Enhanced alleviation of aGVHD by TGF‐β1‐modified mesenchymal stem cells in mice through shifting MΦ into M2 phenotype and promoting the differentiation of Treg cells

Abstract Allogeneic haematopoietic stem cell transplantation (allo‐HSCT) is the only curative method in treating haematologic malignant diseases. Graft‐versus‐host disease (GVHD) is a common complication post–allo‐HSCT, which can be life‐threatening. Mesenchymal stem cells (MSCs) as an adult stem cell with immunoregulatory function have demonstrated efficacy in steroid resistant acute GVHD (aGVHD). However, the outcome of aGVHD treated with MSCs in clinical trials varied and its underlying mechanism is still unclear. TGF‐β1 is a potent cytokine, which plays a key role in immunoregulation. In the present study, we firstly transduced the lentivirus vector containing TGF‐β1 gene with mouse bone marrow‐derived MSCs. Then, we investigated the immunosuppressive effect of TGF‐β1 gene‐modified MSCs on lymphocytes in vitro and its preventive and therapeutical effects on murine aGVHD model in vivo. Murine MSC was successfully isolated and identified. TGF‐β1 was efficiently transduced into mouse MSCs, and high level TGF‐β1 was detected. MSC‐TGF‐β1 shared the same morphology and immunotypic features of normal MSC. In vitro, MSC‐TGF‐β1 showed enhanced immunosuppressive function on lymphocyte proliferation. In vivo, MSC‐TGF‐β1 showed enhanced amelioration on the severity of aGVHD both in prophylactic and therapeutic murine models. Finally, the macrophages (MØs) derived from MSC‐TGF‐β1–treated mice showed a remarkably increasing of anti‐inflammatory M2‐like phenotype. Furthermore, the differentiation of CD4+ CD25+ Foxp3+ Treg cells was significantly increased in MSC‐TGF‐β1–treated group. Taken together, we proved that MSC‐TGF‐β1 showed enhanced alleviation of aGVHD severity in mice by skewing macrophages into a M2 like phenotype or increasing the proportion of Treg cells, which opens a new frontier in the treatment of aGVHD.


| BACKG ROU N D
Allogeneic haematopoietic cell transplantation (allo-HSCT) remains an effective option in treating malignant disease of the haematopoietic system. However, graft-versus-host disease (GVHD) frequently happens after allo-HSCT such that fatal GVHD offsets the benefit of allo-HSCT and hampers development of this treatment. 1,2 Classically, three stages are involved in the development of aGVHD: firstly, tissue damage from conditioning regimen mediates the activation of antigen-presenting cells (APCs); secondly, donor T lymphocytes are then activated by recipient antigens presented by host APCs; thirdly, donor T lymphocytes attack targets tissues and cause damage. 3 aGVHD that does not respond to first-line corticosteroid therapy is associated with a high mortality rate of 90%. 4 Mesenchymal stem cells (MSC) isolated from bone marrow were firstly described by Friedenstein 5 as spindle-shaped, fibroblast-like cells with the potency of differentiating into bone and cartilage in vitro. Based on its capacity of self-renewal and differentiation into tissues including bone, cartilage and adipose, MSC has been widely used in tissue engineering and repair. [6][7][8] MSC can also regulate immunity both by secreting soluble factors and by influencing the biology of immune cells. It is particularly important that MSC expresses few HLA class I and no HLA class II molecules, allowing them to evade allogeneic immune response. This is the so-called 'immunoprivilege', an interesting feature in MSC biology, which makes these cells extremely suitable for both autologous and allogeneic transplantation. 9 Owing to these multiple characteristics, MSC has been extensively researched and clinically applied as second-line therapy for aGVHD. 10,11 From the first study by Le Blanc et al 12 who successfully adopted MSC in the treatment of aGVHD in 2004, the application of MSC in aGVHD has made considerable progress in pre-clinical and clinical research. [13][14][15] However, there are great discrepancies amongst different groups, which could be attributed to the highly variable features of MSC due to the different tissue derivations, culture/experimental conditions and the number of passages of MSC. 13,16,17 As MSC alone is suboptimal for treatment of aGVHD, 18 there is a compelling clinical need for novel approaches to enhance its therapeutic and immunosuppressive property. One rational approach is to combine cell and gene therapy to achieve a greater immunoregulatory effect, by genetically modifying MSC to enhance its activity against aGVHD. 19 The TGF-β family of cytokines is pleiotropic cytokines that play an important role in regulating immune responses. 20 TGF-β1 is the commonest and most studied amongst the three isoforms of TGF-β (β1, β2, β3). As a well-characterized immunosuppressive molecule, it can down-regulate multiple immune responses and participate in the pathological process of immune disorders. 21 TGF-β1 can be secreted by MSC and plays a non-redundant role in the immunomodulatory function of MSC. 22,23 Sławomira KyrczKrzemień showed that low level of TGF-β1 probably being one of the factors contributing to the development of acute GVHD. On the other hand, chronic GVHD symptoms seem to correlate with high TGF-β1 mRNA expression and its serum concentration in patients who underwent bone marrow transplantation for myeloid leukaemia. 24 Taken together, these reports indicate that both MSC and TGF-β1 are potentially active against aGVHD. In this report, we put forward the hypothesis to apply TGF-β1 gene-modified MSC in treating aGVHD for the first time. Firstly, we transduced TGF-β1 into mouse bone marrow-derived MSC (MSC-TGF-β1), checked expression and production level of TGF-β1, and characterized the immunophenotypic profile of MSC-TGF-β1. Secondly, we investigated its inhibitory function on T lymphocyte proliferation in vitro, as T lymphocytes are the main mediators of aGVHD. Thirdly, we examined the in vivo prophylactic and therapeutic efficacy of MSC-TGF-β1 on aGVHD with a mouse model. Finally, we studied the possible underlying mechanism of MSC-TGF-β1 in murine aGVHD. Based upon our comprehensive work, we paved a new way to better understand the characteristics of TGF-β1 gene-modified MSC and their role in treating aGVHD.

| Mice
Female BALB/c (BALB/c, H-2d) mice and male C57BL/6 (B6, H2b) mice aged between 6 and 8 weeks were purchased from Slac Laboratory Animal Co, Ltd. All mice were housed under specific pathogen-free conditions in the animal laboratory centre of Xinhua Hospital.

| Isolation and culture of mouse MSCs
C57BL/6 mice were killed, and the marrow from femurs and tibias was extracted by repeatedly flushing with DMEM complete medium. The washing fluid was filtered through a 40-μm strainer and centrifuged; the cells in the pellet were plated into culture flasks in low-glucose DMEM medium supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 U/mL) and incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 24 hours. After removal of non-adherent cells, the cells remaining were cultured in fresh medium for another 48 hours as the first passage of MSC. Finally, MSC was passaged weekly and used for research after the third passage and before the 10th passage.

| Differentiation of MSCs to adipocytes, osteoblasts and chondrocytes
Mesenchymal stem cells were cultured in completed medium supplemented with certain ingredients for 14-21 days. Osteogenic (glutamine, ascorbate and β-glycerophosphate), chondrogenic (dexamethasone, ascorbate, ITS + Supplement, sodium pyruvate, proline) and adipogenic (glutamine, insulin, IBMX, rosiglitazone) ingredients were added to the culture medium according to the manufacturer's instructions (Cyagen). The differentiated MSCs were fixed with 4% paraformaldehyde and stained with Alizarin red, alcian blue and oil red O, respectively, at proper times.

| MSC transduction
Mesenchymal stem cell was seeded into 6-well plates at a density of 2 × 10 5 per well and transduced with LV-GFP-puro or LV-TGF-β1 at a multiplicity of infection (MOI) of 10 (the viral particles were previously quantified as 2 × 10 8 CFU/mL) and cultured in 2 mL of DMEM complete medium supplemented with 5 μg/mL polybrene. After 24 hours of incubation at 37°C in a humid atmosphere contain 5% CO 2 , the media was changed into fresh complete medium with 15 μg/mL puromycin.
The transduction efficiency was assessed 2 days later through observing under inverted microscope and quantifying by flow cytometry.  Table 1. The monoclonal antibodies used above were either rat antimouse or rabbit antimouse. BD FACSCanto-II (BD Bioscience) was used for acquisition, and the FlowJo software was used to analyse the outcome.

| Real-time PCR
The RNA level expression of TGF-β1 was examined by real-time PCR.
Firstly, total RNA was extracted using TRIzol (Takara). The extracted RNA with the OD260/OD280 nm absorption ratio range from 1.8 to 2.0 was reverse transcribed into cDNA using the PrimeScript RT Master Mix kit (Takara). The transcribed cDNA was used as template for real-time quantitative PCR on a ViiA™7 Real-Time PCR System (Applied Biosystems). Primers were obtained from Sangon Biotech.

| Western blot
Western blot assays were carried out to detect the translation effi-

| Co-culture experiments
Mesenchymal stem cell, MSC-GFP-puro and MSC-TGF-β1 were seeded into 24-well plates (Corning) with a density of 5 × 10 4 / well and incubated in 0.5 mL complete RPMI 1640 medium overnight. Splenocytes were extracted from Balb/c mice (6w). Mouse 1× Lymphocyte Separation Medium (Dakewe) was used to obtain splenic lymphocytes according to the manufacturers' instructions.
The separated lymphocytes were stained with 1 mmol/L of carboxyfluorescein diacetate succinimidyl ester (CFSE, eBioscience) before co-culture and then seeded into the well with MSC, MSC-GFP-puro and MSC-TGF-β1 at a density of 5 × 10 5 /well. The previous medium was changed into 0.5 mL fresh RPMI 1640 culture medium containing 10% FBS, 2 mmol/L glutamine, 100 U/mL penicillin and streptomycin, and 2 μg/mL ConA (Sigma). Lymphocytes labelled with CFSE cultured alone with or without ConA served as controls.

| T lymphocytes proliferation assay
After the cells were co-cultured for 3 days, suspension cells were harvested and labelled with anti-CD3-PerCP-Cy5.5 (eBioscience) as a maker of T cells. The proliferation of CD3 + T cells was analysed by visualizing CFSE fluorescence with a BD FACSCanto-II flow cytometer.

| ELISA
The level of IFN-γ in the supernatant or in the serum was measured by IFN-γ Quantikine ELISA Kit (eBioscience) according to the manufactures' protocols.

| Mouse aGVHD model
The protocol of establishing mouse aGVHD model was according to previously published method. 25

| Clinical and pathologic scoring of aGVHD
Clinical features of aGVHD were monitored every 4 days from day 1 to day 29 post-transplantation based on five separate parameters: weight loss, posture, activity, fur texture and skin integrity, and the detailed description of the scoring system are shown

TA B L E 2 Assessment of clinical GVHD in transplanted animals
in Table 2. 26 Three weeks after transplantation, one mouse from each group on same day 21 after transplant was randomly killed, and histological tissue samples from aGVHD target organs (liver, lung, small intestine and skin) were assessed. The tissues were fixed in 10% formalin, embedded in paraffin, stained with haematoxylin and eosin (H&E) and scored for aGVHD by a pathologist blindly.

| Extraction and analysis of macrophages
We isolated macrophages from the mice by peritoneal lavage with DMEM medium. Briefly, 5 mL of pre-cooled DMEM medium was injected into the peritoneal cavity of mice, and the abdomen of mice was gently massaged for 2-3 minutes. Peritoneal fluid was collected with a syringe avoiding damage to the blood vessels or organs to F I G U R E 1 Mouse bone marrow mesenchymal stem cells (MSC) were isolated successfully. A, Mouse bone marrow-derived MSCs showed morphology consistent with typical MSC (i) and could successfully differentiate to osteoblasts (ii), chondrocytes (iii) and adipocytes (iv). B, Surface markers of MSC, MSC-GFP-puro and MSC-TGF-β1 were analysed by flow cytometry. C, MSC-TGF-β1 morphology was similar to normal MSC (i) and could differentiate to osteoblasts (ii), chondrocytes (iii) and adipocytes (iv). Data are representative of three independent experiments for A-C ensure the absence of contamination from these sources. The procedures above were repeated twice to obtain as many cells as possible.
Finally, the harvest cells were analysed by flow cytometry. Antibody information is listed in Table 1.

| Immunofluorescence staining
Liver and lung tissue specimens were immediately obtained when the mice were killed at day 21 and subsequently fixed in 4% paraformaldehyde for 24 hours at 4°C. Samples were embedded in optimal cutting temperature compound and then sectioned. The Not less than three fields at ×100 magnification per section were randomly selected as representatives for analysis and calculation.

| Statistical analysis
All statistical analyses were performed using SPSS 20.0 (SPSS Inc, Chicago, IL, USA), and graphs were generated using the GraphPad Kiel, Germany) with a α-value of .05, a power of 80%. All the Pvalues are two-sided, and P < .05 is taken as statistically significant.

| Characterization of mice bone marrow-derived MSC
Under inverted light microscopy, the cultured normal bone marrow-derived murine MSCs were all plastic adherent long and MSC-TGF-β1 also could differentiate into osteogenic ( Figure 1C,ii), chondrogenic ( Figure 1C,iii) and adipogenic ( Figure 1C,iv) lineages.
Finally, as normal MSC, MSC-GFP-puro and MSC-TGF-β1 were all plastic adherent long or polygonal cells as showed in Figure 2A. This laid a solid foundation for our following research.

| TGF-β1 successfully transduced MSC resulting in high expression
As ( Figure 2C). Furthermore, we tested TGF-β1 production in the supernatant of cultured cells and found that the MSC-TGF-β1 group was the highest ( Figure 2D). Taken together, target gene TGF-β1 was successfully transduced and ectopically overexpressed in MSC-TGF-β1.

| TGF-β1-transduced MSC manifest enhanced immunosuppressive capacity on T lymphocyte in vitro
To investigate whether MSC-TGF-β1 possess enhanced suppressive function on T lymphocytes, we firstly isolated normal lymphocytes from the spleen of Balb/c mice, and then, we co-cultured MSC/MSC-GFP-puro/MSC-TGF-β1 (5 × 10 4 ), respectively, with the isolated lymphocytes (5 × 10 5 ) which were pre-labelled with CFSE at the ratio of 1:10. The co-culture system was supplemented with additional ConA, which acts as immunostimulant for mice lymphocytes. Cells were incubated at 37°C in a humid atmosphere of 5% In addition, we found that IFN-γ secretion was markedly decreased in the culture medium of MSC-TGF-β1 group by ELISA method ( Figure 3E), which were supported by detecting the RNA quantity in the cultured lymphocytes using real-time PCR ( Figure 3F).

| Prophylactic application of TGF-β1transduced MSC markedly ameliorated the severity of aGVHD and improved overall survival in murine model
Mice

| MSC-TGF-β1 promoted the M2 macrophage polarization in aGVHD mice
To clarify its underlying mechanism of how MSC-TGF-β1 ameliorates the severity of aGVHD and improves survival of murine model, we

| MSC-TGF-β1 increased the proportion of Treg cells in peripheral blood of aGVHD mice
To further investigate other possible mechanism of MSC-TGF-β1 exert effect on murine aGVHD model, we analysed the mononuclear cells from blood of mice at day 21 for detection of CD4 + CD25 + FoxP3 + Treg cells by flow cytometry. We found that the proportion of Treg cells in MSC-TGF-β1-infused mice was much higher than other groups, with the proportion 1.95% for LC group, 3.36% for MSC group, 4.83% for MSC-GFP-puro group and 7.75% for MSC-TGF-β1 group, respectively ( Figure 7A,B). Mean value ± SD of Treg proportion in Figure 7B for BMC+SC, BMC+SC+MSC, BMC+SC+MSC-GFP-puro and BMC+SC+MSC-TGF-β1 groups was This indicated that TGF-β1-transduced MSC ameliorated the severity of aGVHD partly through its effect on Treg cells.

| Therapeutic application of TGF-β1-transduced MSC alleviated the clinical symptoms and improved overall survival in murine model of aGVHD
In order to observe the therapeutic effect of MSC-TGF-β1 on murine aGVHD model, mice were treated with weekly injection of MSC-GFP-puro (3 × 10 5 ) or MSC-TGF-β1 (3 × 10 5 ) after allo-HSCT for consecutive 5 doses. As showed in Figure 8A, compared with the non-treatment control, the median survival of mice in MSC-GFPpuro group was 34 days. Particularly, the MSC-TGF-β1 (3 × 10 5 )treated group showed the best outcome with sixty per cent of mice achieved long-term survival (more than 50 days post-transplantation). Notably, the survival outcome of MSC-TGF-β1 therapeutic group was even superior to that of MSC-TGF-β1 prophylactic group mentioned in Figure 4A. Correspondingly, the clinical symptoms and aGVHD score in MSC-TGF-β1 group were the slightest ( Figure 8B,C). Mean value ± SD of clinical score in Figure 8B for BMC+SC, BMC+SC+MSC-GFP-puro and BMC+SC+MSC-TGF-β1 groups was 28 ± 3.74, 15.4 ± 3.78 and 9 ± 1.58, respectively.

| D ISCUSS I ON
Coupling its immunomodulatory property with its ability of homing to damaged tissue to regulate inflammation and promote repair, MSC-based treatment shows great promise in aGVHD.
While several clinical trials have demonstrated efficacy of MSC in steroid-refractory aGVHD, 13,17,29 there are studies reporting to the contrary. 16 The heterogeneity of the MSC population may be one reason for the inconsistent outcome, 30 which underscores the importance of maintaining and further enhancing its immunoregulatory activity.
In the present study, we aimed to improve MSC-based immu- influencing the biology of immune cells. 9 As a well-characterized immunosuppressive factor, TGF-β1 suppresses immune response either by inhibiting the function of immune cells or by altering their differentiation. 20,21,31 It was documented that TGF-β1 plays a crucial part in the immunoregulatory function of MSC. 32,33 In our study, TGF-β1 gene-modified MSC exerted powerful immunosuppression, which could be attributed to the synergistic effect of these two factors, consistent with a previous study. 34 Another study held that TGF-β1 abolished MSC-mediated immunosuppressive effect on anti-CD3-activated splenocytes, 35   inducing tolerance, facilitating regeneration and repair, and has been proven to be efficient and safe. 38,39 In this study, we proved that MSC-TGF-β1 was superior to MSC in delaying the onset of aGVHD and reducing the severity of aGVHD in mice, thereby offering a novel strategy for treating aGVHD. It must be mentioned here that mice in not only MSC and MSC-GFP-puro infused groups, but also MSC-TGF-β1-infused group succumbed to aGVHD eventually. It is reported that higher proportion of 'ready to differ- Depending on the microenvironment they are exposed to, macrophages exhibit versatility in phenotype and function. 42 43 Previous research showed that MØs play an important role in the pathogenesis of aGVHD through producing TNF-α after exposure to LPS, 44 and human skin lesions infiltrated by CD163+ macrophage were proposed as a predictive factor for refractory GVHD associated with poor overall survival. 45 In contrast, M2-like macrophages reduced the inflammatory response. 46 Considering that different subsets of macrophage play different roles in inflammation process, while GVHD is caused by donor T cell-mediated damage to recipient target organs either directly through cytolytic attack or indirectly through the release of inflammatory mediators. We postulated that macrophages might be involved in the underlying anti-GVHD mechanism of MSC-TGF-β1.
We found that macrophages derived from mice treated with MSC-TGF-β1 showed the highest expression of the M2 marker CD206, and a reduced expression of M1 marker iNOS. Three broad pathways control macrophage polarization: epigenetic and cell survival pathways that prolong or shorten macrophage development and viability, the tissue microenvironment, and extrinsic factors, such as microbial products and cytokines released in inflammation.
Mesenchymal stem cell has been described to shift macrophages to M2-like subsets, 47,48 and MSC-educated macrophages possess the therapeutic potential for GVHD. 48 Furthermore, as demonstrated previously, TGF-β1 can increase the differentiation of M2-like macrophages which function in wound healing and immunoregulation. 49,50 Taken together, these studies partially explain why infusion of MSC-TGF-β1 increased the M2-like macrophages in our murine aGVHD models. However, the precise underlying mechanism how M2 macrophage polarizated and functioned is quite sophisticated and still unknown in our current study, which need further illustration. MSC-TGF-β1 might promote the generation of M2-like macrophages either directly or through its effect on the immune environment. The increased M2-like macrophages can regulate immune response probably by secreting proteins, such as IL-10 and arginase 1 (Arg1). 51 We speculate that these biological behaviours of M2-like macrophages may play an important role in moderating the over-active immune response in aGVHD. Lastly, we also found that MSC-TGF-β1-treated mice showed an increased in the proportion of Treg as compared to other groups. As we know, TGF-β signalling plays a vital role in the development of natural CD4+CD25+Foxp3+ regulatory T cells. 52 The exact mechanism through which Tregs control immune responses has not been fully elucidated. Treg function appears to be cytokine or contact mediated. Several studies showed that IL-10, TGF-β and IL-35 have been implicated in enhancing suppression, whereas CTLA-4, LAG-3, CD39 and granzymes play an important role in the contact-dependent immune control. In a number of different allogeneic HCT animal models, the addition of highly purified CD4+CD25+FoxP3+ Tregs resulted in suppression of GVHD. 53 We hereby speculate plays an important role in immunosuppression procedure. 32,33 In our present study, these two mechanisms were integrated. Soluble factor TGF-β1 could directly inhibit T cell proliferation; on the other hand, macrophage polarization was also observed. This is well in agreement with studies that demonstrated that phagocytosis of MSC induces an immunosuppressive phenotype in monocytes. 55 In our present work, lentiviral vectors were used to delivery target TGF-β1 gene in MSC.
The risk of insertional mutagenesis associated with use of LV vectors should be concerned when it was implemented in clinical trials. In order to avoid such potential detriment, we want to adopt exosomes secreted by TGF-β1 gene-modified MSC instead of whole MSC infusion on our next research plan.

| CON CLUS IONS
In conclusion, we have demonstrated for the first time that TGF-β1 gene-modified MSC showed enhanced alleviation of aGVHD severity in mice by skewing macrophages into a M2 like phenotype or increasing the proportion of Treg cells, which offers a novel option for the prevention and treatment of aGVHD.

ACK N OWLED G EM ENTS
The authors would like to thank all members of the Department of Hematology, Xinhua Hospital, affiliated to Shanghai Jiao Tong University (SJTU) School of Medicine, for their support and to thank Dr Yeh-ching Linn from Singapore General Hospital for English language editing.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest to disclose.

AUTH O R S ' CO NTR I B UTI O N S
LM made substantial contributions to the design of the present study. RW performed the experiments, data collection and data analysis. RW and LM wrote the manuscript. CL, XD and LC provided help in conceiving and designing the study. SH made substantial contributions in data analysis. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article.