Adaptive Immunity, Autophagy Figure 7. Mice deficient in autophagy in macrophages/microglia are susceptible to toxoplasmosis. A, Primary microglia and macrophages were obtained from control and ATG7flox/flox-Lyz-Cre mice. Atg7 and actin expression were assessed by immunoblot. Results shown are those obtained with microglia. Similar findings were observed in primary macrophages. Microglia and macrophages were incubated with a stimulatory anti-CD40 or control mAb, or with IFN-c/TNF-a followed by infection with tachyzoites of RH T. gondii. B–F, ATG7flox/flox-Lyz-Cre and control mice were infected with ME49 T. gondii. Mice (4–5 mice per group) were euthanized at 3 weeks post-infection. B, ATG7flox/flox-Lyz-Cre mice exhibit higher parasite load in the eye and brain than control mice. Numbers of tissue cysts were determined in 100 high power fields. C, D, Compared to control mice (C), infected ATG7flox/flox-Lyz-Cre mice (D) revealed more prominent disruption of retinal architecture and inflammation. PASH; X200. E, F, Compared to control mice (E), infected ATG7flox/flox-Lyz-Cre mice (F) show more prominent areas of inflammation (arrow) and frequent tissue cysts (arrow head) in the brain. PASH X100. G, Eyes and brains were obtained at 3 weeks post-infection and used to isolate RNA. Levels of IFN-c, TNF-a and NOS2 mRNA were examined using real time quantitative PCR and normalized against the levels of 18s rRNA. Each group contained 5 mice. H, Splenocytes were obtained from control and ATG7flox/flox-Lyz-Cre mice 3 weeks post-infection with ME49 T. gondii and were incubated with TLA as described in Materials and Methods. The percentages of CD3+ CD4+ or CD3+ CD8+ T cells that became IFN-c+ or IL-4+ cells were determined by flow cytometry. Results are shown as the mean 6 SEM and are representative of 3 independent experiments. **p#0.01. doi:10.1371/journal.pone.0014472.g007 expression of these mediators does not appear sufficient to prevent ocular and cerebral toxoplasmosis since CD40 as well as the autophagy proteins Beclin 1 and Atg7 are required for protection against the disease. The autophagic machinery can enhance presentation of certain antigens [14,36] and can promote the generation of a Th1 response [14,37]. Our studies indicate that a T cell response against T. gondii can develop in mice with defective autophagy PLoS ONE | www.plosone.org 10 December 2010 | Volume 5 | Issue 12 | e14472