Shown are representative data of 3 independent experiments

Shown are representative data of 3 independent experiments. the presence of OCs. Our data reveal a novel function of human OCs as inducible immunosuppressive cells, and a feedback loop between OCs and activated T cells. Thus, this study provides new insight into the mechanism of the immunosuppressive function of OCs, and may be helpful for developing novel therapeutic strategies for human Sophocarpine diseases involving both the bone and immune systems. value less than 0.05 was considered statistically significant. Unless otherwise indicated, mean s.e.m are shown. Results Bystander effect of OCs on T-cell responses To investigate the effect of OCs as bystanders on T-cell responses, we cocultured OCs with T cells in vitro. The purity of CD4+ T cells isolated from PBMCs was >90% (Supplementary Figure 1). CD4+ T cells were stimulated by allogeneic DCs, TT-pulsed autologous DCs, or staphylococcal enterotoxin B (SEB) in the absence or presence of autologous OCs. We found that T-cell proliferation induced by allogeneic DCs, recalled microbial antigen TT or superantigen SEB was significantly inhibited when OCs were present (Figure 1A-1C). To identify whether this inhibition was contact dependent, we separated allogeneic DC-stimulated T cells and OCs by Transwells during the coculture. As shown in Figure 1A, OCs could still significantly suppress T-cell proliferation when OCs and DC-stimulated T cells were separated by Transwells. However, the inhibitory efficiency on T-cell proliferation in Transwell coculture was significantly lower than that in contact coculture (Figure 1A). This result suggested that both soluble factor(s) and direct contact played important roles in OC-mediated T-cell suppression. To simplify the culture system for the investigation Sophocarpine of the effect of soluble molecule(s) on OC-mediated inhibition, we stimulated CD4+ T cells with Dynabeads coated with CD3/CD28 antibodies in Transwell inserts, in the presence or absence of OCs in the lower chamber of the culture plate. As shown in Figure 1D, the proliferation of T cells was significantly inhibited. These data indicate that OCs suppress T-cell proliferation stimulated by alloantigen, recalled microbial antigen, superantigen and polyclonal T-cell stimuli, and that both soluble molecule(s) and membrane molecule(s) contribute to the inhibition. Open in a separate window Fig. 1 OCs suppress T-cell proliferation in vitro through both soluble molecule(s) and membrane-binding molecule(s)Proliferation of CD4+ T cells stimulated by (A) allogeneic DCs at a T/DC ratio of 10:1, (B) control autologous DCs, or TT-pulsed autologous DCs at a T/DC ratio of 10:1, (C) SEB (5 g/mL) with irradiated PBMCs as accessory cells and (D) -CD3/CD28 Dynabeads at a T/Bead ratio of 2:1 in the absence or presence of autologous OCs for 4C7 d, as measured with CFSE dilution assay. Transwells (pore size: 0.4 m) were used in (A) and (D) to separate stimulated T cells and OCs. Summarized data from 3 to 5 5 independent experiments are shown on the right as mean s.e.m. Tw: Transwells. To exclude the possibility of nutrition consumption mediated T-cell suppression, we measured the viability of OCs and apoptosis of CD4+ T cells (Supplementary Figure 2 and 3). We found that both OCs and T cells survived well during the coculture of OCs and T cells. We also measured the T-cell suppression effect with different ratio of OC:T cells, and on different time points (Supplementary Figure 4). Of note, CD4+ T cells stimulated by allogeneic DCs or -CD3/CD28 Dynabeads in the presence of OCs still expressed activation markers CD25 and CD69, CTLA4, and PD-1 (Figure 2A, 2B). ELISA results showed that activated T cells cocultured with OCs secreted IFN- and IL-2 (Supplementary Figure 5). These results indicate that OCs do not suppress T-cell activation. We then tested the cell cycle of these activated T cells. We found that DC-activated T cells cocultured with OCs contained more G0/G1 phase cells than T cells activated by DCs without OCs (Figure 2C). Similar phenomenon was observed in Dynabeads-activated T cells (Figure 2D). Taken together, these data suggest that T cells inhibited by OCs are still activated T cells, but the proliferation of T cells is inhibited. Open in a separate window Fig. 2 OCs do not suppress T-cell activation, but inhibit cell cycle(A) Flow Sophocarpine cytometry analysis of CD4+ T cells stimulated by allogeneic DCs in the absence or presence of autologous Rabbit Polyclonal to BAG4 OCs. The expression of CD25, CD69, CTLA4, and PD-1 was detected on d6 after the coculture. (B) Flow cytometry analysis of surface markers on CD4+ T cells stimulated by -CD3/CD28 Dynabeads in the absence or presence of autologous.