Immunomodulation by mesenchymal stromal cells: relevance to mesenchymal stromal cells-based therapies

摘要

The clinical benefits of mesenchymal stromal/stem cells (MSC)-based therapies for immune disorders and degenerative diseases are based on their ability to modulate the immune system and secrete growth factors. However, some essential issues concerning the immunosuppressive properties of MSC, such as the differences among MSC derived from different tissues and the influence of MSC on the function of specific immune cell subpopulations (namely the recently described monocyte subsets, and T cell functional compartments) are not well elucidated.Aim: Here, we compared the suppressive effect of human MSC derived from bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM), on lymphocyte activation, proliferation, and mRNA expression of genes with an important role in T cell and NK cell function; investigated the influence of human BM-MSC on monocyte and myeloid dendritic cell (mDC) activation/maturation, and on cytokine expression by peripheral blood mDC, monocyte subpopulations, and the naturally occurring functional compartments of CD4+ and CD8+ T cells, as well as on CD4+ and/or CD8+ T cells producing IL-17, IL-9, and IL-6.Material and Methods: To investigate the ability of MSC to suppress immune cells, peripheral blood mononuclear cells (PBMC) were cultured in the presence or absence of MSC and treated with stimulating agents; cell cultures of non-stimulated PBMC, in the presence or absence of MSC, were also carried out. To compare the effect of MSC derived from BM, AT, and UCM, MSC were co-cultured with PHA-stimulated PBMC; T, B and NK cell activation and proliferation were evaluated by flow cytometry; while RT-PCR was used to quantify mRNA expression on purified T and NK cells’ activation compartments. To evaluate the effect of BM-MSC on mDC, classical, intermediate, and non-classical monocytes, BM-MSC were co-cultured with PBMC stimulated with LPS+IFNγ. Cell activation and cytokine production were assessed by flow cytometry; and cytokine mRNA expression quantified by RT-PCR in purified monocyte subsets and mDC. Co-culture of BM-MSC with PMA+ionomycin-stimulated PBMC was carried out to evaluate MSC effect on CD4+ and CD8+ T cell functional compartments: naive, central memory (CM), effector memory (EM), and effector. Cytokine expression within each functional compartment and the percentage of CD4+ and CD8+ T cells producing IL-17, IL-9, and IL-6 were assessed by flow cytometry; cytokine mRNA expression was quantified in purified CD4+ and CD8+ T cells by RT-PCR.Results: MSC derived from BM, AT, or UCM were able to inhibit CD4+ and CD8+ T cell proliferation and activation; BM- and AT-MSC also prevented B cell proliferation and activation, and CD56dim and CD56bright NK cell activation. AT-MSC always exerted the strongest suppressive action. In turn, UCM-MSC hampered activation of CD56dim NK cells, but had no effect on CD56bright NK cell activation, B cell activation or proliferation. Moreover, MSC co-culture reduced TNF-α and perforin mRNA levels in activated NK cells. In the purified T cell activation compartments, we observed increased mRNA levels of FoxP3 and T-bet by PHA-stimulated T cells in the presence of MSC. BM-MSC effectively inhibited TNF-α and CCL4 protein expression in monocytes and mDC, without affecting CCR7 and CD83 expression. Of note, BM-MSC-driven inhibition was more pronounced for mDC, and the reduction of TNF-α expression was less marked for non-classical monocytes. MSC also decreased mRNA levels of IL-1β and IL-6 in classical monocytes, CCL3, CCL5, CXCL9, and CXCL10 in classical and non-classical monocytes, and IL-1β and CXCL10 in mDC.In the same line, BM-MSC reduced the percentage CD4+ and CD8+ T cells producing TNF-α, IFNγ, and IL-2, as well as TNF-α and IFNγ mean fluorescent intensity (MFI), among all the four functional compartments, except for naive CD4+IFNγ+ T cells, where MSC had no inhibitory effect. While BM-MSC-driven inhibition of TNF-α and IL-2 production was higher for CD4+ T cells, inhibition of IFNγ secretion was more pronounced for CD8+ T cells. Accordingly, a decreased percentage of CD4+ and CD8+ T cells expressing IL-17, IL-17 and TNF-α, and IL-9, and of CD4+IL-6+ T cells, was induced by MSC. Interestingly, BM-MSC also increased IL-4 and TGF-β1, while reduced IL-10 mRNA levels, for CD4+ T cells; and enhanced IL-4, while diminished IL-10 and TGF-β1 mRNA, for CD8+ T cells. Analyzing the functional compartments, we found that, for CD4+ and CD8+ T cells producing TNF-α, EM and effector compartments were the most resistant to MSC suppressive effect; the degree of inhibition of CD4+ T cells producing IL-2 and IFNγ was similar among all the compartments; and EM and effector CD8+ T cells displayed the lowest degree of inhibition for IL-2, and the highest for IFNγ. Conclusions: MSC derived from either BM, AT, or UCM were able to inhibit T cell and CD56dim NK cell activation, and T and B cell proliferation; however, at different extents. In turn, UCM-MSC were unable to inhibit B cell and CD56bright NK cell activation, conversely to BM and AT-MSC. These important differences detected should be taken into account when choosing the MSC source for research or therapeutic purposes. We also found that BM-MSC didn’t impair the expression of maturation markers in monocytes and mDC under our experimental conditions, nevertheless, they hampered the pro-inflammatory function of monocytes and mDC, which may impede the development of inflammatory immune responses. Finally, we reported that the functional compartments of CD4+ and CD8+ T cells were differentially regulate by BM-MSC, which may impact the therapeutic effect of MSC in immune disorders with a distinct distribution of T cells among activation/differentiation compartments. Also, the influence of MSC on IL-9 can extend the research field of MSC in allergic inflammation.