The α_E⁺ Tregs efficiently suppress committed Th1 cells in vivo. (A) FACS analysis of pooled spleen and lymph node CD4⁺ T cells before and after subset sorting. (B) In vitro generated Th1 cells (5 × 105) were injected intravenously together with 5 × 105 FACS-sorted and preactivated T cells into naive BALB/c mice. Twenty-four hours later the DTH response was induced by OVA peptide injection into the footpad. Shown is progression of the inflammatory response monitored by the thickness of footpads injected with OVA/IFA (♦, •, ▴) and control footpads injected with PBS/IFA (♦,○, ▵; mean ± SD; n = 6). The α_E⁺ Tregs showed a significantly higher suppressive capacity than α_E^–CD25+ cells (P < .01, repeated measure analyses). The insert shows the same data set expressed as percent suppression by indicated Treg subsets, in relation to the footpad swelling (OVA/IFA-injected footpad minus PBS/IFA-injected footpad) of mice adoptively transferred with α_E^–CD25^– control cells. (C) In vitro suppressive capacity of indicated T-cell subsets on naive T-cell proliferation was determined after 72 hours of coculture with CFSE-labeled responder cells at a 1:1 ratio. Culture of CFSE-labeled responder cells alone (medium) served as control. Representative histogram plots of CFSE profiles from CFSE⁺CD4⁺ T cells are shown. Quantification of suppressive capacity is based on the CFSE geometric mean of total CFSE⁺CD4⁺ T cells (n = 3; mean ± SD; 1 representative of 2 independent experiments).