hESC NPCs and hfNPCs induced different PBMC proliferative re

hESC-NPCs and hfNPCs induced different PBMC proliferative responses, despite relatively similar expression levels of HLA, co-stimulatory and adhesion molecules. Multiple other factors that are produced by NPCs and may contribute to the regulation of the immune response such as inducible nitric oxide synthase, prostaglandin E2 and Heme Oxygenases (Wang et al., 2009; Bonnamain et al., 2012). In the present study, compared to hESC-NPCs, unstimulated hfNPCs secreted higher levels of TGF-β1, a factor involved in the maintenance of immune tolerance and T-cell homeostasis (Bommireddy and Doetschman, 2007) and also previously reported to balance the immunogenicity of human forebrain derived NPCs (Ubiali et al., 2007). In addition, the higher release of TGF-β2 by hfNPCs may polarize naïve T g protein coupled receptors toward a regulatory function, mediating suppression transplantation and antagonizing adaptive immune responses (Robertson et al., 2007). On the other hand, the release of TGF-β1, TGF-β2 and IL-10 by hESC-NPCs was significantly increased under inflammatory conditions. Ubiali et al. (2007) reported a marked reduction of TGF-β1 release by hNPCs after 3days of inflammatory stimulation, which differs from what we found here. Our findings indicate that the production of immunomodulatory factors and thereby the immunomodulatory potential of hESC-NPCs is promoted when they encounter pro-inflammatory cytokines such as those expressed in neurodegenerative diseases and CNS trauma. A recent study showed that syngeneic mouse NPCs transplanted in a model of focal SCI were able to increase the proportion of Tregs (Cusimano et al., 2012), which are known to promote antigen-specific peripheral tolerance by suppressing the activation and expansion of reactive effector cells. Here we observed that both hESC-NPCs and hfNPCs up-regulated Tregs, defined as the proportion of CD4+CD25+FOXP3+ T cells among CD4+ T cells, after 6days of co-culture when in direct cell–cell contact. Next, we introduced a transwell system to elucidate whether the up-regulation was cell–cell contact dependent or not. The proportion of CD4+CD25+FOXP3+ T cells was not significantly up-regulated in the transwell system. These data suggested that cell–cell contact between hNPCs and PBMCs is an important mechanism to up-regulate CD4+CD25+FOXP3+ T cells. In coherence, Han et al. (2011) showed that the immunosuppressive effects of mouse ESCs and MSCs were mainly mediated by cell–cell contact. The production of immunosuppressive cytokines such as TGF-β1 is one of the major pathways by which Tregs exert their suppressive function. Li et al. (2007) reported that T-cell-specific deletion of TGF-β1 causes T-cell activation and defective Treg function. Bommireddy et al. (2009) showed that FOXP3+ Tregs in TGF-β1−/− mice were unable to prevent the activation of CD4+CD25+ T cells, indicating the expression of FOXP3 alone in Tregs is not sufficient for their function, suggesting that TGF-β1 is required for the suppressive function of Tregs. In the present study, we showed that the release of TGF-β1 was coherent with the up-regulation of CD4+CD25+FOXP3+ T cells in the presence of hfNPCs when allowing direct contact with PBMCs, but not hESC-NPCs. Furthermore, in co-cultures with PBMCs, we showed that hESC-NPCs under the present conditions triggered PBMC proliferation, while hfNPCs did not. We further showed that PBMC proliferative response may be reduced by the exogenous addition of hTGF-β and that hTGF-β1 antibodies may at least partly interfere and result in a higher proliferative response in co-cultures of PBMC and hNPCs. Taken together, these events may suggest that TGF-β1 released at high enough levels by hNPCs may support the induced FOXP3+ Treg population to exert their suppressive effects. Interestingly, after 6days, we observed that TGF-β2 significantly decreased in the transwell system, which may be due to the high level of IL-6 and TNF-α present to inhibit TGF-β2 secretion as previously reported in experimental autoimmune encephalomyelitis (Siglienti et al., 2007).