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Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability

Abstract

Foxp3+ regulatory T cells (Treg cells) are required for immunological homeostasis. One notable distinction between conventional T cells (Tconv cells) and Treg cells is differences in the activity of phosphatidylinositol-3-OH kinase (PI(3)K); only Tconv cells downregulate PTEN, the main negative regulator of PI(3)K, upon activation. Here we found that control of PI(3)K in Treg cells was essential for lineage homeostasis and stability. Mice lacking Pten in Treg cells developed an autoimmune-lymphoproliferative disease characterized by excessive T helper type 1 (TH1) responses and B cell activation. Diminished control of PI(3)K activity in Treg cells led to reduced expression of the interleukin-2 (IL-2) receptor α subunit CD25, accumulation of Foxp3+CD25 cells and, ultimately, loss of expression of the transcription factor Foxp3 in these cells. Collectively, our data demonstrate that control of PI(3)K signaling by PTEN in Treg cells is critical for maintaining their homeostasis, function and stability.

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Figure 1: Characterization of Pten-ΔTreg mice.
Figure 2: Pten-ΔTreg mice develop spontaneous systemic lymphoproliferative disease with age.
Figure 3: Population expansion of PTEN-deficient Treg cells in vivo.
Figure 4: Loss of PTEN abolishes the ability of Treg cells to resolve autoimmune inflammation.
Figure 5: PTEN deficiency skews Treg cell metabolism toward glycolysis.
Figure 6: TSDR-methylation analysis.
Figure 7: Deletion of PTEN leads to instability of Treg cells in vitro.
Figure 8: Deletion of PTEN leads to instability of Treg cells in vivo.

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Acknowledgements

We thank A. Rudensky (Memorial Sloan Kettering Cancer Center) for Foxp3-YFP-Cre mice and Foxp3eGFP–Cre–ERT2Rosa26-YFP mice. Supported by the US National Institutes of Health (R56AI083304 and 01HL018646 to L.A.T.; R21AI105607 to L.A.T. and J.A.B.; T32AI007529 to C.M.B.; and P01AI56299 to P.T.S., A.H.S. and L.A.T.), the Cancer Research Institute (A.H.) and (1S10RR023440).

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Authors and Affiliations

Authors

Contributions

A.H., M.D., B.P., P.T.S., J.Q., N.T. and V.A.G. designed and performed experiments; C.M.B., J.C.R. and A.H.S. designed experiments; A.H., M.D., J.A.B. and L.A.T. designed the study; and A.H. and L.A.T., with assistance from M.D. and J.A.B., wrote the manuscript.

Corresponding author

Correspondence to Laurence A Turka.

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Competing interests

L.A.T. owns equity in and has a family member employed by Novartis.

Integrated supplementary information

Supplementary Figure 1 Deletion of PTEN does not affect the development or phenotype of Treg cells.

(a) Distribution of CD4+ and CD8+ T cell populations in thymus, spleen, pLN and mLN in Pten-ΔTreg mice. Representative of 10 experiments. (b) Expression of Treg-associated markers in Foxp3+ cells isolated from Pten-ΔTreg mice. Representative of 5 experiments.

Supplementary Figure 2 Aged Pten-ΔTreg mice develop non-malignant TH1 inflammation.

(a) Splenocytes from Foxp3-Cre, Pten-ΔTreg and diseased Pten-ΔTreg mice were isolated and assessed for germinal center GL-7+Fas+ B cells. Plots are gated on CD19+. Representative of 2 experiments. (b) Splenocytes from mice as above were isolated and analyzed for CXCR5+ICOS+ T follicular helper (TFH) and T follicular regulatory (TFR) cells. Top panels are gated on CD4+Foxp3 and bottom panels are gated on CD4+Foxp3+. n = 3 per group, representative of 2 experiments; *p = 0.002, **p < 0.05, ***p = 0.003 by one-way ANOVA. (c) Splenocytes from Foxp3-Cre and Pten-ΔTreg mice were analyzed for CXCR3 expression within the Foxp3+ (left) and Foxp3 (right) populations. Representative of 5 experiments. (d) Foxp3+ Treg kidney resident CD4+ cells from Foxp3-Cre, Pten-ΔTreg and diseased Pten-ΔTreg mice were analyzed for CD25 and Foxp3 expression. Representative of 2 experiments. (e) Markers of malignancy in were assessed in diseased Pten-ΔTreg mice. Normal TCR Vβ usage in CD4+ T cells (left) and low CD24 expression (right) indicated non-malignant disease in Pten-ΔTreg mice. Grey = thymocytes. Representative of 10 experiments.

Supplementary Figure 3 Recombination of the Pten locus at the genomic level is confined to Foxp3+ Treg cells in young, healthy Pten-ΔTreg mice.

Genomic DNA was extracted from highly pure sorted cell populations and subjected to PCR to detect wild-type Pten (Pten WT), floxed Pten (Ptenflox), and recombined Pten (PtenΔ4-5). 1: bulk splenocytes, 2: CD4+Foxp3+ Tregs, 3: CD4+CD44loCD62Lhi naïve T cells, 4: CD4+CD44hiCD62Llo activated T cells. Representative of 4 experiments.

Supplementary Figure 4 PTEN-deficient Treg cells maintain a normal Treg cell transcriptional signature.

Transcriptional analysis was performed on Foxp3+CD25+ and Foxp3+CD25 Tregs from Foxp3-Cre and Pten-ΔTreg mice double sorted to high purity. n = 3.

Supplementary Figure 5 Foxp3 expression in CD25+ and CD25 Treg cells.

Cells were isolated from the spleens of Foxp3-Cre mice, young, healthy Pten-ΔTreg mice and diseased Pten-ΔTreg mice, and assessed for expression of Foxp3 within Foxp3+CD25+ and Foxp3+CD25 Treg populations. n = 3, representative of 3 experiments; *p < 0.001, **p < 0.0001 by two-way ANOVA.

Supplementary Figure 6 Differential activation of mTORC1 and mTORC2 in PTEN-deficient Treg cells.

(a) Foxp3+CD25+ Tregs from Foxp3-Cre and Pten-ΔTreg mice were sorted to high purity and cultured in the presence of either the PI3K inhibitor LY294002, IL-2 or both LY and IL-2, and assessed expression of CD25 and Foxp3. (b) CD4+ cells were isolated from Foxp3-Cre and Pten-ΔTreg mice, stimulated with 5 μg/ml of anti-CD3 and anti-CD28 for the indicated times and assessed for pAkt Ser473 (mTORC1, top) and pS6 (mTORC2, bottom) by phospho-flow cytometry. Histograms are gated on Foxp3 (top) or Foxp3+ (bottom) cells. n = 2, representative of 3 experiments; *p < 0.001, **p < 0.05, ***p < 0.01 by two-way ANOVA. Grey = isotype control. (c) Foxp3+CD25+ Tregs from Foxp3-Cre and Pten-ΔTreg mice were sorted to high purity and cultured in the presence of either rapamycin, or either of the PI3K inhibitors LY294002 and Wortmannin, and assessed for CD25 expression. Representative of 3 experiments.

Supplementary Figure 7 Recombination of the genomic Pten locus occurs in activated T cells of diseased Pten-ΔTreg mice.

Genomic DNA was extracted from sorted cell populations from Foxp3-Cre, Pten-ΔTreg and diseased Pten-ΔTreg mice, and subjected to PCR to detect recombined Pten as in Supplementary Fig. 3. 1: bulk splenocytes, 2: CD4+Foxp3+Tregs, 3: CD4+CD44loCD62Lhi naïve T cells, 4: CD4+CD44hiCD62Llo activated T cells.

Supplementary Figure 8 Detection of Foxp3 in Pten-iΔTreg and control iFoxp3-Cre mice.

To avoid complications associated with spectral overlap of the fluorescent reporters, cells were fixed and permeabilized to maintain YFP expression and allow for intracellular staining of Foxp3 with antibody in lieu of eGFP. In the example shown here, using control Foxp3eGFP-Cre-ERT2 Rosa26-YFP mice (iFoxp3-Cre), detection of fluorescence in the FL-1 channel was specific to Rosa26-YFP (top panel, yellow), as cells without tamoxifen treatment only express GFP at low intensity (top panel, green). “+ TAM” (bottom panels) indicates one week after initiation of a 5 day course of tamoxifen as per Materials and Methods. Bottom left displays YFP fluorescence on gated CD4+ cells and bottom right displays Foxp3 Ab staining on gated CD4+YFP+ cells. Representative of 2 experiments.

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Huynh, A., DuPage, M., Priyadharshini, B. et al. Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability. Nat Immunol 16, 188–196 (2015). https://doi.org/10.1038/ni.3077

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