3C). No significant production of IL-2 and IFN-γ was observed with microglia from BSA injected mice even after stimulation (Fig. 3A and B). Together, these

results establish for the first time that, in the absence of infiltrating peripheral and CNS-associated APCs, adult microglia are able to cross-prime ex vivo exogenous Ag to injected naive CD8+ T cells and also highlight that pro-inflammatory signals greatly improve this ability. The brain parenchyma is a highly specialized immune site that likely contributes to continuously downregulate microglial cell activity [1-4]. Sirolimus datasheet We therefore evaluated the capacity of microglia to stimulate naive OT-1 CD8+ T cells in situ. Irradiated mice were cerebrally injected with OVA and, after one day, cerebrally injected with CFDA-SE-labeled OT-1 CD8+ T cells. We then measured the BMS-907351 ic50 proliferation and IFN-γ production by OT-1 T cells. Interestingly, we observed a limited but reproducible proliferation of 40% of the OT-1 CD8+ T cells, among which 20% exhibited at least two cell

divisions (Fig. 4A, middle panel). Co-injection with OVA plus CpG-ODN, GM-CSF and sCD40L resulted in approximately 70% increase of the proliferating rate of OT-1 CD8+ T cells. Among them, 50% exhibited two to four rounds of division (Fig. 4A, right panel). No significant proliferation was observed in mice injected with BSA in the presence of adjuvant (Fig. 4A, left panel). In parallel, injection of irradiated-mice with OVA did not induce IFN-γ Chlormezanone production by OT-1 cells (Fig. 4C). The IFN-γ-producing

OT-1 T-cell frequency was similar in OVA (2.56 ± 0.22% of OT-1 cells; mean ± SD, n = 3) and BSA (2.22 ± 0.77% of OT-1 cells) injected mice. However, the injection of OVA plus CpG-ODN, GM-CSF and sCD40L significantly increased (**p < 0.005) the frequency of IFN-γ-producing OT-1 T cells (7.41 ± 1.64% of OT-1 cells) contrary to BSA plus CpG-ODN, GM-CSF and sCD40L (3.25 ± 0.26% of OT-1 cells). Finally, in order to evaluate the impact of non-microglial APCs in Ag cross-presentation within the brain and also to confirm the absence of non-microglial APCs in the brain of irradiated mice, we compared the capacity of the brain of irradiated and non-irradiated mice to cross-present Ags in vivo. The proliferation of OT-1 cells was higher in the brain of OVA-injected non-irradiated mice than irradiated mice, while their differentiation into IFN-γ-producing cells was not significantly affected (Fig. 4B and C). More precisely, in non-irradiated mice, intracerebral injection of OVA induced a strong OT-1 cell proliferation in the CNS (more than 90% cells exhibited two or more cell divisions) (Fig. 4B, right panel), contrary to BSA even in the presence of adjuvant (Fig. 4B, left panel).