Caspase-8-mediated cell death of CD8⁺ T cells is required for control of Toxoplasma gondii in the brain

Host cell death represents a major mechanism of limiting intracellular pathogens. Toxoplasma gondii is an intracellular protozoan parasite that establishes a chronic infection of the brain and other immune-privileged tissues. We sought to determine whether cell death is a mediator of parasite control in the brain. Numerous types of programmed cell death have been defined and are regulated by distinct molecular pathways. We focused on the role of caspase-8, which is associated with the extrinsic pathway of apoptosis. The deletion of Casp8 alone in mice results in embryonic lethality associated with widespread necrosis. The additional deletion of Ripk3 results in mice that are viable. We began by infecting mice that lacked Casp8 and Ripk3, Ripk3 alone, and WT C57BL/6 controls with 10 cysts of Me49 parasites. We found that Casp8^-/-Ripk3^-/- mice were susceptible to T. gondii infection and survived approximately 50 days with overwhelming parasite burdens in the brain. We found that mice lacking Ripk3 and the ability to trigger necroptosis were resistant to infection and had parasite burdens comparable to WT mice. Intriguingly, Casp8^-/-Ripk3^-/- mice had equivalent or stronger immune responses to T. gondii in the brain with the recruitment of IFN-γ-producing T cells and iNOS-producing macrophages. These results suggested that specific cell types in the brain may be particularly susceptible to T. gondii infection in the absence of Casp8. To this end, we infected WT and Casp8^-/-Ripk3^-/- mice on a cre-reporter background (flox-stop-flox ZsGreen) with a cre-secreting parasite (Pru-cre-mCherry). As previously reported, we found that primarily neurons are infected in wildtype mice. In Casp8^-/-Ripk3^-/- mice we found that many more neurons, astrocytes, and surprisingly CD8⁺ T cells had interacted with parasite. Infection of CD8⁺ T cells by T. gondii in WT mice has been previously reported by several groups. Given that CD8⁺ T cells are highly migratory in the brain, cell death may be required to restrict pathogen spread throughout the brain. To test the role of Casp8 specifically in CD8⁺ T cells, we generated CD8a-cre x Casp8^fl/fl x Ripk3^-/- x Ai6 mice and Casp8^fl/fl x Ripk3^-/- x Ai6 controls. We find that mice with Casp8-deficient CD8⁺ T cells have elevated levels of parasite and CD8⁺ T cells in the brain at six weeks post-infection. These data suggest that cell death is an additional mechanism by which CD8+ T cells restrict parasite in the brain.