The α_E^–CD25⁺ Tregs efficiently prevent naive T-cell proliferation in vivo. (A) In vitro preactivated Treg subsets from DO11.10 mice were radioactively labeled with 111In and injected intravenously into BALB/c mice, in which 24 hours before a DTH response had been induced, followed by the determination of radioactivity in the control and the antigen-draining lymph node (dr LN) after 24 hours using a γ counter. Percentage of total recovered radioactivity is shown (n = 12; mean ± SD; data pooled from 2 independent experiments; **P < .01). (B-D) CFSE-labeled naive CD4⁺ T cells (5 × 105) derived from DO11.10 mice were adoptively transferred into BALB/c recipients, which 48 hours before had received 5 × 105 non-preactivated FACS-sorted Tregs followed by subcutaneous OVA immunization. Three days later the effect of the transferred Tregs on naive T-cell proliferation was assessed by measurement of footpad swelling and by FACS. (B) Measurement of footpad thickness shows the suppressive effect of indicated Treg subsets on the development of effector cells. Significance was determined by one-tailed unpaired Student t test (n = 3; mean ± SD; 1 representative of 2 independent experiments; *P < .05). (C) Representative FACS plots of gated CD4⁺ cells from antigen-draining popliteal lymph nodes show the effect of indicated Treg subsets on the proliferation of CFSE-labeled KJ1.26⁺ responder cells. Numbers indicate the frequency of CFSE⁺KJ1.26⁺ cells, which have undergone 3 or more cell divisions, and the frequency of CFSE⁺KJ1.26⁺ cells in the undivided fraction. Interestingly, during the 5-day in vivo period more than 95% of adoptively transferredα_E^–CD25⁺ Tregs (CFSE^–KJ1.26⁺) did not acquire α_E expression, indicating relatively stable phenotypes under these conditions. (D) The quantification of suppressive capacity is based on the CFSE geometric mean of total CFSE⁺CD4⁺KJ1.26⁺ T cells (n = 3; mean ± SD; 1 representative of 2 independent experiments; **P < .01).