SEARCH

SEARCH BY CITATION

Keywords:

  • Mesenchymal stem cell;
  • T lymphocyte;
  • Dendritic cells;
  • Migration;
  • Acute graft-versus-host disease

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Due to the potent immunoregulatory capacity, mesenchymal stem cells (MSCs) have been used in clinical trials to treat acute graft-versus-host disease (aGvHD), although the detailed in vivo mechanisms remain elusive. In a murine lethal aGvHD model, MSCs delayed the development of the disease. Interestingly, we found that MSC infusion increased the number of T lymphocytes in the secondary lymphoid organs (SLOs). Since the expression of CD62L and CCR7 is prerequisite for lymphocyte migration into SLOs, the in vitro experiments revealed that in the presence of MSCs, T lymphocytes (including CD4+CD25+ regulatory T cells) preferred to take the naive-like phenotype (CD62L+/CCR7+) in mixed lymphocyte reaction and maintained the migratory activity elicited by secondary lymphoid tissue chemokine (SLC). Dendritic cells (DCs) are the initiator of immune response. CCR7 expression is pivotal for their maturation and migration into SLOs. However, CCR7 expression and SLC-driven migratory activity of DCs were remarkably suppressed by MSC coculture. The processes above were realized mainly through secretory mechanism. Consistently, MSC infusion maintained T lymphocytes to take CD62L+/CCR7+ phenotype and decreased the CCR7 expression and proportion of DCs in SLOs of aGvHD mice. In conclusion, the altered migratory properties of T cells and DCs might contribute to the immunosuppressive activity of transplanted MSCs in the setting of aGvHD.

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


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Author contributions: H.L.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; Z.K.G. and X.X.J.: data analysis and interpretation, manuscript writing; H.Z. and X.S.L.: collection and assembly of data; N.M.: conception and design, financial support, administrative support, provision of study material, manuscript writing, final approval of manuscript.

Mesenchymal stem cells (MSCs) are characterized by their intrinsic self-renewal capacity and multilineage differentiation potentials [1]. Under defined inductive conditions, MSCs can differentiate into cells derived from embryonic mesoderm, such as osteoblasts, chondrocytes, adipocytes, and hematopoiesis-supporting stromal cells [2, [3]4]. Recent in vitro investigations have definitely demonstrated that MSCs may also act as a pleiotropic immune regulator to suppress an ongoing immune process through cytokine secretion and/or direct cell-cell contact to affect nearly all immune cells including T, natural killer, B, and dendritic cells (DCs) [5, [6], [7], [8]9]. Their immunomodulatory role was further confirmed by the findings that MSC administration prolonged the mean survival time (MST) of allogeneic skin in baboon [10] and in mice [11]. Moreover, MSCs are being used from bench to clinic (phase III) to treat acute graft-versus-host disease (aGvHD), which is still a major cause of post-transplant mortality [12, 13]. However, further understandings of the mechanisms through which the transplanted MSCs act in vivo are needed for the development of efficacious aGvHD therapy.

It is well known that aGvHD develops when there are antigen incompatibilities between host and donor, and the host is incapable of rejecting the graft that contains immunocompetent cells, which was defined as Billingham's tenets in 1966 [14]. Many clinical observations also indicated that the development of aGvHD requires the migration of effector cells, particular T cells from the secondary lymphoid organs (SLOs) to the target tissues such as liver, intestines, skin, lung, and bone marrow. So the requirement of the effector cells migrating into the target tissues was proposed to the Billingham's classic criteria [15]. Consistently, administration of FTY720, which is capable of preventing lymphocyte egress from SLOs to peripheral organs, inhibited GvHD lethality [16, 17]. All these aroused our enthusiasm to investigate whether MSCs could inhibit aGvHD development by affecting the migratory property of T lymphocytes.

Considering that CD4+CD25+ regulatory T cells (Tregs) play an important immunoregulatory role in graft-versus-host reaction [18, [19]20], that DCs are the most potent initiator of the immune responses, and that the crosstalk between Tregs and DCs determines the occurrence and development of immune reaction [21], we also studied whether MSCs could affect T-lymphocyte migration via DCs.

In a murine aGvHD model, we addressed these questions by comparing the organ pathologic damages and the cell numbers in SLOs of aGvHD mice with those of MSC cotransfer. Further in vitro and in vivo experiments showed that coculture or cotransfer of MSCs altered migratory activity while reserving the “naive”-like phenotype of T lymphocytes and DCs. This suggested that the alteration in the migratory property of T lymphocytes and DCs contributes significantly to the therapeutic benefits of MSCs in aGvHD.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Mice

Inbred C57BL/6 (H2b) and BALB/c (H2d) female mice were purchased from the Laboratory Animal Center, Academy of Military Medical Sciences (Beijing, http://www.bmi.ac.cn). All experiments in this study were performed in accordance with the Academy of Military Medical Sciences Guide for Laboratory Animals.

Culture of Murine Bone-Derived MSCs

MSCs from murine compact bone were isolated and culture-expanded as described in our previous report [11]. Briefly, long bones from 2- to 3-week-old female C57BL/6 mice were digested by collagenase II (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and the remaining bone debris were cultured in α-minimal essential medium (α-MEM; Gibco, Grand Island, NY, http://www.invitrogen.com) containing 10% fetal bovine serum (FBS; StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) in a humidified atmosphere of 5% CO2 at 37°C. The adherent cells at passages 3–5 were used as MSCs in the experiments below.

Murine aGvHD Model [22]

Splenic mononucleocytes from female C57BL/6 mice were prepared by Ficoll gradient centrifugation and an inoculum of 2 × 107 viable cells was intravenously injected into sublethally irradiated (5 Gy) BALB/c mice without or together with graded doses of MSCs (2 × 106, 1 × 106, 2 × 105, 2 × 104) in a total volume of 0.25 ml phosphate-buffered saline (PBS). Mice in aGvHD group received 2 × 107 splenic mononucleocytes only, whereas mice in aGvHD + MSC group received 2 × 107 splenic mononucleocytes and 1 × 106 MSCs. A group of aGvHD mice received 1 × 106 MSCs 3 days later after the injection of 2 × 107 splenic mononucleocytes.

Pathologic Assessment of Tissue

Tissues from the aGvHD and aGvHD + MSC groups were taken for pathologic analysis on day 6 after transplantation. Specimens of the liver, spleen, whole gut, and skin were obtained, fixed in 10% neutral-buffered formalin, and embedded in paraffin, and 6-mm thick samples were sectioned and stained with hematoxylin and eosin.

Flow Cytometry Analysis

Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against mouse CD3, CD4, CD11b, and CD11c; phycoerythrin (PE)-conjugated monoclonal antibodies against mouse CD3, CD25, CD69, CD40, CD80, CD86, and Iab; phycoerthrin-Cy5-conjugated monoclonal antibody against mouse CD62L; and allophycocyanin-conjugated monoclonal antibody against mouse CCR7 (4B12) were all purchased from eBioscience (San Diego, http://www.ebioscience.com). H2b-PE was purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com). For CCR7 detection, cell surface FcγIIIR/FcγIIR were blocked with purified anti-mouse CD16/32 (eBioscience) at a concentration of 5 μg/106 cells for 15 minutes on ice. Without washing, 4B12 antibody was added at 1 μg/106 cells and incubated in a 37°C water bath for 30 minutes. Afterward, the cells were reacted with other monoclonal antibodies at 4°C for 30 minutes at appropriate concentrations in the dark as illustrated in the manufacturer's instructions. Stained cells were washed twice with cold PBS and the events were acquired by FACSCalibur (BD Pharmingen). The collected data were analyzed with WinMDI 2.9 software (Joseph Trotter, Scripps Research Institute, La Jolla, CA, http://www.scripps.edu) after gating for the designated population.

The Number of CD3+ and CD11b+ Cells in the Spleens and Lymph Nodes

At 12 hours, day 2, day 4, and day 6 after transplantation, spleens and lymph nodes (two axillary and two inguinal lymph nodes per mouse) of aGvHD or aGvHD + MSC mice were harvested. The absolute numbers of CD3+ and CD11b+ cells were calculated by multiplying the total numbers of nucleocytes and the proportions of them in nucleocytes shown by flow cytometry analysis (FCM).

Lymphocyte Proliferation Assay

On day 4 after transplantation, splenic mononucleocytes from three mice of aGvHD and aGvHD + MSC groups were harvested and inoculated in 96-well plates at a density of 2 × 106/200 μl RPMI 1640 medium with 10% FBS, 5 × 10−5 M β-mercaptoethanol, and 5 μCi 3H-thymidine per well at 37°C for 18 hours. Then the cells were collected over fiberglass filters, and the β-irradiation was measured on 1450 microbeta Wallac trilux counter (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com). Results were expressed as mean counts per minute (cpm) ± standard deviation (SD).

Cytotoxicity Assay

Cytotoxicity assay was performed with lactate dehydrogenase (LDH) cytotoxicity detection kit (Takara, Otsu, Japan, http://www.takara.co.jp). At day 4 after transplantation, mononucleocytes in spleens of aGvHD and aGvHD + MSC mice were collected and graded numbers of the cells (1 × 106, 5 × 105, 2.5 × 105, 1.25 × 105, 6.25 × 104) were cultured with 1 × 104 P815 (H2b) cells in a total volume of 200 μl RPMI 1640 containing 1% bovine serum albumin (BSA) per well. The wells of background control contain medium alone. The wells of low control contain 1 × 104 P815 cells only in medium and the wells of high control contain 1 × 104 P815 cells in 1% Triton X-100. The absorbance at 492 nm, which represents the values of LDH release, was measured in an enzyme-linked immunosorbent assay (ELISA) reader. The cytotoxicity (%) = (Experiment value-low control)/(High control-low control) × 100.

Mixed Lymphoid Reaction

Splenic T lymphocytes from C57BL/6 mice were obtained by negative selection with magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). MSCs were preplated in 96-well plates (5 × 104/well) and 20-Gy irradiated 24 hours later. T lymphocytes (2 × 105/well) were cultured with graded ratio (0:1, 1:50, and 1:10) of 20-Gy irradiated BALB/c splenic mononucleocytes in the absence or presence of MSCs. The cells were incubated in a total volume of 200 μl/well of 40% RPMI 1640 and 40% α-MEM supplemented with 20% FBS, 2 mM l-glutamine, 100 μM monothioglycerol, 100 U/ml penicillin, and 100 U/ml streptomycin. To assess the activity of the MSC supernatants, cells in mixed lymphoid reaction (MLR) were loaded on the upper chamber of transwell (12-well plate, 6.5 mm in diameter, 0.4-μm pore size; Costar, Cambridge, MA, http://www.corning.com), separating from MSCs preplated on the lower chamber. On days 1, 3, and 5 after incubation, cells were collected for FCM analysis and further in vitro migratory assays.

Measuring FoxP3 mRNA Level by Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction [23]

T cells without or with MSC coculture in MLR were prepared. Total cellular RNA was extracted with TRIZOL Reagent (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) and reverse-transcribed into cDNA using reverse transcriptase kit (Takara). FoxP3 primer pairs for polymerase chain reaction (PCR) were 5′-CCCAGGAAAGACAGCAACC-3′ and 5′-GGGTGGCATAGGTGAAAGG-3′. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. Primer pairs were 5′-TGACCACAGTCCATGCCATC-3′ and 5′-GACGGACACATTGGGGGTAG-3′. SYBR Green PCR kit (Sigma-Aldrich) was used for real-time PCR assay. Primer pairs were 5′-CCCACCTACAGGCCCTTCTC-3′ and 5′-GGCATGGGCATCCACAGT-3′ for FoxP3 and 5′-GGAGCGAGACCCCACTAACA-3′ and 5′-ACATACTCAGCACCGGCCTC-3′ for GAPDH. Foxp3 mRNA levels were normalized relative to GAPDH mRNA expression. Data are presented as the fold change relative to the sample with maximum FoxP3 expression.

DC Induction from Murine Splenic CD11b+ Cells [24]

Splenic CD11b+ cells from C57BL/6 mice were positively sorted with magnetic beads (Miltenyi Biotec). The cells were cultured at a density of 2 × 106/well in 6-well plates in 45% RPMI 1640 and 45% α-MEM supplemented with 10% FBS (HyClone, Logan, UT, http://www.hyclone.com), 10 ng/ml of recombinant mouse granulocyte-monocyte colony-stimulating factor (GM-CSF; Peprotech, Rocky Hill, NJ, http://www.peprotech.com), and 1 ng/ml recombinant mouse interleukin-4 (IL-4; Peprotech) in the absence or presence of MSCs (1 × 106/well preplated, 20-Gy irradiated) for 4 days. Afterward, 10 ng/ml of lipopolysaccharide (LPS; Sigma-Aldrich) was added and the cells were cultured for another 4 days. To assess the activity of the MSC supernatants, CD11b+ cells were seeded on the upper chamber of transwell (6-well plate, 24 mm in diameter, 0.4-μm pore size; Costar), separating from MSCs preplated on the lower chamber (1 × 106 /well, 20-Gy irradiated). The cells were collected at day 4 or day 8 for FCM analysis and further in vitro migratory assay.

In Vitro T-Lymphocyte and DC Migratory Activity Toward SLC

In vitro migratory assays were performed with transwell chambers (12-well plate, 6.5 mm in diameter, 5-μm pore size; Costar) as previously reported [25]. Lymphocytes (1 × 106) cultured in MLR at day 5 (50:1 group) or DCs (5 × 105) induced from CD11b+ cells at day 4 or day 8 were seeded onto the upper chamber in a total volume of 100 μl in RPMI 1640 with 1% BSA. The lower chamber was filled with 600 μl of RPMI 1640 containing 1% BSA and 40 ng/ml SLC (R&D Systems Inc., Minneapolis, http://www.rndsystems.com). After 6-hour incubation at 37°C in a humidified atmosphere, cells per well that had migrated into the lower chamber were counted. The migration ratio = the number of cells migrated into the lower chamber/the number of cells added on the upper chamber initially. All the samples were in duplicates.

Cytoskeleton Organization Stained by F-Actin

Five million T lymphocytes on day 5 in MLR (1:50 group) or DCs induced from CD11b+ cell on day 8 with or without MSC coculture were collected for cytoskeleton detection as previously reported [25]. Briefly, cells were washed twice with PBS containing 1% BSA (PB), permeated with 0.1% Triton X-100 for 20 minutes, and then blocked for 20 minutes with 5% normal sheep serum in PB at room temperature. Cells were labeled with 50 μg/ml FITC-Phalloidin (Sigma-Aldrich) for cytoskeleton staining and 1 μg/ml of 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) for cellular nuclear staining in PB for 40 minutes at room temperature. After thorough washing, cytoskeleton organization pattern was observed. Confocal images were collected by the Zeiss LSM510 Meta (Carl Zeiss, Jena, Germany, http://www.zeiss.com) and were acquired using LSM image browser.

Rac1 and Cdc42 Activity Assay

MSCs were preplated on 6-well plate with α-MEM containing 10% FBS. When confluent, they were further cultured with 50% RPMI 1640 and 50% α-MEM medium without FBS for 24 hours. Twenty million T lymphocytes in MLR or DCs induced from CD11b+ cells with GM-CSF and IL-4 were cultured with or without MSCs. Samples were collected at 15 minutes, 1 hour, and 3 hours after incubation. Rac1/Cdc42 activities were analyzed with Rac1 Activation Assay Kit and Cdc42 Activation Assay Kit (Upstate, Temecula, CA, http://www.upstate.com) by PAK-1 pull-down assay. Briefly, GTP-loaded active Rac1 or Cdc42 was precipitated with PAK-1 p21-binding domain-conjugated agarose beads. Western blotting was performed and visualized with enhanced chemiluminescence reagents (Pierce, Rockford, IL, http://www.piercenet.com).

In Vivo Distribution of Transplanted MSCs

To detect the distribution of transplanted MSCs, cells were infected by an adenovirus vector carrying the enhanced green fluorescent protein cDNA (MSC/eGFP) [26]. MSCs/eGFP (2 × 106) along with mononucleated splenocytes (2 × 107) were injected intravenously into sublethally irradiated BALB/c mice. Forty-eight hours later, samples of the lungs, liver, intestine, spleen, and lymph nodes from the recipients were collected for cryosection examination as previously reported [27]. Briefly, the tissues were fixed in 4% phosphate-buffered paraformaldehyde solution for 2 hours, incubated in 50% paraformaldehyde/sucrose for 12 hours at 4°C, snap frozen in liquid nitrogen, and sectioned into 8 μm by cryoset at −20°C. The sections were then counterstained with 1.0 μg/ml of DAPI in PBS for 20 minutes at room temperature in the dark. Bone marrow cells were collected, and red blood cells were lysed with 0.84% NH4Cl and cytospun onto glass smears. The sections were observed under Olympus CK2 fluorescence microscope (Olympus, Tokyo, http://www.olympus-global.com). The existence ratio of MSCs = The positive sections/All the sections examined per tissue × 100%.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Delayed Development of Lethal aGvHD by MSC Cotransfer

It is well accepted that MSCs display as potent regulators on immune responses in vitro. To determine whether it was the case in an in vivo murine aGvHD model, inocula of graded numbers of cultured MSCs with a constant number of mononuclear splenocytes from C57BL/6 mice have been intravenously injected into sublethally irradiated BALB/c mice. The results showed that the MSTs were significantly prolonged when MSCs were administered at doses of 2 × 106, 1 × 106, and 2 × 105, which meant delayed development of lethal aGvHD. However, when the administration of MSCs (1 × 106) was deferred by 3 days, the MST of the recipient mice was not prolonged. Meanwhile, all the mice of 5-Gy irradiated groups survived after transplantation (Fig. 1A). We selected the respective aGvHD group and aGvHD + MSC group of mice for other experiments.

thumbnail image

Figure Figure 1.. Mesenchymal stem cell (MSC) infusion increases T-lymphocyte number in the secondary lymphoid organs (SLOs) of acute graft-versus-host disease (aGvHD) mice. Survival curves of different groups of mice (A; n = 20). The symptom (B), the representative pathologic changes (C; original magnification ×200), CD3+ cell numbers (D), H2b+ in CD3+ proportions (E), CD3+H2b+ cell numbers (F), splenocyte proliferation (G), and cytotoxicity (H) of aGvHD and aGvHD + MSC mice. *, p < .05; **, p < .01; n = 6.

Download figure to PowerPoint

The Amelioration of the Pathologic Changes of Target Organs and the Enhancement of the Number of T Lymphocytes in the SLOs in aGvHD Mice by MSC Infusion

In agreement with the clinical trials [12, 13], the clinical aGvHD symptomatology including weight loss, ruffled fur, diarrhea, hunched posture, and lethargy were elevated by MSC infusion in this study (Fig. 1B). Organs of aGvHD and aGvHD + MSC mice were collected at the late development stage of aGvHD (day 6) after infusion. Lymphocyte infiltration in the aGvHD target tissues including the liver, jejunum, colon, and skin was milder in aGvHD + MSC recipients compared with that of aGvHD mice. It was noteworthy that there was cellular atrophy, tissue necrosis, and fibrosis in the spleens and lymph nodes of aGvHD mice, whereas large quantities of lymphocytes existed in the aGvHD + MSC mice (Fig. 1C). Further experiments proved that MSC infusion up-regulated the CD3+ cells in spleens of the aGvHD mice and the increased population was mainly donor origin (CD3+H2b+) (Fig. 1D–1F). However, MSC infusion suppressed cell proliferation (Fig. 1G) and cytotoxic effects (Fig. 1H) of T lymphocytes in spleens. The results of the number of T lymphocytes in lymph nodes were similar to those observed in the spleens. All the findings above suggested that MSCs might inhibit aGvHD by enhancing the number of lymphocytes in the SLOs.

Reserved CD62L and CCR7 Expression of CD3+ and CD4+CD25+ Lymphocytes in MLR by MSC Coculture and MSC Supernatants

Since the expression of CD62L and CCR7 molecules is the prerequisite for T-lymphocyte homing into SLOs [28, 29], in vitro experiments were designed and performed to investigate how MSC infusion could have the capacity to lead the enrichment of lymphocytes in SLOs. Our MLR results proved that MSC coculture upregulated the CD62L and CCR7 expression (including CD4+CD25+ Tregs), and the CD69 expression of T lymphocytes was also suppressed in a dose- and time-dependent way (Fig. 2A, 2B). These data indicated that the presence of MSCs downregulated T-lymphocyte activation and maintained their naive-like phenotype. Foxp3, the functional gene of Tregs, was also up-regulated by the MSC coculture detected by PCR (Fig. 2C, 2D) and real-time PCR (Fig. 2E, 2F). Separating MLR from MSCs by transwell with 0.4-μm pores, we found that MSC supernatants play a similarly powerful role in maintaining the CD62L and CCR7 expression on CD3+ cells and Tregs, which indicated that MSCs could exert their function through excreting soluble molecules (Fig. 3A). Moreover, due to the upregulated expression of CCR7, the migratory capability of lymphocyte toward SLC was reserved by MSC coculture and MSC supernatants (Fig. 3B).

thumbnail image

Figure Figure 2.. Mesenchymal stem cell (MSC) coculture reserves CD62L and CCR7 expression on CD3+ and CD4+CD25+ lymphocytes and upregulates FoxP3 expression in mixed lymphoid reaction (MLR). CD62L and CCR7 expression status of CD3+ and CD4+CD25+ lymphocytes in MLR (1:10) of days 1, 3, and 5 (A) and in MLR (0:1, 1:50, 1:10) of day 5 (B). Foxp3 expression detected by polymerase chain reaction (PCR) (C, D) and real-time PCR (E, F). *, p < .05; **, p < .01; n.s., not significant. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PT and MT, the MLR without or with MSC coculture, respectively; 0:1, 1:50, and 1:10, CD3+ cell culture with graded proportions (0:1, 1:50, 1:10, respectively) of 20-Gy irradiated BALB/c spleen mononucleocytes in MLR.

Download figure to PowerPoint

thumbnail image

Figure Figure 3.. Mesenchymal stem cell (MSC) supernatants exhibit comparable effects on the maintenance of T-lymphocyte phenotype and their in vitro migratory activity. (A): CD62L and CCR7 expression status on CD3+ and CD4+CD25+ lymphocyte in mixed lymphoid reaction (MLR) separated from MSCs by transwell membrane on day 5. (B): The migratory activity of those cells towards secondary lymphoid tissue chemokine tested by transwell assay. *, p < .05; n.s., not significant. Abbreviations: PT and PT + MSC, MLR in the absence or presence of MSC coculture, respectively; PT + MSC/trans, separating MLR from MSCs by transwell with 0.4-μm pores.

Download figure to PowerPoint

Potent Role of MSC Supernatants in Inhibiting the CCR7 Expression and Functionality of DCs

Considering that CCR7 determined migratory activity of DCs into SLOs is a critical step for specific cellular immunity, we observed what effects MSCs have on DCs. In vitro experiments were performed, and we found that MSCs suppressed DCs in the expression of CCR7 and other costimulatory molecules, such as CD40, CD80, CD86, and Iab, during induction from CD11b+ cells by IL-4 and GM-CSF for 4 days and during maturation by supplementation with LPS for another 4 days. This suppression effect could be achieved mostly by the supernatants of MSCs when separating them with transwell membrane with 0.4-μm pores (Fig. 4A). Moreover, the migratory activity elicited by SLC was restrained (Fig. 4B). When loading DCs induced from CD11b+ cells with CD3+ lymphocytes in the absence or presence of MSCs, the submature DCs suppressed by MSCs had weak capability to drive lymphocyte activation, which meant MSCs could exert their immunomodulatory function on T lymphocytes via suppressing the maturation of DCs (Fig. 4C).

thumbnail image

Figure Figure 4.. Mesenchymal stem cells (MSCs) affect maturation and functionality of dendritic cells (DCs). Morphologic and phenotypic characteristics of magnetically sorted splenic CD11b+ cells (row Aa) and DCs induced from CD11b+ cells (row Ab–Ag). Original magnification ×300. (B): Migratory activities of those cells. (C): CD62L and CCR7 expression on CD3+ and CD4+CD25+ lymphocytes when loading DCs of day 8 with CD3+ lymphocytes for 3 days. *, p < .05; **, p < .01; n.s., not significant. Abbreviations: DC, DCs induced from CD11b+ cells; DC/MSC, DCs induced from CD11b+ cells with MSC coculture; DC/MSC/trans, DCs induced from CD11b+ cells with the separated MSCs by transwell membrane with 0.4-μm pores; MLR, mixed lymphoid reaction.

Download figure to PowerPoint

Cytoskeleton Reorganization and Rac1 and Cdc42 Activating Inhibition of T Lymphocytes and DCs by MSC Coculture

Recently, family members of the Rho-like GTPases, including Rac1 and Cdc42, were found to play important roles in actin cytoskeleton changes that are required for T-cell activation and DC adhesion and migration [30, [31], [32]33]. Therefore, we examined the cytoskeleton changes and some Rho-like GTPase activating status to further investigate the mechanisms that MSCs had on T-cell activation and DC migratory properties. Our results demonstrated that MSC coculture suppresses the polarization and pseudopodium formation on T lymphocytes (Fig. 5A, 5B) and the polarization and dendriform prominence formation on DCs (Fig. 5C, 5D). Suppressed activation of Rac1 and Cdc42 by MSCs might explain the restrained actin cytoskeleton changes in T lymphocytes and DCs (Fig. 5E, 5F).

thumbnail image

Figure Figure 5.. Mesenchymal stem cells (MSCs) suppress cytoskeleton reorganization and activation of Rac1 and Cdc42. Cytoskeleton pattern of T lymphocytes (A, B) and dendritic cells (DCs) (C, D) observed by confocal microscopy. Fluorescein isothiocyanate (FITC)-Phalloidin (green), 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue); red line = 5 μm. Activation status of Rac1 and Cdc42 in T cells (E) and DCs (F) examined by PAK-1 pull-down assay. Abbreviations: PDC and MDC, DC induction from CD11b+ cells for 8 days without or with MSC coculture, respectively; PT and MT, mixed lymphoid reaction in the absence or presence of MSC coculture, respectively.

Download figure to PowerPoint

Reserved CD62L and CCR7 Expression on Splenic CD3+ and CD4+CD25+ Lymphocytes in aGvHD Mice by MSC Infusion

The data above have demonstrated that MSCs can certainly affect the migratory capacity of T lymphocytes and DCs in vitro. To examine whether it was the same case in vivo, murine aGvHD was developed and the phenotypic change of splenocytes was analyzed at 12 hours, day 2, day 4, and day 6 after infusion. The phenotypic changes of T lymphocytes at the various time points were of the same trend. Data in Figure 6A showed the respective result of day-4 aGvHD; T lymphocytes were almost all donor derived (CD3+/H2b+) in spleen and their proportions were increased by MSC transfusion. Further analysis revealed that the percentages of CD62L+ and CCR7+ subpopulations in T lymphocytes were also increased. MSC infusion did not greatly enhance the proportions of the CD4+CD25+ population that took up only a small portion (approximately 5%). However, when the CD4+CD25+ lymphocytes were further analyzed, the percentages of CD62L+ and CCR7+ cells in the CD4+CD25+ population were dramatically greater in aGvHD + MSC mice than those of aGvHD mice. The phenotype exhibition patterns of cells in lymph nodes were similar to those in the spleens (data not shown).

thumbnail image

Figure Figure 6.. Mesenchymal stem cell (MSC) infusion reserves CD62L and CCR7 expression of CD3+ and CD4+CD25+ lymphocytes and downregulates CCR7 expression and proportion of CD11b+ cells in spleens of acute graft-versus-host disease (aGvHD) mice. CD62L and CCR7 expression on CD3+ and CD4+CD25+ regulatory T cells (A), and CCR7 expression on CD11b+ cells and magnetically sorted CD11b+ cells (B) in spleens at day 4 after transplantation. The proportions (C) and numbers (D) of splenic CD11b+ cells. *, p < .05; **, p < .01. n = 9.

Download figure to PowerPoint

Downregulation of CCR7 Expression and Proportion of CD11b+ Cells in Spleens of aGvHD Mice by MSC Infusion

In vitro experiments proved that MSCs could suppress the maturation and SLC-induced migration of DCs. Additionally, MSCs could suppress T-lymphocyte activation via suppressing DC maturation. To examine whether this is one of the mechanisms through which MSCs ameliorate aGvHD, we measured the proportion and the number of CD11b+ cells in spleens of aGvHD mice. MSC coinfusion decreased the proportion of CD11b+ cells in spleens at various time points after infusion (Fig. 6C). Because of the enhancement of total number of spleen cells, the decrease of the absolute number of CD11b+ cells was obvious only at 12 hours after infusion (Fig. 6D). The phenotypic change of splenocytes was analyzed at 12 hours, day 2, day 4, and day 6 after infusion. The phenotypic changes of DCs at the various time points were of the same trend. Data in Figure 6B showed the respective FCM result of day-4 aGvHD; the CCR7 expression on CD11b+ cells in spleens was downregulated by MSC infusion (left panel). In consideration of the rarity of CD11b+ cells, the cells were magnetically sorted to confirm that result. Almost all the CD11b+ cells expressed CD11c molecule, the hallmark of mature DCs. The CCR7 expression on CD11b+ and CD11c+ cells in spleens was downregulated by MSC infusion (right panel), which might be the reason of the restrained number of CD11b+ cells in spleen.

MSC Distribution In Vivo After Infusion

Many studies suggested that MSCs inhibit immune responses via direct cellular contact and/or cytokine secretion in vitro [6, 34]. It is not clear how MSCs modulate immune response in vivo. If cellular contact is an essential mechanism, one would expect to see a significant number of MSCs in SLOs after transplantation. However, this is not the case. We have systematically examined the tissue distribution of eGFP-labeled MSCs 48 hours after transplantation (Fig. 7A) and found that MSCs are primarily distributed to lung, liver, intestine, bone marrow, and to a much less degree to SLOs (5% ± 10% in the spleen and 0% ± 0% in the lymph nodes) (Fig. 7B). Lack of CD62L and CCR7 expression on MSCs, the signal code to entry into SLOs, might be the main cause for the low distribution of MSCs in SLOs (Fig. 7E, 7F). Together with the in vitro result, this result strongly suggests that MSCs exert their immunoregulatory role mainly by secreting soluble molecules in this murine aGvHD model.

thumbnail image

Figure Figure 7.. Mesenchymal stem cells (MSCs) can be detected readily in various peripheral tissues after infusion, but rarely in secondary lymphoid organs (SLOs). (A): The distribution pattern of MSCs/enhanced green fluorescent protein (eGFP) in vivo at 48 hours after transplantation. MSCs/eGFP: green; 4′, 6-diamidino-2-phenylindole dihydrochloride: blue. (B): The existence ratios of MSCs. Phase contrast and fluorescent images (Ca, Cb) and eGFP expression of MSCs/eGFP (Cc). (Cd–Cf): CD62L and CCR7 expression on MSCs. Abbreviations: FSC, forward scatter; SSC, side scatter.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Despite the improvements in allogeneic stem cell transplantation, aGvHD remains a significant problem after transplantation and a major cause of post-transplant mortality. Since the rebirth of MSCs more than 20 years ago, the cellular and molecular mechanisms through which they act to suppress immune responses have been extensively investigated in vitro and laid a fast foundation under the booming application of conquering aGvHD clinically [12], yet the exact mechanisms through which MSCs act in vivo are largely unknown.

With a murine lethal aGvHD model, we found that MSC infusion could significantly delay the development of aGvHD and lessen the lymphocyte infiltration and pathologic injuries of target organs of aGvHD mice. Noticeably, higher cellularity was evident in the SLOs of MSC cotransferred mice (Fig. 1A–1C). It is accepted that high-grade aGvHD is always accompanied with severe leucopenia in spleen [35]. Consistently, our results also confirmed that MSCs alleviated the cellular decrease in the SLOs, which meant low-grade aGvHD occurred. Further experiments proved that MSC infusion up-regulated the CD3+ cells in spleens of the aGvHD mice and the increased population was mainly donor origin (CD3+H2b+) (Fig. 1D–1F). The increase of donor original T-cell number in SLOs is the integrative consequence of whole body cell migration, local cell proliferation, and cytotoxicity. Our investigations showed that the higher number of donor T lymphocytes in SLOs caused by MSC infusion was not the result of T-lymphocyte proliferation and cytotoxicity (Fig. 1G, 1H). So, it suggests that MSCs inhibited aGvHD by increasing the traffic of T lymphocytes to the SLOs and thus reducing their migration toward target tissues, which is required for the development of aGvHD [15, 36].

The homing of T lymphocytes to SLOs is high endothelial venules (HEVs)-determined and requires definite addressing codes. The interaction between CD62L on lymphocytes and the vascular addressins on HEVs initiates cell rolling on HEVs. SLC, a chemokine that is constitutively presented by HEVs, is able to activate CCR7. SLC/CCR7 cognation elicits rapid Gαi signaling and leukocyte function-associated antigen 1 (LFA-1)-mediated cell arrest; then the transmogrification and extravasation occurs [28, 29]. According to the expression of CD62L and CCR7, T lymphocytes are classified into four different subsets: naive T, central memory T (Tcm), effector memory T (Tem), and effector T (Teff) cells. Naive and Tcm cells express CD62L and CCR7 and therefore can home SLOs. Upon priming and activation into Tem and Teff cells, they lose the expression of CD62L and CCR7, upregulate tissue-specific addressing codes, and emigrate from SLOs into the inflammatory tissues [35]. Our in vitro experiments here showed that the presence of MSCs maintained the expression levels of CD62L/CCR7 molecule on T lymphocytes in MLR and SLC-driven migratory activity. Meanwhile, higher percentages of CD62L+/CCR7+ on CD3+ lymphocytes and higher number of CD3+ cells were also evident in the SLOs of aGvHD + MSC mice. Coculture or cotransfer with MSCs also reduced expression of a T-lymphocyte early activation marker, CD69, further strengthening the observations that MSCs preserved more naive-like lymphocytes in allogeneic immune reaction in vitro and in vivo. These findings suggest that MSCs downregulate T-lymphocyte activation status, promote their homing to and subsequent trapping in SLOs, and consequently, reduce allogeneic lymphocyte infiltration into the aGvHD target tissues.

As a significant part of T lymphocytes, Tregs play important roles in the immune response [37]. Previous studies have demonstrated that ex vivo-expanded Tregs can prevent GvHD by inhibiting activation/differentiation of pathogenic T cells [20]. Furthermore, Tregs mature in the SLOs of the recipients [38]. Recent investigations have showed that only CD62Lhigh but not the CD62Llow Tregs are potent inhibitors of GvHD and bone marrow graft rejection [39, 40], and that CCR7 expression is a prerequisite for their in vivo function [41]. In our study, we found that MSCs also reserved the CD62L and CCR7 expression pattern of Tregs in MLR and in SLOs of aGvHD mice. Therefore, it is plausible to propose that the increased Treg trafficking and homing to SLOs contribute to the immunoregulatory effects of transplanted MSCs.

SLOs are also a strategically positioned collecting station for immune information, where a naive T cell encounters its right partner, an antigen-presenting cell (APC) carrying specific peptide-MHC complexes, and activates and generates armed effectors under the signaling cues from the APC [42, [43]44]. As one kind of professional APC, DCs are the most potent initiator of in vivo immune responses. Interestingly, CCR7 also exerts a key role in DC maturation and migration. Immature DCs express chemokine receptors that ensure their localization in peripheral tissues. Upon their maturation, DCs upregulate CCR7 expression, migrate to SLOs, and localize within the T-cell regions, where they initiate T-cell activation [45]. If the increased quantity of T lymphocytes by MSC infusion counters equal proportion of DCs in SLOs, it can result in forming more armed effector T cells. So we reasonable to suppose MSCs may also have impact on the DCs in SLOs. Our results here demonstrate that the presence of MSCs can significantly inhibit the expression of CCR7 and a series of costimulatory molecules during the induction process of DCs from murine CD11b+ cells in vitro (Fig. 4A). Consistently, MSC administration lowered the CCR7 expression on both CD11b+ and CD11c+ cells, and the proportion and number of CD11b+ in the recipient spleens of aGvHD mice (Fig. 6C, 6D). Therefore, the results indicated that MSCs can alter the migratory capacity of DCs to SLOs and thus, result in the inefficiency of armed Teff transformation from naive T cells and their subsequent infiltration into the targeted tissues.

Recently, family members of the Rho-like GTPases, including Rac1 and Cdc42, have been found to play important roles in actin cytoskeleton changes that are required for cell activation, adhesion, migration, and invasion [28]. T-cell polarization toward and within the cellular interface with an APC plays an important role for effective T-cell activation. It is reported that Rac1 and Cdc42 activity is critical for T-cell polarization and activation. Also, they are required for DC adherence, migration, chemotaxis, and endocytosis and antigen presentation [31, [32]33]. Thus, as shown in Figure 5, the suppressed activation of Rac1 and Cdc42 by MSCs might explain the alteration of cellular actin cytoskeleton recomposition that is essential for T-lymphocyte activation, DC migration, and function exerting.

Many studies have shown that MSCs suppress T-cell immunity in vitro by both direct cell-cell contact and the bioactive substances that MSCs release in the supernatants [6, 34]. Our transwell experiments showed that the MSC supernatant is sufficient for the suppressive activity of MSCs on the phenotypic changes of T cells and DCs (Figs. 3A, 4A) and the chemotaxis activity elicited by SLC (Figs. 3B, 4B). Thus, it is acceptable to deduce that MSCs conserve the migratory properties of both T cells and DCs mainly through the secretory pathway. ELISA examination showed that MSCs secret prostaglandin E2 and trace amounts of IL-4, IL-10, and transforming growth factor β1 (data not show). It is important to further explore which molecule(s) secreted by MSCs plays a crucial role in immune regulation.

Interestingly, when eGFP-expressing MSCs were traced after being intravenously infused into the sublethally irradiated mice, we found that transplanted MSCs could be readily observed in the lung, liver, intestines, and bone marrow. In contrast, the spleens and lymph nodes were the sites where eGFP-MSCs could be scarcely detected in situ by fluorescent microscope [46, 47]. The lack of CD62L and CCR7 expression on MSCs might be the cause of their scarce distribution in SLOs (Fig. 7Ce, Cf) [48, 49]. Consistent with the notion that the capacity of MSCs to secrete soluble factors that alter the tissue microenvironment may play a more prominent role in effecting tissue repair in vivo [50], our finding revealed that the soluble molecules secreted by MSCs are the primary contributor to the altered migratory properties of the immune cells in aGvHD mice.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Our study revealed that in the murine lethal aGvHD scenario, cotransfusion of MSCs can delay the development of lethal aGvHD through inhibiting the migration of effector T lymphocytes to the target tissues. This immunoregulatory activity is realized by downregulating the addressing process of DCs and upregulating the homing activity of Tregs to SLOs, and as a result, more T lymphocytes take the naïve-like phenotype and enrich in the SLOs largely via released soluble molecules of MSCs.

This study provides new clues for clarifying the complexity underlying the in vivo activity of MSCs and may be beneficial to the design of novel therapeutic approaches of MSCs in the setting of aGvHD treatment clinically.

Disclosure of Potential Conflicts of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

The authors indicate no potential conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

We sincerely thank Dr. Sheng Zhou and Yili Yang for critical review of the manuscript. We also thank Li Liao from the Department of Hematopoietic Stem Cell Transplantation, affiliated hospital to Academy of Military Medical Sciences, for her excellent technical assistance in FCM analysis. This study was supported by the National Key Basic Research Program of China (Grant 2005CB522705) and National Natural Sciences Foundation of China (Grants 30730043 and 30600309).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References