Fig. 3.

Inhibition of Treg-DR hypomethylation by NF-κB signaling. (A) Kinetics of Foxp3 CNS2 methylation in CD28(−) iTregs. After CD28(−) iTreg generation (i.e., plate-bound anti-CD3 stimulation in the presence of IL-2 and TGF-β, without anti-CD28) for 24 h from naive CD4⁺ T cells in eFox reporter mice, some cells continued to be cultured for an additional 48 h (Upper Right); other cells were transferred to anti-CD3 noncoated wells and cultured without soluble anti-CD28 mAb (Lower Left), or to anti-CD3 coated wells and cultured with 1 µg/mL soluble anti-CD28 mAb (Lower Center), or to noncoated wells and cultured with anti-CD3/CD28 Dynabeads (Lower Right) for an additional 48 h. GFP⁺ (i.e., Foxp3⁺) cells purified from each culture were assessed for the degree of Foxp3 CNS2 methylation. A representative result (Upper figure) and total results of two (for group a and b) or three (for groups c–f) independent experiments (Lower figure) are shown. (B) Foxp3 CNS2 hypomethylation in highly proliferated (CTV^low) or nonproliferated (CTV^high) Foxp3⁺ cells in the indicated iTreg conditions. Representative of three independent experiments. (C) Screening of inhibitors of Foxp3 CNS2 hypomethylation in CD28(−) iTregs. CD28(−) iTregs were induced in the presence of the indicated antibodies or chemical compounds for 3 d. “Control” indicates without the use of any additional reagents. “37.51” indicates the clone number of anti-CD28 mAb used as positive control. Values are shown as a ratio normalized to nontreated control. A representative result from two independent experiments. (D) FPKM of Foxp3 and NF-κB signaling-related genes from RNA-Seq data. Tact, CD3/28-stimulated naive T convs; nTreg, CD3/28-stimulated peripheral Foxp3⁺ Tregs. Results from two independent experiments are shown. (E) Histogram representation of intracellular c-Rel staining in CD28(+) or CD28(−) iTregs. A representative result of three independent experiments. (F) Ranked enrichment plot of “HALLMARK_TNFA_SIGNALING_VIA_NFKB” (Molecular Signature Database) in gene set enrichment analysis of CD28(+) vs. CD28(−) iTregs. (G and H) CRISPR/Cas9 targeting of STAT5 or NF-κB binding motifs in Foxp3 CNS2. gRNAs were retrovirally induced in Cas9-expressing CD4⁺ T naive cells followed by iTreg induction (see Materials and Methods for details). A representative result from two independent experiments.