Random ODNs also stabilize Foxp3⁺Helios⁺ T cells. (A) Both a TLR9 agonist and a TLR9 antagonist stabilize Helios⁺ Foxp3⁺ Tregs. Cells were isolated as in Figure 5A and stimulated in the presence of 2.5μM CpG ODN or ODN TTAGGG for the indicated time. (B) TLR7 and TLR8 agonists do not stabilize Helios⁺Foxp3⁺ Tregs. Cells were isolated as in panel A and stimulated in the presence of CpG ODN, 10μM of ssDNA40 (CL264, TLR8 agonist), ssDNA40 (TLR7 agonist), or ssRNA41 (negative control for ssDNA40). (C) Random sequence ODNs stabilize Helios⁺Foxp3⁺ Tregs. Left panel, CD4⁺CD127^loCD25⁺ cells were sorted and stimulated for 5 days in the presence of 2.5μM of ODN TTAGGG or ODN NNNGGG. The cells were then expanded with anti-CD3/CD28 beads in the absence of the ODN and stained for Foxp3 and Helios expression 8 days later. Right panel, same conditions as in left panel except that different ODNs were added (4X indicates 4-times repeat [24 mers] of 6 bp nucleotides). (D) Stabilization of Foxp3 and Helios expression by ODN requires physical stability and proper size (> 10 mer) of the ODN. CD4⁺CD127^loCD25^hi cells were stimulated and expanded as same as panel (C) in the presence of phosphorothioate backboned 10 mer of ODN (ODNps10), phosphodiester backboned 25 mer of ODN (ODNpe25), and phosphothioate backboned 25 mer of ODN (ODNps25) at a concentration of 2.5μM. (E) ODNs are localized in cytosol of Tregs. MACS-sorted CD4⁺ T cells were isolated from buffy coat, stimulated with plate-bound anti-CD3/CD28 for 18 hours in the presence of Biotin-5-ODN or unlabeled ODN, and then washed 3 times in FACS staining buffer. Extra and intracellular Biotin-5-ODN in stimulated cells was stained with AlexaFluor 488– or 546–conjugated strepavidins (Invitrogen). For the confocal microscopic analysis, fixed and stained cells were mounted with Vectashield mounting medium with 4,6-diamidino-2-phenylindole (DAPI;blue, Vector Laboratories). Images were taken using a Zeiss LSM 710 Laser Scanning Microscope (Carl Zeiss) equipped with a Plan-Apochromat 63×/1.4 oil-immersion objective at ambient temperature and processed using ZEN 2009 software (Carl Zeiss). The white bar represents 10 μm.