gondii infection. We analysed some possible mechanisms that could explain the Treg cell-mediated immunosuppression described above. Since it was previously reported that during T. gondii-induced suppression, IL-2, RNIs and IL-10 are involved 16, 17, 20, 21, 40, we evaluated the effect selleck chemical of Treg-cell removal on the production of these mediators in vitro. NO2− production was similar in cells from uninfected and infected animals and Treg-cell elimination had no effect in the production of this molecule (Fig. 5), demonstrating that in our system RNIs are not involved in Treg cell-mediated suppression. The role played by IL-10 in T. gondii-induced suppression has been controversial 17, 19–22. However, since it has
been described as a suppressive mechanism of Treg cells, we analysed IL-10 production. As can be observed in Fig. 5, no IL-10 could be detected in culture supernatant of cells from uninfected mice, while cells from infected animals produced highly significant levels of IL-10. Moreover, elimination of Treg cells led to a drastic reduction of the cytokine level. Because this reduction in IL-10 levels correlated with a recovery of T-cell proliferation after Treg-cell removal, we hypothesized that IL-10 produced by Treg cells could be a key molecule involved in the suppression. We thus first analysed IL-10 production by Foxp3+ and www.selleckchem.com/products/VX-770.html Foxp3− cells from infected mice. As can Thymidine kinase be observed in Fig.
6, IL-10 was produced by both Foxp3+ and Foxp3− cells, but after infection, a 3-fold increase in the proportion of
IL-10-producing cells was observed in the Treg-cell population only, suggesting that these cells were the source of the increased amount of IL-10 found in the supernatant. We next carried out in vitro IL-10 neutralization in order to test if this cytokine was responsible of the Treg cell-mediated suppression. Addition of anti-IL-10 mAb did not alter the proliferation of the ungated, the CD4+ and CD8+ subsets from infected mice (Fig. 7A and B) demonstrating that IL-10 was not responsible for the Treg-cell suppressive effect on CD4+ and CD8+ T cells, despite the increased proportion of IL-10-producing Treg cells detected during infection. We finally explored the possibility that the observed suppression by Treg cells was IL-2-dependent. IL-2 levels in culture supernatants of stimulated splenocytes were drastically reduced in the supernatant of cells from infected animals when compared with uninfected animals (Fig. 5), as reported 17, 20, 21, 31, 33. Removal of Treg cells, however, led to a slight but non-significant reduction of IL-2 levels (Fig. 5), suggesting that Treg cells do not suppress IL-2 production. The absence of IL-2 accumulation also indicated that either this cytokine is not involved in Treg cell-mediated immunosuppression or that the Treg and conventional T (Tconv) cells could compete for the reduced IL-2 concentrations.