Treg function and expression of CD69 and CD154 on T effector cells. (A) In contrast to healthy donors, IFN-γ secretion was not suppressed by the addition of autologous Tregs from AA patients to syngeneic T effector cells (Te). HC indicates healthy controls. (B) The relative suppression of Te cells from AA patients (measured by IFN-γ secretion after 2 hours of coculture with Tregs) was highest with healthy Tregs, whereas the Tregs from AA patients were almost unable to suppress IFN-γ secretion. hTr indicates Tregs from health donors; hTe, Te from healthy donors. (C) Similar pattern of suppression was noticed for both TNF-a and IL-2 secretion by Te cells in the presence of healthy Tregs. All tests were repeated with 3 different patients' samples. (E) Coculture of Tregs and Te (in 1:1 ratio), whether from AA patient (autologous Tregs) or from healthy donors (hTr), had no effect on Te expression level of CD69. In healthy donor Te cells, healthy Tregs reduced the level of CD69, whereas the AA Tregs were unable to suppress this marker. (F) Although Tregs from AA patients had minimal or no effect on Te CD154, healthy Tregs were able to suppress this marker both in patients and healthy donors. (G-H) To investigate the effect of cell contact on suppression of Te CD154 and CD69, a transwell coculture of Tregs and Te was set up, and as shown, prevention of cell contact between Te and Tregs by a transwell insert prevented the suppressor effect of Tregs on CD154 and CD69 expression by Te. All tests were repeated on 3 different pretreatment AA samples. u indicates unstimulated; s, stimulated; h, healthy donors; and TW, transwell.