Naïve CD4+ T cells were enriched via negative selection utilizing magnetic-activated cell sorting to deplete cells expressing: CD25, CD19, CD11b, CD11c, NK1.1, F4/80, Ly-6G, CD8α, and Ter119 on MACS LS columns with anti-biotin and anti-APC microbeads (Miltenyi Biotec). Frequency of CD4+IL-17A+ T cells (<1%) was verified using flow cytometry on the LSR II (BD). 5 × 106 cells were injected i.v. into CD45.2 congenic hosts. Recipients were gavaged with cecal contents from Taconic mice 24 hours later. After 10 days, recipients were harvested for assessment of intestinal Th17 differentiation.
For co-transfer experiments, naive Thy1.1+ OT-II cells were mixed at a 1:1 ratio with CD45.1+ cells and a total of 107 cells were injected i.v. After 24 hours, mice were gavaged with SFB-containing cecal contents from Taconic mice and/or fed albumin from chicken egg white (Sigma-Aldrich) in the drinking water (15 mg/ml) for 10 days.
For co-transfer experiments, naive Thy1.1+ OT-II cells were mixed at a 1:1 ratio with CD45.1+ cells and a total of 107 cells were injected i.v. After 24 hours, mice were gavaged with SFB-containing cecal contents from Taconic mice and/or fed albumin from chicken egg white (Sigma-Aldrich) in the drinking water (15 mg/ml) for 10 days.