Figure 5. Resistance to cerebral malaria in Jak3W81R/2 com- pound heterozygotes. Jak3W81R homozygotes were crossed to a mouse line bearing a Jak3 allele (Jak32/2) to create the Jak3W81R/+ compound heterozygotes. Single heterozygotes (Jak3W81R/+, Jak3+/2; Fig. 5A), homozygotes (Jak3W81R, Jak32/2; Fig. 5B), compound heterozygotes (Jak3W81R/2, gray line; Fig. 5A) and C57BL/10J controls were infected with 106 P. berghei ANKA-parasitized RBCs and monitored for survival. All surviving mice were sacrificed on day 15 post-infection (experimental end-point). The data are representative of 2 independent experiments. doi:10.1371/journal.pone.0031012.g005 polarization) (Figures 3 and 4). This immune phenotype closely resembled that of previously described Jak3 knockout mice [28]. What is the mechanism underlying protection against P. berghei- induced CM in Jak3W81R homozygotes? IFN-g plays a critical role in initiating and amplifying pathological inflammatory response during CM, and mouse mutants lacking the IFN-g gene are protected against P. berghei-induced CM (Fig. 1B, [42,43]). Although the dominant cell type(s) responsible for early production of IFN-g in vivo during P. berghei infection has been debated, NK cells, CD4+ and CD8+ T cells have all been implicated [44,45], and all three populations are affected in Jak3W81R homozygotes. In the case of NK cells, results either supporting or excluding a role for these cells in CM pathogenesis have been published. In one study, depletion of NK cells failed to alter the appearance of cerebral symptoms or the outcome of CM in P. berghei infected mice [43], while another study found that IFN-g secretion by NK cells was important for recruitment of CXCR3+ CD4+ and CD8+ T cells to the brain and development of cerebral disease [43,44]. On the other hand, cell depletion and cell transfer experiments in vivo have shown that IFN-g production by CD4+ and CD8+ T cells can both contribute to CM pathogenesis [12,46,47,48]. Results from our adoptive transfer studies in Jak3W81R homozygotes (Figure 6) provide additional insight into this question. We observed that: a) total spleen cells from C57BL/10J mice were A Jak3 Mutation Protects against Cerebral Malaria the only cell population that could fully restore CM-susceptibility in the mutants; b) total T cells and purified CD8+ T cells had a similar effect and caused partial but significant reversion to CM susceptibility in Jak3W81R animals; c) transfer of purified wild type NK cells had no impact on the CM resistance of the Jak3W81R mutants. These results strongly suggest that CD8+ T cells are the major cell type contributing to CM pathogenesis, although other spleen cell populations or other cell:cell interactions, for example T cell dependent NK cell activation [49], appear to be required to observe the full effect. Nevertheless, our results clearly establish a role of the Jak3 kinase in the pathogenesis of cerebral malaria. This participation may reflect the function of Jak3 in the ontogeny of cell populations (NK cells, CD8+ T cells) that produce IFN-g and other soluble mediators of the pathological inflammatory response that are absent in the Jak3W81R mutant. The protective effect of Jak3W81R may additionally involve inhibition of gc chain- dependent cytokine receptor signaling in other cell types, whose ontogeny is not affected by the Jak3 mutation. Nevertheless, our results suggest that pharmacological inhibition of Jak3 may be of therapeutic value in CM. Several small molecule Jak3 inhibitors have been developed and are undergoing clinical evaluation for inflammatory conditions such as rheumatoid arthritis, psoriasis and several autoimmune conditions including autoimmune encephalitis, and rejection of organ transplants [50,51]. Our findings raise the interesting possibility that Jak3 inhibition by some of these molecules may represent a novel strategy for intervention in clinical cases of CM, a proposition that can be tested experimentally. An intriguing finding of our study is the intermediate CM- resistance phenotype characteristic of Jak3W81R/+ heterozygotes, with a proportion of these animals either succumbing late in the cerebral phase or completely surviving the cerebral phase. This was first noticed in haplotype analyses of G3 mice of pedigree 48, with animals heterozygote for the chromosome 8 markers being found in both the CM-resistant and CM-susceptible groups (Figure 2B), and subsequently verified during P. berghei infection of genotyped Jak3W81R/+ hetrozygotes (Figure 5). The effect is not caused by the genetic background of the animals and is specific for Jak3W81R/+ heterozygosity, as is seen when the mutation is introduced onto either B6/B10 or B6/B10-129S1 mixed genetic backgrounds (Figures 2B, S1). The cellular and molecular basis of co-dominance of the Jak3W81R mutation is intriguing. It could be explained either by a partial loss of Jak3 function in a dosage dependent pathway or by a specific dominant negative effect of the Jak3W81R allele. The observation that mice heterozygote for a Jak3 mutation (Jak3+/2) are as susceptible to CM as wild type B6 controls clearly argues for the latter possibility. Interestingly, immunophenotyping results of Jak3W81R/+ heterozygotes show that CM protection in these animals is not associated with alterations in the numbers of NK, T and B lymphocytes, which are all present at normal levels when compared to controls (Figures 3B, 3C). Normal production of IFN-g in response to PMA and ionomycin stimulation under Th1 polarization assay conditions is also seen in Jak3W81R/+ heterozygotes (data not shown). This suggests the possibility of a more subtle dominant negative effect of Jak3W81R on the biochemical properties of Jak3 in cytokine signaling, and that would nevertheless be critical for establishing the inflammatory process during CM. Such a mechanism could take place in the context of sufficient Jak3 activity that would a) allow seemingly normal maturation of different immune cell lineages (NK, B, T cells), but b) not be sufficient to mediate appropriate signaling during an acute inflammatory situation such as CM. The inability of transferred Jak3W81R/+ heterozygote spleen cells to modify CM-resistance of Jak3W81R homozygotes PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31012