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Functional reprogramming of regulatory T cells in the absence of Foxp3

Nature Immunology volume 20, pages 1208–1219 (2019)Cite this article

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Abstract

Regulatory T cells (Treg cells) deficient in the transcription factor Foxp3 lack suppressor function and manifest an effector T (Teff) cell–like phenotype. We demonstrate that Foxp3 deficiency dysregulates metabolic checkpoint kinase mammalian target of rapamycin (mTOR) complex 2 (mTORC2) signaling and gives rise to augmented aerobic glycolysis and oxidative phosphorylation. Specific deletion of the mTORC2 adaptor gene Rictor in Foxp3-deficient Treg cells ameliorated disease in a Foxo1 transcription factor–dependent manner. Rictor deficiency re-established a subset of Treg cell genetic circuits and suppressed the Teff cell–like glycolytic and respiratory programs, which contributed to immune dysregulation. Treatment of Treg cells from patients with FOXP3 deficiency with mTOR inhibitors similarly antagonized their Teff cell–like program and restored suppressive function. Thus, regulatory function can be re-established in Foxp3-deficient Treg cells by targeting their metabolic pathways, providing opportunities to restore tolerance in Treg cell disorders.

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Fig. 1: Inactivation of mTORC2, but not mTORC1, in ∆Treg cells mitigates Foxp3 deficiency.
Fig. 2: mTORC2 blockade endows ∆Treg cells with regulatory function.
Fig. 3: Cell-intrinsic and -extrinsic determinants of the Teff cell–like phenotype of ∆Treg cells.
Fig. 4: mTORC2-dependent and -independent gene expression profiles in ∆Treg cells.
Fig. 5: Contribution of the AKT–Foxo1 axis to the rictor-dependent ∆Treg cell phenotype.
Fig. 6: mTORC2 promotes aerobic glycolysis and OXPHOS in ∆Treg cells.
Fig. 7: Blockade of glycolysis improves the scurfy phenotype of Foxp3ΔEGFPiCre mice.
Fig. 8: mTOR inhibition augments the suppressive function of human FOXP3 mutant Treg cells.

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Data availability

The data presented in the manuscript, including de-identified patient results, will be made available to investigators following request to the corresponding author. Any data and materials to be shared will be released via a material transfer agreement. RNA sequencing datasets have been deposited in the Gene Expression Omnibus with the accession code GSE129472.

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Acknowledgements

This work was supported by National Institutes of Health grants 2R01 AI065617 (to T.A.C.) and RO1 AI102888-01A1 (to M.O.L.).

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Authors and Affiliations

  1. Division of Immunology, Boston Children’s Hospital, Boston, MA, USA

    Louis-Marie Charbonnier, Ye Cui, Emmanuel Stephen-Victor, Hani Harb & Talal A. Chatila

  2. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Louis-Marie Charbonnier, Ye Cui, Emmanuel Stephen-Victor, Hani Harb & Talal A. Chatila

  3. Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, USA

    David Lopez & Matteo Pellegrini

  4. Division of Bone Marrow Transplantation and Immune Deficiency, Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Jack J. Bleesing

  5. Division of Allergy, Immunology, and Rheumatology, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Maria I. Garcia-Lloret

  6. Division of Allergy and Immunology, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT, USA

    Karin Chen

  7. Division of Pediatric Allergy and Immunology, Faculty of Medicine, Marmara University, Istanbul, Turkey

    Ahmet Ozen

  8. Laboratory of Angiogenesis and Vascular Metabolism, VIB-KU Leuven Center for Cancer Biology, Leuven, Belgium

    Peter Carmeliet

  9. Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Ming O. Li

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Contributions

L.-M.C. and T.A.C. designed the experiments and evaluated the data. L.-M.C., Y.C., E.V., H.H. and D.L. performed the experiments. D.L. and M.P. performed the gene expression profiling studies. J.J.B., M.I.G.-L., K.C., A.O., M.O.L. and P.C. provided critical material. L.-M.C. and T.A.C. conceived the project and directed the research. L.-M.C. and T.A.C. wrote the manuscript.

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Correspondence to Talal A. Chatila.

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Supplementary Figure 1 Derivation and characterization of Foxp3∆EGFPiCre mice.

(a) Targeting strategy. An IRES-EGFP-iCre-SV40 poly-A cassette was inserted into an SSPI restriction site in the Foxp3 3’-untranslated sequence in exon 11 immediately downstream of the Foxp3 stop codon and upstream of the poly-A adenylation signal. The PGK-neo cassette was later removed by crossing the founder mice with mice transgenic for FLP recombinase. Neo, Frt PGK-neo cassette ; DT, diphtheria toxin gene ; RV, EcoRV ; S, SspI ; H ; HindIII ; K ; KpnI (b) Southern blot analysis of EcoRV-digested and EcoRI DNA derived targeted embryonic stem (ES) cells revealed by 5’ and 3’ probes respectively. (c) PCR products obtained with cDNA after reverse transcription of mRNA or gDNA isolated from CD4+ T cells of WT or Foxp3∆EGFPiCre mice using forward and reverse primers located respectively on Exon 9 and Exon 10 of the Foxp3 locus. (d) Sanger sequencing of the PCR product obtained with cDNA in (c) from Foxp3∆EGFPiCre CD4+ T cells reveal an aberrant spicing with frt and intron 9 sequences. * represents a premature stop codon. (e) EGFP and YFP or Foxp3 (N-terminal domain) expression of CD4+ T cells from Foxp3EGFPCreR26YFP and Foxp3∆EGFPiCreR26YFP mice. (f) Cropped immunoblots of Foxp3 and Actin in GFP– and GFP+ CD4+ T cells from Foxp3EGFPCre and Foxp3∆EGFPiCre mice. (g) Foxp3 expression of EGFP– and EGFP+ CD4+ T cells from Foxp3EGFPCre (n=7) and Foxp3∆EGFPiCre mice (n=4), normalized on Actin expression and expressed as fold change compared to EGFP+ CD4+ T cells from Foxp3EGFPCre mice. Results are expressed as mean ± SEM and statistical significance was determined by a two-way ANOVA with Sidak’s multiple comparisons (P values as indicated) (h) Methylation status (as defined by the colored bar) of individual or global CpG motifs located at different positions on the Foxp3 Conserved non-coding sequence 2 (CNS2) and the global methylation status of CNS2 CpG motifs (total) of EGFP– and EGFP+ CD4+ T cells from Foxp3EGFPCre and Foxp3∆EGFPicre mice (n=3 mice per group). (i) Foxp3 and YFP expression in CD4+CD8+ double positive (DP), CD4–CD8– double negative (DN), CD4+ or CD8+ single-positive (CD4 SP, CD8 SP respectively) thymocytes or CD4+ or CD8+ splenocytes of Foxp3∆EGFPiCre/+R26YFP mice.

Supplementary Figure 2 Characterization of Rictor- and Raptor-deficient ∆Treg cells.

(a,b) Representative flow cytometric analysis (a) and mean fluorescence intensity (MFI) (b) of phosphorylated 4EBP (p4EBP) and phosphorylated AKT at the Thr308 residue (pT308AKT) expression in unstimulated (US) or anti-CD3 mAb (α-CD3) -stimulated Treg cells from Foxp3EGFPCre and Foxp3∆EGFPiCre mice (n=4 per group). Results represent one of 2 independent experiments. (c) Representative cropped immunoblots and densitometry analyses of Pten, Phlpp1 and Actin in EGFP– and EGFP+ CD4+ T cells from Foxp3EGFPCre and Foxp3∆EGFPiCre mice (n=3 per group). Results represent a pool of 3 independent experiments. (d) Cropped immunoblots and densitometry analyses of pY319-ZAP70 and total ZAP70 in EGFP+ CD4+ T cells from Foxp3EGFPCre and Foxp3∆EGFPiCre mice after 0, 2, 5 and 10 min of anti-CD3 stimulation (1µg/mL) (n=3 per group). Results represent a pool of 3 independent experiments. (e) Rptor gene expression of EGFP– and EGFP+ CD4+ T cells isolated from Foxp3EGFPCre, Foxp3∆EGFPiCre and Foxp3∆EGFPicreRptor∆/∆ mice. The values were normalized for Actin expression and expressed as fold change compared to EGFP–CD4+ T cells from Foxp3EGFPCre mice (n=3 per group). (f) Rictor gene expression of EGFP– and EGFP+ CD4+ T cells isolated from Foxp3EGFPCre (n=8), Foxp3∆EGFPiCre (n=8) and Foxp3∆EGFPiCreRictor∆/∆ mice (n=7), normalized for Actin expression and expressed as fold change compared to EGFP–CD4+ T cells from Foxp3EGFPCre mice. (g) Immunoblots of Rictor and Actin in EGFP– and EGFP+ CD4+ T cells from Foxp3EGFPCre, Foxp3∆EGFPiCre and Foxp3∆EGFPiCreRictor∆/∆ mice. (n=3 per group). (h) Frequencies of Treg cells (EGFP+CD4+) from Foxp3EGFPCre (n=15), Foxp3∆EGFPicre (n=14), Foxp3∆EGFPicreRictor∆/∆ (n=17) and Foxp3∆EGFPicreRptor∆/∆ (n=5) mice. (P values as indicated) Results represent a pool of 5 independent experiments. (i, j) Representative flow cytometric analysis (i) and scatter plot representation (j) of Foxp3, CD25, CTLA-4, ICOS, Helios, Nrp1, GITR and CD73 MFI in Foxp3EGFPCre Teff cells and in Foxp3EGFPCre (n=4, except for CD73, n=8), Foxp3∆EGFPiCre (n=4, except for CD73, n=11) and Foxp3∆EGFPiCreRictor∆/∆ (n=4, except for CD73, n=6) Treg cells. Results represent one of 2 independent experiments, except for CD73 which represent a pool of 2 independent experiments. Statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparisons (P values as indicated). Statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparisons (h, j) or a two-way ANOVA with Sidak’s multiple comparisons (b, c, d, e, f, g) (P values as indicated).

Supplementary Figure 3 Characterization of the peripheral T cell compartment of Foxp3∆EGFPiCreRictor∆/∆ cells.

(a, b) Representative flow cytometric analysis (a) and frequencies (b) of CD62LhiCD44lo, CD62LhiCD44hi and CD62LloCD44hi CD8+ T cells, T-Bet, Gata-3, Rorγt and IL-17, Ki67 and EGFP expression by CD4+ Treg (YFP+) and Teff (YFP–) cells from spleens of Foxp3EGFPCre, Foxp3∆EGFPiCre and Foxp3∆EGFPiCreRictor∆/∆ mice (n=4–8). Results represent one of 3 independent experiments. Statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparisons (P values as indicated). Ex-Treg cells represent the proportion of EGFP– among YFP+ cells.

Supplementary Figure 4 Rictor-deficient Foxp3-sufficient Treg cells have superior in vivo suppressive capacities in CD4 T cell transfer-induced colitis model.

(a) Schematic representation of the protocol of lymphopenia-induced colitis by intraperitoneal adoptive transfer of Teff cells (CD45.1+CD4+CD45RBhiEGFP–) from CD45.1 Foxp3EGFP mice without or with Treg cells (CD45.2+CD4+YFP+) from Foxp3YFPcre or Foxp3YFPcreRictor∆/∆ mice in Rag1–/– mice. (b) Changes in body weight over time are shown. (n=15 for “No Treg cells” group, n=17 for “Foxp3YFPcre Treg cells” group and n=18 for “Foxp3YFPcre Rictor∆/∆ Treg cells” group) (c) Absolute number of CD45.1+ T cells (Teff cell input) and CD45.2+ T cells (Treg cell input) within the entire colon (n=10 for “No Treg cells” group, n=12 for “Foxp3YFPcre Treg cells” group and n=12 for “Foxp3YFPcre Rictor∆/∆ Treg cells” group). (d, e) Disease severity was evaluated by H&E staining of colon sections (n=19 for “No Treg cells” group, n=17 for “Foxp3YFPcre Treg cells” group and n=17 for “Foxp3YFPcre Rictor∆/∆ Treg cells” group) (d) and end point colon length (n=11 for “No Treg cells” group, n=13 for “Foxp3YFPcre Treg cells” group and n=17 for “Foxp3YFPcre Rictor∆/∆ Treg cells” group) (e). (f, g) Representative flow cytometric analysis (f) and absolute number (g) of IFN-γ and IL-17-producing CD45.1+CD4+ T cells from the Teff cell input was quantified within the entire colon (n=11 for “No Treg cells” group, n=13 for “Foxp3YFPcre Treg cells” group and n=13 for “Foxp3YFPcre Rictor∆/∆ Treg cells” group). Statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparisons (c, e, g) or by a two-way repeted mesure ANOVA with Tukey’s multiple comparisons (b) (P values as indicated). Results represent a pool of 3 independent experiments.

Supplementary Figure 5 Contribution of the AKT/Foxo1 axis to the Rictor-dependent phenotype of ∆Treg cell.

(a-c) Gross appearance (a), body weight (b) and survival (c) of WT (n=8 for body weight), Foxp3∆EGFPiCre (n=10 for body weight), Foxp3∆EGFPiCreRictor∆/∆ (n=6 for body weight), Foxp3∆EGFPiCreRictor∆/∆ Foxo1∆/∆ (n=11 for body weight) and Foxp3∆EGFPiCreR26Foxo1AAA (n=10 for body weight) mice. Results represent a pool of 3 independent experiments (d) Frequencies of T-Bet+ and Gata-3+ by CD4+ Treg and Teff cells in spleens of Foxp3EGFPCreR26YFP (n=3), Foxp3∆EGFPiCreR26YFP(n=4), Foxp3∆EGFPiCreRictor∆/∆R26YFP (n=5) and Foxp3∆EGFPiCreR26Foxo1AAA (n=5) mice. Results represent one of 3 independent experiments (e, f) Representative flow cytometric analysis (e) and frequencies (f) of IL-10+ by CD4+ Treg cells in spleens of Foxp3EGFPCreR26YFP(n=4), Foxp3∆EGFPiCreRictor∆/∆R26YFP (n=3) and Foxp3∆EGFPiCreRictor∆/∆ Foxo1∆/∆ (n=3) mice. Results represent one of 2 independent experiments. (g) Gross appearance of WT, Foxp3∆EGFPiCre, Foxp3∆EGFPiCreRictor∆/∆ and Foxp3∆EGFPiCreR26Foxo1AAA mice. (h) In vitro suppression of the proliferation of WT CD4+ Teff cells (Tresp) by Foxp3EGFPCre, Foxp3∆EGFPiCre and Foxp3∆EGFPiCreR26Foxo1AAA Treg (Tsupp) cells (n=3 per group). Statistical significance was determined by a one-way ANOVA with Bonferroni’s multiple comparisons (b) or with Tukey’s multiple comparisons (d, f), two-way ANOVA with Tukey’s multiple comparisons (h) and log-rank test (c) (P values as indicated).

Supplementary Figure 6 Metabolic profiling of Foxp3-sufficient Treg cells and Rictor-sufficient and -deficient ∆Treg cells.

(a) Quantitative PCR analysis of transcripts encoding the glycolysis and pentose phosphate cycle (PPC) enzymes, normalized to Actin expression and expressed as Log2 Fold change, in Foxp3EGFP, Foxp3∆EGFPiCre and Foxp3∆EGFPiCreRictor∆/∆ Treg cells compared to WT CD4+ Teff cells. (b, c) Extracellular acidification rate (ECAR) under glycolysis stress test conditions and Oxygen consumption rate (OCR) under mitochondrial stress test conditions of Treg/∆Treg cells isolated from Foxp3EGFP/EGFP, Foxp3∆EGFPiCre/+ and Foxp3∆EGFPiCre/+Rictor∆/∆ females. (n=4 per group). Results represent a pool of 2 experiments. (d) Lactate release by in vitro cultured resting (No Stim) or stimulated (α-CD3+IL-2) Foxp3EGFP Teff cells and Foxp3EGFP, Foxp3∆EGFPiCre and Foxp3∆EGFPiCreRictor∆/∆ Treg cells. (n=6 per group). Results represent a pool of 2 experiments. (e) Hif1a and Myc gene expression in EGFP+CD4+ T cells from Foxp3EGFP (n=5), Foxp3∆EGFPiCre (n=3) and Foxp3∆EGFPiCreRictor∆/∆ (n=3) mice, normalized to Actin expression and expressed as fold change compared to EGFP+CD4+ T cells from Foxp3EGFP mice. (f) Metabolic pathway analysis showing differentially expressed metabolites between Foxp3∆EGFPiCre and Foxp3EGFP Treg cells, Foxp3∆EGFPiCreRictor∆/∆ and Foxp3EGFP Treg cells and Foxp3∆EGFPiCreRictor∆/∆ and Foxp3∆EGFPiCre Treg cells (n=5 per group). Statistical significance was determined by a one-way ANOVA with Holm-Sidak’s or Bonferroni’s multiple comparisons (e) or two-way ANOVA with Sidak’s or Tukey’s multiple comparisons (c, d) (P values as indicated).

Supplementary Figure 7 Glycolytic blockade and ∆Treg cell-specific Rictor deletion synergize in controlling Foxp3 deficiency disease.

(a) Representative flow cytometric analysis of CD62LloCD44hi CD4+ and CD8+ Teff cells in PBS or 2DG-treated Foxp3∆EGFPiCre mice (Frequencies in Fig. 7h). (b, c) Representative flow cytometric analysis (b) and frequencies (c) of Foxp3∆EGFPiCreRictor∆/∆ mice treated either with PBS or 2DG (2µg/g) from day 14 to day 45 every other day. (d) Representative flow cytometric analysis and frequencies of IFN-γ+ and IL-4+ CD4+ Teff and Treg cells of PBS or 2DG-treated Foxp3∆EGFPiCre mice (Frequencies in Fig. 7h). (e, f) Representative flow cytometric analysis (e) and frequencies (f) of IFN-γ+ and IL-4+ CD4+ Teff and Treg cells of PBS or 2DG-treated Foxp3∆EGFPiCreRictor∆/∆ mice. (g) Representative flow cytometric analysis and frequencies of T-Bet+ and Gata-3+ CD4+ Teff and Treg cells of PBS or 2DG-treated Foxp3∆EGFPiCre mice (Frequencies in Fig. 7h). (h, i) Representative flow cytometric analysis (h) and frequencies (i) of T-Bet+ and Gata-3+ CD4+ Teff and Treg cells of PBS or 2DG-treated Foxp3∆EGFPiCreRictor∆/∆ mice (n=6 per group) Statistical significance was determined by an unpaired t-test (P values as indicated).

Supplementary Figure 8 Characterization of Treg cells from IPEX patients.

Representative flow cytometric analysis of CD25 and CD127 expression (a), and CD4 and FOXP3 expression (b) among CD4+ PBMCs isolated from healthy control subjects (HC1-5) and IPEX subjects (P1-5). (c) Expression of FOXP3, CD25, CD127, CTLA-4 and HELIOS in CD4+CD25loCD127hi T cells of HC (Teff cells HC1-5) and CD4+CD25hiCD127lo population from HC (Treg cells HC1-5) and IPEX patients (Treg cells P1-5).

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 1–4

Reporting Summary

Supplementary Dataset 1

Differentially expressed genes in WT (Foxp3RFP) versus mutant (Foxp3∆EGFPiCre) Treg cells derived from Foxp3∆EGFPiCre/RFP female mice (n = 4 per group).

Supplementary Dataset 2

Differentially expressed genes in single-mutant (Foxp3∆EGFPiCre) versus double-mutant (Foxp3∆EGFPiCreRictor∆/∆) Treg cells derived, respectively, from Foxp3∆EGFPiCre/RFP and Foxp3∆EGFPiCre/+Rictor∆/∆ female mice (n = 4 per group).

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Charbonnier, LM., Cui, Y., Stephen-Victor, E. et al. Functional reprogramming of regulatory T cells in the absence of Foxp3. Nat Immunol 20, 1208–1219 (2019). https://doi.org/10.1038/s41590-019-0442-x

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