Functional reprogramming of regulatory T cells in the absence of Foxp3

Abstract

Regulatory T cells (T_reg cells) deficient in the transcription factor Foxp3 lack suppressor function and manifest an effector T (T_eff) 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 T_reg cells ameliorated disease in a Foxo1 transcription factor-dependent manner. Rictor deficiency re-established a subset of T_reg cell genetic circuits and suppressed the T_eff cell-like glycolytic and respiratory programs, which contributed to immune dysregulation. Treatment of T_reg cells from patients with FOXP3 deficiency with mTOR inhibitors similarly antagonized their T_eff cell-like program and restored suppressive function. Thus, regulatory function can be re-established in Foxp3-deficient T_reg cells by targeting their metabolic pathways, providing opportunities to restore tolerance in T_reg cell disorders.

Figure 1.. Inactivation of mTORC2 but not mTORC1 in ΔT_reg cells mitigates Foxp3 deficiency.

(a,b) Representative flow cytometric analysis (a) and mean fluorescence intensity (MFI) (b) of phosphorylated S6 (pS6) and phosphorylated AKT at Ser473 residu (p_S473AKT) expression of unstimulated (US) or anti-CD3 stimulated (α-CD3) T_reg cells from Foxp3^EGFPCre, Foxp3^ΔEGFPiCre/+ and Foxp3^ΔEGFPiCre mice (n=5 per group). Results represent 1 of 3 independent experiments. (c,d) Body weight at 25–28 days of age (c) and survival (d) of Foxp3^K276X, Foxp3^ΔEGFPiCre, Foxp3^ΔEGFPiCreRictor^Δ/WT, Foxp3^ΔEGFPiCreRictor^Δ/Δ, Foxp3^ΔEGFPiCreRptor^Δ/WT, Foxp3^ΔEGFPiCreRptor^Δ/Δ, Foxp3^ΔEGFPiCreRictor^Δ/ΔRptor^Δ/Δ mice and control littermates. Results represent a pool of 5 independent experiments. (e) Gross appearance of Foxp3^ΔEGFPiCre, Foxp3^ΔEGFPiCreRictor^Δ/Δ and control littermate and their respective spleens and peripheral lymph nodes. (f,g) Representative microscopic pictures of H&E staining (original magnification ×200) (f) and histological scores (g) of skin, lung, colon and liver of Foxp3^ΔEGFPiCre (n=14), Foxp3^ΔEGFPiCreRictor^Δ/Δ (n=7) and control littermates (n=5). Results represent a pool of 3 independent experiments. Statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparisons (c, g), two-way ANOVA with Sidak’s multiple comparisons (b) and log rank test (d) (P values as indicated).

Figure 2.. mTORC2 blockade endows ΔT_reg cells with regulatory function.

(a,b) Representative flow cytometric analysis (a) and frequencies (scatter plots with mean) (b) of CD62L^hiCD44^lo and CD62L^loCD44^hi CD4⁺ T cells or IFN-γ and IL-4 expression by CD4⁺ T_reg (YFP⁺) and T_eff (YFP^–) cells from spleen of Foxp3^EGFPCreR26^YFP (n=5 for CD62L^hiCD44^lo and CD62L^loCD44^hi CD4⁺ T cells, n=4 for IFN-γ and IL-4 expression), Foxp3^ΔEGFPiCreR26^YFP (n=4) and Foxp3^ΔEGFPiCreRictor^Δ/ΔR26^YFP mice (n=5 for CD62L^hiCD44^lo and CD62L^loCD44^hi CD4⁺ T cells, n=4 for IFN-γ and IL-4 expression). Results represent 1 of 4 independent experiments (c) In vitro suppression of the proliferation of WT CD4⁺ T_eff cells (denoted as T responder or T_resp) by Foxp3^EGFPCre T_eff cells pre-treated for 1h with DMSO (Veh WT T_eff cells) or 1μM Rapamycin (Rapa WT T_eff cells), Foxp3^EGFPCre T_reg cells pre-treated for 1h with DMSO (Veh WT T_reg cells) or 1μM Rapamycin (Rapa WT T_reg cells) or Foxp3^ΔEGFPiCre T_reg cells pre-treated for 1h with DMSO (Veh ΔT_reg cells) or 1μM Rapamycin (Rapa ΔT_reg cells) (denoted as T suppressor or T_supp) (n=3 per group). Results represent 1 of 3 independent experiments (d, e) In vitro suppression of T_resp cells by Foxp3^EGFPCre T_reg cells (Foxp3^EGFPCre), Foxp3^ΔEGFPiCre T_reg cells pre-treated for 1h with DMSO (Veh Foxp3^ΔEGFPiCre) or 1μM Rapamycin (Rapa Foxp3^ΔEGFPiCre), Foxp3^ΔEGFPiCreRictor^Δ/Δ T_reg cells pre-treated for 1h with DMSO (Veh Foxp3^ΔEGFPiCreRictor^Δ/Δ) or 1μM Rapamycin (Rapa Foxp3^ΔEGFPiCreRictor^Δ/Δ) (n=3 per group) (Results represent 1 of 3 independent experiments) (d), and Foxp3^ΔEGFPiCreRptor^Δ/Δ T_reg cells pre-treated for 1h with DMSO (Veh Foxp3^ΔEGFPiCreRptor^Δ/Δ) or 1μM Rapamycin (Rapa Foxp3^ΔEGFPiCreRptor^Δ/Δ) (n=3 per group) (Results represent 1 of 3 independent experiments) (e). (f) In vitro suppression of T_resp cells by Rictor-sufficient or -deficient T_eff (CD4⁺CD25⁻ cells from CD4^cre and CD4^creRictor^Δ/Δ) and Foxp3-sufficient T_reg (Foxp3^YFPcre and Foxp3^YFPcreRictor^Δ/Δ) cells (n=3 per group) (Results represent 1 of 2 independent experiments). Results are expressed as mean ± SEM in panels c-f. Statistical significance was determined by a one-way ANOVA (b) or two-way ANOVA with Tukey’s multiple comparisons (c-f) (P values as indicated).

Figure 3:. Cell intrinsic and extrinsic determinants of the T_eff cell-like phenotype of ΔT_reg cells.

(a) In vitro suppression of CD4⁺ T_eff cell (T_resp) proliferation by Foxp3-sufficient T_reg cells from Foxp3^EGFPcre mice and ΔT_reg cells from Foxp3^ΔEGFPiCre/+ and Foxp3^ΔEGFPiCre mice (T_supp) (n=3 per group). Results are expressed as mean ± SEM and represent 1 of 2 independent experiments. (b, c) Representative flow cytometric analysis of Foxp3 and YFP among CD4⁺ T cells (b) and frequencies of ΔT_reg cells (c) among total T_reg cells from Foxp3^ΔEGFPiCre/+R26^YFP (n=12) and Foxp3^ΔEGFPiCre/+Rictor^Δ/ΔR26^YFP (n=10) mice. Results are expressed as scatter plot and mean and represent a pool of 3 independent experiments. (d, e) Representative flow cytometric analysis of IL-4 and IFN-γ among YFP⁺ T_reg cells (d) and frequencies (Scatter plot and mean) of T-Bet⁺, Gata-3⁺, IFN-γ⁺ and IL-4⁺ YFP⁺ T_reg cells (e) from Foxp3^EGFPCreR26^YFP, Foxp3^ΔEGFPiCre/+R26^YFP, Foxp3^ΔEGFPiCre/+Rictor^Δ/ΔR26^YFP, Foxp3^ΔEGFPiCreR26^YFP and Foxp3^ΔEGFPiCreRictor^Δ/ΔR26^YFP mice (n=4 per group). Results represent 1 of 2 independent experiments. (f) CD25, CTLA4, Nrp1 and Helios MFI in WT T_reg and ΔT_reg cells from Foxp3^EGFPCreR26^YFP, Foxp3^ΔEGFPiCre/+R26^YFP, Foxp3^ΔEGFPiCre/+Rictor^Δ/ΔR26^YFP female mice (n=4 per group). ND, Not Determined. Results represent 1 of 2 independent experiments. Statistical significance was determined by unpaired t-test (c), one-way ANOVA (e) or two-way ANOVA (a, f) with Tukey’s multiple comparisons (P values as indicated).

Figure 4.. mTORC2-dependent and -independent gene expression profiles in ΔT_reg cells.

Gene expression profiles of Foxp3^RFP (WT), Foxp3^ΔEGFPiCre and Foxp3^ΔEGFPiCreRictor^Δ/Δ T_reg cells (n=4 per group) isolated from spleen of Foxp3^{RFP/ΔEGFPiCre}R26^YFP and Foxp3^ΔEGFPiCre/+Rictor^Δ/ΔR26^YFP female mice. (a), Gene expression profiles represented as Fold change (Foxp3^RFP vs Foxp3^ΔEGFPiCre) versus Fold change (Foxp3^RFP vs Foxp3^ΔEGFPiCreRictor^Δ/Δ). (b), Heatmap representation, (c), Log₂ expression and (d) volcano plot of differential gene expression in Foxp3^ΔEGFPiCre versus Foxp3^ΔEGFPiCreRictor^Δ/Δ ΔT_reg cells. FDR, false discovery rate; log2FC, log2(fold change). (e), Flow cytometric analysis of IL-10 and Blimp1 expression in T_reg cells of Foxp3^EGFPCre (n=4 and n=8 respectively) and ΔT_reg cells of Foxp3^ΔEGFPiCre (n=4 and n=10 respectively) and Foxp3^ΔEGFPiCreRictor^Δ/Δ (n=4 and n=6 respectively) mice. Results are expressed as scatter plot and mean and represent 1 of 3 independent experiments. (f), Chromatin immunoprecipitation (ChIP) assay for the binding of Blimp1 and control isotype to the Il10 conserved non-coding sequence located 9kb in proximal of il10 promoter (il10 CNS–9) in Foxp3^ΔEGFPiCre (n=5) and Foxp3^ΔEGFPiCreRictor^Δ/Δ (n=13) ΔT_reg cells. Results are expressed as scatter plot and mean and represent a pool of 2 independent experiments. (g), In vitro suppression of T_resp cells by Foxp3^ΔEGFPiCreRictor^Δ/Δ ΔT_reg cells carried out in the presence of either an isotype control or anti-IL-10 mAb (n=3 per group). Results are expressed as mean ± SEM and represent 1 of 2 independent experiments. (h) ChIP assays for the binding of Foxo1 and control isotype to Tbx21 and Ifng promoters in Foxp3^ΔEGFPiCre (n=5) and Foxp3^ΔEGFPiCreRictor^Δ/Δ (n=13) ΔT_reg cells. Results are expressed as scatter plot and mean and represent a pool of 2 independent experiments. Statistical significance was determined one-way ANOVA (e) or two-way ANOVA (f-h) with Tukey’s or Sidak’s multiple comparisons (P values as indicated).

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

(a) Representative flow cytometric analysis and (b) MFI of p₄₇₃-AKT expression in unstimulated (US) and anti-CD3/CD28 mAb-stimulated (α-CD3+α-CD28) T_reg cells from Foxp3^EGFPCre, Foxp3^ΔEGFPiCre, Foxp3^ΔEGFPiCreRictor^Δ/Δ mice (n=3 per group). Results are expressed as scatter plot and mean and represent 1 of 3 independent experiments. (c,d) Representative confocal microscopic merge image of Foxo1 (Red) and DAPI (Blue) (c), and percent of nuclear Foxo1 (d) in unstimulated (US) or 0.1μg/mL anti-CD3 (α-CD3) stimulated T_reg cells from Foxp3^EGFPCre (n=53 for US, n=71 for α-CD3), Foxp3^ΔEGFPiCre (n=74 for US, n=95 for α-CD3), Foxp3^ΔEGFPiCreRictor^Δ/Δ (n=78 for US, n=46 for α-CD3) mice. Results are expressed as scatter plot and mean and represent a pool of 2 independent experiments. (e) Representative flow cytometric analysis and (f) frequencies (scatter plot and mean) of CD62L^loCD44^hi CD4⁺ T_eff cells, and IFN-γ and IL-4 expression by CD4⁺ T_reg and T_eff cells in spleens of Foxp3^EGFPCre (n=10), Foxp3^ΔEGFPiCre (n=7), Foxp3^ΔEGFPiCreRictor^Δ/Δ (n=6), Foxp3^ΔEGFPiCreFoxo1^Δ/Δ (n=3), Foxp3^ΔEGFPiCreRictor^Δ/ΔFoxo1^Δ/Δ (n=4) and Foxp3^ΔEGFPiCreR26^Foxo1AAA (n=5) mice. Results represent a pool of 3 independent experiments. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons (f) or two-way ANOVA with Sidak’s multiple comparisons (b, d) (P values as indicated).

Figure 6.. mTORC2 promotes aerobic glycolysis and OXPHOS in ΔT_reg cells.

(a, b) Extracellular acidification rate (ECAR) under glycolysis stress test conditions (n=4 per group) and Oxygen consumption rate (OCR) under mitochondrial stress test conditions (n=5 per group) of T_reg/ΔT_reg cells isolated from Foxp3^EGFP, Foxp3^ΔEGFPiCre or Foxp3^ΔEGFPiCreRictor^Δ/Δ males (n=4 per group). Results are expressed as mean ± SEM and represent a pool of 2 independent experiments. (c) OCR under mitochondrial fuel test conditions (n=4 per group). Results are expressed as mean ± SEM and represent a pool of 2 independent experiments (d) Pie chart representation of the contribution of glucose, fatty acids and glutamine to the OXPHOS capacities in Foxp3^EGFP, Foxp3^ΔEGFPiCre and Foxp3^ΔEGFPiCreRictor^Δ/Δ ΔT_reg cells. (e) Quantification of metabolites (expressed as scaled index) of glucose metabolism (Phosphoenolpyruvate and lactate), tricarboxylic acid (TCA) cycle (Citrate, fumarate and Malate) and lipid metabolism (Acetylcarnitine (C2), Carnitine, 1-palmitoyl-2-decosahexaenoyl and Phospho-ethanolamine) isolated from Foxp3^EGFP, Foxp3^ΔEGFPiCre and Foxp3^ΔEGFPiCreRictor^Δ/Δ ΔT_reg cells (n=5 per group). Results are expressed as mean ± SEM and represent 1 experiment. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons (e) or two-way ANOVA with Sidak’s multiple comparisons (b) (P values as indicated).

Figure 7.. Blockade of glycolysis improves the scurfy phenotype of Foxp3^ΔEGFPiCre mice.

(a) ECAR under glycolysis stress test conditions and OCR under mitochondrial stress test conditions of ΔT_reg cells isolated from Foxp3^ΔEGFPiCre or Foxp3^ΔEGFPiCrePfkfb3^Δ/Δ males (n=3 per group). Results are expressed as mean ± SEM and represent 1 of 2 independent experiments. (b) Frequencies of CD62L^loCD44^hi CD4⁺ T_eff cells, and IFN-γ and IL-4 expression by CD4⁺ T_reg and T_eff cells in spleens of Foxp3^EGFPCreR26^YFP, Foxp3^ΔEGFPiCreR26^YFP, Foxp3^ΔEGFPiCrePfkfb3^Δ/ΔR26^YFP, Foxp3^ΔEGFPiCreRictor^Δ/ΔR26^YFP, Foxp3^ΔEGFPiCreRictor^Δ/ΔPfkfb3^Δ/ΔR26^YFP mice. Results are expressed as scatter plot and mean and represent a pool of 3 independent experiments. (c) In vitro suppression of CD4⁺ T_eff cell (T_resp) proliferation by Foxp3^EGFPCre, Foxp3^ΔEGFPiCre or Foxp3^ΔEGFPiCrePfkfb3^Δ/Δ T_reg cells (T_supp) (n=3 per group). Results are expressed as mean ± SEM and represent 1 of 2 independent experiments. (d) Frequencies of IFN-γ⁺ and IL-4⁺ Foxp3^ΔEGFPiCreR26^YFP and Foxp3^ΔEGFPiCreRictor^Δ/ΔR26^YFP sorted T_reg cells that were either sham treated or treated ex-vivo with 2-deoxyglucose (2DG) (n=3 per group). Results are expressed as scatter plot and mean and represent 1 of 2 independent experiments. (e) In vitro suppression of CD4⁺ T_eff cell (T_resp) proliferation by Foxp3^ΔEGFPiCre ΔT_reg (T_supp) cells that were pre-treated for 12h with either PBS (Veh ΔT_reg cells) or 2DG (2DG ΔT_reg cells) (n=3 per group). Results are expressed as mean ± SEM and represent 1 of 2 independent experiments. (f, g) Representative hematoxylin and eosin-stained tissue histological sections (f) and tissue histological scores (g) of ears, livers and lungs of Foxp3^ΔEGFPiCreR26^YFP mice treated with PBS (n=) or 2 μg/g 2DG (n=) every other day from day 14 to day 28. Results are expressed as scatter plot and mean and represent a pool of 2 independent experiments. (h) Frequencies of CD62L^loCD44^hi CD4⁺ and CD8⁺ T_eff cells, IFN-γ⁺ and IL-4⁺ T_reg and CD4⁺ T_eff cells and T-Bet⁺ and Gata-3⁺ T_reg and CD4⁺ T_eff cells in Foxp3^ΔEGFPiCreR26^YFP mice treated with PBS (n=6) or 2DG (n=7). Results are expressed as scatter plot and mean and represent a pool of 2 independent experiments. Statistical significance was determined unpaired t-test (g, h), one-way ANOVA with Tukey’s multiple comparisons (b) or two-way ANOVA with Sidak’s multiple comparisons (a, d) or with Tukey’s multiple comparisons (c, e) (P values as indicated).

Figure 8.. mTOR inhibition augments the suppressive function of human FOXP3 mutant T_reg cells.

(a) Schematic representation of FOXP3 illustrating the exons, the protein domains and mapped mutations of five patients. Amino acid changes are referred to by their single letter code. The N-terminal proline rich repressor domain (Repressor), zinc finger (ZF) motif, leucine zipper domain (LZ) and the forkhead DNA-binding domain (FKH DBD) are indicated. (b,c) Mean Fluorescence Intensity (MFI) of pS6 and p_S473AKT in T_reg cells of healthy control subjects (HC) (n=3) and IPEX patients (n=3; P1, P2, P3) stimulated with anti-CD2/CD3/CD28 mAbs (α-CD2/3/28) in the absence or presence of a competitive dual mTOR inhibitor (mTOR inh). Results are expressed as scatter plot and mean and represent 1 of 2 independent experiments. (c) Representative ECAR tracings (HC1 and P1), expressed as mean ± SEM. (d) Evaluation of glycolysis and glycolytic reserve (n=5 each for HC and IPEX groups). Results are expressed as mean ± SEM and represent a pool of 5 independent experiments. (e) Representative OCR tracings (HC2 and P2). Basal respiration, ATP production and maximal respiration in DMSO (Vehicle) or mTOR inhibitor (mTOR inh) treated HC and IPEX T_reg cells (HC n=3 and IPEX: P1, P2, P4). (f) In vitro suppression of third party CD4⁺ T_eff cell (T_resp) proliferation by T_reg (T_supp) cells of a HC or an IPEX subject (P3) that were pre-treated with vehicle or mTOR inhibitor. (g) Compilation of in vitro suppression assay results for HC and IPEX subjects at the ratio of 1:1 T_reg:T_eff cells without or with T_reg cell mTOR inhibitor pretreatment. (h) IFN-γ and IL-4 secretion by vehicle or mTOR inhibitor pre-treated IPEX T_reg cells. Statistical significance was one-way ANOVA with Tukey’s multiple comparisons (h) or two-way ANOVA with Sidak’s multiple comparisons (b-d) or with Tukey’s multiple comparisons (e, f, g) (P values as indicated).