Regulatory T cells require the phosphatase PTEN to restrain type 1 and follicular helper T-cell responses

Abstract

The interplay between effector and regulatory T (T_reg) cells is crucial for adaptive immunity, but how T_reg control diverse effector responses is elusive. We found that the phosphatase PTEN links T_reg stability to repression of T_H1 and T_FH (follicular helper) responses. Depletion of PTEN in T_reg resulted in excessive T_FH and germinal center responses and spontaneous inflammatory disease. These defects are considerably blocked by deletion of Interferon-γ, indicating coordinated control of T_H1 and T_FH responses. Mechanistically, PTEN maintains T_reg stability and metabolic balance between glycolysis and mitochondrial fitness. Moreover, PTEN deficiency upregulates mTORC2-Akt activity, and loss of this activity restores PTEN-deficient T_reg function. Our studies establish a PTEN-mTORC2 axis that maintains T_reg stability and coordinates T_reg-mediated control of effector responses.

Introduction

The interplay between immune regulatory mechanisms and effector T cell responses is a crucial determinant of adaptive immunity. Among multiple regulatory systems, thymus-derived regulatory T (T_reg) cells marked by the transcription factor Foxp3 play a central role in maintaining self-tolerance and preventing autoimmune disease. Recent genetic studies highlight that T_reg cells employ distinct transcriptional programs to control effector T_H1, T_H2 and T_H17 responses^{1, 2}. Specifically, T_reg cells expressing the transcription factors T-bet, IRF4 and STAT3 orchestrate the control of effector T_H1, T_H2 and T_H17 responses respectively^3-5. Owing to their potent suppressive activity and functional diversity, the stability of T_reg cells is actively maintained by Foxp3-dependent and independent mechanisms^{6, 7}. However, under certain inflammatory conditions, T_reg cells could lose Foxp3 expression and lineage stability and acquire effector functions^8-11. Studies also demonstrate the heterogeneity of T_reg cells distinguished by the expression of CD25, CCR7 and other molecules^12-14. Defining the mechanisms involved in the functional diversification and lineage stability of T_reg cells is crucial to understanding immune system regulation.

Follicular helper T (T_FH) cells are a subset of CD4⁺T cells specialized in providing help to B cells for the formation of germinal center (GC) reactions and the development of humoral immunity¹⁵. Excessive T_FH responses, however, lead to the development of autoimmune diseases including systemic lupus erythematosus (SLE)^{15, 16}. T_FH cells are characterized by the preferential expression of the chemokine receptor CXCR5, the co-stimulatory molecule ICOS, the inhibitory molecule PD-1, and the cytokine IL-21. Differentiation of T_FH cells requires the interaction with antigen-presenting cells including dendritic cells and B cells, and is further programmed by lineage-specific transcription factors including Bcl6 and Ascl2^{15, 17}. Further more, T_FH responses are restrained by a specific T_reg cell subset, follicular regulatory T cells (T_FR), in a Bcl6-dependent manner. T_FR cells share phenotypic features with both thymus-derived T_reg and T_FH cells, as evidenced by the concomitant expression of Foxp3, CTLA4, GITR, Bcl6, ICOS, PD-1 and CXCR5, but are functionally distinct from these conventional populations^{18, 19}. The molecular pathways that orchestrate the generation and function of T_FR cells and the interplay with other effector cells have remained unclear.

Emerging studies reveal a central role of mechanistic target of rapamycin (mTOR), a signaling pathway that integrates immune and metabolic cues, in T cell-mediated immune responses^{20, 21}. mTOR signaling is comprised of two distinct complexes: mTOR complex 1 (mTORC1) and 2 (mTORC2), which have unique contributions to effector T cell responses^22-24, and functional fitness of T_reg cells²⁵. Because of the potent effects of mTOR signaling on T cell responses, multiple mechanisms are evolved to actively suppress mTOR signaling²⁰. For instance, loss of the tumor suppressor Tsc1 aberrantly upregulates mTORC1 activity and disrupts T cell quiescence, homeostasis and functions²⁶. T cell-specific deletion of PTEN, an upstream inhibitor of PI3K-Akt signaling, leads to the development of leukemia and autoimmunity^{27, 28}. As a pluripotent molecule, PTEN antagonizes PI3K activity and thus inhibits both mTORC1 and mTORC2 activities²⁰; PTEN also possesses nuclear functions independent of PI3K-Akt activity²⁹. Although PTEN has been implicated in T_reg cells from mice and humans^30-32,T_reg cells deficient in PTEN show largely normal suppressive activity in vitro³³. Therefore, the functional impacts and molecular pathways of PTEN in T_reg-mediated immune homeostasis and function remain to be established.

To investigate the in vivo functions and mechanisms of PTEN in T_reg cells, we have developed a mouse model to delete PTEN selectively in T_reg cells. T_reg-specific loss of PTEN is sufficient to induce a systemic lupus-like autoimmune and lymphoproliferative disease. This is associated with excessive T_FH and GC B cell responses, as well as exuberant interferon-γ (IFN-γ) production and T_H1 reactions. Deletion of IFN-γ considerably rectifies T_FH and autoimmune responses, indicating a crucial role of PTEN in T_reg cells at coordinately controlling T_H1 and T_FH responses. Mechanistically, PTEN deficiency results in the loss of T_reg functional stability and dysregulated transcriptional and metabolic programs, including the balance between glycolytic activity and mitochondrial fitness. Further, PTEN deletion mainly upregulates mTORC2, not mTORC1 activity, in T_reg cells. Depletion of Rictor-mTORC2 activity is sufficient to restore the functional abnormalities observed in PTEN-deficient T_reg cells. Finally, we present evidence that PTEN is haploinsufficient in T_reg cells. Our studies establish a crucial role of the PTEN-mTORC2 axis in mediating T_reg cell stability and homeostasis of the immune system, and highlight that their stability is actively maintained to coordinately regulate the magnitude of T_H1 and T_FH responses.

Results

T_reg deletion of PTEN precipitates an inflammatory disease

To investigate the requirement of PTEN in the homeostasis and functions of Foxp3⁺T_reg cells, a central regulator of immune tolerance, we crossed mice with loxP-flanked Pten alleles (Pten^fl/fl) with Foxp3^YFP-Cre (Foxp3-Cre) mice³⁴ to delete the PTEN conditional alleles specifically in T_reg cells but not naive T cells (hereafter Pten^fl/flFoxp3-Cre) (Supplementary Fig. 1a). To examine whether this alters self-tolerance, we measured the amounts of anti-dsDNA and anti-nuclear antigen (ANA) autoantibodies in the serum by ELISA and binding to fixed Hep-2 slides, respectively. As compared with Foxp3-Cre mice (designated WT), Pten^fl/flFoxp3-Cre mice contained significantly increased titers of circulating anti-dsDNA and anti-ANA antibodies (Fig. 1a,b), indicative of autoimmune reactions. This was associated with considerably elevated titers of serum IgG2a, IgG2c and IgG2b isotypes in Pten^fl/flFoxp3-Cre mice. In contrast, IgG1 and IgG3 titers were reduced, while IgM and IgA titers were comparable (Supplementary Fig. 1b-d). As systemic autoimmunity is frequently associated with renal pathology, we examined the structural integrity of the kidney. The size and cellularity of the glomeruli were greatly increased in the kidney from Pten^fl/flFoxp3-Cre mice, indicative of glomerulonephritis (Fig. 1c). Moreover, glomeruli of the Pten^fl/flFoxp3-Cre kidney contained prominent IgG deposits (Fig. 1d). Therefore, loss of PTEN in T_reg cells results in dysregulation of serum autoantibodies and antibody isotypes and development of renal pathology, indicating a breakdown of immune tolerance in Pten^fl/flFoxp3-Cre mice.

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.

In addition, Pten^fl/flFoxp3-Cre mice spontaneously developed lymphoid hyperplasia, which became prominent when they reached 4-5 months. Specifically, peripheral lymph nodes in Pten^fl/flFoxp3-Cre mice, especially those around the cervical regions, were markedly enlarged. These aged mice also contained a slight increase of the spleen size (Fig. 1e). Moreover, the number and size of lymphoid follicles in Peyer's patches were increased Pten^fl/flFoxp3-Cre mice (Fig. 1f). The development of lymphoadenopathy and Peyer's patch abnormality in Pten^fl/flFoxp3-Cremice indicated an ongoing lymphoproliferative disease.

Altered immune homeostasis upon T_reg-specific loss of PTEN

The development of the autoimmune and lymphoproliferative disease prompted us to examine whether homeostasis of the immune system was altered in Pten^fl/flFoxp3-Cre mice. In our following analyses, we used mice at a young age, prior to the development of the lymphoproliferative disease. The numbers of CD4⁺, CD8⁺, B cells, dendritic cells, and neutrophils were largely comparable between WT and Pten^fl/flFoxp3-Cre mice (Supplementary Fig. 2a-c). However, Pten^fl/flFoxp3-Cre mice contained increased numbers of activated CD62L^loCD44^hi effector/memory T cells in the CD4⁺ and CD8⁺ compartments (Fig. 2a). Moreover, CD44^hi cells from these mice expressed elevated levels of IFN-γ (Fig. 2b). Similarly, expression of CXCR3, a signature chemokine receptor for T_H1 cells, was also increased (Supplementary Fig. 2d). In contrast, IL-17 and IL-4 production was largely normal (Supplementary Fig. 2e). Thus, T cells from Pten^fl/flFoxp3-Cre mice were spontaneously activated in vivo, with a propensity to differentiate into the T_H1 phenotype.

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.

Despite severe autoimmune diseases, Pten^fl/flFoxp3-Cre mice had increased percentage and numbers of Foxp3⁺T_reg cells in the spleen and lymph nodes (Fig. 2c, Supplementary Fig. 2f). To explore the underlying basis for the increased T_reg cellularity, we used caspase-3 staining and BrdU incorporation assays to measure T_reg apoptosis and proliferation, respectively. T_reg cells from WT and Pten^fl/flFoxp3-Cre mice had comparable caspase-3 staining (Fig. 2d), but Pten^fl/flFoxp3-Cre T_reg cells had more BrdU incorporation than WT cells (Fig. 2e), indicative of an elevated rate of proliferation. Therefore, PTEN deficiency causes an increased T_reg cellularity and proliferation.

Uncontrolled T_FH and GC B cells in Pten^fl/flFoxp3-Cre mice

SLE, the prototypical systemic autoimmune disease, is characterized by the heterogeneity of underlying T cell responses. Aside from the roles of the conventional effector (e.g. T_H1 and T_H17) and regulatory responses, recent studies demonstrate a crucial role of T_FH cells in the overproduction of pathogenic autoantibodies and tissue damage in SLE^{15, 16}. The development of an SLE-like symptom in Pten^fl/flFoxp3-Cre mice prompted us to examine whether the T_FH response was altered in these mice. Under steady state, only a small percentage of splenic CD4⁺T cells was stained positive for the T_FH signature molecules CXCR5 and PD-1 in WT mice, but the CD4⁺CXCR5⁺PD-1⁺ population was greatly expanded in the spleen and mesenteric lymph nodes (MLNs) of Pten^fl/flFoxp3-Cre mice (Fig. 3a, Supplementary Fig. 3a). Similar changes were noticed for CXCR5⁺ cells expressing the T_FH-specific co-stimulatory molecule ICOS and transcription factor Bcl6 (Supplementary Fig. 3b,c). The CD4⁺CXCR5⁺PD-1⁺ cells can be further divided into immunostimulatory T_FH and immunoregulatory T_FR cells, as indicated by the absence or presence of Foxp3 expression, respectively^{18, 19}. Both subsets were increased in Pten^fl/flFoxp3-Cre mice (Fig. 3b). Consistent with the increase of CXCR5⁺ PD-1⁺ cells, the spleen and MLNs of Pten^fl/flFoxp3-Cre mice contained 3 fold more GC B cells, denoted by the expression of GC signature markers GL7 and CD95 (Fig. 3c, Supplementary Fig. 3d), even though total B cell numbers were largely unaltered (Supplementary Fig. 2b). Moreover, immunohistochemistry showed that the spleen and MLNs of Pten^fl/flFoxp3-Cre mice contained considerably more and larger Peanut Agglutinin (PNA)-positive GCs than did their WT counterparts (Fig. 3d,e). These results revealed spontaneous T_FH and GC formation in Pten^fl/flFoxp3-Cremice.

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.

We next determined whether PTEN deficiency in T_reg cells affects T_FH and GC responses after T_FH-inducing immunization. After immunization with sheep red blood cells (SRBCs), a strong protein antigen, the formation of T_FH cells and GC B cells was greatly enhanced in Pten^fl/flFoxp3-Cre mice compared with WT mice (Fig. 3f,g). We observed a similar finding after challenging WT and Pten^fl/flFoxp3-Cre mice with a T cell-dependent antigen, NP-OVA precipitated in alum together with LPS (Supplementary Fig. 3e,f). We conclude that deletion of PTEN in T_reg cells results in enhanced T_FH and GC reactions both under steady state and upon immunization.

Coordination of T_H1 and T_FH responses by T_reg signaling

We next investigated whether the increased T_FH response in Pten^fl/flFoxp3-Cre mice was a cell-autonomous defect. To this end, we generated mixed bone marrow (BM) chimeras by reconstituting alymphoid Rag1^−/− mice with a 1:1 mixture of Pten^fl/flFoxp3-Cre CD45.2⁺ (donor) and CD45.1⁺ (spike) BM cells (Pten^fl/flFoxp3-Cre:CD45.1⁺), and as a control, a mixture of WT and CD45.1 cells (WT:CD45.1⁺). The frequency of T_FH cells was considerably increased in both the donor and spike-derived populations in the Pten^fl/flFoxp3-Cre:CD45.1⁺ chimeras, as compared with the frequency of T_FH cells in the WT:CD45.1⁺ chimeras (Fig. 4a). Additionally, Pten^fl/flFoxp3-Cre:CD45.1⁺ chimeras had augmented GC B cells (Fig. 4b). Thus, PTEN deficiency in T_reg cells results in a dominantly acting effect on the T_FH and GC responses.

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.

We therefore explored whether such defect was associated with dysregulated cytokine production. The T_H1 cytokines such as IFN-γ have been implicated in potentiating T_FH responses³⁵, although opposing evidence also exists^{36, 37}, suggesting a context-dependent effect. In the Pten^fl/flFoxp3-Cre:CD45.1⁺ mixed chimeras, IFN-γ production from CD4⁺ and CD8⁺T cells was enhanced irrespective of the source of donor cells (Fig. 4c, Supplementary Fig. 4a). To determine the functional effects of the augmented IFN-γ production, we crossed Pten^fl/flFoxp3-Cre mice with Ifng^−/− mice to generate Pten^fl/flFoxp3-Cre Ifng^−/− double knockout mice. Deletion of IFN-γ did not exert strong effects on the production of IL-17 and IL-4 from conventional CD4⁺T cells (Supplementary Fig. 4b). However, IFN-γ deficiency substantially blocked the elevated frequencies of T_FH and GC B cells (Fig. 4d,e) and the increased formation of GCs in Pten^fl/flFoxp3-Cre mice (Supplementary Fig. 4c,d). Moreover, the increased production of serum ANA antibody and the deposition of IgG in the kidney glomeruli of Pten^fl/flFoxp3-Cre mice were essentially rectified in Pten^fl/flFoxp3-Cre Ifng^−/− mice (Fig. 4f,g). Thus, the increased production of IFN-γ in Pten^fl/flFoxp3-Cre mice largely accounts for the exacerbated T_FH, GC, and autoimmune responses, thereby highlighting the crucial role of PTEN signaling in T_reg cells to coordinate T_H1 and T_FH reactions.

PTEN is crucial in maintaining the stability of T_reg cells

Despite the crucial role of IFN-γ overproduction in disrupting immune homeostasis in Pten^fl/flFoxp3-Cre mice, deletion of IFN-γ did not affect the phenotype of increased T_reg cellularity in these mice (Supplementary Fig. 5a). We therefore explored the direct effects of PTEN deficiency on the homeostasis and functionality of T_reg cells. As compared with WT counterparts, T_reg cells from Pten^fl/flFoxp3-Cre mice showed higher expression of CD44 and CD69 but lower levels of CD62L, indicating an elevated level of activation (Fig. 5a). We next examined T_reg-selective effector molecules. Pten^fl/flFoxp3-CreT_reg cells showed increased levels of ICOS and PD-1, and to a lesser extent, GITR, whereas the expression of CTLA4 was largely normal (Fig. 5b). In sharp contrast to the elevated expression of activation markers and effector molecules, expression of CD25, a signature molecule of T_reg and activated T cells, was markedly downregulated in Pten^fl/flFoxp3-Cre T_reg cells, corresponding to the expansion of the Foxp3⁺CD25⁻ population (Fig. 5c). As compared with Foxp3⁺CD25⁺ cells, the Foxp3⁺CD25⁻ population contained lower levels of Foxp3 expression, as reported previously¹². Consistent with this observation, Blimp1, a transcription factor implicated in CD25 downregulation in CD8⁺T cells³⁸, was upregulated in Pten^fl/flFoxp3-Cre T_reg cells (Fig. 5d). These results indicate that PTEN deficiency in T_reg cells results in dysregulated expression of multiple T_reg activation and phenotypic molecules.

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.

The lineage stability of T_reg cells is a matter of considerable interest and debate^{6, 8-11, 13}, but the signaling mechanisms involved are largely unexplored. To determine whether PTEN deficiency affects the stability of T_reg cells, we crossed Pten^fl/flFoxp3-Cre mice with a Cre recombination reporter allele with the ubiquitously expressed Rosa26 locus containing a loxP site–flanked STOP cassette followed by the gene encoding green fluorescent protein (GFP)²⁵. In this lineage tracing system, Foxp3^Cre-mediated excision of the floxed STOP cassette results in constitutive, heritable expression of GFP, even for those “ex-T_reg” cells that have lost Foxp3 expression (the GFP⁺YFP-Foxp3⁻ population). Pten^fl/flFoxp3-Cre mice contained a striking accumulation of GFP⁺YFP-Foxp3⁻ population (Fig. 5e), indicating the preferential loss of Foxp3 expression upon PTEN deletion. Additionally, T_reg cells from Pten^fl/flFoxp3-Cre mice had increased expression of IFN-γ, whereas IL-17 expression was largely unaltered (Fig. 5f). Moreover, Pten^fl/flFoxp3-Cre T_reg cells upregulated signature molecules characteristic of T_H1 and T_FH cells, including CXCR3 and T-bet, and CXCR5 and Bcl6, respectively, whereas expression of IRF4 and p-STAT3, which are required for T_reg-mediated suppression of T_H2 and T_H17 cells^{4, 5}, remained unchanged (Supplementary Fig. 5b). Consistent with these observations, Pten^fl/flFoxp3-Cre T_reg cells contained an expanded T-bet⁺CXCR3⁺ population that is important for the regulation of type 1 inflammation (Supplementary Fig. 5c)³. Because the development of this T_reg subset is dependent upon IFN-γ signaling³, we examined whether excessive IFN-γ production in Pten^fl/flFoxp3-Cre mice was involved in the dysregulation of T-bet and CXCR3. IFN-γ deficiency had only a partial rescue effect on the expansion of T-bet⁺CXCR3⁺ population in PTEN-deficient T_reg cells (Supplementary Fig. 5c), indicating that the augmented expression of T-bet and CXCR3 upon PTEN deletion is largely cell intrinsic, not simply secondary to the overproduction of IFN-γ. Finally, to directly test the role of PTEN in maintaining T_reg stability, we sorted Foxp3⁺CD25⁺ cells from WT and Pten^fl/flFoxp3-Cre mice and transferred them into congenic mice (CD45.1⁺). The expression of Foxp3 and CD25 was downregulated in PTEN-deficient T_reg cells in various organs examined, as compared with WT cells (Fig. 5g, Supplementary Fig. 5d). These complementary approaches indicate that PTEN deficiency results in a loss of T_reg stability.

Notably, despite the strong effects of IFN-γ at disrupting immune homeostasis (Fig. 4), deletion of IFN-γ in Pten^fl/flFoxp3-Cre mice did not affect the spontaneous development of the Foxp3⁺CD25⁻ population (Fig. 5h). Thus, the enhanced IFN-γ expression from Pten^fl/flFoxp3-CreT_reg cells is associated with the loss of T_reg stability, but is unlikely to be the main driver.

PTEN-dependent transcriptional and metabolic programs

To explore PTEN-dependent molecular mechanisms in T_reg cells, we next used microarrays to compare the transcriptional profiles between WT and Pten^fl/flFoxp3-Cre T_reg cells. Pten^fl/flFoxp3-Cre T_reg cells contained a total of 498 probes (representative of 352 unique genes) with greater than 1.5 fold difference, including 212 upregulated and 286 downregulated probes. Principal component analysis (PCA) showed the clear distinctions between WT and Pten^fl/flFoxp3-Cre samples (Supplementary Fig. 6a). Consistent with the dysregulated stability, loss of PTEN failed to restrain the transcription of genes involved in T_FH differentiation, including Gzmb, Il21, Bcl6, Pdcd1, Il4, Maf and Cxcr5^{15, 39} (Fig. 6a). To identify key networks regulated by PTEN, we did gene-set enrichment analysis (GSEA)⁴⁰ to compare gene expression profiles of WT and PTEN-deficient T_reg cells. Remarkably, the top ten of upregulated gene-sets in Pten^fl/flFoxp3-Cre T_reg cells were all associated with cell cycle pathways (Fig. 6b, Supplementary Fig. 6b). To examine PTEN-dependent canonical pathways in T_reg cells, we next performed the ingenuity pathway analysis (IPA) system by interrogating the differentially expressed genes at the 1.5 fold cut-offs. As shown in Fig. 6c, PTEN deficiency affected multiple canonical pathways implicated in autoimmune diseases, helper T cell differentiation, and immune signaling mediated by transcription factors, co-stimulatory molecules and cytokines. Gene ontology (GO) analysis of these differentially regulated genes also showed that PTEN-deficient T_reg cells significantly upregulated groups of genes involved in autoimmune diseases, such as autoimmune thyroid disease and SLE, as well as in cell cycle regulation (data not shown). The transcriptome analysis therefore highlights an important role of PTEN in the regulation of immune response and cell cycle pathways.

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).

Cellular metabolic programs, especially glycolysis and mitochondrial oxidative phosphorylation, play an important role in shaping T_reg generation and function^{41, 42}, although the molecular mechanisms are unknown. As compared with WT T_reg cells, Pten^fl/flFoxp3-Cre T_reg cells more strongly upregulated glycolysis after TCR/CD28 stimulation (Fig. 6d). We next examined whether PTEN deficiency affects mitochondrial fitness by measuring multiple parameters associated with mitochondria homeostasis and functions. Pten^fl/flFoxp3-Cre T_reg cells showed considerable reduction of reactive oxidative species (ROS), mitochondrial mass, and mitochondrial membrane potential, indicative of impaired mitochondrial fitness (Fig. 6e). Notably, the reduction of these parameters was more prominent in PTEN-deficient Foxp3⁺CD25⁻ T_reg cells than Foxp3⁺CD25⁺ cells (Fig. 6e), indicating that impaired mitochondrial function is associated with T_reg instability. Moreover, the Foxp3⁺CD25⁻ T_reg subset showed a modestly stronger phenotype of BrdU incorporation than Foxp3⁺CD25⁺ cells (Fig. 6f). Altogether, these data establish that PTEN signaling links immune response gene expression, cell cycle progression, and metabolic balance between glycolytic activity and mitochondrial fitness in T_reg cells.

PTEN signals via mTORC2 inhibition in T_reg cells

We determined PTEN-dependent biochemical mechanisms in T_reg cells by examining mTORC1 and mTORC2 activities, as both are under the control of PTEN in multiple cell types²⁰. In response to short-term anti-CD3-CD28 stimulation (5-15 min), Pten^fl/flFoxp3-Cre T_reg cells exhibited slightly elevated phosphorylation of the mTORC1 target S6, as compared with WT T_reg cells (Fig. 7a, Supplementary Fig. 7a). In contrast, Pten^fl/flFoxp3-Cre T_reg cells exhibited much stronger activation of mTORC2 signaling than WT cells, as indicated by the enhanced phosphorylation of Akt at Ser473 and of the Akt downstream target Foxo1 (Fig. 7a). We next determined the effects of stimulating T_reg cells with anti-CD3-CD28 for a longer time (20 h), in the presence or absence of IL-2. Both WT and Pten^fl/flFoxp3-Cre T_reg cells responded to these stimuli with robust phosphorylation of S6 and 4E-BP1, indicative of comparable activation of the mTORC1 pathway under these conditions (Fig. 7b, Supplementary Fig. 7b). In contrast, Pten^fl/flFoxp3-Cre T_reg cells exhibited much stronger activation of mTORC2 signaling than WT cells (Fig. 7b). Therefore, loss of PTEN results in preferential activation of mTORC2 signaling in T_reg cells.

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.

To determine the contribution of mTORC2 activity to Pten^fl/flFoxp3-Cre phenotypes, we crossed Pten^fl/flFoxp3-Cre mice with those lacking Rictor that abrogated mTORC2 activity (Pten^fl/flFoxp3-Cre)²⁵ to generate Pten^fl/flRictor^fl/flFoxp3-Cre mice. Compared with Pten^fl/flFoxp3-Cre T_reg cells with increased cellularity but diminished CD25 expression, T_reg cells in Pten^fl/flRictor^fl/flFoxp3-Cre mice exhibited normal abundance and CD25 expression (Fig. 7c). Other T_reg markers analyzed were also considerably rescued by the deletion of Rictor from Pten^fl/flFoxp3-Cre T_reg cells (Supplementary Fig. 7c). Additionally, T cells in Pten^fl/flRictor^fl/flFoxp3-Cre mice exhibited largely normal distribution of naïve and effector/memory phenotypes (Supplementary Fig. 7d). Also, the spontaneous development of T_FH and T_H1 cells and GC B cells observed in Pten^fl/flFoxp3-Cre mice was blocked by Rictor deletion (Fig. 7d, Supplementary Fig. 7e). Moreover, unlike Pten^fl/flFoxp3-Cre mice, Pten^fl/flRictor^fl/flFoxp3-Cre mice did not contain IgG deposits in the kidney glomeruli (Fig. 7e), and exhibited normal GC numbers in the spleen and MLNs (Supplementary Fig. 7f,g). Therefore, despite the plethora of pathways identified to mediate PTEN functions in various cells²⁰, PTEN acts in an mTORC2-dependent manner in T_reg cells to impinge upon immune homeostasis and tolerance.

PTEN is haploinsufficient in T_reg cells

Given the potent effects of PTEN signaling in T_reg cells, we asked whether partial loss of PTEN function in T_reg cells is physiologically relevant, by analyzing Pten^fl/+Foxp3-Cre mice. As expected, Pten^fl/+Foxp3-Cre T_reg cells, but not other T cell subsets, had partial reduction of PTEN expression at both the mRNA and protein levels (Supplementary Fig. 8a,b). Remarkably, heterozygous loss of PTEN resulted in the expansion of T_reg cells, associated with considerable downregulation of CD25 expression (Fig. 8a). Pten^fl/+Foxp3-Cre mice also contained a striking accumulation of GFP⁺YFP-Foxp3⁻ population in the lineage tracing system (Fig. 8b). Moreover, these mice contained increased CXCR5⁺PD-1⁺ T_FH cells and GL7⁺CD95⁺ GC B cells (Fig. 8c), and more abundance of GCs in the spleen (Supplementary Fig. 8c). These findings prompted us to examine whether heterozygous loss of PTEN in T_reg cells caused autoimmunity and lymphoid hyperplasia as observed in Pten^fl/flFoxp3-Cre mice. Pten^fl/+Foxp3-Cre mice displayed significantly elevated titers of circulating anti-ANA antibodies (Fig. 8d) and augmented IgG deposits in the kidney glomeruli (Fig. 8e), indicative of systemic autoimmunity. Furthermore, Pten^fl/+Foxp3-Cre mice spontaneously developed lymphoadenopathy (Supplementary Fig. 8d). These results demonstrate that PTEN is haploin sufficient in T_reg cells.

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.

Discussion

Extensive progress has been made on the transcriptional and molecular pathways underlying effector T cell diversity and plasticity. In contrast, how these distinct effector T cell responses are controlled by extrinsic mechanisms is poorly understood. Moreover, despite the emerging evidence for the adoption of T_H-specific transcription factors by T_reg cells to suppress the corresponding effector responses^3-5, we have little understanding about the signaling mechanisms that program these suppressive activities. Here we, together with Huynh et al. (companion manuscript), identify PTEN as a crucial molecular pathway in T_reg cells that coordinately regulates T_FH and T_H1 responses (Supplementary Fig. 8e). In particular, loss of PTEN in T_reg cells results in exacerbated T_FH and GC responses and disrupted immune tolerance and homeostasis. Ablation of IFN-γ function reveals the hierarchy between T_reg-mediated control of T_H1 and T_FH responses, with the production of the T_H1 signature cytokine IFN-γ a prerequisite for the potentiation of T_FH responses in Pten^fl/flFoxp3-Cre mice. Further more, the repression of T_H1 and T_FH responses is associated with the active maintenance of T_reg cell stability enforced by PTEN signaling. At the molecular levels, PTEN controls the transcriptional program and metabolic balance in T_reg cells, and it mainly signals via inhibition of mTORC2 activity. Our studies therefore establish that the PTEN-mTORC2 axis acts as a central pathway to orchestrate T_reg cell stability and restrict T_H1 and T_FH responses.

The lineage stability of T_reg cells remains contentious^{6, 8-13} but is an issue of utmost importance, as it directly impinges upon T_reg-mediated control of health and disease and therapeutic strategies². Using lineage tracing and adoptive transfer systems, we show that PTEN-deficient T_reg cells are more prone to lose Foxp3 expression in vivo. Moreover, PTEN deficiency results in apparently hyper-activated T_reg cells, as evidenced by increased cycling, upregulation of activation and phenotypic markers, and aberrant induction of T_H1 and T_FH signature molecules. Consistent with this, PTEN deletion results in loss of CD25 expression, which is normally downregulated in T_reg cells after in vivo activation and proliferation⁴³. While representing only a minor proportion of Foxp3⁺ T_reg cells under steady state, the Foxp3⁺CD25⁻ population likely makes an important contribution, via conversion or selection, to the generation of ex-T_reg cells under inflammatory or lymphopenic environment. Therefore, our studies highlight that the stability of T_reg cells under steady state requires active enforcement by PTEN, which functions, at least in part, by preventing overt T_reg activation and CD25 downregulation.

Generation of T_FH cells requires unique transcriptional programs and is antagonized by T_FR cells^{18, 19}. However, T_FR cells can also promote antigen-specific high-affinity B cell responses¹⁹ and influenza-specific GC reactions⁴⁴. Extensive crosstalk also exists between the differentiation of T_FH cells and other effector lineages¹⁵. Here we show that PTEN signaling in T_reg cells is crucial in the repression of T_FH responses, which is further linked to T_reg stability and T_reg-mediated control of T_H1 responses. Strikingly, loss of PTEN in T_reg cells results in spontaneous T_FH differentiation and GC formation, and the development of SLE-like autoimmune symptoms. Associated with these immune defects is the dysregulated expression of multiple molecules involved in T_FH and T_FR responses, including IL-4, IL-21, Bcl6, Blimp1 and Granzyme B, which likely underlies the loss of proper T_FR functions in PTEN-deficient T_reg cells. Moreover, the uncontrolled T_FH responses in Pten^fl/flFoxp3-Cre mice are dependent upon the T_H1 signature cytokine IFN-γ. Notably, the relationship between T_H1 and T_FH cells is complex and remains controversial. For instance, whereas IFN-γ is essential in driving T_FH cells in the Roquin^san/san autoimmunity model³⁵, it is dispensable for the differentiation of T_FH cells induced by viral infection³⁷. Further, T-bet induction dampens rather than enhances the T_FH differentiation program³⁶. Our studies demonstrate that T_FH and T_H1 responses are coordinately regulated by T_reg cells, with the IFN-γ production required for T_FH and GC reactions. However, we cannot conclude the direct contribution of the exacerbated T_H1 response, independent of T_FH cells, to the autoimmune and lymphoproliferative phenotypes.

As one of the most frequently mutated tumor suppressor genes, PTEN acts in murine T cells to prevent development of leukemia and autoimmunity²⁷. More recent studies unveil that these two effects can be dissociated as they are derived from abnormalities in the thymus and periphery, respectively²⁸. PTEN is also implicated in the regulation of effector T cell responses. Deletion of PTEN in activated T cells enhances effector responses but does not lead to autoimmunity or cancer⁴⁵. miRNAs targeting PTEN expression are also implicated in shaping T_FH responses⁴⁶. We found that deletion of PTEN in T_reg cells leads to the exacerbated effector T cell responses and loss of immune tolerance and tissue homeostasis. PTEN-deficient T_reg cells dominate over WT cells in mediating T_FH and GC responses, which are mediated, at least in part, by dysregulated IFN-γ expression. Moreover, PTEN functions in a haploin sufficient manner in T_reg cells. These results together establish PTEN signaling in T_reg cells as a unique regulator of immune homeostasis and function.

How does PTEN function in T_reg cells at the molecular levels? Our microarray, metabolic and immunological assays reveal the important role of PTEN in linking cell cycle and metabolic machineries and expression of immune effector genes in T_reg cells. Given the opposing effects of glycolytic and mitochondrial metabolism on T_reg cells^{41, 42}, the disrupted balance between these activities in PTEN-deficient T_reg cells is likely an important contributor to the phenotypic alteration, a notion that requires additional investigation. Of note, despite the metabolic functions of PTEN identified in non-immune cells, the underlying mechanisms are highly complex and poorly understood⁴⁷. Here we establish the PTEN-mTORC2 axis as a central determinant of T_reg stability and T_reg-mediated control of effector responses, although the involvement of mTORC2-independent pathways cannot be excluded⁴⁸. Mechanistically, this signaling pathway likely orchestrates both the metabolic and transcriptional programs, such as those mediated by Foxo1 and Blimp1^{7, 38}, in impinging upon T_reg stability and functions. We note that loss of T_reg stability also results from T_reg-specific deletion of neuropilin-1 and Foxo1^{7, 32}, and future work is required to explore the crosstalk between these molecular pathways in T_reg cells.

In summary, our study has unveiled the interplay between the PTEN-mTORC2 signaling axis and the transcriptional and metabolic regulation in T_reg cells as a new mechanism for enforcing T_reg stability and T_reg-mediated repression of T_H1 and T_FH responses. Given the potent effects of selective PTEN deficiency in T_reg cells on immune tolerance and tissue homeostasis, even under steady state conditions or upon a partial loss of function, the identification of this signaling axis provides a new target for therapeutic intervention of systemic autoimmune and lymphoproliferative diseases.

Methods

Mice

Pten^fl/fl, CD45.1⁺, Rag1^−/−, Ifng^−/− and Rosa26^GFP (a loxP-site-flanked STOP cassette followed by the GFP-encoding sequence was inserted into the Rosa26 locus) mice were purchased from the Jackson Laboratory. Rictor^−/− mice have been described previously²⁵. Foxp3^YFP-Cre mice were a gift from A. Rudensky³⁴. Pten^fl/flFoxp3-Cre mice were used at 10-12 weeks old unless otherwise noted, with the age and gender-matched WT mice containing the Foxp3^cre allele as controls. BM chimeras were generated by transferring 7 × 10⁶ T-cell-depleted BM cells into sub-lethally irradiated (5 Gy) Rag1^−/− mice, followed by reconstitution for at least 2.5 months. All mice were kept in a specific pathogen-free facility in the Animal Resource Center at St. Jude Children's Research Hospital. Animal protocols were approved by the Institutional Animal Care and Use Committee of St. Jude Children's Research Hospital.

Flow cytometry

For analysis of surface markers, cells were stained in PBS containing 2% (wt/vol) BSA, with anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-TCRβ (H57-597), anti-CD69 (H1.2F3), anti-CD25 (PC61.5), anti-CD44 (1M7), anti-CD62L (MEL-14), anti-CD45.1 (A20), anti-CD45.2 (104), anti-PD-1 (J43), anti-GL7 (GL-7), anti-CD95 (15A7), anti-ICOS (C398.4A), anti-GITR (DTA-1), anti-CD19 (1D3), anti-CXCR3 (CXCR3-173), anti-MHCII (M5/114.15.2), anti-CD11b (M1/70), anti-CD11c (N418), anti-Ly6G (RB6-8C5; all from eBioscience). CXCR5 was stained with biotinylated anti-CXCR5 (clone 2G8) and streptavid in-conjugated PE (both from BD Biosciences). Intracellular Foxp3 (FJK-16s), T-bet (4B10), IRF4 (3E4), IFN-γ (XMG1.2), IL-4 (11B11), IL-17 (17B7; all from eBioscience), CTLA4 (UC10-4B9; BioLegend), p-STAT3 (4/P-STAT3), PTEN (A2B1), Bcl6 (K112-91; all from BD Biosciences) were analyzed by flow cytometry according to the manufacturer's instructions. Blimp1 (3H2-E8) was purchased from Thermo Scientific. For intracellular cytokine staining, T cells were stimulated for 4 h with PMA plus ionomycin in the presence of monensin before being stained according to the manufacturer's instructions (eBioscience). BrdU and caspase-3 staining was performed as per the manufacturer's instruction (BD Biosciences). For staining mitochondria, lymphocytes were incubated for 30 min at 37°C with 10 nM Mito Tracker Deep Red (Life Technologies) or 20 nM TMRM (tetramethylrhodamine, methyl ester; ImmunoChemistry Technologies) after staining surface markers. ROS were measured by incubation with 5 μMMito SOX™ Red (Life Technologies) after staining surface markers. Flow cytometry data were acquired on LSRII or LSR Fortessa (BD Biosciences) and analyzed using Flowjo software (Tree Star).

Imaging and immunohistochemistry

For cryosections, kidneys and MLNs were freshly frozen in OCT embedding medium. Ten μm thick cryosections were fixed with cold acetone for 5 min prior to rehydration in TBS. Non-specific binding was blocked by incubation in TBS containing 2% BSA and 5% normal donkey serum for 30 min prior to incubation with primary antibodies (2 μg/ml) overnight at 4°C. Slides were washed for 15 min in TBS prior to incubation with AF568-conjugated goat anti-mouse antibody (1 μg/ml; Life Technologies), AF568-conjugated donkey anti-goat antibody (1 μg/ml; Life Technologies), Cy5-conjugated donkey anti-goat antibody (1 μg/ml; Jackson ImmunoResearch), or AF488-conjugated streptavid in (1 μg/ml; Life Technologies) for 1 h at room temperature. Slides were washed for 15 min in TBS prior to mounting in Vectashield hard set with DAPI (Vector Laboratories). Fluorescent images were acquired using a Zeiss Axiovert 200M and 20X EC Plan-NeoFluar objective, detected using a Cascade II EMCCD camera (Photometrics) and analyzed using Slidebook software (3i Intelligent Imaging Innovations). Large image composites were acquired with a Nikon Ti-E inverted microscope with 20X CFI Plan Apochromat Lambda objective and iXon DU897 EMCCD camera, using NIS-Elements software. Visualization of ANA antibodies was performed by staining fixed Hep-2 slides (MBL). Specifically, serum samples were applied to the slide and incubated for 2 h at room temperature followed by 15 min washing in TBS. Bound murine antibodies were detected using AF568-conjugated goat anti-mouse antibody (1 μg/ml; Life Technologies) for 1 h at room temperature, while AF488-conjugated phalloidin (Life Technologies) was utilized to visualize f-Actin and nuclei were detected using DAPI. CD3 (sc-1127 (M-20)) antibody was purchased from Santa Cruz, Biotinylated Peanut Agglutinin (PNA) from Vector Laboratories (B-1075), and IgD (558597) from BD Biosciences.

For paraffin sections, spleen, kidney and Peyer's patches were fixed by immersion in 10% (vol/vol) neutral buffered formalin solution. Fixed tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin, and the clinical signs of autoimmune diseases were analyzed by an experienced pathologist (P. Vogel).

Immunization

For experiments involving antigen-induced T_FH and GC B cell response, antigen for immunization was prepared by mixing NP₁₄-OVA (14 molecules of NP linked to OVA; Biosearch Technologies), 10% KAl(SO₄)₂ dissolved in PBS at a ratio of 1:1, in the presence of LPS (Escherichia coli strain 055:B5; Sigma) and at pH 7. NP-OVA (100 μg) and LPS (10 μg) precipitated in alum was injected intraperitoneally, as described⁴⁶. Alternatively, a fresh preparation of PBS-washed (1 × 10⁹) SRBCs from Colorado Serum Company (31112) was injected intravenously to induce a robust splenic GC response.

Serum antibodies

Autoantibodies (dsDNA) and immunoglobulin subclasses were measured with kits from Alpha Diagnostic International (5110) and Millipore (MGAMMAG-300K), respectively.

Cell purification and adoptive transfer

Lymphocytes were isolated from lymphoid organs (spleen and peripheral lymph nodes that included inguinal, auxiliary and cervical lymph nodes) and naïve and T_reg cells were sorted on a MoFlow (Beckman-Coulter) or Reflection (i-Cyt). For adoptive transfer, CD4⁺CD25⁺Foxp3-YFP⁺ cells from WT and Pten^fl/flFoxp3-Cre mice (CD45.2⁺) were transferred to the congenically marked (CD45.1⁺) recipients. Seven days after the transfer, mice were euthanized for the analysis of Foxp3 and CD25 expression.

RNA and immunoblot analysis

Real-time PCR analysis was performed with primers and probe sets from Applied Biosystems, as described⁴⁹. Immunoblots were performed as described previously⁵⁰, using the following antibodies: p-S6 (2F9), p-4E-BP1 (236B4), p-Foxo1 (9461), Akt phosphorylated at Ser473 (D9E), PTEN (138D6; all from Cell Signaling Technology), and β-actin (AC-15; Sigma).

Glycolysis assay

T_reg cells were stimulated with plate-bound anti-CD3-CD28 for 6 h, and glycolytic flux was measured by detritiation of [3-³H]-glucose, as previously described⁴².

Gene expression profiling and gene-set enrichment analysis

RNA samples from freshly isolated T_reg cells from WT and Pten^fl/flFoxp3-Cremice were analyzed with the Affymetrix HT MG-430 PM Gene Titan peg array, and expression signals were summarized with the robust multi-array average algorithm (Affymetrix Expression Console v1.1). Lists of differentially expressed genes by 1.5 fold or more were analyzed for functional enrichment using the Ingenuity Pathways (www.ingenuity.com). Gene-set enrichment analysis within canonical pathways was performed as described⁴⁰. The microarray data has been deposited into the GEO series database (GSE63625).

Statistical analysis

P values were calculated with Student's t-test (GraphPad Prism). P values of less than 0.05 were considered significant. All error bars represent the s.e.m.