α cd4 clone gk1

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).

We performed cell surface and intracellular staining as previously described^{25 (link)}. We conducted cell surface staining using antibodies, including αCD4 (clone GK1.5; Biolegend, San Diego, CA, USA), αCD19 (clone 6D5; Biolegend), αCD43 (clone S11; Biolegend), αB220 (clone RA3-6B2; Biolegend), αCD8 (clone 53-6.7; Biolegend), αTLR2 (clone QA16A01; Biolegend), and αCD98 (clone RL388; Biolegend).
For intracellular staining, the cells were permeabilized using a Cytofix/Cytoperm fixation and permeabilization solution kit (BD Biosciences, Franklin Lakes, NJ, USA) and stained with antibodies, including α-phospho-mTOR (clone MRRBY; eBioscience, San Diego, CA, USA) and αAkt (clone 55; BD Biosciences).
We detected stained cells using flow cytometry (LSR II, BD Biosciences) and conducted data analysis with FlowJo software (version 10.6.2; Ashland, OR, USA).