(A) Tregs were stimulated for 24 hours, 48 hours, and 72 hours with plate-bound αCD3 and soluble αCD28 at 1 μg/ml respectively with IL-2 (25 U/ml) either in the presence (+NaCl) or absence (media) of an additional 40 mM NaCl prior to being analyzed via qPCR for changes in TBet (TBX21) and IFNG (n = 5). (B) CD4 effector cells were labeled with CFSE, stimulated with αCD2/αCD3/αCD28-coated beads, and cultured alone or cocultured with Tregs. A neutralizing monoclonal antibody for IFNγ was added to the start of the culture at onset at 10 μg/ml (αIFNγ Ab, gray). A normal goat IgG Control was added to the start of culture to account for any nonspecific changes. CFSE dilution was measured by flow cytometry after 5 days in both cell populations. (C) The bar graph depicts a summary of independent experiments (n = 5). Statistical analyses were performed using paired Student’s t test. (D–F) Tregs were stimulated overnight with plate-bound αCD3 (1 μg/ml) and soluble αCD28 (1 μg/ml). The following day, a GFP-tagged viral construct containing shRNA specific for IFNG (shIFNG, gray) and a nontarget viral (control, white) construct were added to the culture at MOI = 5 with IL-2 (25 U/ml). The culture was left for 5 days, and transduced live cells were sorted by GFP⁺ and propidium iodide. IFNG knockdown was confirmed via qPCR (D). Transduced GFP⁺ Tregs were cultured in a CFSE suppression assay at a Treg/CD4 effector cell ratio of 1:3. CFSE dilution was measured by flow cytometry after 5 days (E). The bar graphs depict a summary of experiments demonstrating recovery of suppression in shIFNG in relation to control Tregs (n = 4) (F).