A Jak3 Mutation Protects against Cerebral Malaria atrophied thymus in homozygote mutants (representative of 5 mice per group). (B) FACS density plots of different cell populations in thymus (top), spleen (middle) and bone marrow (bottom) stained for CD4, CD8, CD19, and CD117. The position of the different cell lineages in the scatter plots are identified at the extreme right panel and their numbers are expressed as a percentage (6 SE; n = 5 mice per group) of total cells in this tissue. (C) Flow cytometric analysis of immune cell lineage composition expressed as the absolute number (mean 6 SD; n = 4–6 mice per group) of CD4+ and CD8+ single positive (SP), CD4+CD8+ double positive (DP), B cells (CD19+), granulocytes (GR; Gr1+), hematopoietic stem cells (HSC; lineage 2 CD117+) and NK cells from 105 cells from spleen, thymus and bone marrow. Asterisks (one-way Anova test with Bonferroni post-test) identify significant differences between experimental animals and the C57BL/6J controls: *p,0.05; ** p,0.01; *** p,0.001. Data are representative of two independent experiments. doi:10.1371/journal.pone.0031012.g003 for drug development and therapeutic intervention in CM. Studies in human clinical cases of CM and experiments in mouse models of P. berghei-induced CM have identified excessive TNF-a and IFN- g-driven inflammatory response as a key determinant in CM pathogenesis [2]; novel strategies to blunt this response have shown promise in the prevention and treatment of CM [35,36,37,38,39]. We have implemented a genome-wide muta- genesis screen in mice to systematically identify genes and proteins that mediate pathological inflammatory responses during P. berghei infection in vivo, and whose pharmacological or genetic inactiva- tion may protect from CM. We report on the first mutant identified in this screen. Linkage analyses, genomic DNA sequencing, and complementation studies in double heterozygotes have established that CM protection in this mutant is caused by a mutation (W81R) in the amino-terminal FERM domain of Jak3 (Janus kinase 3; Jak3W81R). Jak3 is a cytosolic tyrosine kinase expressed primarily in the hematopoietic system that plays a critical role in: a) the ontogeny of different myeloid and lymphoid cells, and b) the response of these cells to stimulation by different cytokines. Jak3 interacts with the common gc chain of type 1 and 2 cytokine receptors, which includes IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. This interaction causes recruitment and phosphorylation of STAT family members to trigger downstream transcriptional responses in cells expressing such receptors [40]. In humans, loss of either JAK3 or IL2RG (which codes for the common gc chain) causes autosomal and X- linked T2B+ SCID, respectively [41]. These patients lack T cells and NK cells, have normal numbers of immature and poorly functional B-lymphocytes, a clinical picture in agreement with the established roles of IL-7 (T cell development), IL-2 (peripheral T cell homeostasis and antigen-driven T-cell expansion), IL-15 (differentiation of NK cells), and IL-4 (B-cell maturation and isotype switching) in which associated signaling is impaired in JAK3 mutants [41]. Jak3W81R homozygote mice showed a 2 + SCID in humans, and displayed phenotype that overlaps T B an atrophied thymus, a low number of thymic and splenic CD8+ T cells and NK cells, as well as a near absence of B cells in the bone marrow. CD4+ T cells were present in normal numbers but appeared anergic and did not produce IFN-g in response to simulation with PMA or ionomycin (under conditions of Th1 Figure 4. A W81R mutation in the FERM domain of Jak3 causes resistance to cerebral malaria in pedigree 48. (A) Schematic representation of the Jak3 protein, showing the 7 Jak Homology (JH) structural domains, and associated functional annotation (below). The position of the W81R mis-sense mutation discovered in pedigree 48 is shown. (B) Multiple sequence alignment of the amino terminal portion of Jak3 surrounding W81 shows high conservation across Jak3 relatives (the corresponding species is identified). doi:10.1371/journal.pone.0031012.g004 PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31012