Cd44 bv785

Purified CD4⁺ T cell populations were obtained from spleen and lung using flow-cytometric cell sorting on a MoFlo® Astrios™ with Summit acquisition software (Beckman Coulter, Brea, CA, USA). Cells were cultured with and without PPD-T at 10 µg/ml with the addition of 1 µg/ml anti-CD28 for 3 days at 37 °C/5% CO₂, in co-culture with adherent spleen-derived APC from naïve congenic CD90.1 BALB/c mice. Briefly, spleens were processed as previously and incubated for 4 h/37 °C before removal of non-adherent cells by three aspirations of supplemented DMEM. Antigen and purified CD4⁺ T cells were immediately added to the culture. Any congenic T cells which had failed to be removed from the splenic APC co-culture were excluded from final flow-cytometric analysis through expression of CD90.1. Cell-surface staining was performed as previously with CD90.1-BV421, CD44-BV785, live/dead-Zombie Aqua (all BioLegend) and CD4-APC-H7 (BD Biosciences). After surface staining, cells were washed twice as previously and cold 70% ethanol added to the cell pellet while vortexing. Cells were incubated for 1 h/−20 °C, washed three times, stained for 30 min/room temperature with Ki67-PE (BioLegend) and washed twice before analysis, as previously.

MHC class II tetramer-PE (from the NIH tetramer core facility at Emory, Atlanta, GA), CD4 BUV737 (BD, clone GK1.1), CD8 PerCP-Cy5.5 (Biolegend, clone 53–6.7, cat 100734), CD44 BV785 (Biolegend, clone IM7), CD11b APC (Biolegend, clone M1/70, cat 101212), CD11c APC (Biolegend, clone N418, cat 117310), NK1.1 APC (Biolegend, clone PK136, cat 108710), B220 APC (Biolegend, clone RA3-62B, cat 103212).

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 near-IR 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-Fortessa flow cytometer and analysed with Flowjo software (Treestar). See Figure 1—figure supplement 1 for the gating strategy used to identify mature single positive thymocytes and peripheral naive subsets, and gates to measure Ki67 frequencies.

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+.