Immunomodulation by Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are pluripotent stromal cells that have the potential to give rise to cells of diverse lineages. Interestingly, MSCs can be found in virtually all postnatal tissues. The main criteria currently used to characterize and identify these cells are the capacity for self-renewal and differentiation into tissues of mesodermal origin, combined with a lack in expression of certain hematopoietic molecules. Because of their developmental plasticity, the notion of MSC-based therapeutic intervention has become an emerging strategy for the replacement of injured tissues. MSCs have also been noted to possess the ability to impart profound immunomodulatory effects in vivo. Indeed, some of the initial observations regarding MSC protection from tissue injury once thought mediated by tissue regeneration may, in reality, result from immunomodulation. Whereas the exact mechanisms underlying the immunomodulatory functions of MSC remain largely unknown, these cells have been exploited in a variety of clinical trials aimed at reducing the burden of immunemediated disease. This article focuses on recent advances that have broadened our understanding of the immunomodulatory properties of MSC and provides insight as to their potential for clinical use as a cell-based therapy for immune-mediated disorders and, in particular, type 1 diabetes. WHAT ARE MESENCHYMAL STEM CELLS? Historical perspective, definitions, and tissue localization. More than a century ago, the presence of progenitor cells in the bone marrow with the capability of differentiating to bone were identified (1,2). A series of landmark observations by Friedenstein and colleagues (3,4) led to identification of the clonogenic potential of fibroblast-like cells residing in bone marrow (1). By low-density culturing of bone marrow on plastic culture dishes, Friedenstein and colleagues were able to discard nonadherent hematopoietic stem cells and identify plastic-adherent cells or colony-forming unit fibroblasts, which were later introduced largely by Owen (5) as mesenchymal stromal cells. The term “mesenchymal stem cells” (MSC) appeared in the early 1980s (6) and was largely popularized by Caplan (7). Although studies highlighting the differentiation capabilities of MSC into various cell lineages including bone, cartilage, and adipose tissue have been repeatedly described over the past decade, some investigators argue that the “stemness” of MSCs is lacking, proposing instead to use the term “multipotent mesenchymal stromal cells” (8). While the acronym MSC has become the predominant term used within the literature, no matter what terminology one chooses to use, the field investigating these cells has grown rapidly because of the marked potential in terms of therapeutic exploitation. As noted previously, MSCs are self-renewable multipotent progenitor cells that have the potential to differentiate into various lineages (9). Whereas bone marrow MSCs represent a rare population of cells that make up only 0.001 to 0.01% of total nucleated cells and are 10-fold less abundant than hematopoietic stem cells, they can be readily grown and expanded in culture (10). The frequency of MSCs in postnatal bone marrow has been reported to decline with increasing age (11). Much of our knowledge regarding MSCs has been generated from studies using bone marrow–derived MSCs. However, the source tissue for studies of MSCs has recently been expanded to cells deriving from virtually all tissues including muscle, adipose tissue, and umbilical cord blood (12). It is important to note that the origin of MSCs may determine their fate and functional characteristics (13). Furthermore, whereas the exact functions of MSCs within tissues remain largely unknown, they appear to exert different functions in specific tissues where they reside. For instance, in bone marrow, they are reported to represent the precursor cell for stromal tissues that support hematopoiesis (14). In other tissues, upon receiving appropriate biological signals during tissue injury or inflammation, they may differentiate into specialized cells and play a pivotal role in tissue repair and/or control of inflammation in situ. MSC characteristics and methods for their isolation. From the outset, it is important to note that there is no universally agreed upon set or specific singular marker to identify these cells. As a result, a battery of negative and positive markers is generally used to phenotypically characterize these cells. MSCs generally lack specific cell surface markers of hematopoietic cells (CD14, CD34, CD11a/LFA-1, and CD45), erythrocytes (glycophorin A), and platelet and endothelial cell adhesion molecules (CD31). They express variable levels of CD105 (SH2), CD73 (SH3/4), CD44, stromal antigen 1, and a group of other adhesion molecules and receptors including CD166 (vascular cell adhesion molecule), CD54/CD102 (intracellular adhesion molecule), and CD49 (very late antigen) (15). Finally, their ability to differentiate into several mesenchymal lineages has also been used as an identity marker (1). It is also important to recognize that the specificity of some of these markers vary when discussing MSC derived from humans versus mice (Table 1). To date, identification of MSCs in vivo has also been difficult and challenging. It is interesting that since the first description of MSCs by Friedenstein et al. (3), this method for isolation has largely remained the “standard of practice,” being the adherence of fibroblast-like cells (when isolating cells recovered from bone marrow) to the plastic substrate of a cell culture plate, together with a concurrent lack of adherence of marrow-derived hematopoietic cells (16). Additionally, reports exist proposing negative selection to exclude hematopoietic stem cells, or using positive selections for some of the MSC markers for the purpose of enriching MSCs (9). FUNCTIONAL ACTIVITIES OF MSCs Immunomodulatory properties of MSCs in vitro. The immunomodulatory properties of MSCs were initially reported in T-cell proliferation assays using one of a variety of stimuli including mitogens, CD3/CD28, and alloantigens; settings where the ability of MSCs to suppress T-cell proliferation can readily be determined (17,18). Such suppression occurs irrespective of donor source, including settings in which one uses “third party” MSCs. MSCs also significantly reduce the expression of certain activation markers including CD25, CD38, and CD69 on PHA-stimulated lymphocytes (18); suppress proliferation of both CD4 + and CD8 + lymphocytes; and are able to abrogate the response of memory T-cells to their antigen (19). The immunomodulatory ability of MSCs appears to take effect before the secretion of interleukin (IL)-2, since MSC-mediated anti-proliferative effects on mitogen-stimulated peripheral blood lymphocytes can be reversed (in part) by the addition of exogenous IL-2 (17). Additional studies have noted that supernatants of MSCs were unable to suppress proliferation (15). However, using a transwell culture system with a semipermeable membrane to separate MSC from leukocytes, one effort did note an inhibitory function in terms of suppression, findings that suggest the presence of soluble factors capable of suppression (20). Among the many candidates that could represent such a soluble factor, members of the transforming growth factor superfamily (transforming growth factorβ), hepatic growth factors, prostaglandin E2, and IL-10 secreted by MSCs have all been found to suppress T-cell–mediated antigen responses in vitro (9). Furthermore, Meisel et al. (21) reported induction indolamine 2,3-dioxygenase expression by MSC stimulated with interferon (IFN)-γ. Thus, MSC inhibition of T-cell proliferation could also be due to a depletion of tryptophan (i.e., indolamine 2,3-dioxygenase catalyzes the conversion of tryptophan to kynurenin) and subsequent inhibition of Tcell proliferation. Inducible nitric oxide synthase and heme oxygenase-1 expressed by MSCs have also been implicated for their immunosuppressive properties (22,23). It is likely that these mechanisms are not mutually exclusive and that the relative contribution of each mechanism to modulating immune responses varies in different experimental models. It is also interesting to hypothesize that immunomodulation of MSCs in different tissues may be mediated by different factors. One should note, however, that lack of standardization in isolation and culture conditions and strain-dependent variation has given rise to conflicting findings and interpretations (24). MSC interactions with Tand B-cells, dendritic cells, and natural killer cells. In addition to the soluble factors, MSCs appear to engage themselves in several other pathways regulating T-cell function. The engagement of the inhibitory molecule programmed death 1 (PD-1) to its ligands PD-L1 and PD-L2 has also been demonstrated to be responsible for inhibition of T-cell proliferation via direct contact of MSCs and target cells, leading both to modulate the expression of different cytokine receptors and transduction molecules for cytokine signaling (25). In addition to antigen recognition through the T-cell receptor, T-cell activation requires costimulatory signals involving specific molecules on the surface of both T-cells and dendritic cells. Given the absence of surface expression of key costimulatory molecules by MSCs, it has been proposed that MSCs could also render T-cells anergic, although there is still controversy surrounding the robustness of this hyporesponsiveness (20). Finally, MSCs have been shown to increase either CD4 + CD25 + cells or CD4 + CD25 + FoxP3 + cells in different models and assays (26). Bone marrow–derived MSCs have been found to have inhibitory effects on the proliferation and IgG secretion of B-cells in BXSB mice, a model for systemic lupus erythematosus (27). When MSCs isolated from the bone marrow and B-cells extracted from the peripheral blood of he