CD44^act marks a population of activated Treg cells with superior suppressor activity in vitro. (A) CD4CD25⁺ cells were activated with plate-bound anti-CD3/anti-CD28 for 24 hours and sorted into CD44^hi HA-binding (•) and CD44^hi non–HA-binding (♦) populations, as in Figure 3. Suppressor activity of separated cells and fresh unactivated CD4CD25⁺ cells (▵) was determined by measuring the effect of graded numbers of Treg cells on proliferation of naive CD4CD25^– cells. Data are the mean ± SD of 4 experiments. Statistical differences are shown between CD44^act+ and CD44^act– cells. *P < .005; **P ≤ .01. (B) The average number of unactivated, activated CD44^act+, or activated CD44^act– Treg cells required to give 50% suppression of proliferation. Data are the mean ± SD of 4 experiments. *P < .001 for all pairwise comparisons. (C) Regeneration during the course of a suppressor assay of cells expressing CD44^act from a CD4CD25⁺ population previously depleted of such cells. After 24 hours with plate-bound anti-CD3/anti-CD28 (left), cells were sorted into CD44^act+ and CD44^act– populations as shown (postsort analysis, middle). Sorted populations were PKH labeled and used in an in vitro suppressor assay. After 24 hours in the presence of soluble anti-CD3 plus APCs, cells were restained with CD44-allophycocyanin and Fl-HA and analyzed (right, PKH gated). Continued emergence of an HA-binding population from the originally CD44^act– population is evident. (D) Generation of cells expressing CD44^act from fresh, unactivated CD4CD25⁺ cells during the course of an in vitro suppressor assay. Cells were PKH labeled prior to assay. After 24 hours in the presence of soluble anti-CD3 plus APCs, cells were stained with CD44-allophycocyanin and Fl-HA and analyzed. PKH-gated cells contain a small KM81-blockable Fl-HA–binding population that was not present in the starting population. (E) Degree of suppression correlates with the percentage of CD44^act+ cells in total activated CD4CD25⁺ cells. The numbers of cells required to give 90% suppression (calculated using the slopes of the lines for suppressor assays performed in triplicate with titrated numbers of Treg cells) and the percentage of CD44^act+ cells in each population is shown. The number of CD44^act+ cells within the total activated CD4CD25⁺ population (□) is approximately equal to the number of purified activated CD44^act+ cells (▦) calculated to give 90% suppression in a representative experiment. Corrected value shown in hatched bar.