Transcription factor buffer

Cells were incubated with fixable viability dye (eFluor 455UV; eBioscience, UK) followed by CD16/CD32 antibody (Fc-block; clone 2.4G2, BD Bioscience, UK), then stained with directly conjugated antibodies (10 μg/ml and analysed using an LSR II device (BD Bioscience, UK). The following antibodies were purchased from BD Bioscience, UK unless stated otherwise: MHC class II #553623, CD4 #552051, CD11b #550993, CD11c #550261, CD135 #562537, CD40 #562846, CD86 #560581, CD25 #553866, CCR7 #563596, PDL-1 #564716, Zbtb46 #565832, SIRP-1α #740159, c-kit #560185, CD115 #25–1152-82 (eBioscience) and CX3CR1 #149023 (Biolegend). Transcription factor buffer (BD Bioscience, USA, catalog #562574) was used in accordance with the manufacturer’s instructions to test for transcription factors. FlowJo software (TreeStar, Inc., Ashland, OR, USA) was used for data analysis.

Cells isolated from spleen, lymph nodes, brain, and spinal cord were analysed using a FACS Canto II flow cytometer (BD, Franklin Lakes, NJ) as described^{14 (link)}. The following antibodies were used for phenotypic analysis: mouse anti-NK1.1 IgG2a, hamster anti-CD11c IgG, rat anti-Ly6C IgG2c, rat anti-Ly6G IgG2a, mouse anti-FoxP3 IgG1, mouse anti-CX3CR1 IgG2a, and mouse anti-CD45.1 IgG2a from Biolegend (San Diego, CA) and rat anti-CD4 IgG2a, rat anti-CD8α IgG2a, rat anti-CD25 IgG1, rat anti-I-A^b (MHC-II) IgG2a, rat anti-CD11b IgG2b, mouse anti-CD45.2 IgG2 and rat anti-Gr1 IgG2b from BD Biosciences (San Jose, CA) and rat anti-PD-L1 IgG2a and rat anti-F4/80 IgG2a from eBiosciences (San Diego, CA). Fcγ receptors were blocked by the addition of purified anti-CD16/32 antibodies (Fc Block; 1 μg/10⁶ cells; BD Biosciences) 15 minutes prior to surface staining. For intracellular cytokine staining, GolgiStop (1 μg/10⁶ cells; BD Biosciences), ionomycin (500 ng/ml; Sigma) and PMA (50 ng/ml) were added to the Con A-stimulated splenocyte cultures 5 hours prior to surface antibody staining. After extracellular staining, cells were fixed and permeabilized using Transcription Factor Buffer (BD Biosciences, USA) and stained with rat anti-IFN-γ IgG1 (BD Biosciences) following the manufacturer’s instructions and analyzed using BD FACS Canto II flow cytometer (BD Biosciences).

Data were collected on a LSR Fortessa (BD Biosciences) and analyzed with FlowJo software (Tree Star, Inc.). Cell sorting was performed on an Aria III Fusion (BD Biosciences). Antibodies used in this study can be found in Table S2 in Supplementary Material. For intracellular cytokine staining cells were fixed with 2% paraformaldehyde (15 min, room temperature), then washed in FACS buffer with 0.05% saponin and stained in 0.5% saponin. IRF8 and IRF4 were stained in Foxp3 transcription factor staining set (eBioscience-00-5523-00) and Zbtb46 in transcription factor buffer from BDBioscience-562574. Dead cells were identified with Dapi or live/dead fixable violet (Invitrogen) or zombie UV dye (Biolegend).