Engineering mesenchymal stem cells for immunotherapy

Abstract

Allogeneic hematopoietic stem cell transplantation, after sublethal irradiation of recipient animals, is capable of inducing donor-specific tolerance facilitating subsequent organ transplantation. This approach could reintroduce tolerance in autoimmune diseases and it has been applied to treat autoimmune diseases with, however, a great susceptibility of recurrence. Mesenchymal stem cells (MSCs) present within the bone marrow could be critical to the immunosuppressive effect of the treatment. This tolerance induction may be useful in allogeneic transplantations, where low incidence of graft-versus-host disease was observed when the hematopoietic graft was coinjected with MSCs. In this paper, we discuss the use of MSCs in different therapeutic strategies either as immunosuppressive agents or genetically engineered to express molecules acting against the autoimmune process.

Introduction

Conventional allogeneic bone marrow transplantation is a new strategy to treat a large range of autoimmune diseases. More than 437 patients have been treated with this procedure, including patients with multiple sclerosis (MS), scleroderma, lupus, dermatopolymyositis, rheumatoid arthritis (RA) or juvenile arthritis.1 Autoimmune hematopoietic disorders, including hemolytic anemia, thrombopenia and aplasia, have also been treated. However, the success rate of this approach is lowered by graft-versus-host disease (GVHD), donor graft rejection or incomplete T-cell recovery. Therefore, autologous stem cell transplantation is a preferred therapeutic option, although rapid recurrence has been described. Nevertheless, the immune effect of hematopoietic stem cell transplantation (HSCT) is difficult to distinguish from the effect of the myeloablative regimen, including cyclophosphamide, associated with a low-dose irradiation treatment.

Mesenchymal stem cells (MSCs) are the progenitors of multiple mesenchymal lineages, including bone, cartilage, muscle, fat tissue, marrow stroma as well as astrocytes.2 MSCs are adult stem cells, present in most of the tissues involved in autoimmune diseases. As these MSCs could target the pathological organs, they represent an ideal vehicle to express therapeutic proteins. Moreover, these cells display immunological properties that may enhance their therapeutic potential in autoimmunity. At least, the use of multipotent cells opens the perspective of regenerative medicine after control of the autoimmune process. Since our knowledge of the pathways leading to autoimmunity and chronic tissue inflammation has been improved, new targets for efficient immunotherapy have emerged. This has led to the development of biologics targeting cytokines, the best example being TNF blockade in RA and Crohn's disease, or interferon-β in MS.3,4 Here, we discuss the validity of developing a tissue-specific delivery of cytokine antagonists in order to improve the efficiency of immunotherapy.

MSC characteristics

MSCs or marrow stromal cells are pluripotent cells present in the bone marrow at low quantity (1 out of 104–105 mononuclear cells), which are capable of differentiation into chondrocytes, osteocytes, myocytes and adipocytes.5 MSCs can be expanded more than 104-fold in culture without loss of their phenotype or multilineage potential. Recent studies have revealed that the initial and subsequent plating cell concentrations are a critical parameter.6 Proliferation is increased when low concentrations of cells (<1000 cells/cm2) are seeded, while induction of some differentiation pathways is observed with high cell concentrations, underlining the role of intercellular communications. MSCs are distinct from hematopoietic stem cells as they are not progenitors for the hematopoietic lineages and do not express CD34, CD45, CD14, glycophorin A, T- or B-cell markers.7 MSC potential is routinely determined by the colony-forming unit fibroblast assay (CFU-F) and MSCs are identified by expression of Thy-1 (CD90), endoglin (CD105), vascular cell adhesion molecule (VCAM)-1 (CD106) and hyaluronate receptor (CD44) (reviewed in Noël et al8). Although MSCs are currently recovered from bone marrow, they have also been reported to be isolated from muscle, synovium, cord blood or adipose tissue.9,10,11,12 Standard conditions for MSC generation from bone marrow rely on their adherence capacities among the mononuclear cell population after a 10- to 14-day period of culture, generally using a medium containing fetal bovine serum and, in some reported cases, supplementation with growth factors, such as basic fibroblast growth factor (b-FGF), platelet-derived growth factor (PDGF) or epidermal growth factor (EGF).13 It has recently been shown that MSCs derive from a common progenitor cell, the multipotent adult progenitor cell (MAPC) that gives rise to other lineages as different as endothelium, endoderm and ectoderm.14 The MSCs support the growth of hematopoietic progenitors by secreting a number of hematopoietic cytokines such as macrophage colony stimulating factor (M-CSF), interleukin (IL)-6, IL-7, IL-8, IL-11, IL-12, IL-14, IL-15 and leukemia inhibitory factor (LIF).15 MSCs also have the potential to attract infused hematopoietic stem cells to the marrow by expressing homing receptors and chemokines, such as stromal-derived factor (SDF)-1.

Immunological characteristics of MSCs

It has been suggested that MSCs escape the immune system because they possess a cell surface phenotype which reflects poor recognition by T cells. The absence of MHC II or T-cell costimulatory molecules B7-1, B7-2, CD40 or CD40L could partially explain why human MSCs are not recognized by allogenic T cells.16 Moreover, MSCs have recently been shown to suppress T-cell responses as, in vitro, addition of MSCs to a mixed lymphocyte reaction (MLR) blocked the proliferation of allogeneic T cells.17 This MLR inhibition was dose dependent with an optimal effect at a MSC:T cell ratio of 1:1. The delayed addition of MSCs still inhibited the maximal T-cell response from 96% suppression at day 1 to 73% at day 3. In these conditions, addition of IL-2 partially antagonized the MSC-induced suppression of MLR. Thus, MSCs appeared to be immunosuppressive, probably via the secretion of cytokines such as transforming growth factor (TGF)-β or hepatocyte growth factor (HGF) whereas IL-6 and IL-11 were not involved.18 MSCs failed to induce allogeneic T-cell response in vivo, but they were also shown to induce immune tolerance in the host. Even xenogeneic transplantation of MSCs was well tolerated as demonstrated by the capacity of murine MSCs to colonize and engraft into the injured myocardium of rats after coronary ligation.19 In these last experiments, it must be emphasized that not only allogeneic MSCs were not rejected but they kept their potential of differentiation. These data thus suggest that an universal donor for MSCs may be used in a therapeutic design.

Tissue-homing capacities of MSCs

The capacity of MSCs to adhere to matrix components favors their preferential homing to bone, lung and cartilage when injected intravenously. These homing properties were illustrated in a recent study reporting the systemic infusion of MSCs expressing a marker gene in irradiated syngeneic mice. After 1 month, 8% of bone cells and 5% of lung cells expressed the marker gene.7 In baboons receiving labeled MSCs, cells were still detected in the bone marrow for more than 500 days post-transplantation.20 Moreover, GFP-labeled cells were not detected in other tissues despite the use of a sensitive PCR technique, suggesting that MSCs home preferentially to the bone marrow. Following intracerebral implantation, MSCs were able to proliferate, differentiate and appeared to migrate along the well-defined neural migration pathways.21,22 Moreover, intracerebral injection of marrow MSCs in a mouse model of types A and B Niemann–Pick disease (NPD) revealed that transplanted cells survived at least 6 months after transplant and significantly delayed the Purkinje cell loss characteristic of NPD.23 In another study, patients with osteogenesis imperfecta (a genetic disorder caused by a mutation in the type I collagen gene and characterized by generalized osteopenia) have been treated by systemic injection of allogeneic bone marrow. The treatment resulted in osteoblast engraftment (1.5–2% of donor cells) at 3 months and increase of both total body bone mineral content and skeletal growth.24 In this strategy, the systemic injection of a large amount of MSCs prior to any irradiation was well tolerated. Thus, this set of experimental and clinical results emphasized the therapeutic potential of MSCs.

Hematopoietic and MSCs in autoimmune diseases

Allogeneic HSCT, after sublethal irradiation of recipient animals, is capable of inducing donor-specific tolerance facilitating subsequent organ transplantation. Thus, chimeric mice obtained after allogeneic bone marrow transplantation did not reject cardiac graft from the same donor origin performed 2 months later.25 This state of tolerance allows withdrawal of immunosuppressive medication. Then, it seemed possible that this approach could reintroduce tolerance in autoimmune diseases and it has been applied to treat severe scleroderma, psoriatic arthritis, life-threatening lupus or RA. Different open studies with a low number of patients and short time follow-up were conducted with various clinical results depending on the treated disease. In dermatopolymyositis, for example, patients did not seem to improve significantly.26 In scleroderma, 62 patients have been followed for 3 years with an 8% mortality rate observed after the procedure of HSCT.1 Although a slight improvement was observed, 35% of patients relapsed progressively in the last year of follow-up. In RA, 45% of the patients improved but with a relapse overtime, for half of the patients. Recently, a large controlled study of HSCT was conducted, enrolling 33 patients with refractory RA who were randomized to receive unmodified BMT after standard cyclophosphamide regimen or CD34-selected HSCT.27 The most informative results of this study were that all patients relapsed within 180 days, with no difference whether the patients received highly purified CD34-positive HSCs or unmanipulated bone marrow cells. This suggests that reintroduction of HSCs giving rise to näve T cells will over time lead to aggressive autoimmune cells, depending on the patient genetic background or persistence of the environmental conditions that favor the autoimmune process. In juvenile arthritis, the clinical results seemed to be better, and joint histology showed that local inflammation and T-cell infiltrate decreased as the children improved.28 The benefit of this aggressive regimen has still to be compared with the anti-TNF treatment in terms of efficiency. However, it seems reasonable to include in HSCT protocols patients refractory to TNF-blocking agents. In case of genetically determined autoimmunity, an allogeneic source of stem cells should be necessary to avoid relapse of autoimmunity. However, MSCs or stromal cells present within the bone marrow could be critical to the immunosuppressive effect of the treatment.

Experimental autoimmune diseases, such as adjuvant arthritis in rats collagen-induced arthritis (CIA) or experimental encephalomyelitis in mice, may be cured by bone marrow transplantation.29,30 In these experimental procedures, the total bone marrow transplant consists of both HSCs and stromal cells and among these, MSCs play a central role in inducing tolerance. Allogeneic bone marrow transplantation associated to bone grafts was found to be efficient in the treatment of autoimmune disorders, such as in the MLR/lpr mouse model of lupus.31 At 40 weeks, mice treated with this approach were cured, with a significant decrease in the anti-DNA and antirheumatoid factor (anti-RF) antibody titers in the serum and absence of immunoglobulin deposits in the renal glomeruli. All treated mice survived more than 1 year. In these experiments, stromal cells have been assumed to play a critical role as compared to HSCs. In order to determine the real impact of MSCs in these experiments, the adherent cells were removed from the total bone marrow samples before transplantation. In this case, 75% of the treated animals died within 90 days. In contrast, complementation of adherent cell-depleted bone marrow with stromal cells permitted to cure the autoimmune disease, suggesting that MSCs play a critical role in the therapeutic effect. As an illustration, systemic infusion of autologous MSCs delayed donor cell rejection from 7 to 11 days, in a skin graft murine model.17 In this model, the administration of large amounts of autologous MSCs was shown to be comparable in terms of efficiency to the delivery of cyclosporine conjugated with anti-CD80 therapy. This tolerance induction may be useful in allogeneic transplantations, where low incidence of GVHD was observed when the hematopoietic graft was coinjected with MSCs.32 The tolerigenic effect of MSC delivery was equivalent whatever was the route of cell injection, intravenously or loaded onto an osteo-inductive matrix.

Presence of MSCs has been demonstrated in mesenchymal-derived tissue such as injured synovium in case of chronic inflammation, both in RA patients or in arthritic joints of DBA1 mice with CIA.33,34 In experimental CIA, MSCs were found in the joints of animals at 2 weeks before the onset of the disease. At that time, synovial hyperplasia was associated to an increase of medullar cells in the epiphyseal region and formation of canals between the bone marrow and the joint cavity.34 Histological analysis suggested that MSCs could migrate from the bone marrow. Up to 2% of the cells stained positively for one marker of MSCs, the bone morphogenetic protein receptor (BMPR)-I, and negatively for the HSC-specific CD34 marker. Accumulation of MSCs in the joint was TNFα-dependent, as an anti-TNF treatment could prevent it. The role of MSCs in the arthritic process is yet unsolved, but their ability to produce chemokines and cytokines such as IL-6, IL-12 and IL-15 could suggest an active role in chronic inflammation. Although the origin of these stem cells in the joint is unknown, their presence in the normal synovium has also been demonstrated in humans and in rabbits, where TGFβ stimulation was shown to induce cartilage tissue formation.32,35

Engineering MSCs

MSCs can be used in different therapeutic strategies either as immunosuppressive agents or genetically engineered to express molecules acting against the autoimmune process. As MSCs are not rejected and traffic to injuried mesenchymal tissues, in particular bone, they represent the opportunity to deliver therapeutic proteins. MSCs engineered to express BMP-2, a cytokine involved in bone differentiation, could differentiate into bone when implanted in the muscle.36 They were also capable of inducing the repair of segmental bone defects in mice. In this case, cell-mediated therapy offered the advantages of recruiting MSCs from the recipient (through a paracrine effect) and inducing new endochondral bone formation (autocrine effect). In an experimental SCID/hu model, human MSCs engineered to produce IL-3 were seeded into osteo-inductive ceramic cubes that were then subcutaneously implanted into mice.32 In this study, MSC differentiation occurred inside the cubes and resulted in bone formation and IL-3 secretion into the systemic circulation for at least 12 weeks. MSCs are easily transduced by retroviral vectors, with a gene transfer efficacy between 50 and 85%.37 This allowed long-term transgene expression, above 3 months in rodent models. MSCs may also be used as a vehicle to express cytokines and they have been efficiently transduced to secrete several therapeutic proteins such as human erythropoietin (EPO) or factor IX and in vivo, secretion was detected for up to 90 days following subcutaneous cell implantation.38 As another example, interferon-β-expressing MSCs were able to inhibit the growth of malignant cells in vivo when coimplanted within the tumor cells, but this could not be achieved when MSCs were implanted at a site distant from the tumor.39 Similarly, implantation of MSCs genetically engineered to express the soluble tumor necrosis factor receptor (sTNF-R) II into NOD/SCID mice resulted in detection of the cytokine in the serum and efficacy of the treatment was demonstrated by decreased serum levels of mouse TNFα.40

Conclusion

Engineered MSCs have a great potential in the treatment of autoimmune diseases. Their immunosuppressive effect is in part related to secretion of immunosuppressive cytokines such as TGFβ and HGF that should block the T helper 1 (Th1)-cell-driven response. Moreover, these MSCs may be efficiently genetically modified to express anti-inflammatory cytokines Finally, their homing capacities to mesenchymal tissues, in particular to injured sites, may contribute to their potential use in the treatment of autoimmune diseases.

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Correspondence to C Jorgensen.

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Keywords

  • stem cells
  • immune tolerance
  • arthritis
  • autoimmunity

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