Jef Pinxteren

Application of MultiStem® Allogeneic Cells for Immunomodulatory Therapy: Clinical Progress and Pre-Clinical Challenges in Prophylaxis for Graft Versus Host Disease

The last decade has seen much progress in adjunctive cell therapy for immune disorders. Both corporate and institutional Phase III studies have been run using mesenchymal stromal cells (MSC) for treatment of Graft versus Host Disease (GvHD), and product approval has been achieved for treatment of pediatric GvHD in Canada and New Zealand (Prochymal®; Osiris Therapeutics). This effectiveness has prompted the prophylactic use of adherent stem cells at the time of allogeneic hematopoietic stem cell transplantation (HSCT) to prevent occurrence of GvHD and possibly provide stromal support for hematopoietic recovery. The MultiStem® product is an adult adherent stem cell product derived from bone marrow which has significant clinical exposure. MultiStem cells are currently in phase II clinical studies for treatment of ischemic stroke and ulcerative colitis, with Phase I studies completed in acute myocardial infarction and for GvHD prophylaxis in allogeneic HSCT, demonstrating that MultiStem administration was well tolerated while the incidence and severity of GvHD was reduced. In advancing this clinical approach, it is important to recognize that alternate models exist based on clinical manufacturing strategies. Corporate sponsors exploit the universal donor properties of adherent stem cells and manufacture at large scale, with many products obtained from one or limited donors and used across many patients. In Europe, institutional sponsors often produce allogeneic product in a patient designated context. For this approach, disposable bioreactors producing <10 products/donor in a closed system manner are very well suited. In this review, the use of adherent stem cells for GvHD prophylaxis is summarized and the suitability of disposable bioreactors for MultiStem production is presented, with an emphasis on quality control parameters, which are critical with a multiple donor approach for manufacturing.

Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Fourth Meeting: Lessons Learned from First Clinical Trials

The Fourth Expert Meeting of the Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Consortium took place in Barcelona on October 19 and 20, 2012. This meeting focused on the translation of preclinical data into early clinical settings. This position paper highlights the main topics explored on the safety and efficacy of mesenchymal stem cells as a therapeutic agent in solid organ transplantation and emphasizes the issues (proper timing, concomitant immunossupression, source and immunogenicity of mesenchymal stem cells, and oncogenicity) that have been addressed and will be followed up by the MiSOT Consortium in future studies.

Human multipotent adult progenitor cells are nonimmunogenic and exert potent immunomodulatory effects on alloreactive T-cell responses.

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohns disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.

Clinical-Grade Multipotent Adult Progenitor Cells Durably Control Pathogenic T Cell Responses in Human Models of Transplantation and Autoimmunity

A major goal of immunotherapy remains the control of pathogenic T cell responses that drive autoimmunity and allograft rejection. Adherent progenitor cells, including mesenchymal stromal cells (MSCs) and multipotent adult progenitor cells (MAPCs), represent attractive immunomodulatory cell therapy candidates currently active in clinical trials. MAPCs can be distinguished from MSCs on the basis of cellular phenotype, size, transcriptional profile, and expansion capacity. However, despite their ongoing evaluation in autoimmune and allogeneic solid organ transplantation settings, data supporting the immune regulatory potential of clinical-grade MAPCs are limited. In this study, we used allogeneic islet transplantation as a model indication to assess the ability of clinical-grade MAPCs to control T cell responses that drive immunopathology in human autoimmune disease and allograft rejection. MAPCs suppressed T cell proliferation and Th1 and Th17 cytokine production while increasing secretion of IL-10 and were able to suppress effector functions of bona fide autoreactive T cells from individuals with type 1 diabetes mellitus, including killing of human islets. Furthermore, MAPCs favored the proliferation of regulatory T cells during homeostatic expansion driven by γ-chain cytokines and exerted a durable, yet reversible, control of T cell function. MAPC suppression required licensing and proceeded via IDO-mediated tryptophan catabolism. Therefore, the common immune modulatory characteristics of clinical-grade MAPCs shown in this study suggest that they can be regarded as an alternative source of adult progenitor cells with similar clinical usefulness to MSCs. Taken collectively, these findings may guide the successful deployment of both MSCs and MAPCs for the amelioration of human autoimmunity and allograft rejection.

Heart Grafts Tolerized Through Third-Party Multipotent Adult Progenitor Cells Can Be Retransplanted to Secondary Hosts With No Immunosuppression

Multipotent adult progenitor cells (MAPCs) are an adherent stem cell population that belongs to the mesenchymal‐type progenitor cell family. Although MAPCs are emerging as candidate agents for immunomodulation after solid organ transplantation, their value requires further validation in a clinically relevant cell therapy model using an organ donor‐ and organ recipient‐independent, third‐party cell product. We report that stable allograft survival can be achieved following third‐party MAPC infusion in a rat model of fully allogeneic, heterotopic heart transplantation. Furthermore, long‐term accepted heart grafts recovered from MAPC‐treated animals can be successfully retransplanted to naïve animals without additional immunosuppression. This prolongation of MAPC‐mediated allograft acceptance depends upon a myeloid cell population since depletion of macrophages by clodronate abrogates the tolerogenic MAPC effect. We also show that MAPC‐mediated allograft acceptance differs mechanistically from drug‐induced tolerance regarding marker gene expression, T regulatory cell induction, retransplantability, and macrophage dependence. MAPC‐based immunomodulation represents a promising pathway for clinical immunotherapy that has led us to initiate a phase I clinical trial for testing safety and feasibility of third‐party MAPC therapy after liver transplantation.

Dissection of the Human Multipotent Adult Progenitor Cell Secretome by Proteomic Analysis

Multipotent adult progenitor cells (MAPCs) are adult adherent stromal stem cells currently being assessed in acute graft versus host disease clinical trials with demonstrated immunomodulatory capabilities and the potential to ameliorate detrimental autoimmune and inflammation‐related processes. Our previous studies documented that MAPCs secrete factors that play a role in regulating T‐cell activity. Here we expand our studies using a proteomics approach to characterize and quantify MAPC secretome components secreted over 72 hours in vitro under steady‐state conditions and in the presence of the inflammatory triggers interferon‐γ and lipopolysaccharide, or a tolerogenic CD74 ligand, RTL1000. MAPCs differentially responded to each of the tested stimuli, secreting molecules that regulate the biological activity of the extracellular matrix (ECM), including proteins that make up the ECM itself, proteins that regulate its construction/deconstruction, and proteins that serve to attach and detach growth factors from ECM components for redistribution upon appropriate stimulation. MAPCs secreted a wide array of proteases, some detectable in their zymogen forms. MAPCs also secreted protease inhibitors that would regulate protease activity. MAPCs secreted chemokines and cytokines that could provide molecular guidance cues to various cell types, including neutrophils, macrophages, and T cells. In addition, MAPCs secreted factors involved in maintenance of a homeostatic environment, regulating such diverse programs as innate immunity, angiogenesis/angiostasis, targeted delivery of growth factors, and the matrix‐metalloprotease cascade.

Suppression of IL-7-dependent Effector T-cell Expansion by Multipotent Adult Progenitor Cells and PGE2.

T-cell depletion therapy is used to prevent acute allograft rejection, treat autoimmunity and create space for bone marrow or hematopoietic cell transplantation. The evolved response to T-cell loss is a transient increase in IL-7 that drives compensatory homeostatic proliferation (HP) of mature T cells. Paradoxically, the exaggerated form of this process that occurs following lymphodepletion expands effector T-cells, often causing loss of immunological tolerance that results in rapid graft rejection, autoimmunity, and exacerbated graft-versus-host disease (GVHD). While standard immune suppression is unable to treat these pathologies, growing evidence suggests that manipulating the incipient process of HP increases allograft survival, prevents autoimmunity, and markedly reduces GVHD. Multipotent adult progenitor cells (MAPC) are a clinical grade immunomodulatory cell therapy known to alter γ-chain cytokine responses in T-cells. Herein, we demonstrate that MAPC regulate HP of human T-cells, prevent the expansion of Th1, Th17, and Th22 effectors, and block the development of pathogenic allograft responses. This occurs via IL-1β-primed secretion of PGE2 and activates T-cell intrinsic regulatory mechanisms (SOCS2, GADD45A). These data provide proof-of-principle that HP of human T-cells can be targeted by cellular and molecular therapies and lays a basis for the development of novel strategies to prevent immunopathology in lymphodepleted patients.

Mutual Interaction Between Human Multipotent Adult Progenitor Cells and NK Cells

Human multipotent adult progenitor cells (hMAPCs) are isolated from bone marrow with a more extensive expansion capacity compared to human mesenchymal stem cells (hMSCs) and with the ability to differentiate into endothelium. Like hMSCs, hMAPCs inhibit T-cell proliferation induced by alloantigens. In this study, we tested the interaction between hMAPCs and natural killer (NK) cells. We assessed the susceptibility of hMAPCs to NK cell-mediated lysis and the immunomodulation of hMAPCs on NK cell function during IL-2-driven stimulation and the cytolytic effector phase. Human MAPCs express the ligands PVR and ULBP-2/5/6, which are recognized by activating NK cell receptors. However, they also express MHC class I molecules, which induce inhibitory signals in NK cells. Freshly isolated NK cells at different effector:target ratios did not kill hMAPCs as assessed by an MTT and 51Cr-release assay, while hMAPCs impaired the cytotoxic activity of resting NK cells against the NK-sensitive K562 leukemia cell line. By contrast, IL-2-stimulated NK cells were capable of killing hMAPCs, and preactivated NK cells were not influenced during their cytotoxic effector function against K562 cells by hMAPCs. When added during the 6-day preactivation phase with IL-2, hMAPCs dose-dependently reduced NK cell proliferation in an IDO-dependent manner, but they did not influence the induction of cytotoxic capacity by IL-2. This study indicates that human MAPCs mutually interact with NK cells.

Neuroinflammatory signals enhance the immunomodulatory and neuroprotective properties of multipotent adult progenitor cells

IntroductionStem cell-based therapies are currently widely explored as a tool to treat neuroimmune diseases. Multipotent adult progenitor cells (MAPC) have been suggested to have strong immunomodulatory and neuroprotective properties in several experimental models. In this study, we investigate whether MAPC are of therapeutic interest for neuroinflammatory disorders such as multiple sclerosis by evaluating their capacities to modulate crucial pathological features and gain insights into the molecular pathways involved.MethodsRat MAPC were treated with combinations of pro-inflammatory cytokines that are closely associated with neuroinflammatory conditions, a process called licensing. mRNA expression of immunomodulatory molecules, chemokines and chemokine receptors was investigated. The migratory potential of licensed rat MAPC towards a broad spectrum of chemokines was tested in a Transwell assay. Furthermore, the effect of licensing on the ability of rat MAPC to attract and suppress the proliferation of encephalitogenic T cells was assessed. Finally, neuroprotective properties of rat MAPC were determined in the context of protection from oxidative stress of oligodendrocytes. Therefore, rat MAPC were incubated with conditioned medium of OLN93 cells subjected to sublethal doses of hydrogen peroxide and the gene expression of neurotrophic factors was assessed.ResultsAfter licensing, a wide variety of immunomodulatory molecules and chemokines, including inducible nitric oxide synthase and fractalkine, were upregulated by rat MAPC. The migratory properties of rat MAPC towards various chemokines were also altered. In addition, rat MAPC were found to inhibit antigen-specific T-cell proliferation and this suppressive effect was further enhanced after pro-inflammatory treatment. This phenomenon was partially mediated through inducible nitric oxide synthase or cyclooxygenase-2. Activated rat MAPC secreted factors that led to attraction of myelin-specific T cells. Finally, exposure of rat MAPC to an in vitro simulated neurodegenerative environment induced the upregulation of mRNA levels of vascular endothelial growth factor and ciliary neurotrophic factor. Factors secreted by rat MAPC in response to this environment partially protected OLN93 cells from hydrogen peroxide-induced cell death.ConclusionsRat MAPC possess immune modulatory and neuroprotective properties which are enhanced in response to neuroinflammatory signals. These findings thereby warrant further research to evaluate MAPC transplantation as a therapeutic approach in diseases with an immunological and neurodegenerative component such as multiple sclerosis.

Clinical‐Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes

MultiStem cells are clinical‐grade multipotent adult bone marrow‐derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T‐cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8+ cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL‐mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was—even after major histocompatibility complex class I upregulation—insensitive to alloantigen‐specific CTL‐mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T‐cell activation phase. As a consequence, MultiStem dose‐dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact‐dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T‐cell activation markers CD25, CD69, and human leukocyte antigen‐DR. The MultiStem‐induced CD8−CD69+ T‐cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed‐lymphocyte culture. Finally, the killer activity of activated antigen‐specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical‐grade MAPCs are an immune‐modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs.