biotin (Jackson ImmunoResearch Laboratories). Sections were resolved using a Vectastain ABC kit (Vector Laboratories). Real-time quantitative PCR RNA was obtained from eyes and brains using the RNeasy kit (QIAGEN). After treatment with DNase (Ambion), 0.5 mg of RNA was reverse transcribed to cDNA with Super-Script III reverse transcriptase (Invitrogen) and oligo(dT)12–18 primers. cDNA (2.5 ml) was used as template for RT-PCR using SYBR GREEN PCR Master Mix (Applied Biosystems) and 20 pM (each) primers in 50 ml. Gene expression was assessed using a 7300 Real Time PCR System (Applied Biosystems). Each cDNA sample was run in duplicate. Samples were normalized according to the content of 18S rRNA [55]. Expression of the T. gondii B1 gene was assessed by real-time quantitative PCR. Genomic DNA was isolated from eyes using a DNeasy kit (QIAGEN) and subjected to real-time PCR using SYBR GREEN PCR Master Mix. Each sample was run in duplicate and normalized against L32. Primer sequences for IFN-c [57], TNF-a [57], NOS2 [58], 18S rRNA [55] and T. gondii B1 gene [59] were previously described. Primer sequences for Beclin 1 were forward: 59-GGCCAATAAGATGGGTCTGA-39, reverse: 59-GCTGCACACAGTCCA GAAAA– 39. Primers for the T. gondii B1 gene have been previously described [59]. Primers for L32 were: forward 39-TGTGCAACAAATCTTCACCG- TGC-59; reverse 39-GGATTGGTGACTCTGATGGCC-59. Microglia and in vitro infection with T. gondii Primary brain and retinal microglia as well as mouse brain microglial cell lines BV-2 and N9 (gifts from Drs. Kalipada Pahan, Univ. of Nebraska and Dr. Jun Tan, Univ. of South Florida respectively) were treated overnight with a stimulatory anti-CD40 mAb (1C10; 1 or 10 mg/ml), isotype control mAb or IFN-c (100 U/ml; PeproTech) plus TNF-a (250 pg/ml; PeproTech) [51]. Tachyzoites of the RH or ME49 strains of T. gondii were used to infect monolayers as described [51]. When indicated, microglia were incubated with NG-monomethyl-L-arginine (NMA, 100 mM; Calbiochem) or a neutralizing mAb against mouse IFN-c (XMG 1.2; 10 mg/ml; eBiosciences) in the presence of anti-CD40 mAb, control mAb or IFN-c/TNF-a. 3-methyl adenine (3-MA; 10 mM; Sigma-Aldrich) was added to monolayers 1 hour after infection. Parasite replication was assessed by light microscopy [51]. To isolate brain mononuclear cells from infected mice, brains were digested with papain (15 IU/ml)/DNAse (15 mg/ml; Worthington Biochemicals) for 30 min at 37uC. Tissue was dissociated and cells were loaded onto a gradient made with 30% and 60% isotonic Percoll. Gradients were centrifuged at 10006 g for 30 min. Cells present in the interphase were collected and washed. Microglia/ macrophages were purified using anti-CD11b-coated magnetic beads (Miltenyi Biotec). This resulted in populations that were .95% F4/80+. Lentiviral vectors We constructed an shRNA against mouse Atg5 using the following oligo pair (sense and antisense strands are underlined while the loop is italicized): forward 59-TGGATGAGATAACT- GAAAGATTCAAGAGA TCTTTCAGTTATCTCATCCTTT- TTTC-39; reverse 59-TCGAGAAAAAAGGATGA GATAACT- GAAAGATCTCTTGAATCTTTCAGTTATCTCATCCA-39. An XhoI restriction site was included at the 59-end of the reverse oligo to facilitate cloning. An shRNA against luciferase was used as control [60]. Oligo pairs were annealed and subcloned into the polylinker region of the pLL3.7 vector [61] (American Type Tissue Collection) followed by sequence verification. To generate Adaptive Immunity, Autophagy lentivirus, we co-transfected pLL3.7 containing shRNA and packaging vectors VSV-G, RSV-REV, pMDL-g/p RRE into 293T cells. Supernatants were collected at 24 and 48 h, passed through a 0.45 mm filter, concentrated by ultracentrifugation and stored at 280uC. Cells were transduced by incubation with lentiviral particles at a MOI of 10:1 in the presence of polybrene (8 mg/ml). EGFP+ cells were sorted by FACS at 4 days post- transduction. Efficiency of gene silencing was determined by immunoblot. Transfection of microglia and immunofluorescence Using an Amaxa nucleofector as previously described [6], BV-2 cells were transfected with either control siRNA, Beclin 1 siRNA [62], p21 siRNA [27], Irga6 siRNA [63] (all from Dharmacon), empty vector, or plasmid that encode Beclin 1 [64], p21 [65] or LC3-EGFP (gift from Tamotsu Yoshimori, National Institute for Basic Biology, Okazaki, Japan). The numbers of LC3+ punctae were determined using a Leica DMI 6000 B automated microscope equipped for epifluorescence microscopy. Distinct structures that measure at least 1 mm in diameter were identified as LC3 punctae. Experimental groups had triplicate samples and at least 50 cells per sample were counted. Co-localization of LC3- EGFP around RFP-T. gondii was assessed as described [6]. Immunoblot Membranes were probed with either antibody to Atg5 (ProteinTech Group Inc), Atg7 (Cell Signaling), Beclin 1 (BD Biosciences), p21 (Santa Cruz Biotechnologies), NOS2 (BD Biosciences), TNF-a (Abcam Inc.), Irga6 (Santa Cruz Biotechnol- ogies) or actin (Santa Cruz Biotechnologies), followed by incubation with secondary Ab conjugated to horseradish perox- idase (Santa Cruz Biotechnologies). Bands were developed using enhanced chemiluminescence. In experiments that assessed the effect of CD40 stimulation on Beclin 1, Atg5, Atg7 and p21 expression, microglia were pre-incubated with IFN-c for 18 h to increase the percentage of CD40+ cells. After extensive washing, microglia were incubated with anti-CD40 or control mAb. Pre- incubation with IFN-c was never used in studies of CD40- dependent anti-microbial activity. Flow cytometry Cells were stained with anti-CD3, anti-CD4, anti-CD8, anti- CD11b, anti-CD40, anti-CD45R, anti-CD49d (DX5), anti- Ly6G (Gr-1), anti-F4/80 or isotype control mAbs (all from eBiosciences). For detection of intracellular cytokines, spleno- cytes were incubated with T. gondii lysate antigen (TLA; 10 mg/ ml) for 48 and 72 h. PMA (1 ng/ml; Sigma Chemical), ionomycin (1 mg/ml; Sigma Chemical) and brefeldin A (10 mg/ ml; eBiosciences) were added during the last 5 h of incubation. Cells were permeabilized using IntraPrep permeabilization reagent (Coulter-Immunotech) and stained with anti-IFN-c or anti-IL-4 mAb (eBiosciences). Cells were analyzed using an LSR II (BD Biosciences). Cytokine assays CD4+ T cells were purified from the spleens of infected animals using magnetic beads (Miltenyi). CD4+ T cells (16106/ ml) and were incubated with autologous bone marrow derived macrophages (2.56105/ml) plus either TLA (10 mg/ml) or concanavalin A (5 mg/ml; Sigma Chemical). Supernatants were collected at 48 h. Concentrations of IFN-c in tissue culture supernatants were determined by enzyme-linked immunosor- bent assay (Endogen). PLoS ONE | www.plosone.org 12 December 2010 | Volume 5 | Issue 12 | e14472