Fig. 4.

Activation of CD45RB^lowCD25^– cells in vitro reveals their regulatory properties. (A) Sorted CD45RB^highCD25^– and CD45RB^lowCD25^– cells (25^–45^hi and 25^–45^lo, respectively) maintained for 6 days in culture containing anti-CD3 mAb and IL-2 (IL-2) were washed and cultured with the same number of untreated CD4⁺CD25^– cells, and cultured for another 3 days in the presence of anti-CD3 mAb and antigen-presenting cells. Freshly isolated (Fresh) CD4⁺CD25⁺ (25⁺), CD45RB^lowCD25^– (25^–45RB^lo), and CD45RB^highCD25^– (25^–45^hi) cells were used as controls. (B) Sorted CD45RB^lowCD25^– and CD45RB^highCD25^– cells (25^–45RB^lo and 25^–45RB^hi, respectively) were stimulated with plate-bound anti-CD3 mAb according to Materials and Methods. The evaluation of their suppressor function was performed by adding them at various ratios to untreated CD4⁺CD25^– cells as in A. Freshly isolated CD4⁺CD25⁺ cells (25⁺fresh) were used as control. The percentage of inhibition of naive T cell proliferation is plotted versus the ratio of the population tested/CD4⁺CD25^– cell number at the origin of the culture, as in Fig. 2B.