Immunomodulatory plasticity of mesenchymal stem cells: a potential key to successful solid organ transplantation

Over recent years tremendous progress has been made to understand the basic mechanisms underlying the state of allograft rejection. Regardless of substantial improvements in short-term allograft survival, long-term outcome remains subpar [1‐4]. The current maintenance regimen to support organ transplantation and to reduce transplant-related morbidity includes a combination of immunosuppressive (IS) drugs including calcineurin inhibitors, mTOR inhibitors and anti-proliferative agents [5]. Application of IS drugs has a therapeutic and suppressive effect on host’s immune system. Nevertheless, non-specific immunosuppression produced by IS drugs, also result in instances of undesired immunodeficiency, toxicity to other non-immune cells, cardiovascular disorders and malignancies [6‐11]. In the last decade, extensive research in the field of translational medicine has indicated the use of cell-based therapies complementary to IS drugs for achieving the goal of ultimate IS therapy i.e. a therapy that can induce a balance between maximum efficacy and minimal adverse effects.

Mesenchymal stem cells (MSCs), have recently gained the interest of clinicians and researchers. The likelihood of these MSC based therapies depends upon, their regenerative facets and modulation of the immunological responses engendered through their secreted paracrine mediators [12]. MSCs are recognized for the activation of regulatory immune cells in conjunction with interference in maturation and activation of antigen presenting cells (APCs). As already known, exogenously cultured MSCs upon administration into the patient’s body, interact with the microenvironment in vivo which leads to their activation or licensing. Clinical studies have suggested that this licensing process in vivo is mediated by the presence of soluble factors and cytokines in the circulation. MSCs upon exposure to different concentrations of inflammatory mediators either produce Th1 or Th2 cytokines, growth factors, cell migration factors which assist in tissue maintenance and repair. Along with the inflammatory cytokines, other factors like in vitro culture conditions, Toll-like receptor (TLR) signalling and drug interactions in vivo, may also determine the clinical efficacy of MSCs.

This review aims to describe the influence of microenvironment both in vitro and in vivo on MSC and their implications on various preclinical and clinical studies.

Mesenchymal stem cells originally reported by Friedenstein et al. [13, 14], are multipotent progenitor cells accomplished to differentiate into several specialized cell types. At high density, MSCs, align with each other in a typical spatial pattern and have spindle-shaped fibroblastoid morphology [15]. MSCs righteously referred to as mesenchymal stromal cells, possess trans-differential potential, triggered by, placing MSCs under specific stimuli which advance their development into various lineages namely mesodermal i.e. myocyte, adipocytes, osteocytes, cardiomyocytes, endothelium; ectodermal i.e. neuronal; and endodermal i.e. hepatic, respiratory, pancreatic epithelium [16‐18]. Bone marrow (BM) is considered as a primary source of MSCs while other sources include adult connective tissues such as dental pulp, peripheral blood, adipose tissue and foetal tissues such as Wharton’s jelly, placenta, amniotic fluid, umbilical cord (UC) and umbilical cord blood [19].

Phenotypically, MSCs are recognized by expression of surface markers CD105, CD73, CD90 (mesenchymal lineage markers) and lack of expression of CD34, CD19, CD45, CD11a (hematopoietic lineage markers), CD31 (endothelial lineage marker), HLA-DR (human leukocyte antigen) [18].

Mesenchymal stem cells express intermediate levels of class I major histocompatibility complex (MHC) and do not express class II MHC [18, 20] or other co-stimulatory molecules like B7-1, B7-2, CD80, CD40, CD40L or Fas ligand on their surface [21], which play a crucial role in immune activation. Even though the expression of MHC-II molecules on MSCs is upregulated when stimulated with a low-dose of pro-inflammatory cytokine—interferon (IFN)-γ, no modification in the expression of co-stimulatory molecules is observed [21, 22]. This peculiar profile of MSCs, makes them immune evasive and thus an attractive candidate for cell-based therapies for various clinical conditions.

During transplant rejection, graft from a genetically different donor elicits an allogeneic immune response inside recipient’s body, generated against antigens present on donor graft. The allogeneic immune response is a consequence of an intricate sequence of interactions involving both innate (dendritic cells, macrophages, neutrophils, mast cells and natural killer cells) and adaptive (T and B cells) immune system, ultimately leading to rejection and transplant failure [23‐25]. In order to manage early graft rejections, IS drugs are administered but their effect on controlling long-term graft rejections is questionable [26]. Keeping this in mind, the clinical emphasis recently has shifted towards induction of a state of tolerance towards an organ allograft [27, 28]. Transplantation tolerance is a state marked by the absence of donor-specific inimical immune responsiveness which can be maintained devoid of chronic immune suppression [29, 30]. Although transplant tolerance has been successfully achieved in various animal models, its accomplishment in humans remains incomprehensible [31‐33]. Insights into mechanisms involved in immune activation have led scientists to evaluate cell-based therapies specifically using MSCs for various disease conditions owing to their powerful immunomodulatory potential, without any long-term deleterious effects [34, 35].

Existing data substantiates that MSCs help in instigating a state of tolerance by suppressing the effector cell responses that pose a major threat to the transplanted organ [36]. MSCs possess broad immunoregulatory properties which can modulate the immune responses by strongly inhibiting the differentiation, maturation, function and proliferative responses of immune cells both in vitro and in vivo [37‐39]. Studies have demonstrated the potency of MSCs to induce regulatory T (Tregs) and regulatory B (Bregs) cell activity which further directs suppression of effector and memory immune cell responses [40]. Moreover, MSCs are capable of inhibiting the generation and maturation of dendritic cells (DCs), which impairs their capacity to activate T cells [41]. MSCs can do so by inducing tolerogenic DCs, which produce interleukin-10 (IL-10) and result in an expansion of Treg cells through an indirect mechanism [42]. Similarly, MSCs can also alter the macrophage phenotype from a pro-inflammatory phenotype M1 to an anti-inflammatory phenotype M2 and lead to the generation of MSC-educated macrophages (MEM) possibly using an IL-10 mediated switch [43]. Interestingly, in response to a pathogenic insult, MSCs enhance the microbicidal activity of macrophages by altering the naive macrophages into the inflammatory M1 macrophages, without enhancing their APC function. However, MSCs induce the conversion of already active M1 macrophages into anti-inflammatory M2 macrophages to resolve the hyper-inflammatory state [44]. MSCs have also been shown to suppress the IL-2 mediated proliferation and cytotoxic activity of Natural Killer (NK) cells [45]. These aforementioned immunosuppressive properties inflicted by MSCs possibly lead to induction of a state of peripheral tolerance [46, 47].

For contact-dependent mechanisms, MSCs express a large number of chemokines which lead to chemotaxis of immune cells in the close vicinity of MSCs. As soon as immune cells begin to migrate, MSCs secrete locally active immunosuppressive factors which act on these migrating immune cells in a reciprocal fashion [48]. While paracrine mechanisms are conceded by MSCs through direct secretion of anti-inflammatory factors such as transforming growth factor (TGF-β), hepatocyte growth factor (HGF), nitric oxide (NO), heme oxygenase (HO)-1, indoleamine 2,3-dioxygenase (IDO) and expression of inhibitory co-stimulatory molecules such as TNF-related apoptosis-inducing ligand (TRAIL), programmed death ligand (PD-L1) that work together and influence the effector populations [49]. Through indirect mechanisms, MSCs can either inhibit the maturation of the antigen presenting cells (APCs) or generate regulatory or suppressor cell populations through paracrine signalling [50]. Paracrine signalling pathways are thought to be crucial as MSCs themselves are short-lived due to their susceptibility to lysis by CD8⁺ T cells and NK cells [51, 52]. Interestingly, a recent study has indicated the “apoptotic demise” of MSCs as a key step for activation of effector mechanisms of immunosuppression lead by MSCs [53].

Although a number of factors are known that help in MSC-mediated immunomodulation but every factor serves a different function depending upon the MSC source and its microenvironment. Therefore, further understanding of how MSCs can be directed to produce the “beneficial factors” and how these factors can regulate immune cells, might lead to the achievement of a state of tolerance/partial tolerance through immunomodulation.

Theoretically, the idea of utilizing MSCs as an adjunct immunosuppressive therapy for solid organ transplantation is very alluring as MSCs aid in stimulating tolerogenic immune responses. To test this hypothesis, plenty of preclinical studies in experimental models of organ transplantation have been conducted. The first preclinical study was performed by Bartholomew et al. [54] in a baboon model of skin transplant which illustrated a diminished lymphocyte proliferation with an enhanced graft survival as a result of intravenously infused allogeneic MSCs. Further, studies performed in different experimental transplant models demonstrated that MSC infusion holds the potential to prolong graft survival with minimal rejection rate and this occurred either through suppression of effector T cells or amplification of regulatory cell subsets or both [55‐60]. The data from these preclinical studies have established safety and efficacy of MSCs as a complement to IS therapy which has encouraged their clinical application in patients undergoing transplantation. Till date, over 734 clinical trials investigating the effectiveness of MSC therapy in different immune-mediated or related conditions have been registered on the clinical trial database (clinicaltrials.gov). Using keywords “mesenchymal stem cells” and “transplant”, we were able to find 253 registered trials and out of these only 12 clinical trials are being conducted in solid organ transplant patients. All of these studies are still in Phase 1 or combined Phase1/2 and are dedicated to evaluate safety and efficacy of MSC therapy (Table 1). The first pilot study reporting safety and feasibility of autologous MSC therapy for kidney transplant patients with living related donors surfaced in 2011 [61]. In the following year, Tan et al. [62] demonstrated that autologous MSC infusion in transplant recipients lead to decreased frequency of allograft rejection and opportunistic infections as compared to the control group given anti-IL-2 receptor induction therapy. However, the rejection rates and renal function outcome in MSC treated patients and control group patients were similar at 6 months. Further, a study conducted by Reindeers et al. [63] revealed that a decrease in donor-specific proliferation of peripheral blood mononuclear cells (PBMCs) of MSC-treated patients, 12 weeks post-infusion, but the incidence of viral infections was higher in these patients. Regardless of substantial clinical data, which proved the safety of MSC therapy, there were no considerable supporting reports to confirm the immunological status of treated patients. As the immune profile of patients is the key to foresee the short and long-term graft outcome, subsequent studies focussed on monitoring of both clinical as well as immunological parameters. A pilot study by Peng et al. used a combination of allogeneic MSCs and a low dose of tacrolimus (TAC) to prevent renal allograft rejection [64] and immune profile of these patients was regularly monitored till 1-year post-transplant. The authors remarked the use of allogeneic MSCs as safe and feasible as no incidence of acute rejection was evident in the experimental group. While comparing immune profiles, only a slight change in the percentage of B cells at 3 months post-transplant was observed while there was no change in CD4/CD8 T cells, NK cells or intracellular cytokine expression. Other studies have reported that MSC infusion in a transplantation setting has a Treg cell promoting effect which in return weakens memory and effector T cell responses [61, 65]. A majority of documented studies have explored the use of MSCs in kidney transplant patients and autologous BM aspirates have been used as a preferable MSC source. Although studies in experimental models have indicated that MSCs of both autologous and allogeneic origin display the same immunosuppressive potential still the decision of whether to use donor-derived MSCs or not needs powerful consideration. Allogeneic MSCs have been shown to have enhanced immunogenicity in vivo which might lead to an anti-donor immune response [66, 67]. For the same reason, very few studies (n = 2) have employed allogeneic MSC infusions for patient trials [64, 68].

Data from clinical trials have repeatedly highlighted safety and tolerance of MSCs in humans but given the current scenario, it is difficult to state the long-term therapeutic efficacy of MSCs. Small sample size, the primary cause of the disease in a patient, treatment regime, difference in the immune cell and cytokine profile which decide the effectiveness of a treatment course are some of the factors which make the translation of preclinical findings challenging in human subjects. Moreover, follow-up for a limited time period, different efficacy endpoints and insufficient cellular and molecular findings are some factors which make it difficult to infer anything concrete from these studies.

Although immunosuppression by MSCs is a well-documented notion, its mere understanding does not guarantee a successful outcome in vivo. In vitro studies provide a better control as they allow close monitoring of MSC fate. While after in vivo administration, MSCs become exposed to host immune cells and soluble cytokine/chemokine mediators which modulate their phenotype, thus indirectly controlling their fate, which can either impact the outcome positively or negatively. During disease progression, the role of cytokines is domineering in the acute phase of inflammation. But the inflammatory profile of patients is not stable and it varies from time to time at different stages of disease pathogenesis. These fluctuating cytokine profiles are responsible for the incompetence of MSC therapy in preclinical and clinical studies. Moreover, the drugs used for managing organ transplants and immune-mediated disorders are mostly immunosuppressive in nature which further add to the complexity. The concept of MSC microenvironment has gained the significant attention of the researchers worldwide and may serve as the key element for deciding the success of stem cell therapy. To adapt these cells effectively for clinical applications, in this review we have enlisted relevant factors/conditions which have already been indicated to affect the potency of MSCs.

Mesenchymal stem cells expansion has been established from multiple sources but their properties vary depending upon the site of their isolation [133]. Donor heterogeneity is yet another concern while considering the use of MSC therapy [134]. Age of donor may influence the therapeutic value of MSCs as MSCs derived from old donors have diminished proliferation potential [135] along with an altered membrane glycerophospholipid composition [136]. Moreover, lack of standardized isolation and expansion protocols affect the qualitative properties of MSCs to a great extent. In view of the ongoing trials, there is a lot of variation in the number of cells used for infusion, number of doses, infusion time points and transfusion patterns, which might be a reason behind inconsistent outcomes (Table 1). Therefore, immediate attention is required to deal with these issues for improving the overall therapeutic efficacy and for facilitating the utilization of MSC therapy.

The chief objective of applying MSCs as maintenance immunosuppressive therapy is to augment allograft acceptance and function. Ongoing research has suggested a key role of the microenvironment in defining the fate of MSC therapy. In light of these stimulating findings, we hereby propose different approaches for MSC modifications that can contribute to the success of this therapy.

In past few years, a plethora of studies have theorized and substantiated the immunosuppressive potential of MSCs. Data from clinical trials have assured the safety of MSC based therapies in organ transplant patients. However, the results in terms of efficacy have not been satisfactory which insinuates the need to authenticate these findings further, before implementing this as a global therapy. Although the therapeutic efficacy of engrafted MSCs has not been fully established the microenvironmental cues regulating their plasticity are well indicated, which, if modulated, can result in enhanced efficaciousness. The capability of MSCs to respond differently to variable levels of inflammation, cytokines and immunosuppressive agents have drawn the attention towards their functional plasticity. Understanding and translation of MSC-plasticity mediated immune-regulation can help improvise the foundation of MSC therapy. Moreover, as a part of personalized medicine, it would be beneficial to standardize the protocols for pre-conditioning or genetically modifying MSCs as per the patient’s need to further enhance the applicability and success of these cellular therapies which in future may substitute the current drug therapies.