A Jak3 Mutation Protects against Cerebral Malaria Figure 1. ENU-induced mutation that protects mice against P. berghei ANKA-induced cerebral malaria. (A) Breeding scheme for the production and identification of ENU-induced recessive mutations that convey protection against cerebral malaria (CM). Details of the breeding strategy are described in ‘‘Materials and Methods’’. G3 and F2 pedigrees were phenotyped for the presence of animals resistant to P. berghei-induced CM. (B) Mice were infected with P. berghei ANKA (106 P. berghei ANKA-parasitized red blood cells, i.v.) and survival was monitored over time for individual pedigree P48 G3s (green and blue lines) derived from independent G2 females and a G1 male, and for susceptible C57BL/6J (B6, red line), and resistant mutant mouse strains bearing loss of function mutations in either the IFN-c gene KO (IFN-c KO, black line) or IRF8 (BXH2, brown line). Mice surviving past day 13 post infection were considered to be CM-resistant. doi:10.1371/journal.pone.0031012.g001 appeared unaffected. A similar severe depletion of the CD8+ T cell compartment was also detected upon analysis of spleen cells from these mice (Figure 3B), with an additional reduction in NK cells (Figure 3C). On the other hand, studies of bone marrow cells showed a complete absence of CD19+ B cells in mutant mice. In all tissues examined, there was no effect noted on the myeloid compartment (Gr1+ cells). All alterations in these cell populations were detected only in homozygotes, while heterozygotes showed normal cell numbers, which were similar to C57BL/6J controls. Resistance to cerebral malaria is caused by a mutation in the Jak3 kinase The 17 Mb interval delineating the position of the CM- protective locus on chromosome 8 contains a number of positional candidates that a) have an established role in the immune system, b) are known to be modulated by IFN-c, or c) show IFN-inducible STAT1 binding sites in their promoter. These include Interleukin 12 receptor beta 1 (IL12rb1; 73.3 Mb), interferon gamma inducible protein 30 (Ifi30; 73.2 Mb), Janus kinase 3 (Jak3; 74.2 Mb), heme oxygenase (decycling) 1 (Hmox1; 77.6 Mb) and unc-13 homolog A (Unc13a; 74.1 Mb). Whole genome sequencing of genomic DNA from P48 mutant homozygotes was undertaken, and candidate variants in the above-mentioned genes that were absent from the B6 reference sequence were further validated by re-sequencing. This analysis revealed a unique T-to-A transversion in exon 2 of the Jak3 gene, which causes a W81R substitution in the Band 4.1/Ezrin/Radixin/Moesin (FERM) homology domain at the N-terminus of the Jak3 protein (Figure 4). The FERM domain is involved in mediating interactions of Jak3 with different cytokine receptors in immune cells. The tryptophan at position 81 is absolutely conserved in Jak3 relatives from different species, suggesting a conserved structural/functional role of this residue in Jak3 activity. To confirm that the CM-resistance in P48 mutants was caused by a mutation in Jak3, we carried out complemen- tation testing using Jak32/2 mice (B6.129S4-Jak3tm1Ljb; MGI database, [28]). These animals have previously been shown to PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31012