Tcrβ percp cy5

Single-cell suspensions were incubated in anti-CD16/CD32 Fc block antibody (5 μg/ml; clone 2.4G2; cat # 553141, BD Biosciences, San Jose, CA) and fixable viability dye eFluor 780 (Invitrogen, Carlsbad, CA) for 15 min at 4 °C in cold PBS. Cells were then washed once and stained with combinations of the following antibodies: CD45-BV510 (2 μg/ml; clone 30-F11; cat # 103138, BioLegend, San Diego, CA), TCRβ-PerCP-Cy5.5 (2 μg/ml; clone H57-597; cat # 109228, BioLegend, San Diego, CA), CD4-BUV395 (2 μg/ml; clone GK1.5; cat # 563790, BD Biosciences, San Jose, CA), CD8-af700 (5 μg/ml; clone KT15; cat # MCA609A700, Bio-Rad, Hercules, CA), NKp46-APC (1 μg/ml; clone 29A1.4; cat # 137607, BioLegend, San Diego, CA), CD49b-PE (2 μg/ml; clone DX5; cat # 108908, BioLegend, San Diego, CA), B220-BUV661 (2 μg/ml; clone RA3-6B2; cat # 612972, BD Biosciences, San Jose, CA), and Thy1.2-BUV805 (1 μg/ml; clone 53-2.1; cat # 741908, BD Biosciences, San Jose, CA). Data were acquired on a Symphony flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (FlowJo LLC, Ashland, OR). Gating strategies for the identification of CD8⁺ T cells and NK cells are provided in Supplementary Fig. 13.

Cells were stained with the following monoclonal antibodies and cell dyes: CD45.1 FITC, CD45.2 AlexaFluor 700, TCR-beta APC, CD4 PerCP-eFluor710, CD25 PE, CD44 APC-eFluor780, CD25 eFluor450, CD62L eFluor450 (all eBioscience), TCR $\beta$ PerCP-Cy5.5, CD5 BV510, CD4 BV650, CD44 BV785 (all BioLegend), CD62L BUV737 (BD Biosciences), LIVE/DEAD nearIR and LIVE/DEAD Blue viability dyes (Invitrogen). BrdU and Ki67 co-staining was performed using the FITC BrdU Flow Kit (BD Biosciences) according to the manufacturer’s instructions, along with anti-Ki67 eFluor660 (eBioscience). Cells were acquired on a BD LSR-II or BD LSR-Fortessa flow cytometer and analysed using Flowjo software (Treestar). Subset gates were as follows: CD4 naive: live TCR $\beta$ + CD5+ CD4+ CD25- CD44- CD62L+. CD4 T_EM: live TCR $\beta$ + CD5+ CD4+ CD25- CD44+ CD62L-. CD4 T_CM: live TCR $\beta$ + CD5+ CD4+ CD25- CD44+ CD62L+.