MSCs are multipotent cells that are being clinically exploited as a new therapeutic for treating a variety of immune-mediated diseases.17–19, 44,88 However, frequently used BM derived MSCs are short-living and cannot assure long lasting immunoregulatory function both in vitro and in vivo.89 Consequently, several groups including ours have isolated MSCs from other sources with the hope that these cells could have prolonged lifespan and exert longer-lasting immunoregulatory properties.59–62 Among these alternative sources of MSCs, FL-MSCs seem to be one of the most promising due to their proliferative and differentiation capacity, their low immunogenicity and their immunomodulatory properties.63,64,90,91 We and others demonstrated that even though adult BM and FL-MSCs exhibit similar morphology and phenotypes, FL-MSCs demonstrate a faster growing kinetics, a higher number of cells produced over the same period of time, and a longer inhibition of NK and T cell proliferation compared to BM-MSCs.63,92,93
In this study, to go further in our investigations, we have isolated and compared MSCs from FL and adult BM sources to check which one displays the most efficient immunosuppressive effects on CD3/CD28-stimulated CD3+CD25− murine T convs either directly or indirectly through T reg induction. Before widespread clinical application of human MSCs, it is required to perform a variety of biological validation tests in murine models. Increasing number of recent studies demonstrate that humans MSCs are well tolerated and functional in regenerative and inflammatory mouse models.94–97 Therefore, we chose to perform our investigations by studying the interaction between human MSCs and mice T cells in order to test later their immunological properties in highly inflammatory xenogeneic mice model. We first confirmed that although FL and BM-MSCs share several common characteristics, including a spindle-shaped fibroblast-like morphology, phenotype and differentiation capacities, FL-MSCs bore an outstanding ex vivo expansion ability compared to adult BM-MSCs. Even if both cell types expressed typical MSC markers we observed a higher expression of ICAM-1 in FL-MSCs and conversely a lower VCAM-1 expression in BM-MSCs. Such differences were already observed between FL and adult BM-MSCs.98 Such adhesion molecules may have important roles in the balance between immune activation and immunosuppression. Whether they play an important role in MSC-mediated immunosuppression still remains to be investigated. We also noticed that FL-MSCs contained both CD271bright, and CD271low cells, while adult BM-MSCs cultures contained only CD271 low cells. This finding is consistent with previous studies demonstrating that CD271high cells have a much higher clonogenic capacity than CD271low cells.75–77
To investigate immunomodulatory effects of FL or BM-MSCs toward activated murine T cells we then performed co-cultures assays. Addition of both FL and BM-MSCs to CD3/CD28 activated CD4+CD25− and CD8 + CD25− T cells reduced their proliferation in a dose dependent manner. Interestingly, this immunosuppression was significantly higher when FL-MSCs were used compared to BM-MSCs. We then measured the ability of FL and BM-MSCs to modulate conventional murine T cell activation profile by quantifying the expression of CD25, GITR, ICOS and TNFR2 markers. While both MSC types were able to down-modulate CD4+ Foxp3- and CD8+ Foxp3−T cell activation, this immunomodulatory effect was remarkably stronger with FL than BM-MSCs.
Interestingly, the increase in TNFR2 expression on T cells in the FL-MSC group could reflect the conversion of T cells towards a more anti-inflammatory phenotype. We and others have already showed that many immunosuppressive cells including T regs, MSCs and recently endothelial cells derived from circulating endothelial progenitors are expressing TNFR2. This expression is directly related to their immunomodulatory functions. Moreover, the elevated expression of TNFR2 activation marker has been directly correlated to increase IL-10 and TGFβ anti-inflammatory cytokines production.99,100
We next checked if FL-MSCs, could also exert their immunosuppressive effect indirectly through induction of T regs, as already demonstrated for BM-MSCs.33–40, 101 When CD3/CD28 activated CD3+CD25− murine T cells were co-cultured in the presence of either FL or BM-MSCs we observed CD4+CD25+Foxp3+ and CD8+ CD25+Foxp3+ T regs induction with both MSC types. However, very interestingly, in all conditions tested, FL-MSCs induced a higher percentage of T regs than BM-MSCs.
To better define the phenotypic characteristic of the T reg population induced in the presence of FL or BM-MSCs, we showed that CTLA-4, ICOS, GITR and CD25, markers commonly used to evaluate T reg activation,83,84 were higher expressed in the CD4+Foxp3+ T reg population induced by FL than BM-MSCs. We also checked the expression of TNFR2, one of the most important regulators of T regs activity, that has been shown by us and others to be directly related to their activation and immunosuppressive function81,86,87. We found that the percentage and expression level of TNFR2 was significantly higher on CD4+Foxp3+ iT regs induced by FL-MSCs than with BM-MSCs, reflecting a global activation of the T reg induced population.
Finally, CD4+CD25+ T reg populations acquired from cultures in the presence of the FL-MSCs or BM-MSCs were then assayed in a secondary MLR, to investigate their immune-modulatory effects on activated murine T cells. While both T reg populations induced from FL or BM-MSCs were able to inhibit the proliferation of activated responder T cells, FL-iT reg population were significantly more efficient to inhibit CD4+ or CD8+ T cell proliferation.
Altogether, these results demonstrated that FL-MSCs affected much more murine T cell proliferation and modulate them towards less active phenotypes than adult BM-MSCs. In addition to their substantial suppressive effect FL-MSCs promoted more effectively the transformation of CD3/CD28-activated CD3+CD25− murine T cells into active CD4+CD25+Foxp3+ or CD8+CD25+Foxp3+ T regs and also to a more functional CD4+CD25+Foxp3+ T regs. These results highlight the immunosuppressive activity of FL-MSCs on T cells and show for the first time that one of the main immune-regulatory mechanisms of FL-MSCs is through T regs induction. T reg induction by fetal MSCs not only highlights the existing relation between MSC and T regs31 but also the role played by fetal T regs in tolerance to maternal antigens in utero.102–106 A key challenge of early life is to balance the conflicting demands of generating appropriate, robust immune responses to pathogens and develop tolerance to innocuous and self-antigens. A number of pathways limiting T cell responsiveness in early life have been identified that are either specific or enhanced during early life. In particular, there are increased frequencies of T regs present in the blood and peripheral tissues during fetal life and the function of fetal T regs is enhanced relative to T regs derived from adults.67–70 This abundance of T regs is not reflected in the thymus of equivalent gestational age, where the frequency of CD25+Foxp3+ among CD4+ thymocytes is comparable to the infant thymus.67 This suggests that a significant portion of fetal T regs are derived from expansion of natural T regs or are generated from conventional CD4+Foxp3− T cells in response to antigen. In agreement with this hypothesis, authors demonstrated that when fetal naïve CD4+ T cells are isolated and stimulated with alloantigen, they exhibit a strong predisposition to differentiate into T regs, as compared to adult naïve CD4+ T cells, thereby biasing the immune system toward tolerance.71,72 The data of Mold et al. suggest that hematopoiesis during fetal development occurs in waves, each generating distinct populations of T cells that may coexist for a period of time. CD4+ T cells originating from FL hematopoietic stem and progenitors cells appear to have a propensity to adopt the fate of regulatory T cells. As the source of hematopoiesis switches to the fetal BM, the resulting effector T cell/regulatory T cell ratio gradually moves toward that found in adults.107–109 Interplay between FL-MSC and T reg during fetal life may be a mechanism by which fetal naïve CD4+ T cells preferentially differentiate into T reg. Whether FL-MSC influences T reg conversion and how this interaction contributes to sustained T reg expansion during fetal life warrant further investigation. These results are also in favor of the development of new tools and strategies based on the use of FL-MSCs cells and their derivatives for the induction of immune tolerance. The implications of these findings for clinical application could be especially important if the fetal T reg population induced by FL-MSCs is found to promote tolerance not only to self and non-inherited maternal antigens, but also to foreign antigens encountered. Further insights into the fetal liver-derived T regs, including their unique immunomodulatory properties, could result in novel strategies to regulate allo-immune and autoimmune responses.