On the contrary, no increase of p21 protein level after doxorubicin injury was observed in HC cells despite a higher p53 level, confirming this specific tolerogenic mechanism in stem cells. We did not observe this mechanism operating within SSc–MSCs, the latter already expressing a higher p21 level in the absence of doxorubicin stress, which persisted after drug injury. These results confirmed premature ageing of these cells in SSc and suggested, at molecular level, their inability to escape to any additional stress. Of interest, a recent report showed that SSc–MSCs, although senescent, maintained their ability to suppress in-vitro lymphocyte MK-2206 chemical structure proliferation in mixed lymphocyte reactions [19], but the molecular pathways

involved in this process were not investigated. To understand the possible mechanisms involved in this process, we studied the cytokine profile produced by MSCs both from HC and SSc when co-cultured with PHA-conditioned T lymphocytes. Our results confirmed the inhibitory effect of SSc–MSCs on T cell proliferation, and this activity was associated with a higher IL-6 level in SSc–MSCs when compared to cells from HC. Enhanced IL-6 levels are believed to play a role in triggering the immunosuppressive effect of MSC on T cells [26]. Furthermore,

IL-6 production has been associated frequently with ageing [25], and this production might play a role in preserving the suppressive effect of aged MSCs on T lymphocytes via production of the anti-proliferative click here prostaglandin E2 (PGE2) in these cells [30]. It

is intriguing to speculate that the higher IL-6 production, observed in SSc–MSCs, might potentially cover the progressive loss of function of aged cells, preserving their immunosuppressive ability. MSCs immunomodulation takes place over a multi-stage process involving not only their constitutive ability to suppress T lymphocyte proliferation, but also involving the generation of inducted Tregs [33-35]. This induction requires the presence of TGF-β [50], 4��8C which is considered the major soluble factor associated with MSC promotion of Tregs in vivo [24, 32, 33, 51-54]. It is of interest that, in our setting, a recent report [32] identified a specific role for TGF-β-induced Tregs in MSCs protection against fibrillin-mutated systemic sclerosis, an animal model of the disease. In this regard, in our experiments the higher levels of TGF-β shown in SSc–MSCs, when co-cultured with CD4+CD25– lymphocytes, might allow normal induction and expansion of fully functioning Tregs. Therefore, MSCs from scleroderma patients displayed not only a specific anti-proliferative activity, but also normal ability in promoting the generation of CD4+CD25brightFoxP3+ cells. Notably, we observed a reduced activity of circulating Tregs in our patients and, as already reported, this impaired activity was associated with a decreased surface expression of CD69 on these cells. CD69 is an early membrane receptor, expressed transiently on activated lymphocytes.