(anti-CD3, -CD8, -CD4, -CD19, -CD11c, -CD117) and analyzed by flow cytometry. Adoptive transfer experiments Adoptive transfer was carried out as previously described [12]. Briefly, 8- to-10-week-old wild type (C57BL/10J; B10) or P48 homozygous mutant (P48/P48) mice were injected i.v. with 106 P. berghei ANKA-parasitized erythrocytes (RBC). Five days later, spleens were collected in RPMI-3% FBS, and single cell suspensions of viable cells were prepared. Cells were washed in RPMI-3% FBS by centrifugation, and RBC were lysed by re- suspending the final pellet in red blood cell lysis buffer (Sigma), and incubating for 1 minute at RT. Cells were washed again twice as before and counted. CD8+ T cells, total T cells or NK cells were purified from infected B10 WT splenocytes by magnetic cell sorting (MACS; Miltenyi) according to the manufacturer’s instructions. 5 million CD8+ T cells, total T cells or NK cells from B10 WT infected spleens, or 20 million total WT or mutant splenocytes were transferred i.v. into P48/P48 mutant animals. Two hours later, control and reconstituted mice were infected with 106 P. berghei ANKA parasites and were monitored for appearance of cerebral symptoms and for overall survival. Infection with Mycobacterium bovis (BCG) and Mycobacterium tuberculosis Single cell suspensions of Mycobacterium bovis BCG (strain Montreal) was prepared for in vivo infections as previously described [61]. Briefly, 56104 colony-forming units (CFUs) were inoculated intravenously into 8–12 week-old mice. Six weeks after infection, mice were sacrificed, weighed and the spleen CFUs were determined by homogenization and plating on Dubos oleic agar base. The level of BCG infection was defined as the logarithm of the mean number of viable BCG recovered from spleens. The spleen index was defined as the square root of the spleen weight (6100) divided by the body weight. For Mycobacterium tuberculosis (Mtb) infection, 8–12 week-old mice were infected with 50 CFUs/ lung of M. tuberculosis H37Rv by the aerosol route, and survival was monitored. References 1. WHO website. Available: http://www.who.int/features/factfiles/malaria/en/ index.html. Accessed 2009 March. 2. Hunt NH, Grau GE (2003) Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol 24: 491–499. 3. Bongfen SE, Laroque A, Berghout J, Gros P (2009) Genetic and genomic analyses of host-pathogen interactions in malaria. Trends Parasitol 25: 417–422. 4. Kwiatkowski D (2000) Genetic susceptibility to malaria getting complex. Curr Opin Genet Dev 10: 320–324. 5. Weatherall DJ (2008) Genetic variation and susceptibility to infection: the red cell and malaria. Br J Haematol 141: 276–286. 6. Kwiatkowski DP (2005) How malaria has affected the human genome and what human genetics can teach us about malaria. Am J Hum Genet 77: 171–192. 7. de Souza JB, Hafalla JC, Riley EM, Couper KN (2010) Cerebral malaria: why experimental murine models are required to understand the pathogenesis of disease. Parasitology 137: 755–772. 8. Miu J, Mitchell AJ, Muller M, Carter SL, Manders PM, et al. (2008) Chemokine gene expression during fatal murine cerebral malaria and protection due to CXCR3 deficiency. J Immunol 180: 1217–1230. 9. Grau GE, Heremans H, Piguet PF, Pointaire P, Lambert PH, et al. (1989) Monoclonal antibody against interferon gamma can prevent experimental cerebral malaria and its associated overproduction of tumor necrosis factor. Proc Natl Acad Sci U S A 86: 5572–5574. 10. Grau GE, Fajardo LF, Piguet PF, Allet B, Lambert PH, et al. (1987) Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 237: 1210–1212. 11. Engwerda CR, Mynott TL, Sawhney S, De Souza JB, Bickle QD, et al. (2002) Locally up-regulated lymphotoxin alpha, not systemic tumor necrosis factor alpha, is the principle mediator of murine cerebral malaria. J Exp Med 195: 1371–1377. 12. Campanella GS, Tager AM, El Khoury JK, Thomas SY, Abrazinski TA, et al. (2008) Chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are A Jak3 Mutation Protects against Cerebral Malaria Infection with Citrobacter rodentium Mice were infected at four weeks of age with Citrobacter rodentium strain DBS100. C. rodentium was grown overnight in 3mL Luria- Bertani (LB) broth shaking at 37uC. Mice were infected by oral gavage of 0.1 mL of the overnight culture containing 36108 CFUs. Following infection with C. rodentium, the mice were monitored daily for 30 days post-infection. When any mouse became moribund or reached a clinical endpoint of infection (20% body weight loss, hunching and shaking, inactivity, ruffled fur, anal prolapse, overtly bloody stool, bleeding from the anus and body condition score ,2), it was immediately euthanized. Supporting Information Figure S1 Haplotype map of F2 mice from pedigree 48 for the central portion of chromosome 8 (51.9–84 Mb). F2 mice generated by crossing the G1 male to 129S1 progenitors were genotyped for microsatellite markers (Mouse Genome Informatics Database; www.informatics.jax.org) in the 51.9– 84 Mb interval, and were phenotyped for resistance and susceptibility to P. berghei induced CM. Each row represents the haplotype (A, homozygote B6; H, heterozygote; B, homozygote 129S1) of an individual mouse for the indicated polymorphic markers. (TIF) Acknowledgments The authors are indebted to Patricia D’Arcy, Genevie`ve Perreault, Nadia Prud’homme, Susan Gauthier, Normand Groulx, Mifong Tam, Fiona McIntosh and Lei Zhu for expert technical assistance. Author Contributions Critically revised the manuscript: SEB MB MAB SG SV MS PG. Conceived and designed the experiments: SEB I-GR-G MAB SG SV MS PG. Performed the experiments: SEB I-GR-G JB ST. Analyzed the data: SEB I-GR-G JB PC LL RS MB ML SV MS PG. Contributed reagents/ materials/analysis tools: SAW GAL-T RS MB ML MAB SG SV MS PG. Wrote the paper: SEB PG. required for the development of murine cerebral malaria. Proc Natl Acad Sci U S A 105: 4814–4819. 13. Rudin W, Eugster HP, Bordmann G, Bonato J, Muller M, et al. (1997) Resistance to cerebral malaria in tumor necrosis factor-alpha/beta-deficient mice is associated with a reduction of intercellular adhesion molecule-1 up- regulation and T helper type 1 response. Am J Pathol 150: 257–266. 14. Amante FH, Haque A, Stanley AC, Rivera Fde L, Randall LM, et al. (2010) Immune-mediated mechanisms of parasite tissue sequestration during experi- mental cerebral malaria. J Immunol 185: 3632–3642. 15. McQuillan JA, Mitchell AJ, Ho YF, Combes V, Ball HJ, et al. (2011) Coincident parasite and CD8 T cell sequestration is required for development of experimental cerebral malaria. Int J Parasitol 41: 155–163. 16. Senaldi G, Shaklee CL, Guo J, Martin L, Boone T, et al. (1999) Protection against the mortality associated with disease models mediated by TNF and IFN-gamma in mice lacking IFN regulatory factor-1. J Immunol 163: 6820–6826. 17. Patel SN, Berghout J, Lovegrove FE, Ayi K, Conroy A, et al. (2008) C5 deficiency and C5a or C5aR blockade protects against cerebral malaria. J Exp Med 205: 1133–1143. 18. de Kossodo S, Grau GE (1993) Profiles of cytokine production in relation with susceptibility to cerebral malaria. J Immunol 151: 4811–4820. 19. Bagot S, Campino S, Penha-Goncalves C, Pied S, Cazenave PA, et al. (2002) Identification of two cerebral malaria resistance loci using an inbred wild- derived mouse strain. Proc Natl Acad Sci U S A 99: 9919–9923. 20. Campino S, Bagot S, Bergman ML, Almeida P, Sepulveda N, et al. (2005) Genetic control of parasite clearance leads to resistance to Plasmodium berghei ANKA infection and confers immunity. Genes Immun 6: 416–421. 21. Berghout J, Min-Oo G, Tam M, Gauthier S, Stevenson MM, et al. (2010) Identification of a novel cerebral malaria susceptibility locus (Berr5) on mouse chromosome 19. Genes Immun 11: 310–318. PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e31012