Stable inhibitory activity of regulatory T cells requires the transcription factor Helios

Abstract

The maintenance of immune homeostasis requires regulatory T cells (T(regs)). Given their intrinsic self-reactivity, T(regs) must stably maintain a suppressive phenotype to avoid autoimmunity. We report that impaired expression of the transcription factor (TF) Helios by FoxP3(+) CD4 and Qa-1-restricted CD8 T(regs) results in defective regulatory activity and autoimmunity in mice. Helios-deficient T(regs) develop an unstable phenotype during inflammatory responses characterized by reduced FoxP3 expression and increased effector cytokine expression secondary to diminished activation of the STAT5 pathway. CD8 T(regs) also require Helios-dependent STAT5 activation for survival and to prevent terminal T cell differentiation. The definition of Helios as a key transcription factor that stabilizes T(regs) in the face of inflammatory responses provides a genetic explanation for a core property of T(regs).

Fig 1. Helios^−/− mice develop an autoimmune phenotype

A) Activated CD4 and CD8 T cells (CD44⁺CD62L^lo), GC B (B220⁺Fas⁺) and T_FH (CD4⁺PD-1⁺CXCR5⁺) cells in spleens from (5 mo old) Helios^+/+ and Helios^−/− mice were compared (n=3–6). Representative data from three independent experiments. The mean ± SEM is indicated. *P < 0.05 and **P < 0.01 (Mann-Whitney test). B) Microscopy (200X) of representative hematoxylin and eosin staining of salivary gland, liver, lung, pancreas, kidney sections from (7 mo old) Helios^+/+ and Helios^−/− mice (n=4). Representative data from two independent experiments. C) Generation of autoantibodies was compared in sera from 7 mo old Helios^+/+ and Helios^−/− mice (n=7–10). Representative data from three independent experiments. The mean ± SEM is indicated. *P < 0.05, **P < 0.01 and ***P < 0.001 (Mann-Whitney test). D) Viral infection of Helios-deficient mice. 2 mo and 6 mo old Helios^+/+ and Helios^−/− mice infected i.p. with 2×10⁵ plaque forming units (PFU) LCMV-Armstrong. 30 days later, spleen cells analyzed for GC B and T_FH cells (n=4). Representative data from two independent experiments.The mean ± SEM is indicated. *P < 0.05 (Mann-Whitney test). E) Kidney sections from virus-infected mice were analyzed for IgG deposition and IgG⁺ areas in glomeruli (n=4). Representative data from two independent experiments are shown. The mean ± SEM is indicated. ***P < 0.001 (Mann-Whitney test).

Fig 2. Helios-deficient CD4 and CD8 Treg contribute to autoimmune disease

A) Phenotype of Helios^fl/fl.FoxP3-Cre mice at 6 mo age. The percentage and numbers of activated CD4 T cells (CD44⁺CD62L^lo) in spleen are shown (n=4–5). Data representative of two independent experiments. The mean ± SEM is indicated. ***P < 0.001 (Mann-Whitney test). B) FACS profiles for T_FH and GC B cells from spleens of FoxP3-Cre and Helios^fl/fl.FoxP3-Cre mice (n=4–5). Data representative of two independent experiments. C) Levels of anti-dsDNA Ab in sera of FoxP3-Cre and Helios^fl/fl.FoxP3-Cre mice at 6 mo age (n=4–5). Data representative of two independent experiments. The mean ± SEM is indicated. **P < 0.01 (Mann-Whitney test). D) Lethally irradiated Rag2^−/− mice were reconstituted with hematopoietic progenitors from Helios^+/+, Helios^−/−, Scurfy, Helios^+/+/Helios^−/− (1:1), Helios^+/+/Scurfy (1;1), and Helios^−/−/Scurfy (1;1) mice (n=4–6). Intensity of immune cell infiltration was quantified by scoring immune cell infiltration 7 weeks after reconstitution: >4 (most severe), 2–3 (severe), 1 (mild) and 0 (none). Data representative of two independent experiments. E) Defective inhibitory activity of Helios-deficient CD4 and CD8 Treg. Rag2^−/− hosts received sort-purified Teff cells (CD25⁻CD44^loCD62L^hi, CD45.1) and CD4 Treg (CD3⁺CD4⁺YFP⁺) from spleens of FoxP3-Cre or Helios^fl/fl.FoxP3-Cre mice. Recipients were examined for changes in weight, microscopy of intestine pathology and number of CD11b⁺Gr-1⁺ cells in spleen (n=4). Representative data from two independent experiments. The mean ± SEM is indicated. *P < 0.05 and ***P < 0.001 (Kruskal-Wallis test). F) Rag2^−/− hosts received CD4 T cells (Teff: CD25⁻CD44^loCD62L^hi, CD4 Treg: CD3⁺CD4⁺CD25⁺) from spleens of defined donor mouse strains. Recipients were examined for changes in weight (n=4). Representative data from three independent experiments. The mean ± SEM is indicated. *P < 0.05 (Kruskal-Wallis test). G) WT B and CD25-depleted CD4 T cells were transferred into Rag2^−/− hosts along with Ly49⁺ or Ly49⁻ CD8 T cells from spleens of Helios^+/+ or Helios^−/− mice. Rag2^−/− adoptive hosts were immunized with NP₁₉-KLH in CFA at day 0 and reimmunized with NP₁₉-KLH in IFA at day 10. NP specific IgG1 responses were measured at day 15 (n=3). Data representative of three independent experiments. The mean ± SEM is indicated. *P < 0.05 (Kruskal-Wallis test).

Fig 3. Helios-dependent STAT5 activation and stabilization of CD4 Treg

A) Distribution of genome-wide Helios binding sites in activated FoxP3⁺ CD4 and Ly49⁺ CD8 Treg, B) Number of Helios target genes and overlapping Helios binding sites in CD4 and CD8 Treg, C) DNA motif analysis of Helios bound regions, D) ChiP-Seq analysis of Helios binding and modified histones at STAT5b, Jak2, NFAT5, and Birc2 in CD4 and CD8 Treg. Start sites of each gene locus are indicated. Vertical lines in gene diagrams (bottom) indicate exons. E) Pathway analysis of genes targeted by Helios in CD4 and CD8 Treg. Solid lines represent genes known to have a direct connection. Dotted lines represent a presumptive interaction based on reported studies. Major nodes of genes are marked by red circles. Filled symbols indicate Helios target genes and white symbols indicate neighboring genes that are functionally associated but not included in Helios target genes.

Fig 4. Helios-dependent STAT5 activation and stabilization of CD4 Treg

A) CD4 Treg from spleens of Helios^+/+ and Helios^−/− mice were transferred into Rag2^−/− γc^−/− mice and numbers, apoptosis and the anergic phenotype of recovered CD4 Treg from spleens were analyzed 5 days after transfer (n=4). Representative data from three independent experiments. The mean ± SEM is indicated. *P < 0.05 and **P < 0.01 (Mann-Whitney test). B) Levels of FoxP3 expression were compared between splenic CD4 Treg from 6 mo old FoxP3-Cre and Helios^fl/fl.FoxP3-Cre mice (left panel) and from indicated hosts that were induced for colitis (n=4) (middle, right panels). Representative plots > 3 different experiments. The mean ± SEM is indicated. *P < 0.05 and **P < 0.01 (Mann-Whitney test). C) BM chimeras were generated by reconstituting Rag2^−/− mice with hematopoietic progenitors from Helios^+/+, Helios^−/−, or Helios^fl/fl/FoxP3-Cre mice. After six-eight weeks, IL-2 responsiveness of FoxP3⁺ CD4 cells from spleens was tested as described in Methods. Representative histograms for expression of p-STAT5 from two independent experiments. D) CD4 Treg from Helios^+/+ and Helios^−/− mice were transduced with retrovirus expressing GFP or STAT5-CA/GFP, before stimulation with anti-CD3/CD28 Abs in the presence of IL-2 (0–50 ng/ml) and IL-4 (20 ng/ml). Levels of FoxP3 expression after 5 days at 5 ng/ml IL-2 are shown (left panel). IFNγ production by FoxP3⁺ cells is shown (right panel). Representative data from two independent experiments. The mean ± SEM is indicated. *P < 0.05 and **P < 0.01 (Mann-Whitney test). E) 2 mo old Helios^fl/fl and Helios^fl/fl.FoxP3-Cre mice were immunized i.p. with 8 × 10⁸ SRBC. After 7 days, FoxP3⁺ cells from spleen were analyzed for IFNγ and IL-17A expression (n=4–5). Representative data from two independent experiments. The mean ± SEM is indicated. *P < 0.05 and ***P < 0.001 (Mann-Whitney test). F) OT-II cells (1×10⁶) were transferred into Rag2^−/− hosts along with CD25⁺CD4⁺ T cells (2×10⁵) from spleens of CD45.1⁺ Helios^+/+ or CD45.2⁺ Helios^−/− mice followed by immunization with OT-II peptide (10 μg) in CFA. After 5 days spleen cells from Rag2^−/− hosts were analyzed for FoxP3 expression and effector cytokine expression by CD4 Treg. (n=4). Representative data from three independent experiments. Data are mean ± SEM (error bars). *P < 0.05, **P < 0.01 and ***P < 0.001 (Mann-Whitney test).