Immunomodulation and present clinical applications

The immune phenotype of MSC, described as Major Histocompatibility Complex (MHC) class1⁺ or MHC class1^low, MHC class2^-, CD40^-, CD80^-, and CD86^-, is considered as little immunogenic, and transplantation of MSC into an allogeneic recipient may not require immunosuppression (Bartholomew, 2002). Although they express MHC class1 to some extent, which may activate allogeneic T-cells, the absence of potential co-stimulatory signals inhibits T-cell proliferation and leaves the T-cells in an anergic state (Glennie, 2005). Numerous studies documented inhibition of T-cell activation by MSC in vitro, and multiple mechanisms were suggested.

MSC modulate T-cell activation and proliferation both in an autologous and allogeneic pattern. They inhibit T-cell proliferation in mixed lymphocyte reactions (Le Blanc, 2003). Some studies reported the necessity of cell-to-cell contact to inhibit T-cell responses (Kim, 2007; Krampera, 2003; Nasef, 2007; Selmani, 2008), whereas others described cell-contact independent inhibition and the presence of soluble factors inducing T-cell proliferation arrest (Di Nicola, 2002; English, 2007; Krampera, 2006; Nasef, 2007). T-cells being activated by antigen-presenting cells, further reports described cellular interaction of MSC with antigen presenting cells. This contact was necessary to induce secretion of soluble factors either by MSC previously connected to monocytes (Groh, 2005), or by antigen presenting cells which were primed by contact with MSC (Beyth, 2005). MSC also inhibited the differentiation of monocytes into dendritic cells, the most potent antigen presenting cell type, after MSC-monocyte contacts (Jiang, 2005). MSC can also interact with B-cells inducing their cell-cycle arrest at G0-G1 transition of the cell cycle, thus inhibiting their proliferation (Corcione, 2006; Tabera, 2008). Inhibition of B-cell proliferation was mediated through a soluble factor and decreased expression of multiple

chemokine-receptors on the surface of B-cells, suggesting an impairment of chemotactic migration of B-cells (Corcione, 2006). Finally, MSC also suppress Natural Killer (NK) cell proliferation, cytokine secretion and cytotoxicity against MHC class1 expressing targets, when cultured at low NK/MSC ratios (Sotiropoulou, 2006). Both cell contact-dependent and soluble factor-contact-dependent mechanisms were demonstrated, suggesting multiple pathways for MSC-mediated inhibition of NK cells. At high NK/MSC ratios however, NK cytotoxicity could lyse MSC, implying fine regulation between proliferation/inhibition and activation (Sotiropoulou, 2006).

Multiple mechanisms have been involved in cell-contact inhibition of the immune system by MSC. First, MSC inhibited naïve and memory T-cell activation, which was reported to be contact-dependent and yet independent on antigen presenting cells (Krampera, 2003). Physical contact was required but no specific ligand/receptor candidate was identified. In lymphocyte-MSC interplay, interaction of programmed death 1 (PD1) to its ligands PD-L1 and PD-L2, modulated the expression of TGFbeta1 and its receptor. This interaction also down-regulated transduction molecules for cytokine signaling, such as STAT5a and STAT5b, and thereby inhibited proliferation of T-cells (Augello, 2005; Chang, 2006). Kim et al identified intra-cellular pathways in T-cells that are activated when MSC and T-cells are in close contact. P16^INK4A, p21^waf1 and p27^kip1 were strongly up-regulated, and cyclin A, B, D1, and E were down-regulated, thereby decreasing phosphorylation of retinoblastoma (Rb) gene and inducing cell-cycle arrest (Kim, 2007). Several studies described up-regulation and secretion of TGFbeta and IL-10 in T-cells in the MSC-T-cell contact model (Nasef, 2007; Prevosto, 2007). These factors then stimulate the generation of regulatory T-cells, another indirect mechanism for immunomodulation (Prevosto, 2007). MSC interaction with dendritic cells also induced production of IL-10, and inhibited T-cell proliferation in an indirect way, by converting classical antigen presenting cells into regulatory antigen presenting cells with T-cell suppressive characteristics (Beyth, 2005). A very elegant study allowed to integrate several of these mechanisms: Selmani et al showed that MSC-T-cell contacts induce expression and secretion of non-classic human leukocyte antigen (HLA)-G5 from MSC, which in turn potentiates 10 secretion (Selmani, 2008). Both HLA-G5 and IL-10 block T-cell proliferation in synergistic way and allow the generation and amplification of regulatory T-cells (CD4+CD25^highFOXP3⁺).

Multiple factors secreted by MSC were recognized to contribute to T-cell inhibition, among which several cytokines. In response to different stimuli, such as inflammation, T-cells also produced cytokines, which induce changes in MSC.

Interferon (Ifn) gamma is essentially secreted by T-cells (CD8⁺ or CD4⁺ of Th1 type), and by NK cells upon activation of an immune response (Schoenborn, 2007). Ifn gamma induces upregulation of key molecules in MSC such as B7-H1 (Sheng, 2008), and indoleamine 2,3-dioxygenase (IDO) (Meisel, 2004) that will in turn trigger major effects on inhibition of T-cells, or development of T-cell anergy. IDO is an enzyme that was first described in macrophages (Munn, 1999) and syncytiotrophoblasts of the placenta at the feto-maternal interface (Mellor, 1999;

Munn, 1998). IDO catabolizes tryptophan and creates a local microenvironment in which reduced tryptophan concentration precludes T-cell proliferation (Mellor, 2003).

Therefore, MSC expressing IDO reduce tryptophan levels in culture medium and thereby inhibit T-cell proliferation, in a cell contact independent mechanism (Chang, 2006; Jones, 2007; Krampera, 2006; Meisel, 2004; Suva, 2008). Concomitantly to Ifn gamma, the presence of TNFalpha, IL1-alpha or IL1-beta provokes the expression of inducible nitric oxide synthase (iNOS) and certain chemokines (CXCL-9 (MIG) and CXCL-10 (IP-10)) by MSC (Ren, 2008). In this very elegant study, Ren et al demonstrated that CXCL-9 and CXCL-10 secreted by MSC promote lymphocyte migration into proximity of MSC, which produce large amounts of nitric oxide (NO) and thereby inhibit T-cell proliferation. iNOS is specifically induced in MSC by CD4⁺ and CD8⁺ T-cells, but not by B-cells. Synthesized NO inhibits T-cell proliferation by the Stat5 signal transduction pathway (Sato, 2007). Heme-oxygenase-1 (HO-1), another inducible enzyme with immunomodulatory properties was identified in MSC, and was expressed independently of Ifn gamma treatment (Chabannes, 2007). HO-1 repression in MSC restored T-cell proliferation, demonstrating its specific immunosuppressive activity. The mechanisms of action of HO-1 are not fully elucidated but seem to act through carbon-monoxide accumulation thereby inducing inhibition of T-cell proliferation (Pae, 2004), toll-like receptor (TLR) activation (Ke, 2007; Nakahira, 2006) and/or cytoprotective, anti-oxidant effects (Shen, 2005).

HLA-G secretion by MSC, after MSC-T-cell interaction, inhibited T-cell proliferation but also protected MSC from cytotoxic effects of co-cultured T-cells, making HLA-G a very potent immunosuppressive, CD8⁺ T-cell inhibitor (Morandi, 2008). HLA-G also induced secretion of IL-10, with which T-cell proliferation is inhibited in a synergistic manner (Selmani, 2008).

Cytokines such as IL-6 and IL-10 influenced either directly or indirectly MSC-T-cell interactions. MSC secrete high amounts of IL-6 in co-culture experiments with T-cells. IL-6 acts either directly on T-cells, with partial suppression of proliferation, or indirectly through inhibition of maturation of dendritic cells which do not upregulate MHC class2 and co-stimulatory molecules (Djouad, 2007). Similarly, IL-10, secreted by antigen presenting cells, T-cells or MSC when cultured in contact with each other, induced development of aberrant and immature dendritic cells with a T-cell suppressor activity (Beyth, 2005; Selmani, 2008). Another study also identified IL-10 as an important cytokine for modulation of T-cell proliferation in co-culture with MSC.

Addition of anti-IL-10 neutralizing antibodies decreased proliferation, thus suggesting that IL-10 may have a proliferative effect (Rasmusson, 2005). These controversial results demonstrate the complexity of these systems and the difficulty to analyze the regulation of one particular factor. Interaction between MSC and the immunological key players, that are antigen presenting cells and T-cells, is complex and involves multiple cytokines some of which are secreted only after direct contact, and other through distant soluble factor-dependent interaction. Allogeneic MSC can interact with different type of T-cells (i.e.Th1 and Th2 CD4+ cells), regulatory T-cells, dendritic cells (i.e. DC1 and DC2) and NK cells. With each partner, they can interfere with their function and modulate proliferation and differentiation (Aggarwal, 2005).

Differences in cells numbers and balances of concentrations of cytokines in experimental settings can give opposite results, as in vitro models do only partially reflect in vivo mechanisms.

Immunomodulatory properties of MSC have also been studied in vivo and already used in some clinical settings, such as graft versus host disease (GvHD) after bone marrow / hematopoietic stem cell transplantation. When MSC were transplanted intravenously and concomitantly with donor-matched and third party skin grafts in an allogeneic skin transplantation model in baboons, skin-graft survival was significantly prolonged for both, donor-matched and third party skin, in comparison to control animals (Bartholomew, 2002). Tolerance induction by co-transplanting donor-derived MSC with either solid organs or pancreatic islets were studied by multiple groups, as this approach seems to hold many promises for clinical applications. In conjunction with low doses of clinically used immunosuppressive drugs, donor-derived MSC induced tolerance to allogeneic cardiac transplants in rats (Popp, 2008).

Figure 2: schematic summary of immunomodulatory interventions of MSC

MSC reduce the generation and differentiation of dendritic cells (1). MSC increase the percentage of regulatory T-cells through production of cytokines imparting regulation or promoting the generation of regulatory dendritic cells producing IL-10 (2). MSC engage in cell-to-cell contact through a variety of receptors with T-cells (3,4). In addition, MSC suppress effector T-cells through various growth factors, inducible nitric oxide synthase (iNOS), heme oxygenase (HO)-1, or indoleamine 2,3-dioxygenase (IDO) (5). MSC also act through downregulation of immunoglobulin production by B-cells (6). Finally, upregulation of MHC class II on MSC could lead to downregulation of NK cell cytotoxicity and proliferation (7). DC, dendritic cells; HGF, hepatic growth factor; TGF, transforming growth factor; TNF, tumor necrosis factor. (Adapted from Abdi et al) (Abdi, 2008).

This mechanism was partially induced by IDO, as inhibition of IDO induced graft rejection. Close interaction of recipient dendritic cells with donor MSC turned dendritic cells tolerogenic. Similarly, with low doses of cyclosporine, MSC induced skin graft prolongation in an allogeneic rat skin graft transplantation model, whereas without immunosuppression, MSC infusion induced sensitization, with accelerated premature skin rejection, probably related to TNFalpha secretion (Sbano, 2008).

MSC co-transplanted with HSC and islet cells improved engraftment of HSC and reversed streptozotocin-induced diabetes by a second islets infusion in rats (Itakura, 2007). Mixed hematopoietic chimerism and donor-specific tolerance were achieved with low dose of total body irradiation, anti-thymocyte globulin and 15-deoxyspergualin immunosuppressive treatment. Skin graft survival of skin from MSC-identical donors was also prolonged thereby demonstrating tolerance induced by

MSC infusion (Deng, 2004; Itakura, 2007). Other protocols and studies demonstrated a beneficial effect of MSC-transplantation in a model of autoimmune encephalomyelitis, by in vivo induction of T-cell anergy (Zappia, 2005). In mice deficient for regulatory T-cells with multi-organ autoimmune disorders, MSC transplantation showed MSC engraftment within ancillary and mesenteric lymph nodes, with a decrease of autoimmune enterophathy, through a global immunosuppressive mechanism. This was confirmed by increased CD4+, CD8+

production within the thymus, increased serum levels of IL-10 and decreased Ifn-gamma (Parekkadan, 2008). MSC transplantation thus induce immunomodulatory changes in vivo with specific actions in certain models, broad spectrum immunosuppression in other studies, but also sensitization and accelerated rejection of co-transplanted organs or MSC themselves, in further protocols (Badillo, 2007;

Djouad, 2005; Inoue, 2006; Nauta, 2006). Transplanted MSC seem immunogenic in certain conditions (Badillo, 2007; Nauta, 2006). In regard of these different results and outcomes, the immune balance seem fragile and light immunosuppression before allogeneic MSC infusion, or multiple repeated infusions (Beggs, 2006), seem necessary to prevent early rejection and sensitization. Once they achieve engraftment, immunomodulatory mechanisms can then induce tolerance or treat inflammatory diseases.

MSC were also used with success to improve outcome of allogeneic bone marrow transplantation by promoting hematopoietic engraftment in a clinical setting (Lazarus, 2005). Their immunomodulatory characteristics were thought to improve GvHD, in allogeneic bone marrow transplantation. Animal models gave controversial results on outcomes and real benefit of MSC transplantation to treat GvHD (Badillo, 2008; Li, 2008a; Sudres, 2006; Tian, 2008; Yanez, 2006). In humans, the first successful treatment of steroid resistant GvHD with MSC infusion was reported in 2004 (Le Blanc, 2004). Since then, several studies were performed in patients with acute GvHD (Le Blanc, 2008; Muller, 2008; Ning, 2008; Ringden, 2006). In a multicentric clinical trial, Le Blanc et al analyzed the outcome of 55 patients treated with 1-3 infusions of MSC (HLA-identical, haploidentical or HLA-matched third party) for steroid resistant severe acute GvHD. Fifty four percent of patients had a complete response, associated with lower transplantation-related mortality and increased overall survival, when compared to partial or non-responders to MSC therapy (Le Blanc, 2008).

In summary, immunomodulatory characteristics of MSC have been demonstrated in vitro and several mechanisms described, such as induced and contact-independent (cytokines and other soluble factors) inhibition of T-cell proliferation and induction of regulatory T-cell. In vivo, MSC infusion could prolong allogeneic organ transplantation, facilitating tolerance induction and decreasing inflammation in autoimmune diseases in animal models. Clinical applications were also performed with positive effects of MSC infusion in patients suffering from acute steroid resistant GvHD.

1.5.7. Clinical applications of multipotent mesenchymal stromal cells and