Treg cells require the phosphatase PTEN to restrain TH1 and TFH cell responses

Abstract

The interplay between effector T cells and regulatory T cells (Treg cells) is crucial for adaptive immunity, but how Treg cells control diverse effector responses is elusive. We found that the phosphatase PTEN links Treg cell stability to repression of type 1 helper T cell (TH1 cell) and follicular helper T cell (TFH cell) responses. Depletion of PTEN in Treg cells resulted in excessive TFH cell and germinal center responses and spontaneous inflammatory disease. These defects were considerably blocked by deletion of interferon-γ, indicating coordinated control of TH1 and TFH responses. Mechanistically, PTEN maintained Treg cell stability and metabolic balance between glycolysis and mitochondrial fitness. Moreover, PTEN deficiency upregulates activity of the metabolic checkpoint kinase complex mTORC2 and the serine-threonine kinase Akt, and loss of this activity restores functioning of PTEN-deficient Treg cells. Our studies establish a PTEN-mTORC2 axis that maintains Treg cell stability and coordinates Treg cell-mediated control of effector responses.

Figure 1. Pten^fl/flFoxp3-Cremice develop age-related autoimmune and lymphoproliferative disease

(a) Quantification of dsDNA-specific IgG in the serum of Pten^+/+Foxp3-Cre (WT) and Pten^fl/flFoxp3-Cre mice (2-6 months old). (b) Representative images (scale 60 μm) and quantification of fluorescent intensity (right) of serum ANA IgG autoantibodies detected with fixed Hep-2 slides. (c) Histology images of kidney glomeruli sections stained with H&E (magnification: ×60; scale 50 μm). (d) Immune fluorescence images of kidney glomeruli sections showing IgG deposits (scale 40 μm). (e) Images of spleen and peripheral lymph nodes from WT (upper, ∼5 months old), Pten^fl/flFoxp3-Cre mice prior to the development of lymphoproliferative disease (middle, ∼2.5 months old), and Pten^fl/flFoxp3-Cre mice with lymphoproliferative disease (lower, ∼5 months old). (f) H&E staining of Peyer's patches in the intestine of WT and Pten^fl/flFoxp3-Cre mice (magnification: left, ×4; scale 1mm and right, ×20; scale 200 μm). Data are representative of at least two independent experiments (a-f). *P < 0.05 and **P < 0.001. Data are mean ± s.e.m.

Figure 2. Increased T cell activation and altered immune homeostasis in Pten^fl/flFoxp3-Cre mice

(a) Expression of CD62L and CD44 on WT and Pten^fl/flFoxp3-Cre splenic T cells. Numbers in quadrants indicate percent cells in each. (b) Expression of IFN-γ in CD4⁺ and CD8⁺ T cells of WT and Pten^fl/flFoxp3-Cre mice. (c) Flow cytometry analysis of T_reg cells in WT and Pten^fl/flFoxp3-Cre splenic CD4⁺ T cells. Below, the frequency and numbers of T_reg cells. (d,e) Caspase-3 activity (d) and BrdU staining at 16 h after injection of BrdU (e) in T_reg cells from the spleen and peripheral lymph nodes (MLNs) of WT and Pten^fl/flFoxp3-Cre mice. Data are representative of at least ten independent experiments (a-c) and two independent experiments (d,e). *P < 0.05 and **P < 0.001. Data are mean ± s.e.m.

Figure 3. The aberrant induction of T_FH cell and GC responses in Pten^fl/flFoxp3-Cremice

(a) Flow cytometry analysis of CXCR5⁺PD-1⁺ cells (gated on CD4⁺TCRβ⁺ cells) in the spleen of WT and Pten^fl/flFoxp3-Cremice. Right, the frequency and numbers of T_FH cells. (b) Analysis of conventional T_FH (CD4⁺CXCR5⁺PD-1⁺Foxp3-YFP⁻) and T_FR cells (CD4⁺CXCR5⁺PD-1⁺Foxp3-YFP⁺) in the spleen of WT and Pten^fl/flFoxp3-Cremice. (c) Analysis of GL7⁺CD95⁺ GC B cells (gated on CD19⁺ B cells) in the spleen of WT and Pten^fl/flFoxp3-Cremice. Right, the frequency and numbers of GC B cells. (d) PNA staining of spleen sections of WT and Pten^fl/flFoxp3-Cremice (magnification, ×4; scale 1mm). (e) Immune fluorescence of MAN sections of WT and Pten^fl/flFoxp3-Cremice for the staining of CD3 (red) and PNA (green) (scale 60 μm). Bottom, quantification of germinal center area. (f,g) Analysis of T_FH (f) and GC B cells (g) in the spleen of mice immunized with SRBCs 7 days previously. Data are representative of at least ten independent experiments (a-c) and two independent experiments (d-g). *P < 0.05, **P < 0.01 and ***P < 0.001. Data are mean ± s.e.m.

Figure 4. Analysis of bone marrow-derived chimeras and Pten^fl/flFoxp3-Cre Ifng^−/− mice reveals an important contribution of IFN-γ overproduction to dysregulated T_FH responses in Pten^fl/flFoxp3-Cre mice

(a-c) Sublethally irradiated Rag1^−/− mice were reconstituted with a 1:1 mix of CD45.1⁺ BM and either CD45.2⁺ WT or Pten^fl/flFoxp3-Cre BM cells. Following reconstitution, the mixed chimeras were analyzed for T_FH (a), GC B cells (b), and intracellular staining of IFN-γ in CD4⁺ T cells (c). (d,e) Analysis of T_FH (d) and GC B cells (e) in the spleen of WT, Pten^fl/flFoxp3-Cre, Ifng^−/− and Pten^fl/flFoxp3-Cre Ifng^−/− mice. (f) Representative images and quantification of fluorescent intensity (right) of ANA IgG autoantibodies detected with Hep-2 slides in the serum from WT, Pten^fl/flFoxp3-Cre, Ifng^−/− and Pten^fl/flFoxp3-Cre Ifng^−/− mice (scale 60 μm). (g) Representative images of immune fluorescence imaging of kidney sections showing IgG deposits (scale 300 μm), and quantitative analysis (right). Data are representative of three independent experiments (a-e) and one experiment (f,g; n=3 WT, 6 Pten^fl/flFoxp3-Cre, 2 Ifng^−/− and 3 Pten^fl/flFoxp3-Cre Ifng^−/− mice). Data are mean ± s.e.m.

Figure 5. PTEN deficiency impairs T_reg stability

(a,b) Expression of CD44, CD69, CD62L (a) and ICOS, PD-1, GITR and CTLA4 (b) in T_reg cells from the spleen of WT and Pten^fl/flFoxp3-Cre mice. Mean fluorescent intensity (MUFI) is presented in the plots. (c) Expression of CD25 and Foxp3 (gated on CD4⁺TCRβ⁺ cells) in the spleen of WT and Pten^fl/flFoxp3-Cremice; the numbers above the graphs indicate the mean fluorescent intensity (MUFI) of Foxp3 in CD25⁻ and CD25⁺ subsets. Right, quantification of Foxp3⁺CD25⁻ cells. (d) Expression of Blimp1 in the splenic T_reg cells of WT and Pten^fl/flFoxp3-Cre mice. (e) Foxp3-YFP and GFP expression in CD4⁺ T cells from Pten^+/+Foxp3-Cre Rosa26^GFPand Pten^fl/flFoxp3-Cre Rosa26^GFPmice. (f) Intracellular staining of IFN-γ and IL-17 (right, quantification of IFN-γ⁺ and IL-17⁺ producing cells in T_reg cells), and RNA analysis of IFN-γ in T_reg cells of WT and Pten^fl/flFoxp3-Cre mice (IL-17 RNA was undetectable). (g) WT and Pten^fl/flFoxp3-Cre T_reg (CD45.2⁺) were transferred into CD45.1⁺ recipients, followed by analysis of donor cell percentages (upper) and Foxp3 and CD25 expression (lower). Right, direct overlays of donor-derived WT and Pten^fl/flFoxp3-Cre T_reg cells for Foxp3 (upper right) and CD25 levels (lower right). (h) Flow cytometry analysis of Foxp3-YFP⁺CD25⁻ cells in WT, Pten^fl/flFoxp3-Cre, Ifng^−/− and Pten^fl/flFoxp3-Cre Ifng^−/− splenic T_reg cells. Data are representative of at least three independent experiments (a-f, h) and two independent experiments (g). NS, not significant; *P < 0.05 and **P < 0.001. Data are mean ± s.e.m.

Figure 6. PTEN-dependent gene expression and metabolic programs in T_reg cells

(a) Heat maps display expression, relative to the WT mean (over or equivalent to 1.5 fold), of T_FH-related genes. (b) GSEA reveals the cell cycle mitotic pathway as one of the most extensively upregulated pathways in Pten^fl/flFoxp3-Cre T_reg cells. (c) IPA analysis of canonical pathways controlled by PTEN in T_reg cells. (d) Glycolytic activity of T_reg cells stimulated with α-CD3-CD28. (e) Analysis of ROS production, mitochondrial mass and mitochondrial membrane potential (tetramethylrhodamine, methyl ester, TMRM) in T_reg cells of WT and Pten^fl/flFoxp3-Cremice. (f) Analysis of BrdU incorporation in T_reg subsets of WT and Pten^fl/flFoxp3-Cremice. Data are representative of one experiment (a-c; n=3 WT, 3 Pten^fl/flFoxp3-Cre mice), two independent experiments (d,f) and three independent experiments (e).

Figure 7. Dysregulated mTORC2 activity in PTEN-deficient T_reg cells responsible for the immune tolerance breakdown

(a,b) Immunoblots of phosphorylation of Akt (S473), Foxo1 and S6 in resting and short-term stimulated T_reg cells (a) and in resting and long-term stimulated T_reg cells (b) isolated from WT and Pten^fl/flFoxp3-Cre mice. (c,d) Flow cytometry analysis of WT, Pten^fl/flFoxp3-Cre, Foxp3^creRictor^fl/fl and Pten^fl/flRictor^fl/flFoxp3-Cremice for Foxp3 (upper) and CD25 (lower) expression in CD4⁺ T cells (c), and proportions of FH cells (upper) and GC B cells (lower) (d). (e) Kidney sections showing IgG deposits (scale 300 μm) and quantification (right; quantitative results of WT and Pten^fl/flFoxp3-Cre mice in Fig. 4g are shown here for comparison). Data are representative of two independent experiments (a,b), three independent experiments (c,d) and one experiment (e; n=2 Foxp3^creRictor^fl/fl and 3 Pten^fl/flRictor^fl/flFoxp3-Cremice). Data are mean ± s.e.m.

Figure 8. Heterozygous loss of PTEN in T_reg cells is sufficient to disrupt immune homeostasis

(a) Flow cytometry analysis of Foxp3 and CD25 expression in WT and Pten^fl/+Foxp3-Cresplenic CD4⁺ T cells. (b) Foxp3-YFP and GFP expression in CD4⁺ T cells from Pten^+/+Foxp3-CreRosa26^GFPand Pten^fl/+Foxp3-Cre Rosa26^GFPmice. (c) Analysis of CXCR5⁺PD-1⁺ cells (gated on CD4⁺TCRβ⁺ cells) and GL7⁺CD95⁺ GC B cells (gated on CD19⁺ B cells) in the spleen of WT and Pten^fl/+Foxp3-Cre mice. (d) Representative images (scale 60 μm) and quantification of fluorescent intensity (right) of serum ANA IgG autoantibodies detected with fixed Hep-2 slides. (e) Immune fluorescence images of kidney glomeruli sections showing IgG deposits (scale 100 μm). Data are representative of at least two independent experiments (a-e). *P < 0.0001. Data are mean ± s.e.m.