Loss of epigenetic modification driven by the Foxp3 transcription factor leads to regulatory T cell insufficiency

Abstract

Regulatory T (Treg) cells, driven by the Foxp3 transcription factor, are responsible for limiting autoimmunity and chronic inflammation. We showed that a well-characterized Foxp3(gfp) reporter mouse, which expresses an N-terminal GFP-Foxp3 fusion protein, is a hypomorph that causes profoundly accelerated autoimmune diabetes on a NOD background. Although natural Treg cell development and in vitro function are not markedly altered in Foxp3(gfp) NOD and C57BL/6 mice, Treg cell function in inflammatory environments was perturbed and TGF-β-induced Treg cell development was reduced. Foxp3(gfp) was unable to interact with the histone acetyltransferase Tip60, the histone deacetylase HDAC7, and the Ikaros family zinc finger 4, Eos, which led to reduced Foxp3 acetylation and enhanced K48-linked polyubiquitylation. Collectively this results in an altered transcriptional landscape and reduced Foxp3-mediated gene repression, notably at the hallmark IL-2 promoter. Loss of controlled Foxp3-driven epigenetic modification leads to Treg cell insufficiency that enables autoimmunity in susceptible environments.

Figure 1

Foxp3^gfp NOD mice develop accelerated autoimmune diabetes. (A) Female Foxp3^gfp (n=29), Foxp3^gfp/wt HET (n=19) and WT (n=25) NOD mice in the same colony were monitored weekly for diabetes onset (***p<0.001, **p<0.003; Kalpan-Meier with groups compared using the Log-rank test). (B) Insulitis scoring of Foxp3^gfp and WT NOD mice was performed at 8 weeks of age. (C) Cellular infiltrate in the islets, PLN, and spleen of 8 week old pre-diabetic WT, Foxp3^gfp and FoxP3^GFP-hCre NOD mice were analyzed by flow cytometry (n=6–10 mice per group; *p<0.05, **p<0.01, ***p<0.001). Error bars represent SEM.

Figure 2

Foxp3^gfp nTreg insufficiency in vivo. (A) Thy1.1⁺ Tconv (2×10⁶ CD4⁺CD45RB⁺CD25⁻ from C57BL/6 mice; >99% purity post-sort) were injected into Rag1^−/− mice or mixed with Thy1.2⁺ Tregs (0.5×10⁶) from either WT or Foxp3^gfp C57BL/6 mice (CD4⁺CD45RB^loCD25⁺; >95% purity post-sort, confirmed by Foxp3 intracellular stain). The number of CD4⁺Thy1.1⁺ splenic T cells was determined 7 days later. 11–20 mice per group from 4 separate experiments are shown. Statistical analysis was one-way ANOVA analysis of variance (** p<0.01 and ***p<0.001). (B–C) Foxp3^−/− mice (2–3 day old) were adoptively transferred with 10⁶ sorted Tregs (CD4⁺CD45RB^loCD25⁺) from either WT or Foxp3^gfp C57BL/6 mice (>95% purity by Foxp3 intracellular stain). After 35 days, mice were sacrificed and CD4⁺ T cell numbers and Foxp3⁺ percentages in the spleens and peripheral LNs quantified. Three separate experiments with 5–9 mice per group are shown (*p<0.05, ***p<0.001). Error bars represent SEM. (D) Foxp3^−/− mice treated as in (C) were evaluated over 18 weeks for clinical signs of autoimmunity. When mice reached a clinical score of 5 (out of six), they were euthanized. n=10 mice/group (**p<0.01, ***p<0.001; Kalpan-Meier with groups compared using the Log-rank test).

Figure 3

Impaired Foxp3^gfp iTregs conversion in vitro. (A) CD4⁺CD45RB^hiCD25⁻ Tconv were purified from Foxp3^gfp/wt NOD HET mice and stimulated with anti-CD3/CD28 coated beads, 5ng/ml TGFβ and 100U/ml IL-2. After 5 days, cells were stained with anti-Foxp3. Cells were gated on GFP⁺Foxp3⁺ and GFP⁻Foxp3⁺. A representative flow plot and an average of 5 experiments are shown (**p<0.01 by 2-way analysis paired t-test). (B) CD4⁺CD45RB^hiCD25⁻ T_conv were sorted from Foxp3^gfp and Foxp3^DTR-GFP C57BL/6 HET mice, stimulated and analyzed as in (A). A representative of 6 separate experiments is shown (*** p<0.001 by 2-way analysis paired t-test).

Figure 4

Impaired development of Foxp3^gfp iTregs in vivo. CD4⁺CD45RB^hiCD25⁻ Tconv were sorted (>98% purity) from Foxp3^gfp and WT C57BL/6 or NOD mice and 2×10⁶ cells injected i.v. into Rag1^−/− or NOD.SCID mice, respectively. Mice were sacrificed 21 days post-injection; spleens and LNs harvested and stained for CD4, CD25 and intracellular Foxp3. (A) Representative flow plots. (B) Percentage and total number of CD4⁺Foxp3⁺ cells in spleens and MLN (n=9 mice per group, ***p<0.001, *p<0.03 (C) CD4⁺CD45RB^hiCD25⁻ Tconv (C57BL/6 Foxp3^gfp/wt HET mice, >99% purity; 2×10⁶) were injected i.v. into Rag1^−/− mice and analysed as in (A) (n=9 mice; *p<0.05 by 2-way analysis paired t test). (D) CD45.1⁺ C57BL/6 mice were injected i.v. with a 50:50 mixture of OT-II CD4⁺CD45RB^hiCD25⁻ Tconv (>99.7% Foxp3⁻) from either Thy1.1⁺CD45.2⁺ WT OT-II mice or Thy1.2⁺CD45.2⁺ Foxp3^gfp OT-II mice, and placed on drinking water with OVA antigen. After 6 days, the percentage of Foxp3⁺ iTregs in PP, MLN, spleen and peripheral LN was determined (n=15; **p<0.005, *p<0.05 by 2-way analysis paired t-test).

Figure 5

Foxp3^gfp exhibits selective impaired molecular interactions. CD4⁺CD45RB^loCD25⁺ WT and Foxp3^gfp NOD nTregs (sorted to >93% purity) were analysed by IP/western blot using the Abs indicated. Representative blots of at least three separate experiments are shown. (G) Arrows highlight ubiquitylated Foxp3.

Figure 6

Altered transcriptional landscape in Foxp3^gfp Tregs. FoxP3^GFP-hCre or Foxp3^gfp NOD Treg and Tconv purified by FACS (>99% purity), mRNA isolated and subjected to Affymetrix analysis (4 independent samples per group). (A) PCA analysis. (B–C) Volcano plot comparing FoxP3^GFP-hCre (B) or Foxp3^gfp (C) Tregs with their respective Tconv controls. Highest modulated genes are marked. (D) Most differentially expressed genes in Foxp3^gfp Treg compared to wild type Tregs and Tconv controls are depicted in a heat map. Treg signature genes are in red (Supplemental Figure 14 and Pillai et al., 2011) (E) FoxP3^GFP-hCre/wt or Foxp3^gfp/wt HET Tregs were stained for Itgae (CD103). Data are representative of 5–6 mice (**p<0.01 by 2 way analysis paired t test).

Figure 7

Enhanced IL-2 promoter activity and IL-2 production by Foxp3^gfp Tregs. (A) Jurkat T cells were transfected with an IL-2-luciferase reporter plasmid along with indicated Foxp3-encoded plasmids. Empty vector pMIY was used to maintain equal DNA content for all transfections. Cells were either treated with PMA plus ionomycin or left untreated 12hr post-transfection, prior to analysis of luciferase activity, which was normalized to the Renilla luciferase activity. Data shown are representative of at least 3 independent experiments (mean and SD of triplicate transfections are shown; *p<0.005 by t test). (B) ChIP assay was performed with anti-Foxp3 Ab or IgG control followed by PCR using primers flanking the Foxp3-binding region of the IL-2 promoter. Shown are the mean (±SEM) of at least 3 independent experiments (**p<0.001 by t test). (C) Histone acetylation at the IL-2 promoter was determined by ChiP in the cell subsets indicated. Shown are the mean (±SEM) of at least 3 independent experiments (*p<0.005 by t test). Naive is defined as rested, while Tconv is defined as previously activated by anti-CD3/CD28. (D) CD4⁺CD45RB^loCD25⁺ Tregs from WT, Foxp3^gfp and FoxP3^GFP-hCre NOD mice were sorted (>95% purity), stimulated (0.5×10⁵) with plate-bound anti-CD3 and soluble anti-CD28 for 72h, and IL-2 concentration in supernatants determined by ELISA. Shown are the mean (±SEM) of at least 3 independent experiments (*p<0.05 by 2-way paired t test).