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Fax: +82-2-599-3589; E-mail: [email protected]. Combination Cell Therapy Using Mesenchymal Stem Cells and. Regulatory T-Cells Provides a Synergistic ...
Cell Transplantation, Vol. 23, pp. 703–714, 2014 Printed in the USA. All rights reserved. Copyright  2014 Cognizant Comm. Corp.

0963-6897/14 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368913X664577 E-ISSN 1555-3892 www.cognizantcommunication.com

Combination Cell Therapy Using Mesenchymal Stem Cells and Regulatory T-Cells Provides a Synergistic Immunomodulatory Effect Associated With Reciprocal Regulation of Th1/Th2 and Th17/Treg Cells in a Murine Acute Graft-Versus-Host Disease Model Jung-Yeon Lim,* Min-Jung Park,† Keon-Il Im,* Nayoun Kim,* Eun-Joo Jeon,* Eun-Jung Kim,* Mi-La Cho,† and Seok-Goo Cho*‡ *Laboratory of Immune Regulation, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary’s Hospital, The Catholic University of Korea College of Medicine, Seoul, Korea †Rheumatism Research Center, Catholic Institutes of Medical Science, Seoul St. Mary’s Hospital, The Catholic University of Korea College of Medicine, Seoul, Korea ‡Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Hospital, The Catholic University of Korea College of Medicine, Seoul, Korea

Mesenchymal stem cells (MSCs) have been considered to be an ideal cellular source for graft-versus-host disease (GVHD) treatment due to their unique properties, including tissue repair and major histocompatibility complex (MHC)-unmatched immunosuppression. However, preclinical and clinical data have suggested that the immunomodulatory activity of MSCs is not as effective as previously expected. This study was performed to investigate whether the immunomodulatory capacity of MSCs could be enhanced by combination infusion of regulatory T (Treg) cells to prevent acute GVHD (aGVHD) following MHC-mismatched bone marrow transplantation (BMT). For GVHD induction, lethally irradiated BALB/c (H-2d) mice were transplanted with bone marrow cells (BMCs) and spleen cells of C57BL/6 (H-2b) mice. Recipients were injected with cultured recipient-derived MSCs, Treg cells, or MSCs plus Treg cells (BMT + day 0, 4). Systemic infusion of MSCs plus Treg cells improved clinicopathological manifestations and survival in the aGVHD model. Culture of MSCs plus Treg cells increased the population of Foxp3+ Treg cells and suppressed alloreactive T-cell proliferation in vitro. These therapeutic effects were associated with more rapid expansion of donor-type CD4+CD25+Foxp3+ Treg cells and CD4+IL-4+ type 2 T-helper (Th2) cells in the early posttransplant period. Furthermore, MSCs plus Treg cells regulated CD4+IL-17+ Th17 cells, as well as CD4+IFN-g + Th1 cells. These data suggest that the combination therapy with MSCs plus Treg cells may have cooperative effects in enhancing the immunomodulatory activity of MSCs and Treg cells in aGVHD. This may lead to development of new therapeutic approaches to clinical allogeneic hematopoietic cell transplantation. Key words: Allogeneic bone marrow transplantation; Graft-versus-host disease (GVHD); Mesenchymal stem cells (MSCs); Regulatory T-cell

Introduction Acute graft-versus-host disease (aGVHD) is a major complication of tissue toxicity and organ injury following allogeneic hematopoietic stem cell transplantation (HSCT). Although immunosuppressive drugs have improved the survival rates for allogeneic transplantation, severe grade III or IV aGVHD is not always easily reversed with high doses of steroids (41,42). Additionally, for ­steroid-refractory GVHD, although second-line therapies such as antithymocyte globulin (13,23,31), interleukin 2

(IL-2) receptor antibodies (10), monoclonal cluster of ­differentiation 3 (CD3) antibodies (6), rapamycin (4), and extracorporeal photopheresis (30) have been considered, the clinical outcomes are generally poor with a high mortality rate due to infectious complications and sustained GVHD-related cytopenia and multiorgan failure. Recently, mesenchymal stem cells (MSCs) have been considered to be an emerging alternative to current pharmacological immunosuppressive drugs in the field of transplantation because they can mediate potent immunoregulatory

Received July 1, 2012; final acceptance February 7, 2013. Online prepub date: February 26, 2013. Address correspondence to Professor Seok-Goo Cho, M.D., Ph.D,. Laboratory of Immune Regulation, Convergent Research Consortium for Immunologic Disease, Department of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Hospital, The Catholic University of Korea College of Medicine, 222, Banpo-daero, Seocho-gu, Seoul 137-701, Korea. Tel: +82 2 2258 6052; Fax: +82-2-599-3589; E-mail: [email protected]

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effects on various cell types, regulating both adaptive and innate immune responses (7,9,35). The immunomodulatory properties of MSCs have led to therapeutic trials of MSCs to treat GVHD after HSCT. Many phase I/II trials worldwide have presented the clinical benefits of MSC therapy in GVHD ever since Le Blanc et al. reported successful treatment using third-party haploidentical mesenchymal stem cells in a pediatric patient with severe aGVHD (19,20). Unexpectedly, a recent commercial phase III unpublished trial (Osiris Therapeutics) failed to meet its primary endpoint of a durable complete response (≥28 days); however, the MSC group showed a strong trend of improvement in patients with gastrointestinal or hepatic GVHD and in pediatric patients (2,27). Additionally, the immunomodulatory capacity of MSCs has not been proven to be effective in preventing GVHD in clinical trials or preclinical models, although MSC therapy has shown promising results in established GVHD. The immunosuppressive activity of MSCs can be influenced by environmental parameters related to inflammatory conditions. During acute inflammation, Th1-type cytokines induce the polarization of M1 macrophages, which “license” MSCs to inhibit immune responses. Thus, experimental evidence suggests that MSCs are effective for treating GVHD only if administered when the concentrations of acute inflammatory molecules, such as ­interferon-g (IFN-g) and tumor necrosis factor-a (TNF-a), are elevated in the recipient. These data may explain the clinical trial results. However, a previous study demonstrated that administration of MSCs at the time of HSCT, before any GVHD had developed, did not change the frequency of acute or chronic GVHD (25). Further, MSCs conferred no benefit in mice with collagen-induced arthritis, and the immunosuppressive effect of MSCs on T-cell proliferation was reversed by the addition of TNF-a in vitro (24). Moreover, our previous study showed a negative effect of a single infusion of MSCs in a murine model of collagen-induced arthritis (CIA), as MSCs alone did not prevent proinflammatory cytokines (29). MSCs can generate immunoregulatory cells such as CD4+CD25+Foxp3+ (Forkhead box P3+) T regulatory (Treg) cells, and the immunosuppressive properties of MSCs may depend, in part, on their effects on Treg generation or function. MSCs induce plasmacytoid dendritic cells to produce interleukin 10 (IL-10), a process that may favor the development of inducible Treg cells in vivo. Additionally, powerful regulatory CD4+ or CD8+ lymphocytes are generated in cocultures of peripheral blood mononuclear cells (PBMCs) with MSCs (1,22). These data strongly suggest that Treg cells may amplify the reported MSC-mediated immunosuppressive effect. Induced Treg cells (iTreg cells) can suppress effector T-cell responses in vitro and in vivo, and they have attracted a great deal of attention largely based on

their well-established importance in maintaining peripheral tolerance (32). Although the instability of Foxp3 expression has been reported to limit the usefulness of adoptively transferred iTreg cells as a source of cellular therapy for the abrogation of GVHD, several recent phase I trials indicated that peripheral blood or cord blood­derived Treg cells could decrease GVHD (12,39,40). The immunomodulatory capacity of MSCs was not targeted by the inhibitory effect of Treg cells and vice versa. In addition, Treg cells supported MSC function, as they did not alter the secretion of IFN-g by immune cells in solid organ transplantation (8). Considerable progress has been made in the development of MSC treatment for GVHD. However, MSCs have a number of limitations. MSCs often fail to control GVHD, even with use of a variety of timing and dose protocols. Similarly, MSC treatment of GVHD in clinical trials has inherent constraints from preclinical experiments. Recipient-derived Treg cells initially occupy a niche in posttransplantation recipients, undergo significant expansion, and contribute to the T-cell compartment for an extended period before the production of donor-derived CD4+Foxp3+ T-cells by MSCs. Therefore, the combination of MSCs and Treg cell therapy is a novel method for preventing GVHD, and it was first applied in an animal model. The present study was performed to investigate whether coinfusion of ex vivo-expanded MSCs and iTreg cells could play a complementary role in preventing GVHD. Materials and methods Mice Eight- to 10-week-old female C57BL/6 (B6, H-2b) and female BALB/c (H-2d) mice were purchased from Orient Bio (Sungnam, Korea). The mice were maintained under specific pathogen-free conditions in an animal facility with controlled humidity (55% ± 5%), light (12/12 h light/dark), and temperature (22°C ± 1°C). The air in the facility was passed through a high-efficiency particulate absorption (HEPA) filter system designed to exclude bacteria and viruses. Animals were fed mouse chow and tap water ad libitum. The protocols used in the present study were approved by the Animal Care and Use Committee of The Catholic University of Korea. Isolation and Culture of MSCs Recipient (BALB/c) bone marrow cells (BMCs) were collected by flushing femurs and tibias with Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) containing 2 mM l-glutamine (Gibco), 1% antibiotics [penicillin (10 U/ml)–streptomycin (10 g/ml; Gibco)] and 15% heat-inactivated fetal bovine serum (FBS) with endotoxin level ≤5 EU/ml, hemoglobin level ≤ 10 mg/dl (Gibco). Cell immunophenotypes were persistently positive

COMBINATION CELL THERAPY USING MSCs AND TREGs

for stem cell antigen-1 (Sca-1), CD44, and CD29, but negative for c-Kit, CD11b, and CD34 after more than 15 passages using the antibodies described later in agreement with previous reports (34). Treg Generation Previously, we reported an iTreg generation method using all-trans retinal (Retinal). To obtain Treg cells, isolated CD4+ T-cells from recipients (BALB/c) were cultured with plate-bound anti-CD3 (1 μg/ml; BD PharMingen, San Diego, CA, USA), soluble anti-CD28 (1 μg/ml; Biolegend, San Diego, CA, USA), human recombinant transforming growth factor-b (TGF-b; 5 ng/ml; PeproTech, London, UK), and Retinal (0.1 μM; Sigma-Aldrich, St. Louis, MO, USA) for 3 days (16). The expanded induced Treg cells were sorted by flow cytometry to obtain a ~90% pure CD4+CD25+ population. Mixed Lymphocyte Culture (MLC) A mixed lymphocyte culture (MLC) was prepared in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) containing 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES; Gibco), 2 mM l-glutamine, 5% heat-inactivated FBS, 100 mM sodium pyruvate, and 1% antibiotics [penicillin (10 U/ml)–­ streptomycin (10 g/ml)]. Cells (1 × 106/well) from spleens of C57BL/6 mice were stimulated with 1 × 106 of irradiated (2,000 rad) BALB/c splenocytes, 5 × 104 MSCs, or 1 × 105 Treg cells in 2 ml of culture medium. Cultures were maintained at 37°C in a 5% CO2 atmosphere. Spleen cells were harvested for cytokine detection, and cells were collected for intracellular cytokine assays after 5 days. Proliferation Assay CD4 T-cells (1 × 105/well) from spleens of C57BL/6 mice were stimulated with 1 × 105 irradiated (2,000 rad) T-cell-depleted BALB/c splenocytes, 5 × 103 to 1 × 104 MSCs, and/or 5 × 103 to 1 × 104 Treg cells in 0.2 ml of culture medium. For proliferation analysis, cells were pulsed with 1 μCi [3H]thymidine (GE Healthcare, Piscataway, NJ, USA) per well for the final 8 h of the 72-h culture period. Finally, [3H]thymidine incorporation was determined using a liquid b-scintillation counter (Beckman, Fullerton, CA, USA). Bone Marrow Transplantation and GVHD Induction Recipient (BALB/c, H-2d) mice were exposed to a 800cGy dose of radiation from a Mevatron MXE-2 instrument (Siemens, New York, NY, USA) with a focus-to-skin distance of 100 cm and a rate of 70 cGy/min. Recipient mice were injected intravenously (IV) with 5 × 106 BMCs and 5 × 106 spleen cells from donor mice (C57BL/6, H-2b). Control group was comprised of irradiated mice receiving 5 × 106 T-cell-depleted (TCD) BMCs, which does not

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induce GVHD. T-cells were depleted from BM by using CD90.2 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for negative selection. Survival after bone marrow transplantation (BMT) was monitored daily, and the degree of clinical GVHD was assessed weekly using a scoring system that summed changes in five clinical parameters: weight loss, posture, activity, fur texture, and skin integrity. Combination Cell Therapy of MSCs and Treg Cells Control GVHD Mice were injected IV with 1 × 106 MSCs, 2 × 106 Treg cells, or 1 × 106 MSCs plus 2 × 106 Treg cells twice weekly after BMT (BMT + day 0, 4). Control mice received IV injections of an equal volume of phosphate-buffered saline (PBS; Gibco) at the same time points. Clinicopathological Evaluation Mice were killed on day 12 after BMT for blinded ­histopathological analysis of GVHD targets (skin, liver, and large intestine). Organs were harvested, cryoembedded, and subsequently sectioned. Tissue sections were fixed in 10% buffered formalin (Sigma-Aldrich) and stained with hematoxylin (Sigma-Aldrich) and eosin Y 1% solution (Muto Pure Chemical Co., Ltd., Tokyo, Japan) for histological examination. The scoring system for each parameter denoted 0 as normal, 0.5 as focal and rare, 1 as focal and mild, 2 as diffuse and mild, 3 as diffuse and moderate, and 4 as diffuse and severe, in accordance with previously published GVHD histology (11). Enzyme-Linked Immunosorbent Assay (ELISA) for IL-10 and TGF-b IL-10 and TGF-b concentrations were measured by sandwich ELISA as follows. Anti-mouse IL-10 and TGF-b (R&D Systems, Minneapolis, MN, USA) were added to a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°C. The wells were blocked with blocking solution [PBS containing 1% bovine serum albumin (BSA; Gibco) and 0.05% Tween 20; Bio-Rad, Hercules, CA, USA] for 2 h at room temperature. The test samples and standard recombinant IL-10 and TGF-b (R&D Systems) were added to separate wells of the 96-well plate, and the plate was then incubated at room temperature for 2 h. The plate was washed, biotinylated IL-10 and TGF-b polyclonal antibody (R&D Systems) were added, and the reaction was allowed to proceed for 2 h at room temperature. The plate was washed, 2,000-fold diluted ExtrAvidinalkaline phosphatase (Sigma-Aldrich) was added, and the reaction was allowed to proceed for a further 2 h. The plate was then washed, and 50 ml of p-nitrophenyl phosphate disodium salt (Pierce Chemical Company, Rockford, IL, USA) diluted to 1 mg/ml in diethanolamine­ buffer­ ­(Sigma-Aldrich) was applied. Absorbance was measured

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at 405  nm on an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). Flow Cytometric Analysis Mononuclear cells were immunostained with various combinations of the following fluorescence-conjugated antibodies: CD25–allophycocyanine (APC) (eBioscience, San Diego, CA, USA), CD4–Peridinin Chlorophyll  Pro­ tein Complex (PerCP) (eBioscience), Foxp3–phycoerythrin (PE) (eBioscience), IFN-g–APC (eBioscience), IL-4-PE (BD PharMingen), IL-17–fluorescein isothiocyanate (FITC) (eBioscience), CD44–FITC (eBioscience), cytotoxic T-lymphocyte antigen-4-PE (CTLA-4, BioLegend), glucocorticoid-induced tumor necrosis factor receptor– FITC (GITR, eBioscience), CD103–PE (BioLegend), CD29–FITC (BioLegend), Sca-1–PE  (Ly-6A/E, Bio­ Legend), c-Kit–FITC (CD117, BioLegend), CD11b–APC (BD PharMingen), CD34–PE (BioLegend), major histocompatibility complex (MHC) I–FITC (H-2b, BD PharMingen), MHC I–PE (H-2d, BD PharMingen), MHC II–FITC (I-Ad, BD PharMingen), CD80–PerCP (B7-1, BD PharMingen), and CD86–APC (B7-2, BD PharMingen). Before intracellular cytokine staining, cells were stimulated in culture medium containing phorbol myristate acetate (25 ng/ml; Sigma-Aldrich), ionomycin (250 ng/ml; Sigma-Aldrich), or monensin (GolgiStop, 1 μl/ml; BD PharMingen) in an incubator with 5% CO2 at 37°C for 4 h. Intracellular staining was performed using an intra­ cellular staining kit (eBioscience) according to the manufacturer’s protocol. Flow cytometry was performed on a fluorescence-activated cell sorting (FACS) Calibur cyto­ meter (BD PharMingen) using FlowJo software (TreeStar, Ashland, OR, USA). Statistical Analysis All statistical tests, performed with the use of SAS version 9.2 (SAS Institute, Inc., Cary, NC), were twosided, and values of p