In vivo Cytotoxicity Assay

Cytotoxic activity of CD8⁺ T cells was measured as previously described⁹¹. Briefly, WT and Trem2 ^−/− mice were infected with LCMV WE. Eight days post infection syngeneic splenocytes from uninfected C57BL/6 mice were pulsed with the LCMV immune-dominant epitope GP_33-41 (KAVYNFATC) at a concentration of 10 µM in RPMI 10% FCS with 2-mercaptoethanol for 4 hours. GP_33-41-pulsed and unpulsed cells were fluorescently labelled (Cell Proliferation Dye eFluor 450, eBioscience) at two different concentrations. After labelling, the two populations were mixed in equal ratios. Virus-infected or uninfected control mice were intravenously injected a total of 1.5 × 10^7 cells. Four hours later, the ratio of eFluor^hi (GP_33-41) and eFlour^low (unpulsed) cells was determined in the blood of recipient mice via flow cytometry. Specific killing was calculated as 100 – ([(% eFluor^hi infected mouse/% eFluor^low infected mouse)/(% eFluor^hi uninfected mouse/% eFluor^low uninfected mouse)] × 100).