Peripheral blood mononuclear cells (PBMC) were isolated from blood cones obtained from healthy donors (NHS Blood and Transplant [NHSBT] UK) by LSM 1077 (PAA) gradient centrifugation and then incubated with CD25+ Microbeads to derive CD25+ enriched cells using a LS column (Miltenyi Biotech). CD25+ cells were stained with monoclonal antibodies and Treg cell subsets were isolated by FACS using a BD FACSAria I cell sorter.