Statistical analysis Statistical significance was assessed by Student’s t test and ANOVA. Mortality curves and histopathologic changes were analyzed using Mann-Whitney U test. Differences were considered statistically significant when P was ,0.05. Supporting Information Figure S1 CD40 induces anti-T. gondii activity in microglia independently of IFN-c and nitric oxide. A, B, Primary brain microglia were incubated with a stimulatory anti-CD40 or control mAb, or with IFN-c/TNF-a as indicated followed by infection with tachyzoites of RH T. gondii. Microglia were also treated with a neutralizing anti-IFN-c mAb (A) or NMA (B) prior to addition of anti-CD40 mAb or IFN-c/TNF-a. The numbers of tachyzoites per 100 microglia were determined microscopically at 18 h post- infection. Results are shown as the mean + SEM and are representative of 3 independent experiments. **p,0.01. Found at: doi:10.1371/journal.pone.0014472.s001 (0.11 MB TIF) Figure S2 Autophagy mediates anti-T. gondii activity induced by CD40 in microglia. A, Primary brain microglia from B6 mice were incubated with a stimulatory anti-CD40 or control mAb, or with IFN-c/TNF-a as indicated. 3-MA or vehicle were added 1 hour after challenge with RH T. gondii. The numbers of tachyzoites per 100 microglia were determined microscopically at 18 hours post- infection. B–D, Knockdown of Atg5 abrogates anti-T. gondii activity induced by CD40 stimulation. Primary bone marrow- derived macrophages (B), primary brain microglia (C) or BV-2 cells (D) were transduced with EGFP-encoding lentiviral vectors that contained either shRNA against Atg5 or control shRNA. EGFP+ cells were sorted 4 days after transduction. Protein expression of Atg5 and actin were examined by immunoblot. Cells were then incubated with a stimulatory anti-CD40 mAb, control mAb or IFN-c/TNF-a followed by infection with RH T. gondii. The numbers of tachyzoites per 100 microglia/macrophag- es were determined microscopically at 18 h post-infection. Results are shown as the mean + SEM and are representative of 3 independent experiments. **p,0.01. Found at: doi:10.1371/journal.pone.0014472.s002 (0.17 MB TIF) Figure S3 CD40 induces anti-microbial activity against not only type I but also type II strains of T. gondii, and this effect is independent of Irga6. A, BV-2 cells were incubated with a stimulatory anti-CD40 or control mAb followed by challenge with tachyzoites of the RH strain of T. gondii (type I strain) or the P strain (ME49 clone) of the parasite (type II strain). The numbers of tachyzoites per 100 microglia were determined microscopically at 18 hours post-infection. B, BV-2 cells were transfected with control siRNA or siRNA against Beclin 1. BV-2 cells transfected with control siRNA or siRNA against Beclin 1 were incubated with a stimulatory anti-CD40 mAb, control mAb or IFN-c/TNF-a References 1. Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6: 463–477. 2. Mizushima N, Ohsumi Y, Yoshimori T (2002) Autophagosome formation in mammalian cells. Cell Struct Funct 27: 421–429. 3. Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MC, et al. (2004) Autophagy is defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119: 753–766. 4. Nakagawa I, Amano A, Mizushima N, Yamamoto A, Yamaguchi H, et al. (2004) Autophagy defends cells against invading Group A Streptococcus. Science 306: 1037–1040. 5. Ogawa M, Yoshimori T, Suzuki T, Sagara H, Mizushima N, et al. (2004) Escape of intracellular Shigella from autophagy. Science 307: 727–731. 6. Andrade RM, Wessendarp M, Gubbels MJ, Striepen B, Subauste CS (2006) CD40 induces macrophage anti-Toxoplasma gondii activity by triggering Adaptive Immunity, Autophagy followed by infection with a type II strain of T. gondii (P strain). C, BV-2 cells were transfected with control or Irga6 siRNA. Protein expression of Irga6 and actin were examined by immunoblot. Cells were then incubated with a stimulatory anti-CD40 mAb, control mAb or IFN-c/TNF-a followed by infection with a type II strain of T. gondii (P strain). Results are shown as the mean + SEM and are representative of 3 independent experiments. **p,0.01. Found at: doi:10.1371/journal.pone.0014472.s003 (0.15 MB TIF) Figure S4 CD40 does not affect Beclin 1 mRNA levels. A, Primary brain microglia from B6 mice were incubated with a stimulatory anti-CD40 mAb or control mAb. Serial dilutions of cDNA were used to examine levels of Beclin 1 mRNA by real-time quantitative PCR, which were normalized against the levels of 18s rRNA. B, Four weeks after infection with ME49 T. gondii, microglia/macrophages were purified from brains of B6 or CD402/2 mice. Beclin 1 mRNA levels were examined by real- time PCR. Each bar represents a single mouse. Results are shown as the mean + SEM and are representative of 3 independent experiments. Found at: doi:10.1371/journal.pone.0014472.s004 (0.09 MB TIF) Becn1+/2 Figure S5 mice do not have abnormal lymphoid organ phenotypic composition or defective induction of T. gondii- specific T cell response. A, Splenocytes were obtained from uninfected control, Becn1+/2 and CD402/2 mice. Expression of CD4+ T cells (CD3+ CD4+), CD8+ T cells (CD3+ CD8+), B cells (CD45R + ), NK cells (CD49d+), and granulocytes (Ly6-G+) were examined by flow cytometry. B, Splenocytes were obtained from control, Becn1+/2 and CD402/2 mice 4 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. C, Purified CD4+ T cells were obtained 4 weeks post-infection with ME49 T. gondii and were stimulated with macrophages plus TLA or Concanavalin A (Con A). IFN-c in supernatants was measured by ELISA. Results are shown as the mean + SEM and are representative of 3 independent experiments. Found at: doi:10.1371/journal.pone.0014472.s005 (0.12 MB TIF) Acknowledgments We thank Drs. Tamotsu Yoshimori, George Yap, Louis Weiss, Kalipada Pahan and Jun Tan for providing reagents, parasites and cell lines. Author Contributions Conceived and designed the experiments: JACP CSS. Performed the experiments: JACP GO ER AS JVG KG CSS. Analyzed the data: JACP GO AS CSS. Contributed reagents/materials/analysis tools: MK KT GL BL. Wrote the paper: JACP CSS. autophagy-dependent fusion of pathogen-containing vacuoles and lysosomes. J Clin Invest 116: 2366–2377. 7. Ling YM, Shaw MH, Ayala C, Coppens I, Taylor GA, et al. (2006) Vacuolar and plasma membrane stripping and autophagic elimination of Toxoplasma gondii in primed effector macrophages. J Exp Med 203: 2063–2071. 8. Subauste CS, Andrade RM, Wessendarp M (2007) CD40-TRAF6 and autophagy-dependent anti-microbial activity in macrophages. Autophagy 3: 245–248. 9. Yano T, Mita S, Ohmori H, Oshima Y, Fujimoto Y, et al. (2008) Autophagic control of listeria through intracellular innate immune recognition in drosophila. Nature Immunology 9: 908–916. 10. Shelly S, Lukinova N, Bambina S, Berman A, Cherry S (2009) Autophagy is an essential component of Drosophila immunity against vesicular stomatitis virus. Immunity 30: 588–598. PLoS ONE | www.plosone.org 13 December 2010 | Volume 5 | Issue 12 | e14472