Isolation of Murine T Cell Subsets

T cells were purified from mouse spleens and lymph nodes (LN) as described (5 (link), 20 (link)). Briefly, single cell suspensions were incubated with purified anti-CD11b, -CD8, -B220, anti-Gr-1, -CD16/32, -Ter119 (BD Bioscience, San Jose, CA) and -I-A/E (eBioscience) mAbs. The cells were then washed and incubated with anti-rat dynabeads (Dynal, Invitrogen) according to the manufacturer’s instructions. Bulk CD4⁺ T cells were then purified by magnetic negative selection of the unwanted cells. For Treg isolation, these bulk CD4⁺ T cells were incubated with anti-CD25-PE mAb and CD4⁺CD25⁺ T cells isolated by positive selection using anti-PE microbeads and MS separation columns (Miltenyi Biotec). Purity was assessed by cytofluorometric analysis and was consistently 90–95%. The remaining cells were used as CD4⁺CD25⁻ Tconv.