Cd25 bv650

Single cell suspensions were prepared from the thymus, spleen and lymph nodes of busulfan chimeric mice, wildtype control mice, or germ free mice. Cells were stained with the following monoclonal antibodies and cell dyes: CD45.1 FITC, CD45.2 FITC, CD45.2 AlexaFluor700, TCR- $\beta$ APC, CD4⁺ PerCP-eFluor710, CD44 APC-eFluor780, CD25 PE, CD25 eFluor450, CD25 PE-Cy7, CD62L eFluor450, NK1.1 PE-Cy7 (all eBioscience), CD45.1 BV650, CD45.2 PE-Dazzle, TCR- $\beta$ PerCP-Cy5.5 CD4⁺ BV711, CD44 BV785, CD25 BV650 (all Biolegend), CD62L BUV737 (BD Biosciences), LIVE/DEAD nearIR and LIVE/DEAD blue viability dyes. For Ki67 staining, cells were fixed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set and stained with either anti-mouse Ki67 FITC or PE (both eBioscience). Cells were acquired on a BD LSR-II or a BD LSR-Fortessa flow cytometer and analysed with Flowjo software (Treestar). Conventional CD4⁺ cells were identified as live TCR- $\beta$ + CD4⁺ CD25- NK1.1-, and then CD44 and CD62L were used to identify EM (CD44+CD62L-) and CM (CD44+CD62L+) subsets.

Single cell suspensions were prepared from the thymus, spleen and lymph nodes (cervical, axillary, brachial, inguinal, and mesenteric) of Ki67-mCherry-CreER Rosa26^RYFP double reporter mice. Cells were stained with the following monoclonal antibodies and cell dyes: TCR-β APC, CD4⁺ PerCP-eFluor710, CD44 APC-eFluor780, CD25 PE, CD25 eFluor450, CD25 PE-Cy7, CD62L eFluor450 (all eBioscience), TCR- β PerCP-Cy5.5 CD4⁺ BV711, CD44 BV785, CD25 BV650 (all Biolegend), CD62L BUV737 (BD Biosciences), LIVE/DEAD nearIR and LIVE/DEAD blue viability dyes. Cells were acquired on a BD LSR-Fortessa flow cytometer and analyzed with Flowjo software (Treestar). Gating strategy to identify DP, mSP and naive peripheral T cells was identical to that we have employed previously for analysis of mice expressing Ki67-RFP fusion protein (14 (link)). Briefly, DP thymocytes are identified as CD4⁺CD8⁺ CR^lo CD5^lo, CD4 mSP are CD4⁺CD8⁻CD44^loCD25^loCD62L^hi, CD8 mSP are CD4⁻CD8⁺CD44^loTCR^hiCD62L^hi, peripheral naive CD4⁺T cells are TCRR^hiCD4⁺CD44^loCD62L^hi, and peripheral naive CD8⁺T cells are TCR^hiCD8⁺CD44^loCD62L^hi.

Fluorochrome-conjugated anti-mouse monoclonal antibodies CD3-AF700 (cat # 100216), CD4-BV785 (cat # 100453), CD8-PB (cat # 100725), CD25-BV650 (cat # 102038), GITR-PECy7 (cat # 120222), ICOS-PECy5 (cat # 107708), IFNg-PeCy7 (cat # 505826), and IL-2-PB (cat # 503820) were purchased from Biolegend. CD44-Percp-cyanine5.5 cat# 45-0441-80, CD62L-APCeFL780 (cat # 47-0621-82), Foxp3-APC (cat# 17-5773-82), Eos-eFL660 (cat # 50-5758-80), Helios-PeCy7 (cat # 25-9883-42), Aiolos-PE (cat # 12-5789-80),and bCatenin-eFL660 (cat # 50-2567-42) were purchased from Thermo Fisher Scientific. PD-1-PECy7 (cat# 25-9985-80), CXCR5-BV421 (cat # 562889), Bcl-6-PE (cat # 569522), and phospho-STAT5-PE (pY694) (cat # 612567) were procured from BD Biosciences.