α lfa 1 h155 78

Isolated splenocytes at 6 dpi were stained with LIVE/DEAD Aqua followed by Pb‐1‐specific tetramer and surface markers staining. The following antibodies were used: α‐CD62L (clone MEL‐14, Biolegend, 1:200 dilution), α‐CXCR4 (clone 2B11, ebioscience, 1:200 dilution), α‐CD43 (clone 1B11, Biolegend, 1:200 dilution), α‐CD44 (clone 1M7, BD Biosciences, 1:200 dilution), α‐CCR5 (clone HM‐CCR5, Biolegend, 1:200 dilution), α‐CXCR3 (clone CXCR3‐173, ebioscience, 1:200 dilution), α‐LFA‐1 (H155‐78; Biolegend, 1:200 dilution), and VLA‐4 (clone R1‐2, Biolegend, 1:200 dilution). Levels of surface expression were determined by mean fluorescence intensity (MFI).

Mice were sacrificed and perfused by intracardial injection of PBS at 6 dpi. Subsequently, the brain and spleen were extracted and processed to obtain leukocytes as previously described (Claser et al, 2011; Teo et al, 2013). Isolated leukocytes were stained with LIVE/DEAD Aqua (Life Technologies), then blocked in 100 μl of blocking buffer consisting of a mix of 1% of rat and mouse serum (Sigma‐Aldrich) in FACS buffer [1% BSA, 2 μm EDTA in PBS]. Next, cells were incubated with PE‐labeled SQLLNAKYL‐H‐2D^b (Pb‐1) tetramer (Howland et al, 2013) on ices before addition of conjugated antibodies for another 20 min of incubation. Cells were fixed in IC fixation buffer (ebioscience) for 5 min before acquisition using a LSR II flow cytometer (BD Biosciences). Conjugated antibodies used were as follows: α‐CD45 (clone 30‐F11, BD Biosciences, 1:400 dilution), α‐CD3 (clone 17A2, BD Biosciences, 1:200 dilution), α‐CD4 (clone GK1.5, Biolegend, 1:400 dilution), α‐CD8 (clone 53–6.7, BD Biosciences, 1:400 dilution), α‐LFA‐1 (H155‐78; Biolegend, 1:200 dilution), α‐NK1.1 (clone PK136, ebioscience, 1:200 dilution), α‐CD11b (clone M1/70, Biolegend, 1:400 dilution), and α‐Ly6G (clone 1A8, Biolegend, 1:400 dilution). Gating strategy of T‐cell infiltrate in the brain is shown in Appendix Fig S7. Majority of the T‐cell infiltrate in the brain of the infected mice express LFA‐1 marker (Appendix Fig S7).