Mesenchymal Stromal Cells for Graft-Versus-Host Disease

Abstract

Mesenchymal stromal cells (MSCs) have been shown to mediate immune responses in vitro and in vivo. These observations have led to clinical trials of MSC administration to ameliorate acute graft-versus-host disease (GVHD), the most serious complication arising after allogeneic hematopoietic cell transplantation. Clinical data suggest a benefit in approximately two-thirds of patients with steroid-resistant acute GVHD. Preliminary studies have been reported on the use of MSCs to treat de novo acute GVHD, for prophylaxis of the condition, and more recently, in the management of chronic GVHD. Although preclinical data inferred a possible role of MSCs in affecting GVHD mechanisms, more robust animal models became available only after numerous clinical trials with these cells had been undertaken. Further clinical trials, the development of more appropriate animal models and an effective means of tracking and imaging the introduced cells in real time in patients, are required to better define their role in this important area of medicine.

Introduction

T he study of mesenchymal stromal cells (MSCs) has gone through several phases over the past 30 years. There has been a progression in focus (although not linear) from the support of hematopoiesis (Keating et al., 1984) to gene delivery strategies (Keating et al., 1990) to, more recently and very actively, tissue repair (Prockop, 2009) and most recently, and perhaps the most exciting of all, to an investigation of MSCs as a means of modulating immune responses (Aggarwal and Pittenger, 2005; Uccelli et al., 2008). Here, we review the use of MSCs in ameliorating the major clinical drawback of allogeneic hematopoietic cell transplantation: graft-versus-host disease (GVHD).

Pathophysiology of Acute GVHD

Clinical GVHD is a severe inflammatory condition and the main immune complication of allogeneic hematopoietic cell transplantation. Three preconditions for GVHD were proposed by Billingham (1966). First, immunologically competent cells (i.e., mature T lymphocytes) need to be present in the allograft. Second, the recipient needs to be immunocompromised. This situation is created by the conditioning regimen of intensive chemotherapy or chemoradiotherapy delivered before transplantation to make space for the new donor hematopoietic system and to reduce the likelihood of host immune rejection of the allograft. Third, the recipient must express tissue antigens different from the donor tissue antigens (or alternatively, express host self-antigens that are recognized inappropriately). Such human leukocyte antigens (HLAs), encoded by the genes within the major histocompatibility complex (MHC) cluster, are expressed on all nucleated cells in the human body. When the host cells recognize donor cells as foreign, they initiate activation of allogeneic T cells by host antigen-presenting cells, such as dendritic cells (Krensky et al., 1990).

Clonal expansion of donor T cells in GVHD is preceded by physical injury, especially to the gastrointestinal tract (Hill and Ferrara, 2000). This is a consequence of the conditioning regimen used before transplantation, when extensive cell death is accompanied by the secretion of inflammatory cytokines, such as tumor necrosis factor-α or interleukin (IL)-1. Donor T cell immune recognition of host antigens and activation of allogeneic T cells mediate further expression of cellular and inflammatory factors (e.g., from activated mononuclear phagocytes) that collectively amplify the local tissue injury and general inflammatory response.

Although GVHD is a multiorgan, destructive disorder, the clinically relevant target organ damage involves epithelial cell necrosis and thus is most obvious on the skin, in the gastrointestinal tract, and in the liver. A maculopapular rash often begins on the palms and soles, and may become generalized erythoderma with desquamation and bullae. Lower gastrointestinal tract involvement manifests in abdominal pain, and voluminous secretory and bloody diarrhea. Upper gastrointestinal disease is characterized by anorexia, dyspepsia, nausea, and vomiting. Hepatobiliary dysfunction presents typically with hyperbilirubinemia and jaundice.

Without intervention almost all allotransplant recipients develop significant acute GVHD (Sullivan et al., 1986), accompanied by profound immunosuppression and risk of fatal bacterial, viral, and fungal infections. Multiple drugs, typically in combination (e.g., cyclosporine A, tacrolimus, methotrexate, and mycophenolate mofetil), or strategies to deplete T cells ex vivo or in vivo, are used to prevent donor anti-host immunological complications of allotransplantation. Virulent cases of acute GVHD require therapy, typically with corticosteroids as the first-line therapy. If the GVHD is severe or unresponsive to steroids, anti-thymocyte globulin, a polyclonal immunoglobulin prepared by injecting rabbits or horses with lymphocytes, or other approaches such as extracorporeal photopheresis are used. Even with dramatic increases in the understanding of the pathobiology of GVHD over the last half-century, true progress in the clinical care of individuals with GVHD has been limited.

Relevant Preclinical Studies with MSCs

The rationale for clinical studies in acute GVHD was based on the immunomodulatory properties of MSCs identified by numerous in vitro assays and in animal models (reviewed by Keating, 2006, 2008; Uccelli et al., 2008; Tolar et al., 2010). Key observations in vitro included the ability of MSCs to suppress lymphocyte proliferation in mixed lymphocyte cultures or by mitogens (Di Nicola et al., 2002; Le Blanc et al., 2003a; Maitra et al., 2004), the inhibition of proliferation of cytotoxic T cells (Ren et al., 2008), and escape from the recognition of alloreactive lymphocytes (Le Blanc et al., 2003a,b; Maitra et al., 2004). Several reports have shown that MSCs exert potent immunosuppressive effects in vivo as well, especially the delayed rejection of histoincompatible skin grafts in a baboon model (Bartholomew et al., 2002) as well as in rodents (Guo et al., 2006; Aksu et al., 2008).

Although perhaps not unusual in the annals of clinical research, especially in the field of blood and marrow transplantation, there was a paucity of preclinical models demonstrating the efficacy of MSCs in ameliorating acute GVHD before clinical studies commenced. In fact, the most convincing preclinical models were developed after the demonstration of clinical efficacy. For example, Tisato and colleagues (2007) generated a xenogeneic model of acute GVHD using nonobese-diabetic/severe combined immunodeficient (NOD/SCID) mice transplanted with human peripheral blood mononuclear cells. Circulating proliferating human T cells (CD45⁺CD3⁺) were found and were also detected in tissues known to be clinical targets of GVHD, including mesenteric lymph nodes, liver, lungs, and peritoneal fluid but not the skin. After treatment with weekly doses of MSCs of cord blood origin, the mice showed a significant decrease in human T cell proliferation, had reduced tissue damage, and improved survival. None developed GVHD. There was no therapeutic effect when MSCs were infused as a single dose or when administered only at the onset of GVHD, suggesting in this model that MSCs have a greater role in prophylaxis than treatment. These data underscore a previous observation that the immunosuppressive effects are exerted more on T cell proliferation than on effector function.

In an inbred murine model (C57BL/6 and BALB/c) of lethal GVHD in which animals were transplanted with splenic mononuclear cells from C57BL/6 mice, infusion of MSCs delayed the development of GVHD and improved survival (Li et al., 2008). The authors observed that MSC infusions increased the number of T lymphocytes in secondary lymphoid organs. In the presence of MSCs, T lymphocytes acquired a distinct immunophenotypic profile (CD62L⁺CCR7⁺) and dendritic cells decreased their expression of CCR7. The results suggest that MSCs inhibited GVHD by downregulating T lymphocyte activation and promoted trafficking of T cells to secondary lymphoid organs, consequently reducing allogeneic lymphocyte infiltration and GVHD.

Two studies explored the in vivo cytokine environment that likely mediates anti-GVHD effects of MSCs. The studies suggested that interferon (IFN)-γ is required to initiate the anti-GVHD effect of MSCs (Polchert et al., 2008); Murine recipients of IFN-γ^–/– T cells did not respond to MSCs and died of GVHD. When MSCs were incubated with IFN-γ before infusion, they become activated and suppressed GVHD, resulting in a marked improvement in survival. The degree of MSC activation and anti-GVHD effects were directly proportional to the degree of IFN-γ exposure (Polchert et al., 2008). Another murine model of GVHD did not respond to MSCs except when the lethally irradiated mice were transplanted and injected with MSCs transduced with IL-10 (Min et al., 2007). There was significantly lower mortality in recipients of the IL-10-engineered MSCs (70 vs. 10% survival) and decreased serum levels of INF-γ 7 days after MSC injection.

Not all studies give concordant or positive results. For example, several murine models of GVHD with MHC-mismatched donor–recipient pairs failed to show any therapeutic benefit of MSC infusions (Sudres et al., 2006; Badillo et al., 2008; Prigozhina et al., 2008). Several factors are likely to contribute to these discrepancies, including the tissue source of MSCs (bone marrow, cord blood, or adipose tissue), the method of isolation to remove myeloid precursors (Friedenstein et al., 1968), the immunodepletion process (Baddoo et al., 2003), or the dose and scheduling of MSC administration (e.g., coadministration or injection after the allogeneic graft).

Another possible explanation for the lack of an anti-GVHD effect in some preclinical studies is that, despite the putatively immunoprivileged features of MSCs, the cells are rejected. In some studies, MSCs generated sufficient immunity to induce a memory response and their rapid elimination (Eliopoulos et al., 2005; Nauta et al., 2006). The injection of MHC-mismatched MSCs engineered to release erythropoietin failed to increase the hematocrit level whereas the hematocrit level was markedly increased when syngeneic MSCs were used (Eliopoulos et al., 2005). However, immune rejection of allogeneic MSCs may not be a concern clinically in the setting of highly immunosuppressive conditioning regimens.

Treatment of Steroid-Resistant Acute GVHD with MSCs

As noted previously, the greatest clinical challenge to effective allogeneic hematopoietic cell transplantation is acute and chronic GVHD (Kernan et al., 1993). Front-line management of established GVHD includes steroids, but only up to one-half with acute GVHD will respond (Deeg, 2007). There is no established therapy for steroid-resistant acute GVHD and although numerous agents have been tried, prognosis is poor, with as few as 16% surviving 2 years (Deeg, 2007). Moreover, almost all patients with treatment-resistant grade IV acute GVHD have a fatal outcome weeks to months later.

In this context, the administration of MSCs to manage this life-threatening condition was both novel and feasible, despite the limited preclinical data available. The approach was supported by the following lines of evidence: demonstration in vitro of the ability of MSCs to suppress the proliferation of activated alloreactive lymphocytes (Di Nicola et al., 2002; Le Blanc et al., 2003a), the short-term safety of the coinfusion of autologous MSCs and hematopoietic cells in patients undergoing intensive chemotherapy for breast cancer (Koc et al., 2000), and highly preliminary data suggesting reduction in acute and chronic GVHD in patients with advanced hematological malignancies undergoing HLA-identical sibling transplants and receiving a coinfusion of bone marrow or peripheral blood allografts and same-donor MSCs (Frassoni, 2002).

In a landmark case report, Le Blanc and colleagues (2004) described the successful therapy of a 9-year-old boy with treatment-resistant grade IV acute GVHD, using third-party MSCs. The report is notable for the following reasons: The MSCs were from his haploidentical mother and not the original HLA-matched unrelated donor; and a sustained, complete response occurred only after the second infusion of MSCs and reversal of severe gut and liver GVHD was documented. Encouraged by this proof of principle, eight additional patients with steroid-refractory GVHD were treated, six of whom had a favorable clinical response (Ringdén et al., 2006).

A European Blood and Marrow Transplant Group (EBMT)-sponsored phase II trial was next conducted in 55 patients with steroid-resistant, severe acute GVHD (Le Blanc et al., 2008). A median dose of 1 × 10⁶ cells/kg was administered. Patients received MSCs from HLA-identical allograft donors, haploidentical family donors, or unrelated HLA-mismatched donors. Twenty-seven patients had one MSC infusion whereas 28 had two or more. No side effects of MSC administration were documented. A complete response was documented in 30 (55%). There was no correlation between response rate and donor HLA match. Patients achieving a complete response had lower 1-year treatment-related mortality than the others (37 vs. 72%; p = 0.002) and superior survival (53 vs. 16%; p = 0.018).

On the basis of these promising results a prospective randomized trial, again with EBMT sponsorship, is underway to assess the role of MSCs in managing treatment-resistant acute GVHD.

Results of a company-sponsored randomized, placebo-controlled multicenter phase III trial for steroid-resistant severe acute GVHD have been reported in abstract form (Martin et al., 2010). In a 2:1 randomization, 163 patients received multiple infusions of third-party MSCs and 81 were given placebo. No difference was observed in achieving the primary end point of a durable complete response for ≥28 days (35 vs. 30%). Subset analysis, however, showed that patients with gut and liver involvement had improved complete or partial response rates at 100 days (82 vs. 68% and 76 vs. 47%, respectively; p < 0.05). Infusional toxicity, infection rates, and incidence of recurrent malignancy were similar in the two arms. The overall response rates were confounded by the lack of an effect on skin GVHD (100-day complete and partial responses of 78 vs. 77%).

Increased clinical studies in this area have brought several refinements that are likely to be reflected in subsequent trials. For example, EBMT studies suggesting that MSCs from unrelated HLA-mismatched donors give results similar to those of MSCs from the same donor have led to earlier intervention in the treatment of acute GVHD: previously cryopreserved and readily available third-party MSCs can be more rapidly deployed than HLA-matched MSCs, which need to be procured from allograft donors. Indeed, a phase II study (sponsored by Osiris Therapeutics) of third-party non-HLA-matched MSCs added to steroids for the therapy of grade II–IV acute GVHD showed a 77% 28-day complete response rate in 31 evaluable patients (Kebriaei et al., 2009). Patients were randomized to receive low-dose (2 × 10⁶ cells/kg) or high-dose (8 × 10⁶ cells/kg) MSC infusions. The infusions (62 in 31 patients) were well tolerated, ectopic tissue was not detected by computed tomography up to 2 years later, and the cell dose did not appear to affect response.

In another development, fetal bovine serum (FBS) can be replaced with human platelet lysate in MSC culture medium to circumvent the potential of an immune response to FBS proteins attached to MSCs (Horwitz et al., 2002) and to avoid infectious complications associated with xenogeneic products while maintaining or even enhancing cell growth (Schallmoser et al., 2007; Muller et al., 2008; von Bonin et al., 2009).

Role of MSCs in Acute GVHD Prophylaxis

Single-arm trials have been conducted to assess the role of MSCs in the prophylaxis of acute GVHD, based on the putative efficacy of preclinical models of MHC-mismatched allogeneic murine transplantation in reducing acute GVHD (Chung et al., 2004) and on the availability of cryopreserved functional MSCs. Protocols involve cotransplantation of hematopoietic cells and third-party or same-donor MSCs. Results showing short-term safety of the procedure and reduced nonrelapse mortality (Lazarus et al., 2005; Baron et al., 2010) should be interpreted with caution because of the small numbers of subjects and the lack of control cohorts. As a further cautionary note, one study raises the possibility of an excess relapse rate in HLA-identical sibling-matched allotransplant recipients with hematological malignancies cotransplanted with same-donor MSCs (Ning et al., 2008). In this open label, randomized trial of a total of 25 patients, whereas the incidence of grade II–IV acute GVHD was less in the MSC group (1, or 11.1%) than in the non-MSC cohort (8, or 53.3%) of evaluable patients, 6 (60%) versus 3 (20%) relapsed, yielding survivals of 30 and 66.7%, respectively, at 3 years. Here, too, outcomes cannot be regarded as statistically robust, given the small number of patients. These data, nonetheless, suggest that further studies are required to investigate the effects of MSCs on graft-versus-leukemia (GVL) in patients who do not develop acute GVHD. It should be remembered that the GVL study conducted by Horowitz and colleagues (1990) identified a cohort that exhibited a GVL effect but had neither acute nor chronic GVHD.

Treatment of Chronic GVHD with MSCs

Chronic GVHD also represents major morbidity and mortality after allogeneic hematopoietic cell transplantation and treatment remains unsatisfactory (Weisdorf, 2005). One report suggests that MSCs may also ameliorate sclerodermatous chronic GVHD (Zhou et al., 2010). A more recent study evaluated 19 patients with refractory chronic GVHD treated with MSCs (Weng et al., 2010). Outcomes were similar to those obtained for steroid-resistant GVHD and were excellent in reducing not only liver and gastrointestinal manifestations but also skin disease. Results are sufficiently promising to warrant a prospective randomized trial for intractable disease. This observation, however, also raises the question of whether the well-established mechanism of GVL is preserved in patients whose chronic GVHD has been effectively treated (Horowitz et al., 1990). Further research is needed, both preclinical and correlative in the context of carefully controlled trials, to help address this important issue.

Caveats to the Clinical Use of MSCs

In addition to concerns regarding the attenuation of a GVL effect in the event of suppression of GVHD, MSCs may also provide a sanctuary for leukemia blasts, thereby providing a setting for disease relapse that may occur late. Early laboratory studies demonstrate inhibition of apoptosis among leukemic cells that interact closely with MSCs (Konopleva et al., 2002). MSCs also produce transient cycle arrest of tumor cell lines, both hematopoietic and nonhematopoietic (Ramasamy et al., 2007). When human malignant cell lines are coinjected with MSCs into immune-deficient mice, tumor cell growth is more rapid (Ramasamy et al., 2007). Further studies are required to better understand the factors affecting malignant cell quiescence versus proliferation during MSC–cancer cell interactions. This is particularly relevant given that MSCs are frequently administered to patients with known malignancies. These observations underscore the importance of employing relevant preclinical models and conducting insightful correlative studies, in the context of controlled clinical trials.

Also, although the possibility of malignant transformation of MSCs exists (Tolar et al., 2007), culture conditions under which this can be avoided are now well established (Prockop et al., 2010). Nonetheless, the possibility of long-term adverse effects such as tumorigenicity, ectopic activity, the transfer of infectious agents, and other rare events can only be addressed by a database registry of consecutively reported cases provided by many cell therapy centers, as has been pioneered by the Center for International Blood and Marrow Transplant Research (CIBMTR) for hematopoietic cell transplantation.

Future Considerations

Outcomes of randomized controlled trials will help considerably to better define the role of MSCs in the management of GVHD, but are unlikely to be definitive. The limitations are inherent in the current status of cell therapy and are not restricted to the use of MSCs.

We have limited understanding of the dose and scheduling of MSCs (or of any cell therapy component except hematopoietic progenitor cells for autologous or allogeneic transplantation) because of the absence of real-time tracking in vivo in patient recipients (in contrast to animal models, for which superb tracking methodologies exist). Consequently, it has been difficult to conduct studies correlating cell dose/kinetics with desired end points. Moreover, uncertainty remains as to whether or not persistence or location of MSCs is important in affecting outcome. Improvements will come specifically from imaging and tracking experiments in patients. Conducting studies of this nature should have high priority.

More definitive studies will be necessary to determine prospectively any benefit to subgroups or by disease location (e.g., viscera vs. skin). One approach that was particularly successful during the earlier days of blood and marrow transplantation was the use of relational database registries to identify determinants of outcome unlikely to be established by prospective trials. Analysis of MSC trials from consecutive cases reported to the EBMT and CIBMTR are likely to be helpful in this regard, especially regarding cell dose, infusion schedule and frequency, MSC tissue source, and culture conditions among other factors.

Another attractive feature of using MSCs for the treatment of GVHD is their ability to mediate tissue regeneration (Tolar et al., 2010). Although there have been several reports suggesting the contribution of donor cells to repair tissue damaged by GVHD or by the transplant intensive therapy regimen itself (Le Blanc et al., 2004; Ringdén et al., 2007), further work, including the development of animal models, needs to be conducted.

Last, and especially in conjunction with relevant animal models, genetic modification of MSCs holds considerable promise, as evidenced by the murine IL-10-transduced MSC experiments. Our own study with MSCs engineered to express a mutant thymidylate monophosphate kinase (TMPK) gene aiming to obviate the risk of increased recurrence of malignancy is a case in point (administration of azidothymidine to the patient will eliminate the cells expressing the TMPK transgene) (Neschadim et al., 2007). It is likely that safely engineered MSCs may provide more targeted, and hence more effective, cell therapy of GVHD.

Footnotes

Acknowledgment

Armand Keating holds the Gloria and Seymour Epstein Chair in Cell Therapy and Transplantation at the University Health Network and the University of Toronto.

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