Lfa 1

Brains and spleens were harvested at the indicated days after infection following perfusion with cold PBS. Spleen single-cell suspensions were prepared by homogenization through a 70 µm cell strainer (BD Falcon). Brains were digested with 50 U/mL of type II and IV collagenase (Gibco, Invitrogen) in RPMI, and mononuclear cells isolated on a 33% Percoll density gradient (GE Healthcare). After lysing red blood cells and determining the total cell concentration, cells were incubated with an FcR blocker (Miltenyi Biotech), labelled for the extracellular antigens CD3, CD8, CD4 (BD Pharmingen), CD69, LFA-1, CD62L, CXCR3, CD25 (all from Biolegend), fixed with 4% paraformaldehyde (PFA) and stained for the intracellular markers Granzyme B (Biolegend) and Foxp3 (eBioscience) using the Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer’s instructions. Single fluorochrome-labelled cells were used to compensate for spectral overlap. Isotype-matched fluorescence minus one controls were used to set gate cutoffs. Acquisition was performed using a BD Fortessa and data analysed using FlowJo (Tree Star Inc.).