Lsrfortessa 2

Immunophenotypic analysis was performed using one of the following instruments: BD FACSCanto II, BD LSRFortessa II, BD FACSymphony A5, Beckman Coulter Cytoflex S or Beckman Coulter Cytoflex LX. Antibodies used for immunophenotypic analysis are listed in Supplementary Data 7. Cells were incubated with a human (Miltenyi Biotec, #130-059-901, dilution 2:100) and mouse FcR blocking reagent (BD #553141, dilution 1:100) prior to staining with antibody cocktail. All steps were performed at 4 °C in PBS 2% FBS. Data were analyzed with FCS Express 6 and 7 (DeNovo Software). Staining for cell cycle analysis was performed by fixing and permeabilizing cells with the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set as per manufacturer’s indications, followed by 30 min incubation at +4^oC with human FcR blocking reagent (Miltenyi Biotec, #130-059-901, dilution 2:100) and overnight staining at +4^oC with Ki67-AF647 (BD #558615, dilution 2:100). Samples were acquired on the FACSymphony A5 with low flow rates after addition of Hoechst 33342. Staining for EdU incorporation was performed with the Click-iT™ Plus EdU Pacific Blue™ Flow Cytometry Assay Kit (TermoFisher Scientific #C10636) following the manufacturer’s indications.

Spleen single-cell suspensions were incubated with a Fc-receptor blocking antibody 2.4G2 (kindly provided by Dr. Louis Boon, Bioceros BV, Utrecht, The Netherlands) and the percentage of spleen cell populations (macrophages, B cells, Dendritic cells, T cells, monocytes) were determined using the following antibodies: F4/80 (BM8) (Invitrogen, Bleiswijk, The Netherlands), CD45R (B220) (eBioscience, Vienna, Austria), CD11c (HL3) (BD Biosciences), CD4 (GK1.5) (eBioscience, Vienna, Austria), CD8α (53-6.7) (eBioscience, Vienna, Austria), CD3e (145-2C11) (eBioscience, Vienna, Austria), Lineage (Lin) (B220, NK1.1, CD90, CD49. Ly6G) (ebioscience, Vienna, Austria) CD45.1 (ebioscience, Vienna, Austria). Samples were analyzed with a LSR Fortessa II (Beckman Coulter) and the FlowJo software (Tree Star Inc., Ashland, The United States). The different spleen cell populations were defined as follow: DCs (CD11c+MHCII+), macrophages (F4/80+), B cells (CD45R+MHCII+), CD4+ (CD3+CD8-CD4+), CD8+T cells (CD3+CD8+CD4-) and monocytes (CD45+Lin-F4/80-CD11c-MHCII-CD11b+Ly-6C+). Percentages were reported to the total number of splenocytes of each mouse to calculate the number of cells per population (Figure S2 and S3).