genes have shown that host-driven inflammation plays a central role in CM pathogenesis [2,8]. Indeed, local production of pro- inflammatory cytokines such as IFN-c [9], TNF-a [10] and LT-a [11], upregulation of chemokine ligands [12] and cell adhesion molecules [13], as well as sequestration of immune cells (CD4+, CD8+, NK cells) [12,14,15] and parasites [15] in the brain microvasculature have all been shown to play a role in CM. On the other hand, inactivating mutations in genes coding for pro- inflammatory molecules [16,17], and generation of anti-inflam- matory cytokines like IL-10 [14] and IL-4 [18] have been associated with protection from CM. Genome-wide linkage studies in informative crosses derived from mouse strains showing varying degrees of susceptibility to P. berghei- induced CM have detected at least six quantitative trait loci (QTL) - designated berghei resistance (Berr) loci – as modulating response to infection including survival from acute infection: Berr1 (chromosome 1), Berr2 (chromosome 11), Berr3 (chromosome 9), Berr4 (chromo- some 4), Berr5 (chromosome 19), and the Cmsc locus mapping to the H-2 region of chromosome 17 [19,20,21,22]. The Berr5 locus co- localizes with three other immune loci, including Trl-4 (tuberculosis resistance), Tsiq2 (T-cell secretion of IL-4), and Eae19 (experimental allergic encephalitis 19) suggesting the possibility of a common genetic effect underlying these phenotypes. Nevertheless, the modest effects of these individual loci, the relatively large size of the chromosomal regions mapping underneath the QTL peaks, and the large number of positional candidates have precluded the positional cloning of the genes involved. ENU mutagenesis is a powerful experimental tool used to introduce random mutations in the mouse germ-line. Such mutations can be propagated in informative pedigrees, where they can be bred to homozygosity, and where their effect on a given physiological system or host pathway can be investigated. In high throughput screening experiments, such mutations may manifest themselves as rare pheno-deviant pedigrees displaying unique disease-associated phenotypes. The positional cloning of the mutant gene, facilitated by the de novo nature of the mutation (absent from the reference sequence), may in turn identify novel proteins that play a role in the specific phenotype and associated pathology. This strategy has been used successfully to identify genes, proteins and pathways in a broad range of disease states, including susceptibility to infections [23], obesity [24], muscle development and function [25], cardiomyopathy [26] and thrombocytopenia [27]. In this study, we implemented a large scale ENU mutagenesis strategy to identify genes that play an important role in the pathogenesis of cerebral malaria. Intravenous infection of C57BL/ 6J and C57BL/10J mice with 106 P. berghei-parasitized erythro- cytes is uniformly lethal with all animals developing cerebral symptoms by day 5–6 and succumbing to infection by days 7–10. We searched for recessive mutations that would protect mice form P. berghei-induced CM and associated lethality, and that would confer survival to this otherwise lethal infection. We aimed to identify novel protein and biochemical pathways that may constitute novel targets for small molecule inhibition and therapeutic intervention in this lethal infection. In a first example of this screen, we report the identification of a pheno-deviant pedigree that displays segregation of a CM-resistance phenotype. We demonstrate that this resistance is phenotypically expressed as a severe depletion of several immune cell compartments including CD8+ T cells, B cells and NK cells, and caused by a mutation in the Jak3 gene. Resistance to CM in this mutant is associated with an impaired Th1 response, which is concomitant with increased susceptibility to infection with mycobacteria (M. tuberculosis), and Citrobacter. A Jak3 Mutation Protects against Cerebral Malaria Results Identification and characterization of a cerebral malaria resistant ENU mutant To identify genes, proteins, and cellular pathways important for the pathogenesis of cerebral malaria (CM), we screened pedigrees derived from ENU-mutagenized mice, looking for the appearance of CM-resistant pheno-deviant pedigrees on the otherwise CM- susceptible genetic background of C57Bl/6J (B6). Such pedigrees are believed to segregate protective mutations fixed for homozy- gosity, and affecting genes that are important for CM pathogenesis including host-driven detrimental effects. In our protocol, mutagenized B6 males were crossed to C57Bl/10J (B10, to facilitate subsequent genetic mapping), and the resulting G1 males were backcrossed to B10 (Figure 1A); the resulting G2 females (two per G1 male) were backcrossed to their G1 father to produce G3 pedigrees where mutations are fixed to homozygosity in 25% of the animals. These G3 pedigrees were infected with P. berghei ANKA, and we monitored the presence of pheno-deviant progeny that fail to develop cerebral symptoms and survive this infection. When such positive pedigrees were detected, additional G3 animals from the same G2 females and G1 father were generated and phenotyped to validate the presence of a protective mutation. Screening a total of 3967 G3 mice from 153 pedigrees identified several such pheno-deviant pedigrees. One of these pedigrees, #48 (P48), displayed a fairly high percentage of resistant animals (,31% resistance), with both G2 females producing CM-resistant offspring (Figure 1B), and was chosen for further analysis. A genome-wide scan was carried out in 44 G3 animals from P48 (15 CM-resistant and 29 CM-susceptible) using 131 polymorphic markers informative for B6 and B10. Linkage analysis (using R/ qtl) identified a 17 Mb region on the central portion of chromosome 8 (95% Bayesian credible interval: 60.4 Mb– 77.4 Mb) as regulating differential CM-resistance in this pedigree, with a logarithm of odds (LOD) score of 5.8 (Figure 2A). Haplotype analysis revealed that, as expected, resistance to CM at this locus was associated with homozygosity for B6-derived alleles (from mutated G0 male), while homozygosity for B10 alleles was associated with CM-susceptibility, and with B6/B10 heterozygotes being present in both resistant and susceptible groups (Figure 2B). These findings suggested that the CM-protective effect of this locus is co-dominant in this cross. The P48 G1 male was also outcrossed to CM-susceptible 129S1 WT females, and the resulting offspring were intercrossed to generate a total of 211 F2 mice, which were phenotyped by infection with P. berghei ANKA (Figure 2C). Approximately 25% of these F2 survived the cerebral phase, indicating that the resistance trait associated with P48 is fully penetrant on the distinct genetic background of 129S1. Genotyp- ing of these animals verified that CM resistance was controlled by the chromosome 8 locus, and also showed a co-dominant mode of inheritance of B6 protective alleles in this cross (Supplementary Figure S1). Immunological phenotyping of P48 mutants To gain insight into the cell population phenotypically expressing the CM-protective mutation, we examined different lymphoid and myeloid organs, and monitored specific cell populations within them in mice fixed for homozygosity for B6- derived alleles at the chromosome 8 locus. Macroscopic examination readily identified severe thymic atrophy in homozy- gotes (Figure 3A), while heterozygotes were normal, suggesting a possible thymus development defect. FACS analysis further identified a severe depletion of the CD8+ T cell compartment in thymus of these mice, while the CD4+ T cell compartment PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31012