Cd45 1 percp cy5

CD4 T cells were enriched using the naïve CD4 T cell isolation kit (Milteni Biotec, Cat. 130–104-453) from mononuclear cells isolated from the spleens and lymph nodes of congenically marked, wild-type (CD45.1) and CCR7-deficient (CD45.2) mice. A mixture of CD45.1-naïve CD4 T cells (3×10⁶) and CD45.2-naïve CD4 T cells (1.5×10⁶) were labeled with carboxyfluorescein succinimidyl ester (CFSE) and transferred into GF, B. thetaiotaomicron WT-monocolonized, or BtSULT Δsult-monocolonized B6 mice by retro-orbital injection. After 24h, recipient mice were sacrificed, and immune cells were isolated from the spleens and MLNs and stained with the following antibodies: CD45-Pacific blue (1:300, Biolegned), CD4-BV605 (1:300, Biolegend), CD25-PE (1:200, ebioscience), CD44-APC (1:300, ebiosience), CD62L-PE/Cy7 (1:300, Biolegend), CD45.1-PerCP/Cy5.5 (1:200, ebioscience) and CD45.2-APC/Fire750 (1:200, Biolegend). The ratio of donor CD4 T cells in either MLNs or spleens was first determined by dividing the number of CFSE+ CD4+ T cells (donor cells) by that of CD45+ cells (recipient cells) in each tissue. We then determine the relative migration of donor cells into the MLNs over the spleens of recipient mice by calculating the normalized ratio. Gating strategy and representative data are shown as Supplementary Figures 16b and 17c.