Vα2 b20.1

Single-cell suspensions isolated from indicated mouse organs were stained on ice with antibodies specific for CD4 (RM4–5; eBioscience), CD5 (53–7.3; eBioscience), CD8α (53–6.7; eBioscience), CD8β (YTS 156.7.7; Absolute Antibody), CD25 (3C7; BioLegend), CD44 (IM7; eBioscience), CD62L (MEL-14; BioLegend), CD69 (HI.2F3; BioLegend), CD122 (5H4; BioLegend), CD132 (TUGm2; BioLegend), and Vα2 (B20.1; BioLegend). For the staining with antibody specific for CCR7 (4B12; eBioscience), cells were incubated at 37 °C for 30 min before the staining with other antibodies. To isolate naïve CD8⁺ T cells, spleen cells were stained with biotinylated antibodies specific for CD4, CD44, and B220 (RA3–6B2; BioLegend), and cells that did not bind to the antibodies were enriched with magnetic bead conjugated streptavidin (Miltenyi Biotec). Multicolor flow cytometry and cell sorting were performed on FACSVerse and FACSAriaII (BD Biosciences), respectively.

Antibodies used for staining were as follows: anti-CD3, -CD4, -CD8, -CD25, -CD44, -CD45, -CD62L, -ICOS, -PD1, -TIM3, -Ly51, -MHC-II A/E, -Vα2 (B20.1), and -Vβ5 (MR9-4) (all BioLegend); anti-Fgl2 (Bioss); anti-CCR2, -CXCR3, -EBI3 and -ST2 (all R&D); anti-Foxp3 (eBiosciences); anti-Ki67, -DM (BD Pharmingen); and anti-DOb (M-15), -A^b/CLIP (30.2) (Santa Cruz). Intracellular expression of Foxp3, Ki67 and DM and DOb was determined using the Intracellular Fixation & Permeabilization buffer set (eBiosience) according to the manufacturer’s protocol. EdU detection was done after the last wash with permeabilization buffer following the Click-iT EdU kit (Molecular Probes) instructions. Flow cytometric analysis was performed on an LSR II, sorting on a FACSAria (BD Bioscience), or on a MoFlo (Beckman Coulter) and data were analyzed using FlowJo software (Tree Star).