Efficacy of Bone Marrow-Derived Mesenchymal Stem Cells in ... - Core

Efficacy of Bone Marrow-Derived Mesenchymal Stem Cells in the Treatment of Sclerodermatous Chronic Graft-versus-Host Disease: Clinical Report Hong Zhou,1,* Mei Guo,2,* Chunjing Bian,1 Zhao Sun,1 Zhuo Yang,1 Yang Zeng,1 HuiSheng Ai,2* Robert Chunhua Zhao1,* The success of treatment for sclerodermatous chronic graft-versus-host disease (ScGVHD) remains disappointing. The immunomodulatory ability of bone marrow (BM)–derived mesenchymal stem cells (MSCs) shows promise in treating GVHD, especially given its previous success in treating patients with acute GVHD (aGVHD). The potential efficacy and safety issues for treating cGVHD, particularly ScGVHD, remain to be clarified, however. Here, we report 4 patients with ScGVHD who received MSCs expanded ex vivo from unrelated donors by intra-BM injection. After MSC infusion, the ratio of helper T lymphocyte (Th) 1 cells to Th2 cells was dramatically reversed, with an increase in Th1 and a decrease in Th2 achieving a new balance. Correspondingly, symptoms gradually improved in all 4 patients. During the course of MSC treatment, the patients’ vital signs and laboratory results remained normal. At the time of this report, none of the 4 patients had experienced recurrence of leukemia. Although this study alone cannot guarantee the application of MSCs in ScGVHD, our findings strongly suggest that this treatment is therapeutically practicable, with no detectable side effects. This approach may provide new insight into the clinical treatment of ScGVHD, with the aim of greatly increasing the survival rate in patients with leukemia who undergo allogeneic BM transplantation (BMT). Biol Blood Marrow Transplant 16: 403-412 (2010) Ó 2010 American Society for Blood and Marrow Transplantation

KEY WORDS: Bone marrow–derived mesenchymal stem cell, Chronic graft-versus-host disease, Sclerodermatous chronic graft-versus-host disease, Allogeneic hematopoietic cell transplantation

INTRODUCTION Chronic graft-versus-host disease (cGVHD) is a serious and common long-term complication of allogeneic hematopoietic cell transplantation (AHCT), From the 1Institute of Basic Medical Sciences & School of Basic Medicine, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China; and 2Department of Hematology and Transplantation, Affiliated Hospital of Academy of Military Medicine Science, Beijing, People’s Republic of China. Financial disclosure: See Acknowledgments on page 411. * Both authors contributed equally to this work. Correspondence and reprint requests: Robert Chunhua Zhao, MD, PhD, Center of Excellence in Tissue Engineering, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, PR China. Huisheng Ai, MD, Department of Hematology and Transplantation, Affiliated Hospital of the Academy of Military Medicine Sciences, Beijing, PR China. (e-mail: [email protected] or [email protected]). Received August 28, 2009; accepted November 8, 2009 Ó 2010 American Society for Blood and Marrow Transplantation 1083-8791/10/163-0013$36.00/0 doi:10.1016/j.bbmt.2009.11.006

occurring in 25%-40% of patients who survive for more than 100 days after AHCT [1]. The immunopathogenesis of cGVHD is mediated in part by helper T lymphocyte 2 (Th2) cells, with a syndrome of immunodeficiency and an autoimmune disorder [2,3]. cGVHD affects almost all organs and tissues, manifested by a marked increase in collagen deposition and a lack of T lymphocyte infiltration in the target organ tissues. The appearance of sclerotic skin lesions is the most common pathological change in patients with cGVHD; this condition is termed sclerodermatous cGVHD (ScGVHD). The main clinical manifestations of ScGVHD include lichenoid lesions, sclerodermatous lesions, disorders of pigmentation (eg, areas of hypopigmentation and hyperpigmentation), and leopard-skin eruptions (widespread, well-delimited, hyperpigmentated macules) [4-6]. Despite the widely used combination treatment of steroids and cyclosporine A(CsA) or tacrolimus, the outcome in patients with extensive ScGVHD remains unsatisfactory. Other agents that have been studied include etritrenate, thalidomide, hydroxychloroquine, extracorporeal photopheresis, ultraviolet (UV) B light, and psoralen 1 UVA light [7], but these agents have 403

Extensive Skin, intestinal tract II CsA-TAC Sib/sister ALL M 4

38

MM M 3

43

AML/M4 F 2

43

M 1

AML indicates acute myelogenous leukemia; MM, multiple myeloma; ALL, acute lymphoblastic leukemia; CsA, cyclosporine A; GVHD, graft-versus-host disease; aGVHD, acute graft-versus-host disease; cGVHD, chronic graft-versus-host disease; Sib, HLA-identical sibling; MMF, mycophenolate mofetil; MTX, methotrexate; TAC, tacrolimus; TDM, thalidomide; PDN, prednisone.

37.3 No response

4.5 No response

TDM(200)-PDN (30)-TAC(30) CsA(75)-TDM (150)-PDN(15)MMF(2000) Extensive Liver I CsA-MTX

Extensive None 0 CsA-MMF Sib/brother

Sib/sister

12 No response

No response

TDM(150)-PDN (30)-TAC(35) CsA(75)-PDN(30) Extensive Skin II CsA Sib/sister

Nonmyeloablative AHCT Nonmyeloablative AHCT Nonmyeloablative AHCT Nonmyeloablative AHCT

Therapeutic Effect Site GVHD Prophylaxis Diagnosis Age, Years Sex

Table 1. Baseline Patient Characteristics

The study group comprised 4 patients with ScGVHD (3 males, 1 female; median age, 41 years; range, 38 to 43 years) presenting at the Affiliated Hospital of Academy of Military Medicine Science between September 2006 and August 2008. Informed consent was obtained according to institutional guidelines in accordance with the Declaration of Helsinki. The clinical protocol was approved by the institution’s Ethics Committee. Baseline patient characteristics are summarized in Table 1. aGVHD and cGVHD grading was performed according to classical criteria. All 4 patients underwent nonmyeloablative (NMA) AHCT for a primary hematologic malignancy and developed ScGVHD at a median time of 11.5 months (range, 4-19 months) after transplantation. One of the patients had a chest skin biopsy, the pathology report of which supported the diagnosis of ScGVHD (Figure 1). All

Graft

Patients

AML/M4b

PATIENTS AND METHODS

Donor

Bone Marrow Transplantation

Grade

aGVHD

Class

Immunosuppression before MSC Therapy, mg/Day

ScGVHD

Duration before MSC Infusion, Months

been found to carry serious side effects. In addition, infliximab, a humanized chimeric antibody against tumor necrosis factor (TNF); etanercept, a TNF antagonist [8]; and rituximab, an anti-CD20 chimeric monoclonal antibody (mAb) [9,10] have been used to treat cGVHD. Further study of this innovative approach is underway with the efficacy in the air. There remains no effective, specific therapy to block the development of cGVHD. ScGVHD, the most severe form of the disease, is frequently refractory to standard treatment modalities, further limiting therapeutic options for affected patients [11-13]. Numerous studies have shown that bone marrow (BM)–derived mesenchymal stem cells (MSCs) have profound immunomodulatory functions both in vitro and in vivo. MSCs are known to modulate the proliferation, activation, and maturation of T and B lymphocytes in vitro in a dose-dependent and time-limited manner [14,15]. Our laboratory first reported the immunomodulatory effects of MSCs on the abnormal immune system of BXSB mice that were born with an immunologic deficiency and developed lupus naturally. The results showed that MSCs can significantly down-regulate Th2 cells in pathological conditions and further inhibit the abnormal activation of humoral immunity to maintain the original balance. Furthermore, the renal function of BXSB mice exhibited significant and lasting improvement [16]. MSCs also can suppress the differentiation from Th0 cells to Th1 cells and further affect cytoimmunity, making this approach effective in treating acute GVHD (aGVHD) [17-20]. But is MSC therapy effective in patients with ScGVHD? To explore this question, we treated 4 patients with ScGVHD with intra-BM injection (IBMI) of MSCs.

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Figure 1. Biopsy specimen of chest skin in patient 2 before MSC infusion. Stratified squamous epithelium showing mild hyperkeratosis with parakeratosis, basal cell liquefaction degeneration, and T lymphocytes infiltrating into the superficial layer of the derma, with visible manipulus of tissue cell–phagocytized hemosiderin. (Hematoxylin and eosin staining; original magnification 100.)

patients received standard immunosuppressant medications for ScGVHD, such as cyclosporine A(CsA), thalidomide, prednisone, and tacrolimus, at a median time of 17.3 months (range, 4.5-37.3 months) before MSC treatment, but the condition did not improve. Ex Vivo MSC Culture Expansion MSCs were collected from unrelated donors in accordance with informed consent principles and cultured as described previously [21,22]. In brief, donor BM aspirates were collected, and mononuclear cells (MNCs) were fractionated over a 1.077-g/mL Ficoll-Paque density gradient. The interface was collected and resuspended in Dulbecco’s modified Eagle’s medium (low-glucose DMEM/F12) and 40% MCDB-201 medium (Sigma-Aldrich, St Louis, MO), supplemented with 2% fetal bovine serum (FBS; Gibco Life Technologies, Paisley, UK), 1-insulin transferring selenium (Gibco Life Technologies), 1029 M dexamethasone (Sigma-Aldrich), 1024 M ascorbic acid 2-phosphate (Sigma-Aldrich), 10 ng/ mL of epidermal growth factor (Sigma-Aldrich), and 10 ng/mL of platelet-derived growth factor BB (Sigma-Aldrich), and then plated in culture flasks (1 106 cells/mL). The culture was maintained at 37 C in a humidified environment containing 5% CO2. After 24-48 hours of culture, nonadherent cells were removed, and the adherent layer was cultured until it reached 70%-80% confluence. Cells were then detached with 0.05% trypsin and 0.01% EDTA and replated. MSCs were harvested and used at passages 3-6. All cell culture steps were performed in the in-house Good Manufacturing Practices facility.

MSCs Alleviate ScGVHD

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(PBS) containing 0.5% bovine serum albumin (BSA; Sigma-Aldrich), and incubated with primary antibodies for 30 minutes at 4 C. To detect intracellular antigens, cells were fixed in 2% paraformaldehyde for 15 minutes at 4 C, and then permeabilized with 0.1% saponin (Sigma-Aldrich) for 1 hour at room temperature. The working concentration for primary antibodies against human Flk-1 (Santa Cruz Biotechnologies, Santa Cruz, CA), CD29, CD31, CD34, CD44, CD45, CD105, CD106, and HLA-DR (all from BD Biosciences, San Diego, CA) was 20 ng/mL. Same-species, same-isotype irrelevant antibodies were used as negative controls. Cells were incubated with fluorescein isothiocyanate–conjugated secondary antibodies for 30 minutes at 4 C after being washed with PBS containing 0.5% BSA. After another 3 washes, cells were resuspended in PBS and analyzed with a fluorescence-activated cell sorter (FACS; Vantage SE; BD Biosciences). Multilineage Differentiation of MSCs To identify the capacity for MSC multilineage differentiation, MSCs were cultured under differentiation conditions. For adipocyte differentiation, MSCs were cultured at 2-3 104/cm2 in DMEM with 10% FCS, 1026 M dexamethasone, 50 mg/mL of ascorbic acid, and 100 mg/mL of 1-methyl-3-isobutyl-xanthine (all from Sigma-Adrich). After 14 days, Oil Red O staining was performed to show adipocyte differentiation. For osteoblast differentiation, MSCs were cultured at 1 104/cm2 in DMEM with 10% FBS, 10 mM b-glycerophosphate, 1027 M dexamethasone, and 0.2 mM ascorbic acid (all from Sigma-Aldrich). After 21 days, the cells were stained with von Kossa’s stain to reveal osteogenic differentiation. For chondrocyte differentiation, spheroids of 2 105 MSCs were formed by centrifugation at 800 rpm in 15-mL centrifuge tubes. After incubation at 37 C and 5% CO2 in chondrogenic induction medium (DMEM supplemented with 2% FCS, 10 ng/ mL transforming growth factor b1, and 50 nM ascorbic acid [all from Sigma]) for 4 days, the spheroids were transferred to 96-well U-bottomed plates. After 21 days, the spheroids were harvested and fixed in 10% buffered formalin for 2 hours at room temperature. The fixed spheroids were then dehydrated by treatment with a series of graded alcohols, cleared by treatment with xylene, and infiltrated with paraffin. Paraffin sections (8 mm) were stained with 0.1% Safranin O to detect mucopolysaccharide synthesis. Mitogen Proliferative Assays

Cell Surface Antigen Analysis For immunophenotyping analysis, the expanded cells were washed with phosphate-buffered saline

In the mitogen proliferative assays, triplicate wells containing responder 1 105 MNCs were cultured with 50 mg/mL of phytohemagglutinin (PHA; Roche,

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Mannheim, Germany) in a total medium volume of 0.1 ml at 37 C in 5% CO2, and MSCs were added on day 0. Irradiated MSCs (30 Gy) were cocultured with the MNCs at a MSC: MNC ratio of 1:10. Control wells contained only MNCs. Cultures were pulsed with 1 mCi/well of tritiated thymidine (Shanghai Nucleus Research Institute, China) on day 2, and harvested 18 hours later with a Tomtec automated harvester (Wallac, Gaithersburg, MD). Thymidine uptake was quantified using a liquid scintillation and luminescence counter (TRILUX; Wallac).


Table 2. Summary of MSC Infusion Patient

Frequency

1

1 2 3 4 5 6 7 8 1 2 3 4 1 2 3 4 1 2 3 4 5 6 7

2

MSC Infusion and Therapeutic Effect Evaluation The patients received MSC infusion at a dose of 1.0-2.0 107 cells by IBMI from the anterosuperior iliac spine. MSCs from the same donor for the same patient were infused more than once. Because of differences in the patients’ condition, family circumstances, and hospital and other factors, the number of infusions ranged from 4 to 8. Concomitant medications for ScGVHD were individualized for each patient, but all were current standard medicines, and the doses were tapered significantly (Table 2). The physical examination included skin scoring and range-of-motion assessment. The modified Rodnan skin (RS) score was used to assess cutaneous sclerosis changes [23]. The RS scale grades skin thickness from 0 to 3 as follows: 0, uninvolved skin; 1, skin involved with ability to pinch; 2, inability to pinch; 3, inability to move. The pinch is done in a single movement of moderate intensity, using only the fingertips. All affected areas were assessed, and the results of all areas were added to give a final overall score. Immunofluorescent Staining of Intracellular Cytokines and Flow Cytometric Analysis Stimulation of Peripheral Blood Mononuclear Cells (PBMCs) MNCs were prepared from patients’ peripheral blood (PB) by Ficoll-Paque density gradient centrifugation and suspended in RPMI 1640 medium supplemented with 10% (vol/vol) FCS, 2 mM l-glutamine, 0.1 mM nonessential amino acids (Life Technologies, Carlsbad, CA), 1 mM sodium pyruvate, 100 U/mL penicillin, 100 mg/mL streptomycin, 1% HEPES buffer, and 10 mM 2-mercaptoethanol. To stimulate interleukin (IL)-2 and interferon (IFN)-g production, PBMCs were stimulated for 6 hours with 5 ng/mL phorbol myristate acetate (PMA; Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) in the presence of a protein transport inhibitor containing monensin (GolgiStop; Sigma-Aldrich). To stimulate IL-10 production, PBMCs were stimulated for 24 hours with 1.0 mg/ mL of lipopolysaccharide (LPS; Sigma-Aldrich) in

3

4

Inversion Time, Day

Dose, Cells/2 mL

Concomitant Medications, mg/Day

0

1.2 107

PDN(20)-TAC(19) -TDM(50)

+9 +14 +22 +28 +35 +49 +52 0 +14 +39 +47 0 +8 +15 +22 0 +7 +14 +28 +38 +45 +52

2.0 107 1.0 107 2.0 107 2.0 107 2.0 107 2.0 107 1.5 107 1.0 107 2.1 107 1.1 107 1.0 107 1.8 107 1.5 107 1.0 107 2.0 107 2.2 107 1.7 107 1.4 107 2.1 107 2.0 107 1.9 107 1.0 107

PDN(5)

TAC(19)

PDN(5)-MMF(0.25)

MSC indicates mesenchymal stem cells; TAC, tacrolimus; TDM, thalidomide; PDN, prednisone.

the presence of GolgiStop. For stimulating IL-4 production, PBMCs were stimulated for 2 days with 50 mg/mL of PHA (Roche), washed, and then cultured in medium containing 10 ng/mL of rhIL-2 and 20 ng/mL of rhIL-4 (both from Sigma-Aldrich) for 3 days. Finally, the cells were harvested and restimulated for 4 hours with PMA and ionomycin in the presence of GolgiStop. Immunofluorescent Staining of Intracellular Cytokines and Flow Cytometric Analysis The stimulated cells were washed with PBS containing 0.5% BSA (Sigma-Aldrich), fixed in 2% paraformaldehyde for 15 minutes at 4 C, permeabilized with 0.1% saponin (Sigma-Aldrich) for 1 hour at room temperature, and then incubated with phycoerythrin (PE)-conjugated primary antibodies for 30 minutes at 4 C. The working concentration for primary antibodies against human IL-2, IFN- g, IL-10, and IL-4 (BD Biosciences) was 20 ng/mL. Same-species, same-isotype irrelevant antibodies were used as negative controls. After 3 washes, cells were resuspended in PBS and analyzed using a Vantage SE FACS (BD Biosciences). Statistics All statistical analyses were performed using SPSS version 13.0 (SPSS Inc, Chicago IL). The paired-sample t-test was used to test the probability of significant differences between samples. Statistical significance was defined as P \ .05.


RESULTS


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demonstrated by Safranin O staining for detecting the synthesis of mucopolysaccharide (Figure 2D).

Characteristics of MSCs Morphology and Phenotype Analysis MSCs from donor BM gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast-like morphology (Figure 2A). MSCs could be ex vivo expanded by successive cycles of trypsinization, seeding, and culture. The phenotype of MSCs was Flk1-, CD29-, CD44-, CD106-, and CD105-positive, and CD34-, CD31-, and HLA-DR– negative (Figure 2E), in accordance with our previous findings. Adipogenic Differentiation When MSCs were cultured in adipogenic medium for 2 weeks, more than 80%-90% of cells differentiated into lipid-laden cells that were stained by Oil Red O (Figure 2B). Osteogenic Differentiation When MSCs were cultured in osteogenic medium for 3 weeks, more than 80% of cells could be induced into osteogenic lineage, as demonstrated by von Kossa staining (Figure 2C). Chondrogenic Dfferentiation When spheroids of MSCs were cultured in chondrogenic medium for 3 weeks, more than 80%90% of cells differentiated into cartilage cells, as

Immunomodulatory Effect on T Cell Proliferation To explore the mechanisms of MSCs in alleviating ScGVHD, we examined the role of MSCs in T cell proliferation. Irradiated MSCs were added to mitogen-stimulated T cells in a proliferation reaction. MSCs clearly inhibited the T cell proliferation. In mitogen-sitmulated T cell proliferation, the proliferation of T cells at a MSC: MNC ratio of 1:10 was significantly inhibited, to about 80%. This result was confirmed by subsequent experiments (Figure 3), suggesting that MSCs may modulate the immune system through inhibition of T cell proliferation. Outcome of MSC Treatment for ScGVHD The patients’ responses to MSC therapy and their outcomes are summarized in Table 3. With a median overall follow-up of 14.1 months (range, 4.6-23 months) from time of initiation of MSC therapy, all 4 patients exhibited improved RS score (.70%) and decreased signs and symptoms. Patient 1 experienced effective pain relief in the lower extremities on day 110 after the first MSC infusion. Skin sclerosis and edema of the extremities were slightly alleviated; a yellow callus without exudates formed on the surface of the foot ulcer. On day 116, skin lesions (ie, desquamation and edema of the extremities, and ulcers and exudation on the right palm and both feet) were further

Figure 2. In vitro differentiation and typical phenotypes of MSCs. (A) Cell morphology of MSCs at the third passage. (B) Oil Red O staining of MSCs after adipogenic induction. (C) von Kossa staining of MSCs after osteogenic induction. (D) Safranin O staining of MSCs after chondrogenic induction. (E) Phenotypes of MSCs at the third passage.

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Figure 3. Effect of MSCs on T lymphocyte proliferation in mitogen proliferative assays. Shown are nonstimulated T cells (T0), PHA-stimulated T cells (Ts), and PHA-stimulated T cells cocultured with MSC at a MSC:T cell ratio of 1:10. Data are given as the mean 6 standard deviation of 3 independent experiments. *P \.05 versus T0; **P \.05 versus Ts.

improved, with both hands dermatoglyph-visible. Following repeated intermittent MSC infusion, local skin gradually became soft with the ability to be pinched, and diffuse decrustation and newborn skin were observed. Joint mobility increased, and pain in the extremities vanished. Patient 2 exhibited marked improvement in skin condition after 4 rounds of MSC infusion. Skin sclerosis on the trunk and extremities was alleviated, and joint mobility was greatly improved, allowing the patient to walk without assistance. But, because the patient suffered from psoriasis, her local skin (especially in the lower extremities and hands) remained desquamative, with concomitant hyperpigmented and hypopigmented lesions. In patient 3, edema of the hands and feet was alleviated on day 16 after the first MSC infusion. On day 115, the patient reported local itching. By day 122, desquamation decreased, and less desquamative skin was produced. In addition, no ulcers or pyorrhea were observed. Patient 4 demonstrated the most striking improvement. On day 114 after the initial MSC infusion, the sclerotic and hyperpigmented skin in some areas exhibited significantly improved elasticity and complexion; the ulcer on the right ankle was healing, with decreased exudate. By day 128, skin sclerosis was further alleviated, the patient’s complexion was shallowed noticeably, fine leg hairs were increased,

and the patient complained of intolerable itching of the partial skin. On day 133, skin hairs were thicker, and the patient complained of more frequent itching. As immunity improved, the frequency of fever was markedly reduced. After the 3 subsequent MSC infusions, the patient’s condition improved; desquamation and edema disappeared, skin elasticity recovered somewhat, sweat gland and follicular orifice function returned to normal, the ankle ulcer formed a scab and healed, and joint mobility improved significantly (Figure 4). In addition, MSC therapy allowed for a significant tapering of standard medications (Tables 1 and 2). Our findings indicate that MSCs begin to exert their therapeutic effects during the first 2 weeks of therapy, resulting in significant clinical improvement. Furthermore, the efficacy of MSCs can be sustained by repeated intermittent MSC infusion. Change in T Lymphocyte Subset after MSC Infusion To evaluate the change in the Th1 cell:Th2 cell ratio before and after MSC infusion, PBMCs were extracted and stimulated by polyclonal activators at multiple times before and after MSC infusion. The IFN-g–, IL-2–, IL-10–, and IL-4–producing cell proportions were analyzed by flow cytometry. The results show significantly higher proportions of IL-10– and IL-4–producing cells compared with IL-2– and IFNg–producing cells, indicating an apparent imbalance of the 2 T lymphocyte subsets. That is, Th0 cells experienced a differentiation imbalance and tended to be Th2 cells before MSC treatment. Nevertheless, after repeated intermittent MSC infusion, the proportions of IL-10– and IL-4–producing cells gradually decreased, whereas the proportions of IL-2– and IFNg–producing cells gradually increased. This overall trend was consistent in all 4 patients (Figure 5A-D). The flow cytometry data were analyzed by statistical methods to detect significant changes in the 4 patients (Figure 5E and F). Specifically, the results indicate that MSC infusion plays a role in down-regulating excessive Th0-to-Th2 differentiation and increasing the proportion of Th1 cells, thereby reversing the Th1 cell–Th2 cell imbalance and achieving a new equilibrium point in patients with ScGVHD.

Table 3. Outcome after MSC Therapy

Patient

MSC Therapy Duration at Last Follow-Up, Days

Side Effects

RS Score before MSC Therapy

RS Score at Last Follow-Up

Overall Follow-Up, Months

Status at Last Follow-Up

1 2 3 4

52 47 22 52

None None None None

28 24 21 35

7 3 6 8

23 20.3 8.5 4.6

Alive Alive Alive Alive

MSC indicates mesenchymal stem cell; RS, Rodnan Skin.



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Figure 4. The change process of skin lesions before and after MSC infusion in patient 4. Shown are changes in the skin lesion of the neck (A), left arm (B), back (C), and right ankle (D) on days 0, 114, 128, 133, 140, 154, 176, 192, and 1123 after initial MSC infusion. Over time, the skin became soft, desquamation was reduced, skin color was shallowed, edema disappeared, and the ankle ulcer formed a scab and healed.

Safety of MSC Infusion The 4 patients involved in this trial were all closely monitored for changes in body temperature, pulse, respirations, blood pressure, oxyhemoglobin saturation, and electrocardiography readings. Blood biochemical examination of liver and kidney function was conducted as well. No abnormalities were found. In addition, with a median overall follow-up of 14.1 months (range, 4.6-23 months), none of the 4 patients experienced a recurrence of leukemia. Overall, the results suggest that MSC infusion is safe and reliable.

DISCUSSION Of the 3 major cell subsets, Th0 cells can differentiate into Th1 or Th2 cells in response to certain chemical signal stimuli, Th1 cells mostly secrete IFN-g and IL-2, and Th2 cells mainly secrete IL-10 and IL-4. The Th1 cell:Th2 ratio is considered a major factor reflecting immune status [24]. Th cell imbalance is an important pathological mechanism that is

receiving increasing attention in the study of immune system disorders; for example, an increase in Th1 cells is reportedly associated with aGVHD, and an increase in Th2 cells is an important factor in the development of fibrosis [25,26]. Some studies have suggested that cGVHD is an immunodeficient and autoimmune disease induced by an increasing proportion of Th2 cells [1,2], and here we observed an increasing proportion of Th2 cells in all 4 patients with ScGVHD, in accordance with a previous report [27]. MSCs have an high proliferation capacity, multilineage differentiation potential, and low immunogenecity. Previous investigations have shown that MSCs may modulate the immune system through various mechanisms (eg, inhibiting T cell proliferation through direct contact and cytokine secretion). Some studies also have found that MSCs can affect the differentiation of Th cells, but this effect may be inconsistent in normal and abnormal immune systems. In an earlier study, we found that in the presence of a physiological condition, MSCs mainly inhibit Th0 cell differentiation to Th1 [28]. Similarly,

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Figure 5. Percentage of intracellular cytokines producing T lymphocyte subsets detected by flow cytometry. (A) Patient 1: polygram detected by flow cytometry on days 0, 114, 128, and 149 of MSC infusion. (B) Patient 2: polygram detected by flow cytometry on days 0, 114, 139, and 147 of MSC infusion. (C) Patient 3: polygram detected by flow cytometry on days 0, 18, 115, and 122 of MSC infusion. (D) Patient 4: polygram detected by flow cytometry on days 0, 17, 114, 128, and 135 of MSC infusion. (E) Patient 4: histogram of IL-2–, IFN-g–, IL-10–, and IL4–producing T lymphocyte subsets on days 0 and 135 of MSC infusion. (F) Comparison of the 4 intracellular cytokines producing cells between before infusion of MSCs and the final infusion of MSCs. The difference is statistically significant (*P \.05).


in increased Th1–related diseases like aGVHD, MSC treatment also could reduce the proportion of Th1 cells, thereby improving the patient’s condition; this effect has been confirmed in clinical applications [17-20]. Nonetheless, the fact is that for some increased Th2–related immune disorders, such as acute-phase fibrosis, MSC transplantation also exerts a good therapeutic effect [29,30]. Thus, MSC therapy may be a therapeutic option for ScGVHD; clinical trials are needed, however. In normal BM, MSCs are very rare (1 out of 1045 10 MNCs). Thus, under normal conditions, MSCs may not initiate the immune response to foreign antigens, which is consistent with findings in vitro. In both PHA-stimulated T cell proliferation reactions and mixed lymphocyte reactions (MLRs), inhibition of MSCs to T cell proliferation disappeared at an MSC:MNC ratio of 1:100. Furthermore, MSCs’ immunomodulatory capacity in a normal immune system in vivo is dose-dependent and disappears when the number of infused MSCs drops to 104 [14]. But, MSCs can proliferate easily in vivo, and because of low immunogenicity, MSCs can be transplanted abundantly to treat immune system disorders. In addition, our previous study in animal models showed that MSCs’ immunomodulatory capacity was intense during the first 1-2 weeks after infusion, was obviously weakened after the third week, and almost disappeared after the fourth week [28]. Because ScGVHD is a chronic disease, we needed to repeatedly intermittently infuse MSCs, with the time interval controlled at 1-2 weeks. Furthermore, in accordance with our previous research [31,32], we controlled the dose at an order of magnitude of 107. Gao et al. [33] reported that mouse MSCs given intravenously were blocked in the lungs, and consequently the homing of MSCs to BM was disturbed [34]. Injecting MSCs directly into the BM might markedly reduce their peripheral residence in the lungs and liver, an advantage of this route; thus, the 4 patients in this trial received their MSC infusions through the anterosuperior iliac spine. After intermittently repeated MSC infusions, the ratio of Th cells changed dramatically: Th1 increased and Th2 decreased, and a new Th cell balance was achieved. Correspondingly, patient symptoms also gradually improved, suggesting, an association between ScGVHD and Th cell imbalance, which MSC can reverse. Furthermore, after MSC therapy, all standard medications that the patients took could be tapered significantly. The 4 patients involved in this trial all underwent NMA AHCT after the mitigation of primary leukemia and subsequently developed cGVHD. Previous studies have shown that MSC transplantation can increase the risk of leukemia recurrence [35]. Some studies have claimed that AHCT recipients who develop GVHD


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experience fewer leukemia recurrences and have a longer life span compared with those who do not develop GVHD; these positive effects are achieved via the graft-versus-leukemia (GVL) reaction [36]. GVHD and GVL are usually concomitant; that is, the GVL effect is subdued when GVHD is attenuated, leading to leukemia relapse. However, in this trial, from the beginning of the MSC infusion to the end of the follow-up period (median time, 14.1 months), none of the 4 patients experienced recurrence of leukemia. This suggests that the immunomodulatory effect of MSCs is selective and controllable. In addition, no MSC infusion–related adverse reactions or side effects were observed, suggesting that MSC infusion is safe and reliable. To confirm the safety of isolated and cultured MSCs in vitro for clinical applications, we designed a preclinical Phase I trial to evaluate the feasibility and security of intravenous infusion of these cells after ex vivo expansion. Infusions of autologous MSCs cultured in vitro at doses of 5 104/kg, 5 105/kg, and 2 106/kg were safe and well tolerated in healthy volunteers [31]. In summary, despite its limited size, the present study suggests a benefit of MSC infusion therapy in treating ScGVHD. Further prospective investigations are warranted.

ACKNOWLEDGMENTS Financial disclosure: This work was supported by grants from the 863 Project of the Chinese Ministry of Science and Technology (2006AA02A109 and 2006AA02A115), the National Natural Science Foundation of China (30830052), the Beijing Ministry of Science and Technology (D07050701350701), and the Cheung Kong Scholars program. Authorship Statement Hong Zhou was involved in study conception and design, data analysis, interpretation, and manuscript writing. Mei Guo provided study materials or patients and interpreted data. Chunjing Bian was involved in manuscript writing. Zhao Sun, Zhuo Yang, and Yang Zeng provided study materials or patients. HuiSheng Ai was involved in study conception and design and provided study materials or patients. Robert Chunhua Zhao was involved in study conception and design, securing of financial and administrative support, provision of study materials or patients, data collection and/or assembly, data analysis and interpretation, manuscript writing, and final approval of the manuscript. Financial disclosure: The authors have no conflicts of interest to disclose.

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