C57BL/6 mice were co-cultured withirradiated (15Gy) HESC (3 � 105) and human fibroblasts (HFib)(3 � 105), respectively, in culture medium [Roswell Park MemorialInstitute (RPMI)-1640 supplemented with 5% heat inactivated fetalcalf serum, 2 mmol/l L-glutamine, 100 �g/ml streptomycin, 100IU/ml penicillin and 5 � 10-5 mol/l 2-mercaptoethanol] in humid-ified air containing 5% CO2 at 37°C for 24 h. The DC were thenirradiated (15Gy). The mixed leukocyte reactions (MLR) was per-formed exposing naive CD4� T-cells (5 � 104 cells per well) tosyngenic DC (2 � 104 cells per well) alone or syngenic DCco-cultured with HESC or HFib (2 � 104 cells per well). To studyif the regulatory T-cells from mice treated with costimulation block-ade or its isotype control reagents could specifically down-modulatethe immune response induced by HESC, the previously separatedCD4�CD25� T-cells (5 � 104 cells per well) were added to thewells. Five wells per culture were set up in 96-well round-bottomedmicrotiter plates (Corning Life Sciences, Acton, MA, http://www.corning.com/lifesciences) and incubated in humidified air contain-ing 5% CO2 at 37°C. Each culture was labeled with 2 �Ci [3H]thymidine (Amersham, Buckinghamshire, U.K., http://www.amersham.com) for 20 hours prior to harvest. The proliferation of the responderswas measured at day 6 as counts per minute (cpm) and each experimentwas repeated three times. Two mice from each group were used foreach MLR.

Statistical Analysis

Data are presented as mean � SD. For statistical analysis of MLRdata, Wilcoxon Signed Rank Test was used as a nonparametric testfor two related samples. A p � .05 was considered significant.

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RESULTS

HESC Transplanted into the TestisThe HESC injected into the testis of SCID mice (n � 6) andcostimulation blockade treated C57BL/6 mice (n � 6) devel-oped into teratoma in all animals when studied histologically attwo months after transplantation. This was in contrast to thefindings in the isotype control treated C57BL/6 mice (n � 6),where no surviving HESC was found. The teratoma did notdiffer between the SCID mice and the costimulation blockadetreated C57BL/6 mice. They formed exophytic cystic tumors,where histological analysis revealed neuroepithelium, broncho-epithelium, and bone, hence typical histological features repre-sent the three germ layers (Fig. 1A–C). The cells in the teratomawere of human origin as demonstrated by FISH staining (Fig.1D), and the normal testis tissue was compressed toward thecapsule. There was no sign of any acute CD4� T-cell-mediatedinflammatory response in the teratoma of the costimulationblockade treated C57BL/6 mice, and the only CD4� T-cellspresent were mouse CD4�CD25�Foxp3� regulatory T-cells(Fig. 2A). These cells were clustered at the capsule of the testisclose to the interface of normal tissue and teratoma from wherethey seemed to migrate into the peripheral parts of the teratoma.No CD4�CD25�Foxp3� regulatory T-cells were seen in thecentral parts of the teratoma. In the isotype control treatedC57BL/6 mice no FISH positive cells could be identified after 2months. There were no signs of an ongoing inflammatory re-sponse and only a small scar could be seen at the site of theinjection.

HESC Transplanted into the HeartIn SCID mice, HESC engrafted in the heart in two of five miceformed expansive cystic tumors. These were mainly composedof different epithelial structures and fibrous stroma (Fig. 1E, F).Although their growth characteristics were similar to teratoma,

they do not fulfill the criteria of a teratoma because cells fromall three germ layers were not found. We have thus termed thesetumors teratoma-like. The cells in the teratoma-like tumors didnot stain positive for Desmin or �-actinin, indicating that theHESC were not cardiomyocytes and thereby were not influ-enced in their differentiation by the surrounding myocardium. Inthe other three SCID hearts the injection site was clearly presentbut there was no surviving HESC. This is in contrast to the testiswhere the HESC developed into teratoma in all animals tested(n � 6). In the costimulation blockade treated and isotypecontrol treated C57BL/6 mice we could not find any survivingHESC after 1 (n � 2 in each group) and 2 months (n � 6 in eachgroup), but the histological features differed between the twogroups. In the isotype control treated group there was only a scarleft in the myocardium, with no sign of an inflammatory re-sponse. In the costimulation blockade treated group, the heartsshowed signs of lymphocytic infiltrates at 1 month after trans-plantation (Fig. 2B–F), which was also seen in one of the heartsafter 2 months (Fig. 2G, H). The site of implantation of HESCwas infiltrated by activated macrophages (CD11b�) and theT-cell response was characterized by both CD4� and CD8�

cells. These histological finding were similar to the acute im-mune response seen during the first days after transplantation ofHESC to untreated immunocompetent mice hearts [14]. Withcostimulation blockade this immune response was delayed. Inthe other five hearts removed at 2 months a scar with no FISHpositive cells was found at the site of injection (Fig. 2I).

In an attempt to induce tolerance toward these HESC-derived progeny, we injected another group of immunocompe-tent mice with a second round of treatment with costimulationblockade 3 weeks after transplantation (costimulation blockadex2 treated, n � 5) or with isotype controls (isotype control x2treated, n � 5). As expected, no surviving HESC was found inthe isotype control x2 treated group after two months, and theonly histological finding was a scar at the site of injection. Inone of the hearts (n � 1/5) in the costimulation blockade x2

Figure 1. Teratoma formation 2 months after injection of undifferentiated HESC into testis and heart of mice. HESC injected into the testis ofC57BL/6 mice treated with costimulation blockade developed into teratoma (n � 6). Hematoxylin and eosin staining of cross sections revealed tissuesrepresenting the three germ layers: (A) neuroepithelium, (B) bronchoepithelium with goblet cells (arrow), and (C) bone. (D): FISH staining of arepresentative teratoma showed that all the cells in the tumor were derived from HESC (red cells). (E): Hematoxylin and eosin staining of an expansivegrowing teratoma-like tumor in the heart of a SCID mouse (n � 2/6). (F) FISH staining reveals that the tumor originates from the HESC (red cells).Panel F is a higher magnification of the square plotted in panel E. Bar represents: 100 �m in panels A–C, F; 200 �m in panel D and 1,000 �m inpanel E.

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treated group we found surviving HESC-derived cells after 2months (Fig. 3). These cells were encapsulated in the myocar-dium and they had not developed into any teratoma-like struc-ture. Furthermore, the HESC-derived cells stained negative forTRA 1–60 and the cardiomyocyte markers Desmin and �-acti-nin, indicating that they were no longer undifferentiated HESCor myocytes. In the tissue surrounding the engrafted HESC-derived cells there was no sign of inflammatory response norcould we identify a significant amount of regulatory T-cells inthe tissue surrounding the engrafted HESC-derived cells.

The Functional Impact of Regulatory T-Cells inInducing Tolerance Toward HESCMLR were designed to mimic the clinical setting of HESCtransplantation where the host DC present antigens from pro-cessed HESC to host CD4� T-cells. Naive CD4� T-cells fromC57BL/6 mice were cultured together with syngenic DC,previously co-cultured with either HESC or with HFib.CD4�CD25� T-cells were separated from the spleens of syn-genic C57BL/6 mice previously transplanted with HESC intothe heart and sacrificed after 2 months. The influence that theCD4�CD25� T-cells separated from the costimulation blockadeor isotype control groups had on naive CD4� T-cell prolifera-tion was measured. As shown in Figure 4, the CD4�CD25�

T-cells separated from the costimulation blockade group signif-icantly downregulated naive CD4� T-cell proliferation with a

mean of 22% (p � .05), whereas CD4�CD25� T-cells from theisotype control group did not inhibit proliferation of naiveT-cells. Furthermore, the downregulatory effect of the immuneresponse mediated by CD4�CD25� T-cells was specific towardundifferentiated HESC, because they did not inhibit the prolif-eration of naive T-cells toward HFib.

Figure 2. Immunological response to HESC transplanted into the testis and myocardium. (A): CD4�CD25�Foxp3� T-cells (green cells) at theinterface of normal tissue and teratoma in the testis of a costimulation blockade treated mouse two months after transplantation. (B): Hematoxylinand eosin staining of a cross section of a representative heart showing cellular infiltration one month after transplantation of HESC into themyocardium of costimulation blockade treated mice. This immune response was characterized by (C) activated macrophages (CD11b�, green cells),(D) T-cells (CD3�, green cells), where the T-cell response is characterized by both (E) CD4� cells (green cells) and (F) CD8� cells (green cells).The same immune response was seen in one of the hearts (n � 1/6), 2 months after transplantation with (G) activated macrophages (CD11b�, greencells) and (H) CD3� T-cells (green cells). (I) In the majority of the hearts there was only a scar left in the myocardium two months after transplantation(n � 5/6). Bar represents: 100 �m in panel A–H; 200 �m in panel I. Abbreviation: DAPI, diamidino-2-phenylindole.

Figure 3. HESC 2 months after transplantation into the myocardium.HESC were transplanted into the myocardium of C57BL/6 mice andreceived treatment with costimulation blockade directly after transplan-tation and then again 3 weeks later (n � 5), which led to the acceptanceof these cells in one recipient as demonstrated by (A) hematoxylin andeosin staining of the heart. There were no signs of inflammatory re-sponse as demonstrated by anti-CD3� and anti-CD11b� staining aroundthe engrafted cells. (B): FISH-staining showing that the cells were ofhuman origin (red cells). Bar represents 200 �m. Abbreviations: DAPI,diamidino-2-phenylindole; FISH, fluorescence in situ hybridization;HESC, human embryonic stem cells.

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DISCUSSION

The results presented herein clearly demonstrate that HESC areimmunogenic and are rejected when transplanted over the xe-nogeneic barrier into the testis or the myocardium of immuno-competent C57BL/6 mice. The environment of the testis isconsidered an immune-privileged area [32], nonetheless, HESCwere rejected when transplanted to the testis. The HESC lineused has to a minimal degree been exposed to animal compo-nents during the derivation and culturing process. Altogetherthis means that the immune response seen in these experimentswas induced by the HESC and not by contaminating animalproducts. These findings further emphasize that HESC are notimmune-privileged and confirms our previous findings [14].

In order to circumvent immune rejection without usingchronic immunosuppression, we attempted to induce a state ofimmunological tolerance toward the engrafted cells. Ever sinceMedawar�s pioneering experiments [34], the “Holy Grail” of thetransplant community has been to induce tolerance to trans-planted tissues and organs. The use of costimulation blockadehas been heralded as a great leap toward achieving this goal [31,35–39]. The strategy that has attracted the greatest attentiontargets the costimulatory molecules, CD40L, and B7 molecules,expressed on T-cells and DC under inflammatory circum-stances. By blocking these pathways with anti-CD40L andCTLA4Ig (a fusion protein of a high affinity receptor for B7 andthe Fc component of human IgG1) given during the first weekafter transplantation, indefinite acceptance of vascularizedmouse and rat cardiac transplants in mice can be achieved [40,41]. Double costimulation blockade (anti-CD40L/CTLA4Ig)

has a varied outcome depending on the type of tissue trans-planted and the strain of the recipient mouse [42]. By simulta-neously blocking LFA-1, CD40L-independent immune re-sponses can be inhibited, which has been shown to beadvantageous in the transplantation of discordant xenogeneiccellular transplants [31]. This triple costimulation blockade (an-ti-CD40L/CTLA4Ig/anti-LFA-1) given during the first 8 daysafter transplantation induced long-term acceptance and im-proved pig islet graft function in diabetic mice when comparedwith double costimulation blockade (anti-CD40L/CTLA4Ig)[31]. Using the same triple costimulation blockade regime,HESC engrafted in the testes of all immunocompetent miceforming exophytic growing teratoma at 2 months after trans-plantation, with similar growth characteristics as in the SCIDmice. There was no sign of CD4� T-cell-mediated immunerejection. The development of CD4�CD25� Foxp3� T-cellsindicate that costimulation blockade had induced immunologi-cal tolerance toward the HESC-derived cells. These results arein contrast to those obtained when HESC were injected into themyocardium of C57BL/6 mice treated with the same shortcourse of costimulation blockade. The HESC induced an acuteCD4� T-cell-mediated immune response one month after injec-tion, causing rejection of the implanted HESC. Nonetheless,CD4�CD25� T-cells specific to undifferentiated HESC withthe capacity to downregulate naive T-cell activation developedin these recipients. This could imply that the host was tolerant ofundifferentiated HESC but, as the cells differentiate, their geneexpression changes [43], and thereby new antigens, which thehost has not been tolerated toward, are expressed. These newantigens could thereby activate other clones of T-cells whichwere not present at the time or place of tolerization and, subse-quently, induce rejection.

In order to study the possibility to induce tolerance towardthe in vivo differentiating HESC, we repeated the costimulationblockade treatment three weeks after transplantation. In one offive mice, the HESC-derived cells were found in the myocar-dium after 2 months, but did not develop into teratoma, sincecells representative of all three germ layers were not present.Instead, the engrafted cells were encapsulated in the myocar-dium, and there was no sign of inflammatory response.

The difference in survival of HESC-derived cells betweenthe testis and the heart of the costimulation blockade treatedmice may also be due to nonimmunological factors. Injection ofHESC into the hearts of SCID mice only induced tumor growthin two of five mice, while teratoma formations were seen in thetestes of all SCID mice. The difference in tumor developmentbetween the heart and testis thus seems to be due to factorspertaining to engraftment in the myocardium, similar to thefindings when nonpurified mESC-derived cardiomyocytes wereinjected into the heart [5].

CD4�CD25�Foxp3� T-cells were seen clustered at theinterface between normal tissue and teratoma in the testes ofthe costimulation blockade treated mice. These findings supportthe hypothesis that costimulation blockade induces peripheraltolerance mediated by regulatory T-cells. In contrast, no regu-latory T-cells were seen around the engrafted HESC-derivedcells in the myocardium, which is in accordance to the findingswhen pig islets were transplanted into mice using the samecostimulation blockade protocol [31]. To further evaluate therole of regulatory T-cells in maintaining peripheral tolerance toimplanted HESC, we performed MLRs. In the experimentsdescribed in this report, 95% pure populations of naive CD4�

T-cells were isolated and exposed to syngenic DC co-culturedwith HESC or HFib, in order to imitate the clinical situation asclosely as possible. To study if the regulatory T-cells from micetreated with costimulation blockade or isotype control reagentscould specifically down-modulate the immune response induced

Figure 4. Peripheral tolerance toward undifferentiated HESC mediatedby regulatory T-cells. Naive CD4� T-cells were cultured together withirradiated DC presenting antigens from either HESC or HFib. Theproliferation of CD4� T-cells (cpm), was measured at day 6.CD4�CD25� T-cells were separated from both the costimulation block-ade treated (Costim Block) and isotype control treated (Isotype Control)mice previously transplanted with HESC into the heart. TheseCD4�CD25� T-cells were then added to naive CD4� T-cells respond-ing to DC presenting antigens from either HESC or HFib. TheCD4�CD25� T-cells separated from the mice in the costimulationblockade group significantly down-regulated naive CD4� T-cell prolif-eration with a mean of 22% (p � .05, *), while the CD4�CD25� T-cellsfrom the isotype control group did not, implying that CD4�CD25�

T-cells were specific toward the HESC. The asterisk (*) indicates asignificant down-regulation of the immune response. One representativeMLR is demonstrated. The experiment was conducted three times andtwo mice from each treatment group were used for each MLR. Values �mean � SD. Abbreviations: CPM, counts per minute; DC, dendriticcells; HESC, human embryonic stem cells; HFib, human fibroblasts;MLR, mixed leukocyte reactions.

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by HESC, regulatory T-cells from the different groups wereadded to the ongoing indirect immune response. RegulatoryT-cells isolated from the costimulation blockade group down-modulated the immune response induced by HESC significantlyby 22%, whereas regulatory T-cells from the isotype controlgroup did not downregulate the immune response at all. Theeffects mediated by the regulatory-T-cells also seemed to bespecific toward HESC, since the immune response induced byHFib was not affected. These data indicate that, even though thegrafts were not maintained in the heart, the recipient nonethelesswas tolerized to undifferentiated HESC.

The tumorigenicity of mESC in the heart of syngeneic micehas been well-characterized [5], but the tendency of HESC toform teratoma in different compartments is still a relativelyunexplored area of research. To study the tumorigenicity ofHESC, we injected undifferentiated HESC into both the myo-cardium and the testes of SCID mice. In the testis, teratomaformations were seen in every mouse, while in the myocardiumteratoma-like formations were seen in two of five mice, 2months after transplantation. This indicates that HESC are astumorigenic as mESC and from this standpoint, it seems logicalthat pure HESC-derived cardiomyocyte populations should beused for cardiomyoplasty.

An interesting observation is that the engrafted HESC-derived cells in the myocardium of costimulation blockadetreated mice did not develop into teratoma. This may be due tothe fact that repeated treatment with costimulation blockade hasinduced tolerance to one type of HESC-derived cell, whereasother cells with another gene expression are rejected. From thispoint of view, costimulation blockade might reduce the tumor-igenicity of HESC by empowering the immune system with the

capacity to remove cells that are not of the same phenotype asthe cells to which tolerance toward is desired.

CONCLUSION

Altogether, these findings indicate that it is possible toinduce immunological tolerance toward undifferentiated HESCtransplanted into the testis and to induce regulatory T-cells toundifferentiated HESC when transplanted into the heart. Thesertoli cells of the testis express Fas ligand (FasL, CD95) whichinduces apoptosis of reactive T-cells toward intratesticulargrafts [44]. This capacity, when combined with costimulationblockade, tips the balance from rejection to the induction ofregulatory T-cells and tolerance induction [32]. In the heart,HESC differentiate, changing their antigen expression andthereby they are no longer protected from rejection by theregulatory T-cells induced at the time of tolerization. By usinghighly purified HESC-derived cardiomyocytes already differen-tiated into the desired phenotype, it might be possible to induceperipheral tolerance with costimulation blockade and reduce therisk of teratoma formation. Until these goals have been reached,clinical trials with HESC-derived cells should not be performed,because they are both immunogenic and tumorigenic.

DISCLOSURE OF POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.

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DOI: 10.1634/stemcells.2008.0111 2008;26;1850-1857; originally published online May 8, 2008; Stem Cells

Strömberg, Henrik Ekberg, Outi Hovatta, Christer Sylvén and Matthias Corbascio Christian Danielsson, Agneta Månsson-Broberg, Göran Dellgren, Anne-Marie

Karl-Henrik Grinnemo, Rami Genead, Makiko Kumagai-Braesch, Agneta Andersson, and Induces Regulatory T-Cells to HESC Transplanted into the Heart

Costimulation Blockade Induces Tolerance to HESC Transplanted to the Testis

This information is current as of July 19, 2009

& ServicesUpdated Information

http://www.StemCells.com/cgi/content/full/26/7/1850including high-resolution figures, can be found at:

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