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.
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Acknowledgements
The authors thank J. Wei, D. Bastardo Blanco and S. Brown for help with immunological assays; C. Cloer and B. Rhode for animal colony management; A. Rudensky for Foxp3YFP-Cre mice and the St. Jude Immunology FACS core facility for cell sorting. Supported by US National Institutes of Health (AI105887, AI101407, CA176624 and NS064599 to H.C.), the American Cancer Society (to H.C.) and the Crohn's and Colitis Foundation of America (to H.C.), and the Arthritis Foundation (to K.Y.).
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S.S. and K.Y. designed and performed cellular, molecular and biochemical experiments and contributed to writing the manuscript; C.G. did imaging assays; P.V. did histopathology analysis; G.N. did bioinformatic analyses; and H.C. designed experiments, wrote the manuscript and provided overall direction.
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Supplementary Figure 1 Deletion of PTEN and dysregulation of immunoglobulins in Ptenfl/flFoxp3-Cre mice.
(a) Analysis of Pten mRNA expression in Treg and naive CD4+ T cells (left) and PTEN protein expression in CD4+YFP+, CD4+YFP− and CD8+ T cells from the spleen of WT and Ptenfl/flFoxp3-Cre mice (right). (b-d) Quantification of IgG subclasses (b), IgM (c) and IgA (d) in the serum of WT and Ptenfl/flFoxp3-Cre mice (the IgG2a binding antibody shows cross-reactivity with IgG2c). Data are representative of at least two independent experiments (a) and one experiment (b-d; n = 22 WT and 24 Ptenfl/flFoxp3-Cre mice). NS, not significant; *P < 0.05 and **P < 0.01. Data are mean ± s.e.m.
Supplementary Figure 2 Analysis of immune cells in Ptenfl/flFoxp3-Cre mice.
(a-c) Analysis and quantification of CD4+, CD8+ (a), B cells (b), conventional dendritic cells (MCHII+CD11c+) and neutrophils (Ly6G+CD11b+) (c) in the spleen of WT and Ptenfl/flFoxp3-Cre mice. (d) Expression of CXCR3 on CD4+ T cells from the spleen of WT and Ptenfl/flFoxp3-Cre mice; numbers above graphs indicate MFI. (e) Expression of IL-4 and IL-17 in CD4+ and CD8+ T cells from WT and Ptenfl/flFoxp3-Cre mice after in vitro stimulation for 4 h. (f) Flow cytometry analysis of Treg cells in CD4+ T cells from peripheral lymph nodes (PLNs) and mesenteric lymph nodes (MLNs) of WT and Ptenfl/flFoxp3-Cre mice. Data are representative of three or more independent experiments (a,b,d-f) and two independent experiments (c). NS, not significant. Data are mean ± s.e.m. Numbers indicate percentage of cells in quadrants or gates.
Supplementary Figure 3 Analysis of TFH and GC B cells.
(a) Flow cytometry of CXCR5 and PD-1 expression (gated on CD4+TCRβ+ cells) in the MLNs of WT and Ptenfl/flFoxp3-Cre mice. (b,c) Flow cytometry of CXCR5 and ICOS (b), and CXCR5 and Bcl6 (c) expression (gated on CD4+TCRβ+ cells) in the spleen and MLNs of WT and Ptenfl/flFoxp3-Cre mice. (d) Flow cytometry of GL7 and CD95 expression (gated on CD19+ cells) in the MLNs of WT and Ptenfl/flFoxp3-Cre mice. (e,f) Analysis of TFH (e) and GC B cells (f) in the spleen of mice immunized with NP-OVA 7 d previously. Data are representative of three independent experiments (a-d) and one experiment (e,f; n = 4 WT, 4 Ptenfl/flFoxp3-Cre mice). *P < 0.05. Data are mean ± s.e.m. Numbers indicate percentage of cells in gates.
Supplementary Figure 4 Regulation of Treg cells in mixed chimeras and Ptenfl/flFoxp3-Cre Ifng−/− mice.
(a) Sublethally irradiated Rag1−/− mice were reconstituted with a 1:1 mix of CD45.1+ BM and either CD45.2+ WT or Ptenfl/flFoxp3-Cre BM cells. Following reconstitution, the mixed chimeras were analyzed for intracellular staining of IFN-γ in CD8+ T cells. (b) Cytokine production of IFN-γ, IL-17 and IL-4 by CD4+ T cells from WT, Ptenfl/flFoxp3-Cre, Ifng−/− and Ptenfl/flFoxp3-Cre Ifng−/− mice after in vitro stimulation for 4 h. (c) PNA staining of spleen sections (magnification, x2; scale bars, 2 mm). (d) Immunofluorescence of MLN sections for the staining of CD3 (red), PNA (green) and IgD (white) (scale 500 μm). Right, quantification of germinal center area. Data are representative of three independent experiments (a,b) and one experiment (c,d; n = 3 WT, 6 Ptenfl/flFoxp3-Cre, 2 Ifng−/− and 3 Ptenfl/flFoxp3-Cre Ifng−/− mice). Data are mean ± s.e.m. Numbers indicate percentage of cells in quadrants or gates.
Supplementary Figure 5 Analysis of Treg cells in Ptenfl/flFoxp3-Cre Ifng−/− mice and Ptenfl/flFoxp3-Cre Treg cell phenotypes under steady state and after adoptive transfer.
(a) Flow cytometry analysis of Treg cells in WT, Ptenfl/flFoxp3-Cre, Ifng−/− and Ptenfl/flFoxp3-Cre Ifng−/− mice. (b) Expression of CXCR3, CXCR5, T-bet, Bcl6, IRF4 and p-STAT3 in Treg cells from the spleen of WT and Ptenfl/flFoxp3-Cre mice; numbers above graphs indicate MFI. (c) Analysis of CXCR3 and T-bet expression in Treg cells in the spleen of WT, Ptenfl/flFoxp3-Cre, Ifng−/− and Ptenfl/flFoxp3-Cre Ifng−/− mice. (d) Analysis of Foxp3 (left) and CD25 (right) expression in PLNs and MLNs of CD45.1+ recipients after transferring WT and Ptenfl/flFoxp3-Cre Treg (CD45.2+) cells; numbers above graphs indicate MFI. Data are representative of at two independent experiments (a-d). Numbers indicate percentage of cells in gates.
Supplementary Figure 6 The transcriptional profiles controlled by PTEN in Treg cells.
(a) PCA mapping of WT and Ptenfl/flFoxp3-Cre Treg cells. (b) The list of top 10 gene sets upregulated in Ptenfl/flFoxp3-Cre Treg cells by GSEA. NES, normalized enrichment score.
Supplementary Figure 7 PTEN represses mTORC2 signaling in Treg cells to maintain immune homeostasis.
(a,b) PTEN expression in resting and short-term stimulated Treg cells (a) and phosphorylation of 4E-BP1 and PTEN in resting and long-term stimulated Treg cells (b) isolated from WT and Ptenfl/flFoxp3-Cre mice. (c) Expression of CD69, CD62L, CXCR5, ICOS and PD-1, and measurements of ROS, mitochondrial mass, and mitochondrial membrane potential (TMRM) in Treg cells from the spleen of WT, Ptenfl/flFoxp3-Cre, Rictorfl/flFoxp3-Cre and Ptenfl/flRictorfl/flFoxp3-Cre mice; numbers above graphs indicate MFI. (d,e) Flow cytometry analysis of WT, Ptenfl/flFoxp3-Cre, Rictorfl/flFoxp3-Cre and Ptenfl/flRictorfl/flFoxp3-Cre mice for CD62L and CD44 expression on CD4+ and CD8+ T cells (d), cytokine production of CD4+ T cells (e). (f) PNA staining of spleen sections (magnification, x2; scale bars, 2 mm). (g) Immunofluorescence of MLN sections for the staining of CD3 (red), PNA (green) and IgD (white) (scale 500 μm) in WT, Ptenfl/flFoxp3-Cre, Rictorfl/flFoxp3-Cre and Ptenfl/flRictorfl/flFoxp3-Cre mice (quantitative results of WT and Ptenfl/flFoxp3-Cre mice in Supplementary Fig. 4d are shown here for comparison). Data are representative of two independent experiments (a,b), three independent experiments (c-e) and one experiment (f,g; n = 2 Rictorfl/flFoxp3-Cre and 3 Ptenfl/flRictorfl/flFoxp3-Cre mice). Data are mean ± s.e.m. Numbers indicate percentage of cells in quadrants.
Supplementary Figure 8 PTEN expression and immune dysregulation in Ptenfl/+Foxp3-Cre mice and the model describing PTEN signaling in Treg cells.
(a,b) Analysis of Pten mRNA (a) and protein (b) expression in Treg, naive CD4+ (CD62L+CD44−), and CD8+ T cells from WT and Ptenfl/+Foxp3-Cre mice. Numbers below the PTEN lanes indicate band intensity relative to that of β-actin (b). (c) Immunofluorescence of spleen sections of WT and Ptenfl/+Foxp3-Cre mice for the staining of CD3 (red) and PNA (green) (scale bars, 60 μm). Right, quantification of germinal center area. (d) Images of peripheral lymph nodes from WT and Ptenfl/+Foxp3-Cre mice (~5 months old) with lymphoproliferative disease. Data are representative of two independent experiments (a-d). *P < 0.001. Data are mean ± s.e.m. (e) Schematics of PTEN signaling in Treg cell functions and immune tolerance. Loss of PTEN in Treg cells dysregulates mTORC2 signaling and metabolic and transcriptional programs, leading to the disrupted stability of Treg cells (as evidenced by the downregulation of CD25 expression, as well as other abnormalities not depicted here). Associated with loss of Treg cell stability and the ensuing TH1 responses and IFN-γ production is the aberrant induction of TFH responses, spontaneous formation of germinal center responses, and development of autoimmune and lymphoproliferative disease.
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Shrestha, S., Yang, K., Guy, C. et al. Treg cells require the phosphatase PTEN to restrain TH1 and TFH cell responses. Nat Immunol 16, 178–187 (2015). https://doi.org/10.1038/ni.3076
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DOI: https://doi.org/10.1038/ni.3076
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