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

Abstract

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-2^d) mice were transplanted with bone marrow cells (BMCs) and spleen cells of C57BL/6 (H-2^b) 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-γ ⁺ 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.

Keywords

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 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-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), 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-α 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 blood-derived 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-γ 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-2^b) and female BALB/c (H-2^d) 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 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-β (TGF-β; 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 β-scintillation counter (Beckman, Fullerton, CA, USA).

Bone Marrow Transplantation and GVHD Induction

Recipient (BALB/c, H-2^d) mice were exposed to a 800-cGy 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-2^b). Control group was comprised of irradiated mice receiving 5 × 10⁶ T-cell-depleted (TCD) BMCs, which does not 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-β

IL-10 and TGF-β concentrations were measured by sandwich ELISA as follows. Anti-mouse IL-10 and TGF-β (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-β (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-β 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 ExtrAvidin-alkaline phosphatase (Sigma-Aldrich) was added, and the reaction was allowed to proceed for a further 2 h. The plate was then washed, and 50 μl 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 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 Protein Complex (PerCP) (eBioscience), Foxp3–phycoerythrin (PE) (eBioscience), IFN-γ–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, BioLegend), c-Kit–FITC (CD117, BioLegend), CD11b–APC (BD PharMingen), CD34–PE (BioLegend), major histocompatibility complex (MHC) I–FITC (H-2^b, BD PharMingen), MHC I–PE (H-2^d, BD PharMingen), MHC II–FITC (I-A^d, 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 intracellular staining kit (eBioscience) according to the manufacturer's protocol. Flow cytometry was performed on a fluorescence-activated cell sorting (FACS) Calibur cytometer (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 two-sided, and values of p < 0.05 were considered statistically significant. The comparisons between groups were analyzed statistically using the Kruskal–Wallis test. Pairwise group comparisons used the Mann–Whitney U test, and p values were adjusted for multiple comparisons using Bonferroni's method to determine the statistical significance of these comparisons. The experimental values are expressed as mean ± SEM, and so the data have not been represented nonparametrically.

Results

Phenotypes of Culture-Expanded MSCs and Treg Cells

For immunophenotypic characterization of culture-expanded BALB/c MSCs, surface protein expression was examined using flow cytometry at the 15th passage. Culture-expanded MSCs showed typical fibroblast-like morphology and were uniformly positive for Sca-1, CD44, CD29, and MHC I (H-2^d) but negative for cKit, CD11b, CD34, MHC II (I-A^d), CD80 (B7-1), and CD86 (B7-2) (Fig. 1A) (34). Retinal-induced BALB/c Treg cells expressed several regulatory T-cell phenotypic markers, including CTLA-4, CD103, GITR, and CD44 (Fig. 1B).

Figure 1.

Characterization of host-derived mesenchymal stem cells (MSCs) and regulatory T (Treg) cells. (A) MSCs were distinguished from hematopoietic cells by negative expression of the cell surface markers c-kit, cluster of differentiation 11b (CD11b), CD34, major histocompatibility complex (MHC) II (I-Ad), CD80, and CD86, but positive expression of stem cell antigen (Sca)-1, CD44, CD29, and MHC I (H-2d). (B) Treg cells were analyzed by fluorescence-activated cell sorting (FACS) for intracellular fork-head box P3 (Foxp3) and surface expression of the indicated cellular markers [CD103, glucocorticoid-induced tumor necrosis factor receptor (GITR), cytotoxic T-lymphocyte antigen-4 (CTLA-4) and CD44] in the gated T-cell populations. The reported percentages are those of double-positive cells. Results are representative of four independent experiments.

Effects of MSC and Treg Cell Coculture on the T-Cell Proliferative Response

To test the suppressive effects of MSCs and Treg cells on T-cell proliferation in vitro, we cultured C57BL/6 CD4⁺ T-cells in the presence of irradiated T-cell-depleted BALB/c splenocytes and MSCs with or without Treg cells by [³H]thymidine uptake. As expected, very high [³H]thymidine incorporation was observed in the allogeneic setting, but the addition of MSCs plus Treg cells resulted in a marked reduction of splenocyte proliferation compared with single cells. Even when injecting the same amount of MSCs (1 × 10⁴; n = 3), Treg cells (1 × 10⁴; n = 3), or MSCs plus Treg cells (5 × 10³ + 5 × 10³ respectively; n = 3), the MSC plus Treg group was more effective in its suppressive function. However, between Treg and MSC plus Treg group, effects were not statistically significant. Additionally, in the proliferation assay, the effect of Treg cells was dose dependent; increased Treg cells (5 × 10³, 1 × 10⁴) showed higher suppression efficiency, whereas the effect of MSCs (5 × 10³, 1 × 10⁴) was less dose dependent. Joo et al. reported that T-cell proliferation was inhibited in a dose-dependent manner until a 1:1 MSC/splenocyte ratio, and then there were no significant increases in T-cell suppression upon addition of further MSCs (17). Thus, we decided to use an MSC/Treg ratio of 1:2 based on our observation that increasing the amount of Treg cells rather than MSCs more strongly inhibited T-cells (Fig. 2). This ratio was used throughout in both in vivo and in vitro experiments. These data indicate that the MSC/Treg ratio is an important regulator of post-BMT splenic T-cell proliferation in the alloresponse.

Figure 2.

Effect of mesenchymal stem cells (MSCs) and regulatory T (Treg) cells on the proliferation of alloreactive T-cells. Effector T-cell suppression in response to MSCs and Treg cells was measured using [³H]thymidine incorporation in spleen cells isolated from C57BL/6 mice. Briefly, CD4⁺ T-cells were stimulated with T-cell-depleted irradiated BALB/c splenocytes in the presence of MSCs with or without Treg cells. The cells were cultured for 3 days, with the addition of [³H]thymidine for the final 8 h before harvesting. The incorporation of [³H]thymidine into CD4⁺ T-cells was determined using a liquid β-scintillation counter (cpm = counts per minute). Control T-cells were those stimulated with irradiated T-cell-depleted BALB/c splenocytes. Data are shown as the mean ± SEM; results are representative of four independent experiments. *p < 0.05; **p < 0.01.

The Combination of Recipient-Derived MSCs Plus Treg Cells Results in Expansion of Donor-Type Foxp3⁺ Treg Cells in the Alloresponse

To confirm the immunoregulatory functions of MSCs and Treg cells associated with helper T-cells, we analyzed cytokine expression by flow cytometry. We determined whether coculture of recipient-derived MSCs plus Treg cells increase donor-derived CD4⁺CD25⁺Foxp3⁺ T-cells in the alloresponse. To predict the increase in Foxp3 expression of the MSC and Treg combined group compared with each single group in GVHD mouse models, we combined MSCs and Treg cells during an in vitro alloresponse to detect Foxp3 expression levels. When we analyzed the FACS results, instead of detecting donor-derived Treg cells, we first gated H-2^b and then examined the change in CD4⁺CD25⁺Foxp3⁺ Tregs to determine the change solely in the donor cells (H-2^b). The results showed that treatment with combined MSCs plus Treg cells resulted in a higher increase in Foxp3 expression levels (Fig. 3A). The bars show that the percentages of donor-derived CD4⁺CD25⁺Foxp3⁺ Treg cells in each group were assessed by FACS (Fig. 3B). Our results demonstrated that the combination of MSCs plus Treg cells displayed protective effects against aGVHD-related morbidity and mortality by increasing Foxp3⁺ T-cell expansion in vitro.

Figure 3.

Induction of Foxp3⁺ regulatory T (Treg) cells and changes in immunosuppressive cytokine production by donor splenic T-cells stimulated in vitro with host spleen cells following coculture with MSCs and Treg cells. The responder C57BL/6 splenocytes (1 × 10⁶) and stimulated BALB/c splenocytes (1 × 10⁶) were cocultured with 5 × 10⁴ of irradiated (2,000 rad) MSCs with (1 × 10⁵) or without (1 × 10⁵) irradiated Treg cells. Cells were collected for intracellular cytokine assays after 5 days. (A, B) Treg cells were identified by their expression of CD4, CD25, and intracellular Foxp3 in splenocytes using fluorescence-activated cell sorting (FACS). (C, D) The supernatant levels of interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) in alloresponse cultures after differentiation for 3 days were measured by enzyme-linked immunosorbent assay (ELISA). The purity of all cell subsets was >95% as determined by fluorescence-activated cell sorting (FACS) analysis. Data are shown as the mean ± SEM; results are representative of four independent experiments. *p < 0.05; **p < 0.01.

Effects of MSC and Treg Cell Coculture on Cytokine Production

To confirm the immunoregulatory functions of MSCs and Treg cells associated with helper T-cells, we measured cytokine expression levels by ELISA. The coculture of MSCs plus Treg cells group and MSC group exhibited markedly increased proportions of IL-10 (Fig. 3C) and TGF-β (Fig. 3D) in culture supernatants in the alloresponse. Another research group demonstrated that MSCs exhibited immunosuppressive functions through the secretion of TGF-β and induction of IL-10 (21). This indicates that Treg cells did not increase IL-10 and TGF-β secretion of soluble molecules by MSCs during the alloresponse. However, the elevated levels of IL-10 and TGF-β were observed only in cultures containing MSCs. Therefore, we suggest that the elevated levels of IL-10 and TGF-β were produced by MSCs and not by lymphocytes. Hence, cellular interactions, as opposed to the secretion of soluble factors, regulate the immunomodulatory effects of MSCs and Treg cells in coculture.

Long-Term Immunosuppressant Therapy and GVHD Treatment with a Combination of MSCs Plus Treg Cells

We established a model of aGVHD following fully MHC-mismatched HSCT to assess the effects of MSCs and Treg cells on GVHD, engraftment, and immune reconstitution. In this model, lethally irradiated BALB/c (H-2^d) recipients received BMCs and spleen cells from C57BL/6 (H-2^b) donor mice. Following transplantation, recipients were injected with a combination of MSCs with or without Treg cells twice a week. Initial studies of MSCs in the murine aGVHD model demonstrated that a single infusion of MSCs at the time of hematopoietic stem cell transplantation (HSCT) had no effect on the prevention of aGVHD (36). We also could not prevent GVHD by a single administration of MSCs (data not shown). Using this protocol, the MSC plus Treg group survived significantly longer than the other groups, and 80% survived beyond 60 days (Fig. 4A). Recipient mice were monitored for GVHD clinical scores and weight. The coinfusion of MSCs and Treg cells significantly reduced GVHD scores (Fig. 4B) and increased weight (Fig. 4C). The results showed that coinfusion of MSCs and Treg cells could prevent GVHD lethality in recipients and significantly reduced weight loss more effectively than either the MSC or Treg single groups. We concluded that MSC plus Treg cell therapy contributed to the T-cell-induced cooperative effects on alleviation of GVHD.

Figure 4.

Clinicopathological improvement in acute graft-versus-host disease (GVHD) following combination cell therapy with mesenchymal stem cells (MSCs) and regulatory T (Treg) cells. Following transplantation, recipients were administered host bone marrow-derived MSCs plus Treg cells (n = 6), MSCs alone (n = 6), or Treg cells alone (n = 6) by IV injection on days 0, 4. (A) Increase in the survival rate of GVHD mice after MSCs and Treg cells in allogeneic transplantation. All animals were monitored for (B) clinical signs and (C) mean serial weight measurements. Effects were accentuated with MSC plus Treg cell therapy. Improvements in the clinical GVHD scores after MSC and Treg cell transplantation were evaluated by weight loss, posture, activity, fur texture, and skin integrity. Improvement in the histologic GVHD scores after coinfusion of MSCs and Treg cells in allogeneic transplantation. (D) Histological GVHD scores were evaluated in GVHD, MSC, Treg cell, and MSC plus Treg cell mice from the skin, small intestine, large intestine, and liver tissues at 12 days after transplantation, with individual criteria for each specific tissue. Coinfusion of MSCs plus Treg cells significantly reduced the total GVHD histological scores compared with GVHD (control) group. TCD-BM = T-cell-depleted bone marrow cells (negative control). p Values compared to GVHD (control) group. Data are shown as the mean ± SEM; results are representative of three independent experiments. *p < 0.05; **p < 0.01.

Clinicopathological Improvement of GVHD

The combination therapy of MSCs and Treg cells inhibited the expansion of alloreactive CD4⁺ T-cells in vivo and protected against GVHD-related tissue damage to a greater extent than either the MSC or Treg single groups. Histological analysis of the skin, liver, and large intestine from the GVHD control group mice showed scattered slight vacuolation of individual epidermal cells (Fig. 4D). The MSC plus Treg group revealed a remarkable reduction in histology. GVHD histological grading of the large intestine showed effacement and blunting of the villous architecture, mucous cell depletion, and sloughing of epithelial cells, with patchy mucosal ulceration. However, the differences in large intestine histology between the MSC group and the MSC plus Treg group were unremarkable. The liver tissue in the MSC plus Treg group did not show lobular inflammation, but many lymphocytes were seen infiltrating the liver cell plates. These findings were improved by coinfusion of MSCs and Treg cells compared with the MSC or Treg single groups (Fig. 4D).

Coinfusion of MSCs and Treg Cells Modulate Th1, Th2, Th17, and Treg Differentiation in the GVHD Model

The balance among Th1, Th2, and Th17 cells plays an important role in regulating T-cell immune responses and causes organ-specific tissue damage (44). To address the roles of MSCs and Treg cells in the development of GVHD, we first examined the effects of coinfusing MSCs in the presence or absence of Treg cells after myoablative BMT in mice. We investigated changes in cytokine secretion profiles by these cells to understand the mechanisms involved in the observed MSC plus Treg group T-cell-mediated immunosuppressive effects. The percentage of cells of donor origin in recipient mice in each group was full chimerism of 100% in peripheral blood 12 days after irradiation at a dose of 800 cGy and injection of both BMCs and splenocytes from donor mice. Expression levels of donor-type IL-4 (Fig. 5A) and Foxp3 (Fig. 5B) were higher in recipients of MSCs plus Treg cells than in recipients of either MSCs or Treg cells alone. In contrast, spleen cells from the MSC plus Treg cell group showed decreased expression levels of donor-type IFN-γ (Fig. 5A) and IL-17 (Fig. 5B). However, each group was not statistically significant in IL-17 cells. These results indicate that donor CD4⁺ T-cells can reciprocally differentiate into Th1, Th2, Th17, and Treg cells that mediate organ-specific aGVHD. Th1 and Th17 cells in murine GVHD were associated with evidence of severe GVHD, and in situ quantification of the Th1/Th2 and Treg/Th17 ratio was a specific marker for GVHD in a murine model. The Th1/Th2 cell ratio was significantly lower in spleens of the MSC plus Treg-treated group compared with that in each single group in the mouse model of GVHD (Fig. 5C). Inhibition of IL-17 in the MSC plus Treg group was associated with increased donor-type Foxp3 expression, suggesting that coinfusion cellular therapy with MSCs plus Treg cells reciprocally affects both donor-type Treg cell induction and Th17 differentiation (Fig. 5D). Taken together, these data might explain why combination cell therapy with MSCs and Treg cells synergistically inhibits aGVHD in allo-BMT.

Figure 5.

Immunological effects of combination cell therapy with mesenchymal stem cells (MSCs) and regulatory T (Treg) cells in graft-versus-host disease (GVHD) induction. The combination of MSCs and Treg cells results in a significant reduction in Th1 and Th17 cells, but enhancement in Th2 cells and Treg cells. Coinfusion of MSCs and Treg cells led to a greater increase in (A) interleukin 4 (IL-4) and (B) Foxp3 levels and a greater decrease in interferon-γ (IFN-γ) levels than treatment with MSCs or Treg cells alone in a murine model of GVHD. The degree of IL-17 inhibition in the Treg cell single group or MSC plus Treg cell combination group was higher than that in the MSC single group. However, statistical analysis was not significantly different. (C) Data are presented as the ratio of Th1/Th2 among CD4⁺ T-cells, which was calculated as the ratio of IL-4⁺ CD4⁺ T-cells divided by the percentage of IFN-γ⁺ CD4⁺ T-cells. (D) Data are presented as the ratio of Treg/Th17 among CD4⁺ T-cells, which was calculated as the ratio of IL-17⁺ CD4⁺ T-cells divided by the percentage of Foxp3⁺ CD4⁺ T-cells. The purity of all cell subsets was >95% as determined by fluorescence-activated cell sorting (FACS) analysis. Data are shown as the mean ± SEM; results are representative of four independent experiments. *p < 0.05; **p < 0.01.

Discussion

Recently, MSCs have been actively used to treat GVHD, chronic inflammatory disease, and autoimmune diseases. However, the therapeutic effects of MSCs need to be clearly demonstrated in basic research and clinical trials. Considerable progress has been made in understanding the mechanisms by which MSCs exert immunomodulatory functions. MSCs are not constitutively inhibitory; they acquire their immunosuppressive functions after being exposed to the inflammatory environment mainly provided by IFN-γ (25). That is, the immunological effects of MSCs may be determined by cytokines released from the main subtype of helper T lymphocytes during inflammation. Therefore, we assumed that the Th17-centered inflammatory response associated with organ injuries could not be regulated by MSC treatment alone. Alternatively, the combination of MSCs and Treg cells could play a role in enhancing the immunomodulatory capacity of MSCs based on the following rationale. Although GVHD has been classically assumed to be a Th1-mediated response based on findings from animal models (14,37), a recent report demonstrated that in vitro-differentiated Th17 cells mediate lethal aGVHD. Further, combined targeted disruption of the transcription factors T-box expressed in T-cells (T-bet/TBX21), and retinoic acid receptor-related orphan receptor γ (RORγt) resulted in defective differentiation of donor CD4⁺ T-cells toward Th1 and Th17 and ameliorated GVHD after allogeneic BMT (5,28,38,45). Systemic infusion of MSCs alone did not suppress the development of autoimmune arthritis and joint inflammation in autoimmune diseases, such as arthritis, which could be mainly induced by Th17 responses despite the contribution of the Th1 response (26,29,36). In contrast, TGF-β-transduced MSCs suppressed development of autoimmune arthritis and joint inflammation by inducing an endogenous increase in Treg cells and inhibiting the Th17 cell response. These data suggest that the immunomodulatory capacity of MSCs might be effective in controlling Th1 response-mediated disease, but not Th17 response-mediated diseases, such as autoimmune arthritis and certain conditions of GVHD. Therefore, a regulation strategy for the pathologically elevated Th17 response may be required to treat these intractable inflammatory conditions. MSCs demonstrate partial therapeutic effects in autoimmune diseases. Many of these experiments have been performed in mice with endogenous Treg cells present. Although MSCs show therapeutic effects in established GVHD, the effects are unclear when coinfused as a preventative measure at the time of BMT. This is because a temporal gap exists until the endogenous donor-derived Treg cells are induced after the myeloablative-conditioning regimen. In addition, the immunomodulatory capacity of MSCs has not been proven to be effective for preventing GVHD in clinical trials or preclinical models; however, MSC therapies have shown promising results in established GVHD.

Recent reports have demonstrated that the addition of Treg cells may improve GVHD in basic research and clinical trials. However, due to their plasticity, Treg cells may lose their suppressive capability and even differentiate into pathogenic T-cells, particularly in inflammatory environments (18,33,43). To maintain stable activation of Treg cells, a large amount or a high purity of Treg cells must be administered. It is difficult to increase the purity of exogenous CD4⁺CD25⁺Foxp3⁺ Treg cells. Additionally, induced Treg cells can convert to effector T-cells. In the present study, we used recipient-derived CD4⁺CD25⁺ Treg cells to suppress GVHD. The myeloablative conditioning regimen induces tissue damage and proinflammatory conditions, and the presence of IL-6 under such proinflammatory conditions results in conversion of natual (n)Treg to Th17 cells. However, iTregs are resistant to this cytokine and therefore might retain their suppressive function at GVHD sites (15). Therefore, we used nonspecifically induced Treg cells that were generated using our protocols. Recent studies have shown that recipient-derived Treg cells initially occupy a niche in transplantation recipients, undergo significant expansion, and contribute to the T-cell compartment for an extended period before donor-derived CD4⁺Foxp3⁺ T-cells eventually predominate (3).

The immunological effects of MSCs depend on the balance between cytokines released by helper T lymphocytes during inflammation (25). In the present study, we assumed that our MSCs could mainly control the Th1 response, but not the Th17 response that was regulated by Treg cells or Treg cells plus MSCs. We previously evaluated CIA in IFN-γ-knockout mice, which have a high IL-17 and low IFN-γ cytokine profile (46). This model allows for clear observations of the results of Treg therapy. IFN-γ-knockout CIA mice, which were adoptively transferred with Treg cells, showed less inflammation and reduced IL-17 production compared with CIA control mice (16). The Th17 response is unlikely to be regulated by MSCs alone. Thus, these must act in combination with an additional factor. Absence of IFN-γ in donor CD4⁺ T-cells resulted in augmented Th17 differentiation and exacerbated Th17-cell-mediated GVHD (44). Thus, administration of CD4⁺CD25⁺ Treg cells may be helpful, as GVHD is an immune response mediated by both Th1 and Th17.

The present study provides evidence that MSCs and Treg cells do not impair each other's respective functions (8); therefore, the use of MSC plus Treg cellular therapy is possible for the prevention of GVHD after allo-BMT. The therapeutic effects of MSCs result from soluble factors and cell-to-cell contact. The molecules produced by MSCs can promote the induction of Treg differentiation and, in turn, the cytokines produced by these Treg cells can promote activation of MSCs. The results showed that the combined MSC plus Treg group displayed a greater increase in Foxp3 levels compared with each single group (Fig. 3A, B). Conversely, we observed that Treg cells did not increase the secretion of IL-10 and TGF-β by MSCs during the alloresponse (Fig. 3C, D). Therefore, combined MSC- and Treg-mediated immunomodulatory functions depend primarily of cellular interactions rather than soluble factors.

The combination MSC plus Treg cell therapy group most effectively decreased IFN-γ-producing Th1 cells and IL-17-producing Th17 cells compared with the MSC or Treg single group. Additionally, the combination therapy group most effectively increased IL-4-producing Th2 cells and Treg cells for tolerance induction in vivo compared with the MSC or Treg cell group. Statistical analysis was not significantly different in IL-17 cells. However, the degree of IL-17 inhibition in the Treg cell single group or MSC plus Treg cell combination group was higher than that in the MSC single group. This suggests that Treg cells contribute principally to the regulation of Th17 responses, and that the combination of MSCs and Treg cells has a cooperative effect in prevention of aGVHD during the early posttransplant period. It has been reported recently that Th17 plays less of a role than Th1, but synergizes with Th1 during GVHD induction (38,45). The induction of Treg cells, specifically the induction of Foxp3, was noticeably higher in the MSC plus Treg cell group compared with the Treg cell or MSC groups. This suggests that the increase in CD4⁺Foxp3⁺ Treg cells was not only due to the administration of Treg cells themselves but also to the induction of additional endogenous Treg cells and prevention of negative conversion by MSCs. Coculture of MSCs and Treg cells markedly increased IL-10 (Fig. 3C) and TGF-β (Fig. 3D) secretion compared with Treg cells alone in splenocyte culture supernatants during the alloresponse. However, it is likely that the elevated levels of IL-10 and TGF-β were produced by MSCs and not by lymphocytes. These observations suggest that both donor- and recipient-derived MSCs function synergistically in prevention of GVHD. Further studies are necessary to determine the effects of recipient-derived MSCs in allogeneic transplantation.

In summary, the combination cell therapy of MSCs and Treg cells induced long-term survival and significantly reduced clinicopathological manifestations in an aGVHD model. It is expected that coadministration of MSCs and Treg cells will improve the shortcomings of MSC or Treg cell therapy alone. Thus, combined cell therapy is a novel therapeutic approach for preventing aGVHD. We conclude that combination therapy with MSCs and Treg cells contributes to the alleviation of GVHD induced by T-cells.

Footnotes

Acknowledgments

This study was supported by a grant from the Korean Health Technology R&D Project, Ministry for Health & Welfare, Republic of Korea (A092258). The authors declare no conflicts of interest.

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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

Abstract

Keywords

Introduction

Materials and Methods

Mice

Isolation and Culture of MSCs

Treg Generation

Mixed Lymphocyte Culture (MLC)

Proliferation Assay

Bone Marrow Transplantation and GVHD Induction

Combination Cell Therapy of MSCs and Treg Cells Control GVHD

Clinicopathological Evaluation

Enzyme-Linked Immunosorbent Assay (ELISA) for IL-10 and TGF-β

Flow Cytometric Analysis

Statistical Analysis

Results

Phenotypes of Culture-Expanded MSCs and Treg Cells

Effects of MSC and Treg Cell Coculture on the T-Cell Proliferative Response

The Combination of Recipient-Derived MSCs Plus Treg Cells Results in Expansion of Donor-Type Foxp3+ Treg Cells in the Alloresponse

Effects of MSC and Treg Cell Coculture on Cytokine Production

Long-Term Immunosuppressant Therapy and GVHD Treatment with a Combination of MSCs Plus Treg Cells

Clinicopathological Improvement of GVHD

Coinfusion of MSCs and Treg Cells Modulate Th1, Th2, Th17, and Treg Differentiation in the GVHD Model

Discussion

Footnotes

Acknowledgments

References

The Combination of Recipient-Derived MSCs Plus Treg Cells Results in Expansion of Donor-Type Foxp3⁺ Treg Cells in the Alloresponse