Pdca 1

Single cells from thymic glands, PDLNs and spleen were stained with the following surface antibodies: CD11b (M1/70, BioLegend, San Diego, CA, USA), F4/80 (BM8, BioLegend), MHC-II (M5/114.15.2, BioLegend), CD11c (N418, BioLegend), B220 (RA3-6B2, BioLegend) and PDCA-1 (927, BioLegend). The cells were then permeabilized and fixed overnight at 4 °C with Fixation and Permeabilization Buffer (eBioscience, San Diego, CA, USA). The next morning, single cells were stained with antibodies to Arginase 1 (A1exF5, ThermoFisher, Auburn, AL, USA), TNF-α (MP6-XT22, BioLegend), Ebi3 (355022, R&D Systems, Minneapolis, MN, USA) and IL-12p35 (27537, R&D Systems). Fc block (#553142 BD BioSciences, San Jose, CA, USA) was used for both surface and intracellular staining. Fixable Viability Dye eFluor^TM 780 (Thermofisher) was used for detecting live cells. All the samples were analyzed using a BD LSR Fortessa at the BioVis Platform (Uppsala University, Uppsala, Sweden). Data from flow cytometry were analyzed using the Flowlogic version 8.6 software (Inivai Technologies, Mentone, Australia) and FlowJo (Ashland, OR, USA). Gating strategies used for analysis are shown in Supplementary Figures S2 and S3.

Inguinal and popliteal lymph node were harvested and 12, 20, or 24 h post-immunization (p.i.), incubated 15 min with collagenase D (Roche) at 37°C and mechanically disrupted by glass-teasing. Cell suspensions were filtered through a 150 μm sieve and stain with biotinylated anti-mouse antibodies for lineage depletion (TCRβ, CD3ε, CD19, B220, NK1.1, Ly6G, PDCA-1) (Biolegend) for 20 min on ice. After washing, cells were incubated with anti-biotin microbeads (Miltenyi) for 15 min at 4°C. DC fraction enrichment was obtained by MACS negative selection (Miltenyi). After spin down, cells were stained with directly conjugated antibodies (CD45, CD11c, CD11b, I-Ab, lineage). Cells were acquired with LSR II Fortessa (BD) for analysis or facs-forted using FACSAria Fusion (BD). FlowJo (version 10, TreeStar) was used for post-acquisition analysis of the data.

Single cell suspensions were prepared from spleen and lymph nodes, treated with red blood cell lysis (R&D) and blocked with Fc-Block prior to staining in cold PBS with 1% FBS with antibodies anti-CD11c (Biolegend), IAb (Biolegend), CD8 (Biolegend), CD11b (Biolegend), CD86 (Biolegend), PDL2 (Biolegend), PDL1 (Biolegend), B220 (Biolegend), PDCA-1 (Biolegend), Thy1.1 (Biolegend), CD44 (Biolegend), CD4 (Biolegend), PD1 (ebiosciences), CXCR5 (BD Biosciences), GL7 (ebiosciences) and CD138 (Biolegend). The Foxp3 staining kit (eBiosciences) was used for intracellular staining of Foxp3 (eBiosciences). Data were acquired by FACSCanto (BD Biosciences) and analyzed using FlowJo Software (Tree Star).