Sample processing for the flow cytometry analysis

To obtain single-cell suspensions, lymphocytes were isolated from the thymus, spleen, inguinal lymph nodes, lungs, and livers as described before³⁵. The prepared cells were stained with the following antibodies: anti-CD4-BV605 (RM4–5, BioLegend), anti-PD-1-BV421 (RMP1–13, BioLegend), anti-CD2-PE-Cy7 (RM2–5, BioLegend), anti-CD23-PerCP-Cy5.5 (B3B4, BioLegend), anti-CD21/35-BV421 (7E9, BioLegend), anti-CD43-PE (1B11, BioLegend), anti-IgM-APC (RMM-1, BioLegend), anti-IgD-BV605 (11-26c.2a, BioLegend), anti-CD8-PerCP-Cy5.5 (53-6.7, eBioscience), anti-CD25-FITC (PC61.5, eBioscience), anti-CD3-PE-Cy7 (145-2C11, BD Biosciences), anti-CD44-V450 (IM7, BD Biosciences), anti-CD45R/B220-FITC (RA3-6B2, BD Biosciences), anti-CD62L-FITC (MEL-14, BD Biosciences), anti-CD69-PE (H1.2F3, BD Biosciences), and anti-TCR-β-FITC (H57-597, BD Biosciences). For the staining of transcription factor FoxP3, the cells were stained using anti-FoxP3-APC (FJK-16s, eBioscience) after permeabilization/fixation step. In all experiments, cells were labelled with the Live/Dead fixable Stain Kit (eBioscience) to discriminate the live and dead cells. The data were acquired by FACS CANTO II (BD Biosciences) and analysed by FlowJo software (Tree Star).