Anti cd86biotin

The spleens of mice were collected at various time points post-infection, as detailed in figure legends. Splenic cells were passed through a 70 mm cell strainer (Fisher Scientific), centrifuged, and then treated with Ammonium-Chloride-Potassium (ACK) red blood cell lysing buffer (Gibco). Following washes, 4 x 10⁶ total spleen cells were incubated with anti-mouse CD16/CD32 (Fc Block, BD Biosciences; Mississauga, ON, Canada) for 15 min on ice according to manufacturer’s instructions. Afterwards, surface staining with the following antibodies: anti-CD95^PE, anti-CD86^biotin, anti-CD19^PE-Cy7, anti-GL-7^AF647 and anti-CD184^BV421 (all BD Biosciences) was performed for 30 min on ice. Following washing, cells were incubated with Brilliant Stain Buffer (BD Biosciences) and stained with Streptavidine^BV605 (BD Biosciences) for 10 min on ice. Cells were then washed and immediately analyzed on a BD FACSaria Fusion. The data collected was analyzed on FlowJo version 10. All antibodies used are listed in S1 Table.

For fluorescence-activated cell sorting (FACS) analysis, cells were labelled with saturating amounts of antibodies in FACS buffer (PBS containing 0.1% BSA and 0.02% NaN₃) to stain cell-surface antigens for 15 min on ice, followed by a fixation/permeabilization step (Fix/Perm buffer, eBioscience) for 30 min at room temperature and intracellular staining for 45 min at room temperature in permeabilization buffer (eBioscience). Stained cells were analysed on an LSR II flow cytometer (BD Biosciences).
For the staining of PBMC, the following Abs were used: Anti-CD3-PE-Cy7, anti-CD4-PerCP, anti-CD4-Pacific Blue, anti-CD4-FITC, anti-CD4-Alexa Fluor 700, anti-CD8-PE, anti-CD25-APC, mIgG₁-APC, anti-CD45RA-PE, anti-CD45RA-PErCP-Cy5.5, anti-CD127-PE, anti-Foxp3-APC anti-Foxp3-Pacific Blue, anti-CCR7-Alexa Fluor 488, anti-Ki-67-Alexa Fluor 700, anti-CTLA-4-PE, anti-CTLA-4-PE-Cy7 (all Biolegend), anti-CD25-PE, anti-CD8-FITC, PE-Cy5-streptavidin, anti-CD86-biotin (all BD Bioscience) and viability dye (Thermo Fischer).
For di-4-ANEPPDHQ (ANE) staining, PBMC were incubated with 4 mM of ANE (Invitrogen) together with anti-CD4 APC-Cy7, anti-CD45RA BV510 and anti-CD25 APC (all from Biolegend) in RPMI medium for 30 min at 37°C and were immediately analysed by FACS.